A comprehensive review on RNA interference-mediated targeting of interleukins and its potential therapeutic implications in colon cancer

Abstract

Colon cancer is the world’s fourth leading cause of death. It is cancer of the latter part of the large intestine, i.e. the colon. Chronic inflammation over a long period also leads to the development of cancer. Cancer in the colon region is arduous to diagnose and is detected at a later stage when it metastasizes to other parts of the body like the liver, lungs, peritoneum, etc. Colon cancer is a great example of solid tumours associated with chronic inflammation. Although conventional therapies are effective, they lose their effectiveness beyond a certain point. Relapse of the disease occurs frequently. RNA interference (RNAi) is emerging as a great tool to specifically attack the cancer cells of a target site like the colon. RNAi deals with epigenetic changes made in the defective cells which ultimately leads to their death without harming the healthy cells. In this review, two types of epigenetic modulators have been considered, namely siRNA and miRNA, and their effect on interleukins. Interleukins, a class of cytokines, are major inflammatory responses of the body that are released by immune cells like leukocytes and macrophages. Some of these interleukins are pro-inflammatory, thereby promoting inflammation which eventually causes cancer. RNAi can prevent colon cancer by inhibiting pro-inflammatory interleukins.

Introduction

Colorectal cancer, or CRC, is the third most prevalent cancer worldwide (Favoriti et al. 2016). In women, it is the second most prevalent cancer, and in men, it is the third most commonly diagnosed cancer. Roughly 9% of all cancer diagnosed is colorectal cancer worldwide (Torre et al. 2015). It is more prevalent in developed countries, being around 55% of all cancers diagnosed. The incident rates of colorectal cancer are higher in males than in females (Torre et al. 2015).

Inflammation is the body’s natural defence system’s reaction to damaging stimuli such as damaged cells, microbes, or an irritant. In chronic inflammation of the intestinal epithelial cells, individual cells are targeted by the body's defence system, where the leukocytes, immune mediators, interleukins, and cytokines promote inflammation which leads to the progression of cancer. Chronic inflammation of the intestinal epithelial cells can also lead to colorectal cancer. Pro-inflammatory interleukins like IL-1, IL-6, IL-17A, IL-33, IL-4, IL-8, IL-11, IL-22, and IL-23 are responsible for tumour progression, which ultimately leads to cancer.

Colorectal cancer is caused by mutations in the oncogenes, genes related to DNA repair mechanism, and tumour suppressor genes (Mármol et al. 2017). Conventional treatments like chemotherapy, radiotherapy, hormonal therapy, immune therapy, and surgery are available to treat colorectal cancer. These treatments work in some cases, but the majority of the cases have seen a relapse of the disease. Moreover, these treatments come along with their share of debilitating side effects. Recently, advanced treatments with fewer side effects are gaining much more importance, though there is a scope to work more on these.

RNA interference (RNAi) is a method of gene silence that occurs after the transcription of a gene and has been biologically conserved through evolution to protect against foreign double-stranded RNA, like exogenous pathogenic and endogenous parasitic nucleic acid, and also modulates the expression of protein-coding genes. This ability of sequence-specific gene silencing can be exploited well for the well-being of humankind and is currently being used in therapeutic intervention, functional genomics, agriculture, and other sectors (Han 2018). RNA interference is achieved by siRNA, miRNA, etc. Small interfering RNAs (siRNAs) are dsRNA molecules with two strands that are approximately 21 nucleotides long. They help in defending the cell from foreign nucleic acids and also contribute to the regulation of posttranscriptional gene expression by degrading the messenger RNA (mRNA) transcript complementary to the target gene (Clark and Rager 2020). Gene silencing is easily carried out by Small interfering ribonucleic acid (siRNA). They can effectively silence the gene as they have high affinity and can bind to the complementary base sequence of the target mRNA (Dykxhoorn et al. 2006). They are a powerful tool for gene knockdown which in turn will suppress tumour, cell migration, and cancer proliferation (Allahyari et al. 2021). siRNA is delivered through a carrier because of its unstable nature in the bloodstream. Safe and efficient transfection of siRNA into the cytoplasm of the cell is essential for the siRNA to act efficiently (Cho et al. 2008). Recently, synthetic siRNA has been exploited to treat various types of cancer (Hattab et al. 2021).

MicroRNA (miRNA) are single-stranded non-coding RNA molecules having approximately 22 nucleotides. They generally form a cluster and target mRNAs of a common response pathway of a cell, e.g. proliferation, apoptosis, etc. As they form a cluster, they can easily regulate the entire pathway (Tran & Montano 2017).

Interleukins are chemicals released by cells that are damaged, infected, or in stress. Activation and differentiation of immune cells are achieved by interleukins. They also have a role in cell proliferation, maturation, adhesion, and migration. During inflammatory and immune responses, interleukins regulate growth, differentiation, and activation because of their pro-inflammatory and anti-inflammatory properties (Justiz Vaillant and Qurie 2021). These are mainly synthesised and released by helper T cells, monocytes, macrophages, etc. (Fig. 1). The inflammatory cells while receiving chronic stimuli produce more pro-inflammatory interleukins which stimulate tumour-promoting events like proliferation, resistance to apoptosis, hypoxia response, angiogenesis, and metastasis.

Fig. 1.

Different types of pro-inflammatory interleukins like IL-6, IL-17A, IL-1, IL-4, IL-8, IL-11, IL-22, IL-23, IL-33, etc. produced by the inflammatory cells on being exposed to chronic stimuli which leads to tumour promoting events such as Proliferation, resistance to apoptosis, hypoxia response, angiogenesis, and finally invasion into the other tissues, thus, leading to cancer

This review discusses the effect of RNA interference therapy, targeting interleukins in colorectal cancer. By targeting and degrading the mRNAs of pro-inflammatory interleukins via RNAi, a reduction in inflammation can be achieved which in turn will inhibit the further development of colon cancer due to chronic inflammation.

Suppressing tumour growth by targeting IL-6

One of the most dominant cytokines present in the tumour microenvironment is IL-6. It is produced by tumour cells, and this helps the tumour cells to grow, proliferate, survive, invade, metastasise, be angiogenic, and be resistant to drugs (Masjedi et al. 2018). In multiple cancers, it has been reported that there is an increase in the level of IL-6, both locally and systemically (Kozłowski et al. 2003). An increased amount of IL-6 leads to an increase in tumour size, advancement in tumour stages, and metastasis, and this, in turn, has a low survival rate in colorectal cancer patients (Galizia et al. 2002a, b).

Cancer cells have exploited various immune suppression mechanisms and have successfully evaded the immune system because of which anticancer immunotherapy is ineffective (Vijayan et al. 2017). Expression of cytokine CD73 occurs through the binding of the promoter of the NET5 gene. The activation of the IL-6/STAT3 pathway has been recently studied, which became the basis of the selection of the genes in this study (Zeng et al. 2020). Antitumour response of the immune cells can be promoted by suppressing cancer progression which is achieved by using siRNA packaged in nanocarriers to target CD73 and IL-6 or by delivering other molecules along with them. Hyaluronate-N,N,N-trimethyl chitosan nanoparticles were synthesised and encapsulated with siRNA against IL-6 and STAT3. This blocked the IL-6/STAT3 pathway, thereby inhibiting cancer cell progression, angiogenesis, migration, etc. (Masjedi et al. 2020). Folic acid-conjugated chitosan lactate nanoparticles were synthesised which co-delivered anti-CD73 siRNA and dinaciclib and halted tumour progression (Hallaj et al. 2020). siRNA against IL-6 was co-delivered with BV6 siRNA via hyaluronate-conjugated PEG chitosan–lactate nanoparticles which showed increased expression of apoptosis gene and downregulation of genes related to migration and metastasis (Salimifard et al. 2020). TAT peptide–chitosan–superparamagnetic iron oxide nanoparticles (SPION) were used to co-deliver siRNA against CD73 and (hypoxia-inducible factor 1, subunit α) HIF-1α which successfully inhibited cancer progression (Hajizadeh et al. 2020). Therefore, it can be concluded that RNA interference (RNAi) of CD73 and IL-6 can be a therapy for the prevention of cancer (Kozielski et al. 2019).

IL-6 causes the development and progression of tumours during inflammation (Grivennikov and Karin 2011). Apoptosis resistance is a great hurdle that is faced when therapeutic approaches against tumour growth are concerned. Upregulation of Inhibitors of apoptosis (IAPs) is one of the major factors which a cancer cell uses to evade apoptosis. There are all 8 members of IAPs, among which CIAP1, CIAP2, and XIAP are of utmost importance as they inhibit the caspase proteins (Nikkhoo et al. 2019). Recently, it has been reported that BV6, which is the bivalent second mitochondria-derived activator of caspase (SMAC) mimic, promoted cell death in many cancer cell lines (Nikkhoo et al. 2020). In cancer cells, it suppresses metastasis and angiogenesis. Therefore, S. Salimfard et al. targeted IL-6 via siRNA and also encapsulated BV6 in a nanoparticle. This combination therapy was able to suppress tumour growth and also increase survival time in tumour (CT26 (colon) and 4T1 (breast)) bearing mice (Salimifard et al. 2020). So, suppressing IL-6 expression through siRNA inhibits tumour growth and development into colon cancer (Fig. 2).

Fig. 2.

Silencing of IL-6 via siRNA in the epithelial cells of the colon where it enters the cell through endocytosis. After escaping the endosome, it forms a RISC-mRNA complex, cleaving the targeted mRNA thereby achieving gene silencing and inhibiting proliferation of colon cancer

Sirtuin 1 (SIRT1) knockdown suppresses IL-1β

One of the major pro-inflammatory cytokines which are abundant in the tumour microenvironment is interleukin 1β (IL-1β). The oncogenic behaviour of IL-1β has been demonstrated by several studies (Song et al. 2003). It has been observed that IL-1β is overexpressed in colon adenocarcinoma (Elaraj et al. 2006). Immune cells like dendritic cells, macrophages, neutrophils, and monocytes produce IL-1β on being stimulated by inflammatory stimuli (Jang et al. 2021). Tumour growth, invasiveness, metastasis, and angiogenesis are promoted when IL-1β signalling is hyperactive (Jang et al. 2021; Chen et al. 2020). Attenuation of tumour progression and enhanced tumour suppressive immunity is observed when IL-1β signalling is inhibited (Apte et al. 2006). IL-1 receptor (IL-1R) is the receptor for active IL-1β, which binds to it after getting released and triggers signalling pathways that helps with the progression of tumour invasion, immune evasion, and malignancy (Litmanovich et al. 2018). In the treatment of human neutrophils with IL-1β, there was a transient elevation in the expression of the IL-1β gene as well as the half-life of the IL-1β mRNA (Mantovani et al. 1991). Adaptive response to environmental stress, ageing, and cellular senescence, all these physiological events have SIRT1 common in them; it is a mammalian deacetylase that is dependent on NAD⁺. The production of SIRT1 is greater in colon cancer cells. IL-8 and IL-6 are some pro-inflammatory interleukins that are produced due to the upregulation of SIRT1 which is induced by IL-1β. J. Jung et al. have successfully prevented the nuclear accumulation and phosphorylation of c-Jun which is induced by IL-1β by the knockdown of SIRT1 via siRNA (Jung et al. 2021). Since IL-1β and SIRT1 regulate the expression of one another reciprocally, knockdown of SIRT1 by siRNA leads to the downregulation of IL-1β.

Downregulating IL-4 prevents tumour progression

In the formation and progression of CRC, the inflammatory milieu around the tumour plays a critical role (Todaro et al. 2006). CRC metastasis is induced by a variety of invading leukocytes as well as other cells in the microenvironment that produce pro-inflammatory cytokines (Todaro et al. 2007). Cytokines such as interleukin (IL)-6, IL-8, IL-13, and IL-17 have a role in the pathogenesis of CRC metastasis (Leon-Cabrera et al. 2017; Li et al. 2008; Zhang et al. 2006; Fang et al. 2015). In CRC cells that express the IL-4 receptor (IL-4R), activation of IL-4 lowers the production of the cell adhesion molecule epithelial (E-)cadherin, suggesting that the cytokine stimulates growth by IL-4R, while a reduction in cell growth and enhanced cell death may be involved in the epithelial–mesenchymal transition (EMT) (Vizcaíno et al. 2015). IL-4 initiates signal transduction by binding to IL-4R, which involves activating the Janus kinase (JAK)1 and JAK3, which phosphorylates and dimerises signal transducer and activator of transcription 6 (STAT6). Lastly, STAT6 homodimers reach the nucleus and bind to the promoters of IL-4-responsive genes, resulting in a wide range of biological effects, and inducing a variety of biological actions (Hedrick et al. 2016). As a result, via modulating signal transduction, STAT6 regulates the cell-mediated response to IL-4 activation. However, IL-4-induced aberrant STAT6 activation enhances pro-metastatic activities in cancer cells, such as migration and infiltration, growth, and mortality. The IL-4/STAT6 signalling system, for example, by increasing the nuclear survivin pool, colorectal cancer stem cells (CR-CSCs) can resist cell death (Wierstra 2008). Moreover, Liu et al. (2008) reported that the IL-4/STAT6 signalling cascade increases nicotinamide–adenine dinucleotide phosphate oxidase 1 expression, which enhances CRC cell proliferation. Although (E2F transcription factor 1) E2F1 is known to be a powerful apoptosis driver in all forms of human cancer after chemotherapy-induced DNA damage, research suggests that E2F1 is linked to cancer development. Increased E2F1 levels in cancer cells have been found to activate cytokine receptor signalling pathways, triggering invasion and metastasis. E2F1 expression was observed to alter the aggressive phenotype of CRC cells with high E2F1 expression when it was knocked down. E2F1 was discovered to have a new role as an enhancer in the IL-4/STAT6 signalling pathway, in this study. The results supported the idea that E2F1 can enhance STAT6 production by increasing the expression of specificity protein 3 (SP3), a transcription activator of the STAT6 gene seen in CRC cells. A strong reaction to IL-4 stimulation was followed by an increase in total STAT6 protein, as shown by a good level of phosphorylation of STAT6. Ultimately, as targets of activated STAT6, zinc finger E-box-binding homeobox (Zeb)1 and Zeb2 increased EMT and aggressiveness of CRC cells. In HCT116 and HT-29 cells, knockdown of E2F1 by siRNA significantly reduced the expression of STAT6 at the transcription and translational level, whereas STAT6 was shown to be significantly increased in RKO and DLD1 cells following E2F1 overexpression (Murata et al. 1996). IL-4 appears to decrease cancer-directed immunosurveillance and promote tumour spread, according to several lines of data. IL-4, for example, can boost autocrine development in pancreatic cancer cells while still inhibiting immunological responses via paracrine activities on invading immune cells (Cao et al. 2016). Furthermore, it has been discovered that malignant or cancerous cells from the lung, bladder, and colon secrete IL-4, imparting resistance to cell death caused by chemotherapy (Terzić et al. 2010; Shawki et al. 2018). Resistance to oxaliplatin in CR-CSC cells is dependent on autocrine IL-4 production (Wang and Karin 2015). It was identified in the current study that the procured EMT-like properties of numerous colorectal cancer cell lines are activated by IL-4. However, other colorectal cancer cell lines (RKO and HCT116) with differing amounts of STAT6 expression responded to IL-4 in very different ways. STAT6, a significant signalling transducer in the IL-4 circuit, may have an impact on the aggressiveness of CRC cells produced by IL-4. A deficiency of STAT6 was previously found to reduce inflammation and prevent the early stages of colitis-related CRC (Ullman & Itzkowitz 2011). Additionally, as HT-29 cells with an active phenotype of STAT6 were compared to Caco-2 cells with a deficient phenotype of STAT6, the HT-29 cells exhibited more aggressive metastasis than the Caco-2 cells had (Waldner et al. 2012). Furthermore, knocking down STAT6 in HT-29 cells lowers growth and causes apoptosis (Lee et al. 2012). As a result, the STAT6 level may be a determinant of CRC progression aided by inflammatory cytokines. E2F1, a crucial transcription factor for colorectal cancer formation that promotes CRC cell motility and invasion, was the subject of a prior study (Barderas et al. 2012). In the present investigation, the concentration of STAT6 protein was discovered to be linked with E2F1 in several CRC cell lines. STAT6 transcription is E2F1-dependent, according to additional in vitro investigations (Murata et al. 1996). SP3, a part of the Specificity protein (SP) transcription factor family, is involved in the expression of the STAT6 gene. SP transcription factors, particularly SP3, were discovered to be upregulated in cancers, although SP levels in non-tumour tissues are often low (Hyun et al. 2012). Oncogenes like survivin, p65 (NF-κB), B-cell lymphoma 2, EGFR, c-MET, and other receptor tyrosine kinases are triggered by SP transcription factors which in turn are associated with tumour development, survival, migration, angiogenesis, and invasion, according to functional studies (Kanai et al. 2000). SP1 and SP3, which are overexpressed in CRC cells, are essential for migration and invasion (Koller et al. 2010). SP1 and SP3 share more than 90% DNA sequence similarity within their DNA-binding domains and bind to the same DNA region with similar affinity (Bankaitis and Fingleton 2015), while, in the present investigation, SP3 rather than SP1 was discovered to promote the activation of transcription for STAT6 gene in colorectal cancer cells. More critically, elevated SP3 expression appears to be dependent on E2F1, supporting the E2F1/SP3/STAT6 axis as a potential STAT6 signalling regulation route in colorectal cancer. IL-4R and IL-13R1 subunits are found on top of the surface layer of several solid tumours. IL-13 may bind to and activate IL-4R, even though IL-4 has a greater propensity for binding (Di Stefano et al. 2010). In human CRC cells, previous investigations have shown that IL-4 and IL-13 initiate a cascade of signals which turns on the JAK/STAT pathway (especially STAT6) (Liu et al. 2017). By transactivating (Zinc Finger E-Box binding Homeobox 1) ZEB1, a very well-studied EMT core regulator that drives cancer progression, IL-13/STAT6 signalling causes the aggressive characteristics of HT29 and SW480 cells, according to a recent work by Cao et al. (Chen et al. 2018a, b). EMT-related transcription factors such as Slug, Zeb1, Zeb2, Twist1, and Snail1 had their expression patterns studied. Even though STAT6 operates as a shared transducer in IL-4 and IL-13 signalling, it also induces different expression patterns of downstream EMT drivers in response to IL-4 and IL-13 stimulation (Murata et al. 1996). Thus, the knockdown of IL-4 via siRNA will induce cell apoptosis and restrict colon cancer growth.

Inhibiting IL-8 effects RNF183 and NF-kB expression

The RING finger (RNF) protein family is a broad set of proteins that each have a 40–60 amino acid RING finger domain (Nakamura 2011). Over two hundred RNF family genes have been reported, several of which are engaged in pathological and biological processes (Ho et al. 2014). Numerous members of the RING finger family have been implicated in carcinogenesis and its development.

RNF183 is increased in IBD patients' intestinal epithelial cells and TNBS-induced colitis mice (Yu et al. 2016). In CRC cell lines and tissues, RNF183 is abnormally overexpressed. RNF183's ability to induce cell migration is reduced when NF-kB is inhibited with an inhibitor which is a small molecule when IL-8 is depleted using siRNA. RNF183 overexpression enhances tumour cell proliferation, invasion, and metastasis, whereas RNF183 knockdown tests showed the opposite, showing that RNF183 functions as an oncogene in the pathological process of colorectal cancer. RNF183 was found to be overexpressed in both inflammatory bowel disease and colorectal cancer, suggesting that RNF183 plays a role in the transition from inflammation to malignancy. IL-8 production was boosted by RNF183 in CRC cells in an E3 ubiquitin ligase-dependent manner. The transcription factors NF-kB and AP-1 (Collins et al. 2000) govern the expression of IL-8 and they discovered that RNF183 enhances the amount of P65 which is an NF-kB transcription factor as well as its abundance at the IL-8 promoter in colorectal cancer cells. RNF183's tumour-promoting function is eliminated when NF-kB is inhibited and IL-8 is depleted, according to their findings (Geng et al. 2017). Downregulation of IL-8 using siIL8 will result in cell apoptosis and inhibit cell migration of colon cancer cells.

Silencing IL-10 stimulates EBV lytic infection

A gammaherpesvirus known as the Epstein-Barr virus (EBV), thrives in epithelial cells and human B lymphocytes. EBV infection occurs in two phases: latent and lytic, and primary lytic infection typically results in long-term latent infection (Küppers 2003; Taylor and Blackbourn 2011). During latency, EBV only expresses a few genes, including the latent membrane protein LMP1, LMP2A, and short RNA EBERs (Hammerschmidt and Sugden 2013). The two viral immediate–early gene products, BZLF1 (also called ZTA or Z) whose expression can cause EBV to reactivate from latent to lytic phase BRLF1 (called RTA or R) (Kenney and Mertz 2014). EBV-positive tumours are virtually entirely made up of cells that have a latent EBV infection. Death of tumour cells occurs when the transformation of a latent Epstein-Barr virus infection into a lytic form takes place (Feng et al. 2002; Westphal et al. 2000). As a result, techniques for inducing lytic EBV infection in tumour cells are all being investigated as prospective EBV-positive tumour treatment (Feng et al. 2002; Feng et al. 2004; Ambinder et al. 1996; Gutiérrez et al. 1996).

The influence of the Epstein-Barr virus in colon cancer is well known, ranging from 4 to 18 per cent of gastrointestinal carcinomas in numerous studies (Liu et al. 2003). Even though there are numerous similarities in pathogenesis and histology in both gastric and colorectal carcinomas, there have been few studies examining the link between EBV and colorectal cancers. However, an abundance of research shows an aetiologic role for EBV in cancer development in EBV-positive patients. In addition, recent studies have shown that lymphoblastoid cell lines transformed by the Epstein-Barr virus exhibit different methylation patterns than peripheral blood leukocytes. Epstein-Barr virus-expressed transcripts can activate the proto-oncogene c-myc, resulting in cellular damage in a variety of methods including cell cycle regulation, metabolism, protein synthesis, apoptosis, cellular connections, and angiogenesis; this broad spectrum of ramifications results in the activation of colorectal cancer (Karpinski et al. 2011; Liu et al. 2003). In a new study, Liu and colleagues used PCR to detect Epstein-Barr virus in patients with colorectal cancer in China. EBV DNA was detected in Twenty-six samples of One hundred and thirty cases of colorectal cancer, and EBV prevalence was diagnosed to be higher among men with cancer than women. They also identified Epstein-Barr virus carcinogenic factors in colorectal cancer (Liu et al. 2003). Using PCR, carcinomas, polyps, and non-cancerous tissues were examined for the presence of EBV DNA in this study. Sixty per cent of colorectal carcinoma specimens contained viral DNA, compared to thirty-five per cent of colorectal polyps and forty per cent of the non-malignant control group (Abdulhassan et al. 2021).

IL-10 is a multifunctional cytokine generated by a variety of cells. IL-10 expression is high in EBV-associated tumour lesions in previous research (Pachnia et al. 2017; Galizia et al. 2002a, b). The presence of a high amount of IL-10 in the tumour microenvironment is expected to play a role in Epstein-Barr virus latency and Epstein-Barr virus-related malignancies. Initially, IL-10 inhibited both mitogen and recall Ag-induced T cell proliferation and (interferon) IFN release, as well as Th1 and cytotoxic reactions against the dormant protein LMP1 (Marshall et al. 2003). Secondly, to Epstein-Barr virus-infected B cell lines, IL-10 acts as an autocrine growth factor, and neutralisation of IL-10 can drastically reduce EBV-infected B cell lines' proliferation in vitro (Stuart et al. 1995; Beatty et al. 1997). Furthermore, IL-10 could be instrumental in the development and persistence of EBV latency in EBV-infected cells by stimulating the LMP1 protein, which is required for B cell transformation and proliferation in vitro (Kis et al. 2006). Researchers found that knocking down IL-10 via siRNA caused MAPK-NF-κB signalling pathways. Additionally, it has been observed in a mouse model, knocking down IL-10 combined with the chemotherapeutic drug Doxorubicin to activate EBV and treat EBV-positive lymphomas. IL-10 knockdown increased EBV lytic infection in both B-cell and epithelial cell lines. The PI3K-p38 MAPK-NF-κB axis can trigger EBV lytic infection when IL-10 is knocked down. Furthermore, they discovered that siIL-10 induces VEGF-A, which is crucial for PI3K activation and lytic infection of EBV. It was discovered that suppressing IL-10 increased the in vitro and in vivo efficacy of Doxorubicin in killing EBV-positive tumour cells (Gao et al. 2019). Thereby, it can be concluded that siIL-10 knockdown causes the death of colon cancer cells since EBV lytic cycle is stimulated.

MicroRNAs and their role in silencing the gene

A set of single-stranded, phylogenetically conserved small non-coding Ribonucleic acids (RNAs) of 19–25 nucleotide long, it adheres to a target mRNA sequence’s 3'-untranslated region (UTR) and regulates the post-transcriptional gene expression by inhibiting translation or enhancing mRNA degradation are known as microRNAs (miRNAs) (Bartel 2004; Zeitels et al. 2014). Physiological processes such as the development of the immune system and its function are regulated by the miRNAs (Garo and Murugaiyan 2016). Since miRNAs can regulate the extent of inflammation, inflammatory diseases such as inflammatory bowel disease and cancer can be treated with the help of miRNAs (Kalla et al. 2015; Li et al. 2016). MiRNAs are now being studied as possible biomarkers that could bring fresh insights into CRC biology as well as potential treatment options (Wang et al. 2017).

Silencing of IL-17 inhibits MAPKs and NF- κB

Several cancers like colorectal cancer (CRC) are caused due to chronic inflammation which promotes their initiation and progression (Rhodes and Campbell 2002; Francescone, Hou and Grivennikov 2015). Recently, it has been found that Interleukin 17 (IL-17), an inflammatory cytokine has been involved with both sporadic and inflammation-associated colon cancer models, including human CRC (Murugaiyan and Saha 2009; Chae et al. 2010; Kathania et al. 2016; Wang et al. 2014; Wang et al. 2009; Correction 2011; Chung et al. 2013; Wu et al. 2009). A negative correlation has been reported between the elevated levels of IL-17 to the survival of CRC patients. High level of IL-17 has been also linked to the resistance of CRC patients to classical cytotoxic drugs and also to the targeted therapeutics (Wang et al. 2014; Correction 2011; Chung et al. 2013; Lotti et al. 2013). CRC development and progression are promoted by IL-17. The IL-17 receptor (IL-17R) signalling pathway in the intestinal epithelial cells (IECs) promotes the growth and survival of premalignant tumours by activating mitogen-activated protein kinases (MAPKs) and the nuclear factor κ-light-chain-enhancer of activated B (NF- κB) (West et al. 2015). The microenvironment in the gut consists of γδ⁺ T cells, CD4⁺ Th17 cells, and innate lymphoid cells (ILCs), which produce IL-17 (Mucida and Salek-Ardakani 2009). In response to microbial products, myeloid cells like macrophages, dendritic cells produce cytokines like interleukin-1β, IL-23, and IL-6 which induce IL-17 (Ermann et al. 2014). Secretion of IL-17 enhances cytokines by the dendritic cells following the NOD2 signalling pathway (Chae et al. 2013).

miR-146a is a polymorphic miRNA. It has been reported that CRC patients have altered levels of miR-146a (Omrane et al. 2014; Zeng et al. 2014; Garo et al. 2021). IL-17 modulation via miR-146a has been identified to have a critical role in the prevention of colonic inflammation and tumorigenesis. Two interlinked mechanisms are followed by miR-146a to prevent intestinal inflammation and CRC, those are: (a) in IECs tumorigenesis is inhibited by IL-17R and (b) it also limits the production of IL-17 by the myeloid cells. CRC and inflammation of the colon can be ameliorated by the direct inhibition of miR-146a or by the administration of miR-146a mimic. RIPK2 and TRAF6 are the targets of miR-146a, inhibiting these targets will nullify the susceptibility of a person to CRC (Taganov et al. 2006). miR-146a can control NOD2 signalling and is also a suppressor for toll-like receptor (TLR)-mediated inflammation (Garo and Murugaiyan 2016; Quinn et al. 2013). CRC development and progression in tumour cells is modulated by miR-146a as it regulates PGE2. It has been reported that the rate of survival of CRC patients is worse when they have elevated levels of PGE2 and Cox-2, which is an upstream inducer of PGE2 (Grivennikov and Karin 2011). Therefore, we can say that tumour growth and proliferation can be limited by miR-146a. IL-17 can be downregulated by miR146a which further inhibits tumour growth and proliferation in colon cancer.

IL-6 induces EMT pathway

IL-6 is a pro-inflammatory cytokine that promotes tumour and is produced by immune cells and carcinomas (Knüpfer and Preiss 2010). The decreased survival rate of CRC patients, increase in the tumour size, and metastasis; all have been linked with elevated levels of IL-6 in the serum (López-Novoa and Nieto 2009). Inflammation for a prolonged period promotes epithelial-to-mesenchymal transition (EMT), where the phenotypes of the cells switch between epithelial and mesenchymal; the reverse of this process is the mesenchymal-to-epithelial transition (MET) of the cells (Kalluri and Weinberg 2009)(Lujambio and Lowe 2012).

We know that miRNAs play an important role in regulatory functions like proliferation, invasion, metastasis, and apoptosis (Brabletz 2012). By promoting MET and reversing EMT, miRNAs from the miR-200 and miR-34 families suppress metastasis (Hermeking 2012; Hermeking 2007). p53 strongly induces the family members of miR-34 (Siemens et al. 2011). MiR-34a and miR-34b/c suppress the production of the EMT-inducing transcription factor (EMT-TF) SNAIL, thus suppressing the EMT pathway (Kim et al. 2011) and (Iorio and Croce 2012). miRNAs directly or indirectly target the transcription factors which they regulate, therefore forming a feedback loop (Ebert and Sharp 2012; Ptashne 2009). DNA sequences remain the same when there is a change in the epigenetic state, the expression pattern, and the cellular phenotypes, enabling this kind of self-assembling links to be the main component of epigenetic switches (Hitchler and Domann 2009). Inflammation acts as a stimulus for initiating the event of an epigenetic switch, after which the new cells inherit the same phenotype even in the absence of the stimuli and thereafter are maintained by the self-stabilising feedback loop (Rokavec et al. 2012). It has been reported that this type of epigenetic switching promotes cancer initiation (Iliopoulos et al. 2009; Yu et al. 2009). STAT3 can be activated via multiple pathways like the deregulated receptor tyrosine kinases in cancer cells; therefore, there may be a difference in the stimulation of IL-6R/STAT3/miR-34a loop in the tissue and cellular level (Thiery et al. 2009). It has been reported that the cells forming the primary tumour can switch from epithelial to mesenchymal cells which helps them to migrate and then they again switch back to epithelial cells and initiate a new tumour on a new region (Tsai et al. 2012; Rokavec et al. 2014). IL-6 induces IL-6R/STAT3/miR-34a loop activation which in turn induces EMT, thereby shifting the cellular phenotype from epithelial to mesenchymal aiding in the process of invasion, intravasation, and extravasation for the cells to metastasize. p53 interferes with this loop and induces MET, thereby turning the cells back into epithelial cells for colonisation, outgrowing metastasis. Since the IL-6R/STAT3/miR-34a loop is reversible, several treatments aimed at preventing its pro-metastatic activity are possible (Christoffersen et al. 2010). Apart from p53, ELK-1 can also directly induce miR-34a and the same can be inhibited by HSF-1 and STAT3 (Feng et al. 2014; Xiong et al. 2012). Because of the circuit's positive feed-forward character, it can remain active indefinitely once triggered. While the components of the circuit are interrelated in terms of constitutive activation, each component activates its very own collection of downstream genes, all of which contribute to cancer growth. Numerous identified targets of the IL-6R/STAT3/miR-34a loop components, including STAT3's capacity to directly activate the EMT activator ZEB1, might well be important for cancer progression (Lyons et al. 2008). Moreover, it has been reported that SNAIL, a well-known EMT inducer, is a direct miR-34a target (Rose-John 2012)(Becker et al. 2004). SNAIL was found to be activated by IL-6, suppressed by knockdown of IL-6R or STAT3, and was found in significant concentrations in MiR34a^–/– mouse tumours. SNAIL also causes the production of interleukin 6, supplying further responses that may perpetuate the loop (Matsumoto et al. 2010). The above-mentioned findings imply that SNAIL plays an essential role in the IL-6R/STAT3/miR-34a loop. The s–IL-6R, which includes IL-6 trans-signalling and is likewise controlled by miR-34a, was shown to be overexpressed in the mesenchymal cells in colorectal cancer cell lines, which solely contain tumour cells (S. Grivennikov et al. 2009). During tumour formation in the AOM/DSS CAC mice model, m–IL-6R expression declines while s–IL-6R expression increases (Dmitrieva-Posocco et al. 2019)(Q. Zhang et al. 2017a, b). In mice with mir34a-deficiency, it was observed that an even greater concentration of s–IL-6R, implying that miR-34a inhibits s–IL-6R and thereby slows tumour progression.

Interleukin-6 and IL-1b, which are produced by macrophages and neutrophils, in mutant cells, trigger their receptor signalling pathways, increasing the changed clone's survival and proliferative potential (Grivennikov and Karin 2010; Yang et al. 2013). Researchers discovered that STAT3 and nuclear factor κB (NF-κB) signalling in the cells of the immune system and intestinal epithelial cells in the lamina propria (LP) play critical roles in the start and progression of CRC. NF-κB forms a bond with a group of proteins that is inhibitory in nature known as the Inhibitor of κB (IκB) family, which includes IκBa, in unstimulated cells. The initial stage in nuclear factor κB activation is for the Inhibitor of κB kinase (IKK) to phosphorylate IκB, which leads to NF-κB breakdown (Kota et al. 2009). Nuclear factor κB activation in the lamina propria myeloid cells, primarily macrophages, could result in the release of pro-inflammatory cytokines, which can attract additional cells of the immune system and induce inflammation in the intestine. All the recruited inflammatory cells stimulate the production of a range of cytokines, including IL-6, IL-11, and IL-22, which activate the proliferation of premalignant intestinal epithelial cells. These cytokines promote proliferation in IECs by activating the STAT3 pathway, which then works in tandem with NFκB to boost the production of proliferation and survival genes (Zhao et al. 2013). The colon, placenta, thymus, small intestine, and testes are just a few of the tissues where miR-26a is found. Across all vertebrates, miR-26a is entirely conserved. It functions as a tumour suppressor in myc-induced lymphoma (Witwer et al. 2010), colon cancer (Fasseu etal. 2010), hepatocellular carcinoma (Ji et al. 2009), nasopharyngeal carcinoma (Yu et al. 2013), breast cancer (B. Zhang et al. 2011), lung cancer (Li et al. 2014), and other cancers, as well as a tumour promoter in cholangiocarcinoma (Zhang et al. 2012) and glioma (Huse et al. 2009). MiR-26a is also involved in the regulation of inflammatory signals. In human and macaque cells, miR-26a may affect the reaction of the innate immune system to viral infections by directly regulating IFNβ expression (Zhang et al. 2021). In the macrophages present in rats, miR-26a inhibits the expression of Toll-like receptor 3 (TLR3) and reduces the severity of pristane-induced arthritis in rats. In hepatocarcinoma, modulation of signalling pathways associated with IL-6 and NF-kB is connected to the downregulation of miR-26a (Yang et al. 2013). Other research discovered that miR-26a levels are higher in the mucosa of CD and UC patients, implying that miR-26a might play a role in colitis (Chen et al. 2016). miR-26a inhibited the phosphorylation of STAT3 and IkB-a by LPS, implying that miR-26a serves a role in decreasing pro-inflammatory cytokine secretion by adversely regulating STAT3 and NF-kB signalling (Jiang et al. 2014). By targeting IL-6, miR-26a reduced STAT3 and NF-kB signalling in macrophages (Zeitels et al. 2014). A segment in miR-26a adheres to the 3' UTR (Untranslated region) of IL-6 in HCC cells, and this binding region is the same that has been observed earlier. miR-26a inhibits TNFα-mediated IL-6 activation by negatively regulating the NF-kB-related proteins MALT1 and HMGA1 (Du et al. 2020). miR-26a directly interacts with TLR3 and reduces the production of downstream cytokine in NR8383 cells, a macrophage cell line generated from normal rat alveolar macrophage cells (Gracie et al. 2003). According to studies, miR-26a lowers colon cancer by reducing (phosphatase and tensin homolog) PTEN in the intestinal epithelium (Uhlen et al. 2017). MiR-26a reduces oral keratinocyte death by directly targeting (protein kinase c-delta) PKCδ (Yoshimura et al. 2001). PTEN, PKCδ, and TLR3 production suppress the increase in vulnerability to CRC and colitis, according to a dual-luciferase reporter experiment that was conducted. The impact of suppression of miR-26a in CRC and colitis was identified by its direct targeting of PTEN, IL-6, PKCδ, and TLR3 (Gatault et al. 2015). Both miR-34a (Fig. 3) and miR-26a inhibit IL-6 production by targeting different pathways which result in the slowing down of tumour progression in colon cancer.

Fig. 3.

Silencing of IL-6 via miR-34a in colon epithelial cells. The miRNA is endocytosed into the cell and when it escapes the endosome it forms a complex with RISC-AGO-mRNA, degrading the targeted mRNA, achieving translational repression leading to reduced tumour growth and proliferation

Enhancement of the activity of NK cells by IL-18

The interleukin-1 superfamily consists of IL-18 and its regulatory role in the innate and acquired immune systems is becoming more widely recognised (Christensen et al. 2013). IL-18 production is predictive in colorectal cancer, with high levels of IL-18 supporting its function in immune system control and gastrointestinal tract protection (Angius et al. 2019). Recombinant IL-18 has been shown to have antitumor action, but it has also been shown to have antitumor activity with the IL-18 receptor (IL-18 R), IL-18 receptor accessory protein (IL-18 RAP), and IL-18 binding protein (IL-18BP). IL-18 R and RAP enhance the antitumor effect of IL-18, whereas IL-18BP is a secretory antagonist that binds to IL-18 and suppresses its function. Although recombinant IL-18 can affect anticancer activity by activating NK and T cells (Ashraf et al. 2017), other investigations have revealed that IL-18 can have an immediate antitumour effect on cancer cells (Li et al. 2019).

miR-362-3p suppresses the progression of the cell cycle by targeting protein tyrosine phosphatase non-receptor type 1 (PTPN1), upstream transcription factor 2 (USF2), and E2F1, which are all related to the relapse of CRC (Zhang et al. 2019), and miR-425-5p has been shown to play a role in the aetiology of Kristen rat sarcoma viral oncogene homolog (KRAS)-mutated CRC, increasing tumour aggressiveness (Zhou et al. 2013). MiRNA-574-3p has been linked to a variety of cancers, including epithelial ovarian cancer, prostate cancer, and colorectal cancer (Zhang et al. 2017a, b; Lee and Margolin 2011; Dinarello et al. 2013). In gastric cancer, TGF-1 can cause considerable overexpression of miR-574-3p (Li et al. 2021; Mizuno et al. 2018). IL-18 has a critical role in immune system modulation and immune response acceleration (Le et al. 2017). IL-18, in particular, can trigger natural killer cells to kill cancerous cells, rendering it a possible cancer immunotherapy target (King et al. 2011). Due to its extensive transcription in numerous cell types throughout the gastrointestinal tract, scientists explored the possible anticancer activity of IL-18 in colorectal cancer and observed that IL-18 can also stimulate NK cells to suppress cell growth and cause cell death in colorectal cancer. Li et al. discovered five miRNAs that were influenced by IL-18 therapy after evaluating miRNA production in HCT116 cells after and before co-cultivation with natural killer cells and administration of interleukin-18 (Xu et al. 2016). Let-7c is the most studied miRNA in colorectal cancer, having been found in C. elegans (Wang et al. 2017). In CRC, as well as other malignancies such as breast cancer, ovarian cancer, and pancreatic cancer, let-7c is normally downregulated (Li et al. 2016). Let-7c has been associated with cell proliferation, cell cycle regulation, tumour cell motility, and chemoresistance in previous studies. Reduced expression of let-7c, in particular, has been linked to a grim prognosis in individuals with colorectal cancer, suggesting that let-7c functions as a tumour suppressor (Tezcan et al. 2019). Moreover, decreased expression of let-7c in mice caused a spike in tumour occurrence (Li et al. 2016), demonstrating that let-7c takes part in the proliferation of cancer in the intestine of mice. Following co-culturing with natural killer cells and interleukin-18 therapy, let-7c was further downregulated (Xu et al. 2016). PCR experiments have previously shown that TGF-1 promotes the production of miR-574-3p in gastric cancer cells (Mizuno et al. 2018). SMAD4 was discovered to be involved in the expression of miR-574-3p. TGF-1 production was considerably negatively regulated when miR-574-3p was highly expressed, even though there was no notable change in miR-574-3p expression when TGF-1 was abundantly expressed, suggesting that co-culturing with natural killer cells and interleukin-18 therapy may have activated an alternative regulatory pathway in HCT116. The SMAD4 signalling pathway is among the most well-learned downstream pathways of miR-574-3p activity (Feng et al. 2018). In vitro, IL-18 therapy paired with NK cells caused apoptosis and reduced cell growth. Furthermore, after co-culturing with natural killer cells and treatment with interleukin-18, miRNA expression profiling indicated significant suppression of miR-574-3p production. Similarly, Zhou et al. analysed miRNAs from 847 gastrointestinal cancer patients and discovered that miR-574-3p was downregulated (Li et al. 2021). TGF-1 has been shown to greatly increase the production of miR-574-3p (Li et al. 2021; Mizuno et al. 2018). SMAD4 knockdown significantly decreased miR-574-3p overexpression which is induced by TGF-1 in AGS cells, according to Zhang et al. (Mizuno et al. 2018). The miR-574-3p/TGF-1 axis boosts natural killer cells' antitumor potential when co-cultured with HCT116 cells. Interleukin -18 did not affect HCT116 cells alone, but it increased the anticancer activity of natural killer cells in co-culture with HCT116 cells via the miR-574-3p/TGF-1 axis, implying that IL-18 might be a potential target for colorectal cancer immunotherapy (Xu et al. 2016).

IL-1β activates NLRP3

miR-22, a 22-nt long miRNA, is encoded on human chromosome 17p13.3. miR-22 has been shown to function as a suppressor of tumour proliferation in pancreatic and breast cancer, and it has been expressed in a downregulated manner in various cancer lines. It has also been shown to delay tumour progression by influencing growth, migration, and infiltration, and it may be used to treat breast and cervical cancer symptoms (Feng et al. 2018). Furthermore, the production of miR-22 in colorectal cancer tissues is less when compared to that of normal tissue, and silencing of hypoxia-inducible factor 1 was reported to stop CRC progression. NLRP3 (recombinant NLR family, pyrin domain-containing protein 3) is accountable for the development and production of pro-inflammatory cytokines such as interleukin-1 β (IL-1β) and IL-18 as a result of endogenous or pathogenic signals (Liu et al. 2018). In gastric cancer and oral squamous cell carcinoma, according to research, miR-22 directly binds to the 3′ untranslated regions (3′-UTR) of NLRP3 to inhibit cell growth (Olive 2012; Carneiro et al. 2009). MiR-22, through attacking HuR, suppresses cell migration and proliferation in CRC cells in vivo and in vitro studies (Davis et al. 2011). Pattern recognition receptors (PRRs), which have been linked to the function of several inflammasomes, assist the immune system in coping with some other kinds of degradation (Mangan et al. 2018). NLR (nucleotide-binding domain and leucine-rich repeat-containing) proteins are key PRRs that play a significant role in the body (Chung et al. 2019). The inflammasome-modulated innate immune function, which leads to the activation of the IL-1 family of cytokines, is activated by the NLRP3 inflammasome (Shao et al. 2020). Increased NLRP3 inflammasome stimulation has been shown to promote tumour spread in CRC in recent studies (Nayak et al. 2021). It was reported that when miR-22 was overexpressed, NLRP3 expression was low, but when miR-22 was silenced, NLRP3 expression was high, demonstrating that miR-22 effectively suppressed NLRP3 expression. NLRP3 was verified to be a miR-22 target gene using dual-luciferase reporter experiments. NLRP3 is linked to tumour growth, TNM stage, infiltration, and metastasis of lymph nodes in CRC, possibly through activation of the process of EMT (Rutz et al. 2014). MMPs are a major protease family that regulates extracellular matrix turnover and are associated with chronic inflammation (Cong et al. 2020). Cong et al. also looked at the expression of MMP-2, e-cadherin, MMP-9, N-cadherin, and vimentin to see how miR-22 affects MMPs and EMT in CRC by targeting NLRP3. miR-22 overexpression lowered vimentin, MMP-9, N-cadherin, and MMP-2 protein quantity by regulating NLRP3 (Rutz et al. 2014). Thus, miR-22 can regulate IL-1β production by inhibiting NLRP3 which hampers the growth and development of colon cancer.

IL-20 regulates STAT3

IL-19, IL20, IL22, and IL24 are included in the interleukin-20 (IL20) cytokine subfamily, that controls signals via the same heterodimeric receptors (IL20 receptor -subunit (IL20Rα), IL20 receptor -subunit (IL20Rβ), IL10 receptor -subunit (IL10Rβ), and IL22 receptor 1 subunit (IL22Rα1) (Lee et al. 2013). Epithelial and immunological cells have a lot of these receptors. They principally regulate STAT3 and the downstream JAK-STAT signalling pathway. The cytokines of the IL20 subfamily have been found to enhance malignant cell growth, movement, infiltration, and remodelling (Mo et al. 2016; Mo et al. 2015).

In humans, the chromosomal region Xq28 encodes MicroRNA 452 (MIR452, also known as miR- 452), which is grouped with miR-224 within the gamma-aminobutyric acid. A receptor epsilon subunit (GABRE) gene. miR452 has been reported to be elevated in both colitis (J. S. Mo et al. 2019) and CRC tissues in recent research (Benderska et al. 2015). By decreasing vascular endothelial growth factor A (VEGF-A) production, it affects cell growth, migration, and angiogenesis in colorectal cancer tissues (Veerla et al. 2009). miR-26B could be used as a biomarker for colorectal inflammatory processes. This is because miR-26B expression has increased in patients with ulcerative colitis-associated colorectal carcinoma (UCC) and ulcerative colitis (UC), but it is controlled in sporadic colorectal cancer (Fonseca-Camarillo et al. 2014). In urothelial carcinoma (Veerla et al. 2009), oesophageal cancer (Liu et al. 2013), and colorectal cancer (Veerla et al. 2009), miR-452 expression was shown to be significantly higher. Using dual-luciferase reporter assays, miR-452 was found to be a direct target of IL20RA. IL20RA is an interleukin receptor for IL19, IL20, and IL24 that regulates tissue homeostasis, autoimmune disorders, host defence, and oncogenesis (Chen et al. 2018a, b). IBD has been linked to elevated IL19, IL20, and IL24 transcription and protein levels in previous investigations (Fonseca-Camarillo et al. 2014; Fonseca-Camarillo et al. 2013; Andoh et al. 2009). MIR452 overexpression dramatically reduced the amounts of IL20Rα mRNA and protein in CRC cells, according to the study. The results of siIL20Rα therapy in CRC cells are consistent with the previously reported results. These findings suggest that IL20Rα levels in CRC tissues are inversely linked with MIR452 levels. When compared to healthy tissues, the expression of IL20Rα was considerably reduced in CRC tissue. IL19, IL20, and IL24 all display signals via the same receptors for their biological processes on their target tissue. As a result, it is fair to believe that IL20Rα is involved in the regulation of inflammatory bowel disease and colorectal cancer oncogenesis. MIR452 overexpression dramatically reduced the expression of JAK1, STAT1, and STAT3 proteins in colorectal cancer cell lines, mice colitis, and CRC tissues. In CRC cells, the colitis model, or CRC tissues, however, siIL20Rα did not affect STAT1 expression. These findings suggest that the IL20Rα pathway regulates STAT3, but not STAT1. As a result, STAT proteins may play a dual role in the initiation and progression of cancer. STAT3's role in various types of cancer may be receptor-specific and perhaps dependent on its target.

Depending on MIR452 and siIL20RA transfection in colorectal cancer cells, their findings show that STAT1 and STAT3 have distinct expression patterns. Through the IL20RA and JAK1 pathways, the STAT3 protein is anticipated to have a key role in regulating proliferation, migration, apoptosis, metastasis, and angiogenesis. In CRC cell lines, however, siIL20RA did not affect STAT1 expression. MIR452 regulates the JAK1/STAT1 signalling pathway in colorectal cancer cells directly or indirectly, regardless of its particular gene (IL20Rα) receptor route, according to the findings (Lamichhane et al. 2021). miR-452 targets IL20Rα and regulates the progression of colon cancer.

siRNA and miRNA were synthesised to target different interleukins (Table 1) that inhibits colon cancer progression.

Table 1.

Overview of RNA interference-mediated components targeting specific interleukins in colon cancer

S. no.	Type of RNAi (siRNA/miRNA)-mediated gene silencing	Targeted interleukins	Protective effects on colon cancer	References
1	siRNA	IL-8	Prevents proliferation and metastasis	(Ning et al. 2011)
2	siRNA	IL-22	Protects from inflammation	(Ziesché et al. 2007)
3	siRNA	IL-6	Cancer progression, angiogenesis, and migration	(Masjedi et al. 2020)
4	siRNA	IL-4	Inhibits progression and migration	(J. Chen et al. 2018a, b)
5	siRNA	IL-8	Decrease in tumour growth and proliferation	(Conciatori et al. 2020)
6	miRNA	IL-17	Limits proliferation and metastasis	(Garo et al. 2021)
7	miRNA	IL-6	Inhibits inflammation	Zhang et al. (2021)
8	miRNA	IL-18	Promoted antitumour ability of NK cells	(Y.-P. Li et al. 2021)
9	miRNA	IL-1β	Supresses cell proliferation	(Cong et al. 2020)
10	miRNA	IL-20R	Reduces cancer progression, chronic inflammation and pathogenesis	(Lamichhane et al. 2021)

Delivery methods for RNA interference therapy

The commonly used techniques for delivering RNAi are viral transduction and lipid-mediated transfection. It varies with the type of cell line being investigated and also if stable or transient knockdown is favoured to determine which of these methods to employ. The most prevalent application is the employment of cationic lipid-based particles for transient transfection of unmodified siRNAs or altered RNAi complexes since they are best suited for carrying RNAi molecules to a wide variety of cell lines. Viral vectors are frequently used for cell types that cannot be transfected via lipid-mediated methods. Adenoviral vectors are effective for transient delivery in many cell types; nevertheless, lentiviral vectors are the optimal delivery method for challenging cell lines, such as non-dividing cells, and for stable RNAi expression. Recently, polymers are also being used to deliver RNA molecules.

In Table 2, lipid and polymer-based delivery complexes are used for delivering siRNA targeting interleukins in colonic inflammation. These complexes have different administration routes, namely oral, intravenous, and intra-rectal.

Table 2.

Therapeutic mechanisms and delivery methods of siRNA-based drug formulations in colonic inflammation

siRNA targeted specific markers	Anti-inflammatory mechanisms of targeted siRNAs	Mode of administration of siRNA drug formulations	Delivery complex	References
Interferon regulatory factor 8 (IRF8)	siRNA against IRF8 has been introduced in RAW 264.7 cells via lipid-based nanoparticles. The downregulation of IRF8 in turn reduces the secretion of TNFα, IL-6, and IL-12/23, thereby reducing the inflammation in inflammatory bowel diseases which may lead to colon cancer	Intravenous administration	Lipid-based nanoparticles	(Veiga et al. 2019)
The cluster of differentiation 98 (CD98)	siRNA targeted against CD98 was encapsulated in PEI-loaded nanoparticles, which successfully reduced colonic inflammation	Oral administration	Poly-lactic acid (PLA) nanoparticles	(Laroui et al. 2014)
p65	Silica-coated calcium phosphate nanoparticles were synthesised which encapsulated p65 siRNA which attenuates NF-κB and subsequent proteins, to alleviate ulcerative colitis	Intravenous administration	Silica-coated calcium phosphate nanoparticles	(Müller et al. 2022)
Enhancer of zeste homolog 2 (EZH2)	siRNA against EZH2 was introduced in colon cancer cell line and downregulation in the expression of EZH2 was observed while upregulation in necroptotic marker, RIP1, and RIP3	Intra-rectal administration	Lipofectamine 3000 nanoparticles	(Lou et al. 2019)
TNFα	TNFα siRNA loaded in galactosylated chitosan PLGA (GCP) nanoparticles were administered orally. Downregulation of TNFα reduced inflammation in DSS-induced colitis in mice	Oral administration	Galactosylated chitosan PLGA (GCP) nanoparticles	(Huang et al. 2018)

miRNAs targeting different interleukins (Table 3) can be delivered either intravenously, intra-colonic, intra-tumoral, etc. with lipofectamine, polymers as delivery vehicles.

Table 3.

Therapeutic mechanisms and delivery methods of miRNA-based drug formulations in colonic inflammation

miRNA targeted specific markers	Anti-inflammatory mechanisms of targeted miRNAs	Mode of administration of miRNA drug formulations	Delivery complex	References
Yes-associated protein 1 (YAP)	miR-590-5p reduces colonic inflammation also inhibits cell proliferation, and migration, and induces apoptosis, thereby suppressing colon cancer, by targeting YAP	Tail vein administration	Lipofectamine 2000	Yu et al. (2016)
Interleukin 6 (IL-6)	miR-26a inhibits NF-κB/STAT3 pathway, suppressing the production of the pro-inflammatory cytokine, IL-6, which in turn reduces inflammation and subsequent colon cancer progression	Intraperitoneal administration	LNA-anti-miR26a was delivered directly	Zhang et al. (2021)
Cellular- Master Regulator of Cell cycle entry and proliferative metabolism (c-myc)	miR-145 targeted c-myc which inhibited tumour growth in colon cells	Intra-tumoral administration	PLGA/PEI/miRNA-145/HA ((poly(d,l-lactide-co-glycolide)/polyethylenimine) Hyaluronic acid) complex	Li et al. (2018)
Ras Homologous A (RhoA)	miR-31-3p is introduced in colon cells through the help of an adenovirus. It targets the RhoA gene and inhibits inflammation in colon cells	Intra-colonic administration	Anti-miRNA was directly delivered	Fang et al. (2018)
pro-apoptotic protein Bcl2-interacting protein (BIM)	miR-148a was knocked down in Th1 cells which resulted in the alleviation of inflammation	Intravenous administration	Direct delivery of anti-miRNA	Maschmeyer et al. (2018)

Owing to their negative charge and high molecular weight, RNAs cannot infiltrate colon cancer cells. This barrier can be circumvented by utilising delivery mechanisms. Multiple delivery techniques are now being utilised to enhance the effectiveness of RNA therapies. The kinds of delivery vectors are viral as well as non-viral. Viral vectors, like lentiviral, adenovirus, retroviral, and adeno-associated viruses, have a significant transfection efficiency, but their usage is limited by possible cytotoxicity, insertional mutagenesis, and immunogenicity (Thomas et al. 2003). Non-viral vectors based on nanoparticles are dendrimers, liposomes, inorganic nanoparticles, and polymersomes, which offer a viable option for RNA carriers in therapies due to them having low pathogenicity, cost-effectiveness, and ease of manufacture in large quantities (Mintzer and Simanek 2009). Owing to their diameters, nanoparticles can infiltrate tumour tissue in a passive manner. Furthermore, the improved penetration and retention effect permits prolonged agglomeration inside cancerous tissue, hence enhancing the efficacy of the treatment (Fang et al. 2011). For the therapy of colorectal cancer, targeting is crucial. Death receptor 5 (DR5, also known as TRAIL receptor 2) (Perraud 2011), Carcinoembryonic antigen (CEA) (Tiernan et al. 2013), epithelial growth factor receptor (EGFR) (Repetto et al. 2005), folate receptor-α (FR-α), and tumour-associated glycoprotein (TAG)-72 (Kim et al. 2008; Repetto et al. 2005) are the most frequently utilised biomarkers in CRC. Widespread use has been made of the conjugation of ligands to the surface of nanocarriers for the targeted delivery of RNA therapies to CRC cells. Pi et al.(2018) revealed that folate-displaying extracellular vesicles (EVs) can efficiently inhibit colorectal cancer growth in a patient-derived colorectal cancer xenograft mice model by delivering survivin siRNA to CRC cells. Kim et al. have created anti-TAG-72 PEG-immunoliposomes (PILs) to target CRC cells that overexpress TAG-72. Anti-TAG-72 PILs rapidly accumulated in tumour tissues after intravenous treatment, demonstrating that immunoliposomes have considerable promise as gene delivery vehicles for human CRC cells. RNAi-based Nanocarriers can be improved in the aspect of their immunogenicity and target substrate by surface functionalisation and using biocompatible natural polymers.

According to a clinical trial conducted on patients with a solid tumour, a lipid nanoparticle known as ALN-VSP0 that contains siRNA against vascular endothelial growth factor (VEGF) and kinase spindle protein (KSP) has antitumor activity (Tabernero et al. 2013). TKM 080301, a lipid nanoparticle containing anti-siRNA Pololike Kinase 1 (PLK1), exhibited antitumor properties in clinical trials with adrenocortical carcinoma (ACC) tumours (Demeure et al. 2016). In clinical trials, the lipoplex particle Atu027, which contains siRNA against protein kinase 3, was found to inhibit the growth of prostate and pancreatic solid tumours (Schultheis et al. 2014).

Conclusion

Pro-inflammatory interleukins are responsible for the inflammation of a particular tissue like the colon. These interleukins if present in a larger quantity than required and also for a long time eventually lead to the formation of cancer which later metastasises and invades other tissues in the body. Many treatments in terms of antibodies specific to particular interferon are there, but are highly costly and have side effects as well. Therapies that target these proteins, miRNAs, or siRNAs, which have yet to be completely investigated, may be implicated in reducing tumour microenvironment invasion, dissemination, and transformation into CSCs. Although studies into the use of small RNAs as medicinal interventions have extended the preclinical and clinical applications, small RNA-targeted therapy, including miRNAs, is hard due to broad off-target effects. Onpattro (patisiran) was recently licenced by the FDA for polyneuropathy in patients with familial amyloidosis, marking the first FDA approval for a siRNA medication. Similarly, adults with acute hepatic porphyria are treated with givosiran. Acute hepatic porphyria is an uncommon hereditary condition that can result in life-threatening nervous system assaults. As a result, miRNAs, siRNAs, and newly discovered regulatory proteins could be used as possible targeted drug delivery systems in future clinical trials.

Future prospects

RNA interference therapy has come to the limelight because of the ease of manipulation of genes which was very difficult earlier. RNAs being sensitive and easily degradable in the body has made it a challenge to be used fervently. This challenge, however, is being overcome by the use of nanotechnology. Nanotechnology along with medicinal chemistry is coming together to meet the immediate demand. More research and clinical trials should be conducted for RNA interference therapy and implied in the field of medicine. Colon cancer or colorectal cancer has become so prevalent, the cases are increasing day by day. Thus, we require effective treatments which are specific with fewer side effects, unlike conventional cancer therapies. Delivering siRNA, miRNA, etc. via some delivery vehicles like nanoparticles made of polymer, peptide, etc. can be more targeted and effective for such a debilitating disease.

Author contributions

All authors contribute to the conceptualisation of the article. The literature search and the final draft of the manuscript was carried out by Sagari Sil. The outline of the review was prepared by Janet Bertilla and approved by S. Rupachandra.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Data Availability Statement

The raw/processed data required to reproduce these findings cannot be shared at this time as the data also forms part of our future research study.

Declarations

Conflict of Interest

The authors have no conflicts of interest to declare that are relevant to the content of this article.

Ethical Approval

This article does not contain any studies with human participants or animals performed by any of the authors.