Hsa_circ_0005571 promotes the proliferation and invasion of colorectal cancer cells

Abstract

Dysregulated expression of circular RNAs (circRNAs) has been implicated in the initiation and progression of various diseases, including cancer. In this study, we investigated the role of hsa_circ_0005571 in modulating the biological characteristics of colorectal cancer (CRC) cells. Initially, the circRNA expression profiles of CRC tissues and corresponding normal tissues were analyzed using bioinformatics to identify differentially expressed circRNAs. Subsequently, quantitative real-time PCR (qRT‒PCR) was used to validate the expression levels of hsa_circ_0005571 in both CRC tissues and cell lines. In vitro assays, including the Cell Counting Kit-8 (CCK-8), colony formation, 5-ethynyl-2′-deoxyuridine (EdU) incorporation, wound healing, and Transwell invasion assays, were employed to assess the effects of hsa_circ_0005571 on CRC cell proliferation and invasion. Additionally, tumorigenic potential was examined through in vivo tumorigenesis experiments in nude mice. Bioinformatic predictions and experimental evidence indicated that hsa_circ_0005571 may function as a sponge for miR-520f-3p, which was found to be downregulated in CRC cells. Moreover, LRP8 was predicted and confirmed as a direct downstream target of miR-520f-3p, with both the mRNA and protein levels of LRP8 significantly elevated in CRC cells. Overall, our results suggest that hsa_circ_0005571 enhances CRC proliferation and invasion by modulating the miR-520f-3p/LRP8 axis. Furthermore, elevated hsa_circ_0005571 expression in CRC tissue samples and cell lines relative to that in normal controls was positively correlated with metastasis (p = 0.029) and advanced clinical stage (p = 0.006). Survival analyses revealed that CRC patients with high levels of hsa_circ_0005571 expression had significantly lower overall survival rates than did those with low expression (p < 0.05). Functional assays confirmed that hsa_circ_0005571 facilitates CRC cell proliferation and migration, whereas its knockdown results in reduced migration, proliferation, and tumorigenic potential in vivo. Finally, Western blot analyses demonstrated that LRP8 expression varied in accordance with the levels of hsa_circ_0005571 and miR-520f-3p, further confirming the involvement of the hsa_circ_0005571/miR-520f-3p/LRP8 regulatory axis in CRC. Our study suggested that hsa_circRNA_0005571 promotes proliferation and migration through the miR-520f-3p/LRP8 axis in CRC. Consequently, hsa_circ_0005571 may represent a promising novel gene target for the diagnosis and treatment of CRC.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-16450-2.

Introduction

CRC is an invasive neoplasm that affects the colon or rectum and is among the most prevalent gastrointestinal malignancies globally¹. As living standards and lifestyles have evolved, the global incidence of CRC has risen markedly, with approximately 1.48 million new cases reported each year. Mortality rates, particularly in developing nations, continue to increase annually². Despite significant advancements in modern diagnostic and therapeutic techniques, including early detection of CRC, surgical interventions, and chemotherapeutic treatments, the five-year survival rate for patients with metastatic CRC remains approximately 14%^3,4. Consequently, there is an imperative need to investigate the molecular mechanisms of CRC further to increase survival rates, a pursuit that constitutes a central focus of contemporary CRC research.

CircRNAs represent a distinct class of noncoding RNAs characterized by their circular conformation, which notably lacks both a 3′ polyadenylated tail and a 5′ cap structure⁵. Due to technological advancements, numerous functional circRNAs characterized by attributes such as abundance, endogenous origin, stability, specificity, and conservation have been identified in mammals⁶. Studies have shown that circRNAs are involved in not only normal physiological processes but also pathological conditions, including cardiovascular diseases and cancers⁷. They modulate gene expression through the sequestration of microRNAs, the binding of functional proteins, or the coding of proteins⁸. For example, circHERC4 inhibits CRC progression via the miR-556-5p/CTBP2/E-cadherin pathway⁹, whereas circPLCE1 is downregulated in CRC and may attenuate CRC progression by facilitating the ubiquitin-dependent degradation of RPS3¹⁰. CircMETTL15 acts as a ceRNA for miR-374a-5p to regulate ESCO2 expression, thereby promoting the biological behavior of LoVo cells¹¹. CircSATB1 facilitates the RNF25-mediated ubiquitylation and degradation of FKBP8, releasing its inhibitory effects on mTOR signaling¹². The distinct circular conformation of circRNA confers resistance to ribonucleases, facilitating its detection in human blood, tissues, and metabolic waste¹³. Although research on the role of circRNAs in tumorigenesis is expanding, the regulatory network of circRNAs in CRC remains inadequately understood¹⁴. Further research into circRNAs is warranted to evaluate their potential as biomarkers and therapeutic targets for CRC.

Recent studies have increasingly highlighted the clinical potential of circRNAs as noninvasive biomarkers in CRC. Owing to their covalently closed structure, circRNAs exhibit remarkable stability in body fluids such as blood and exosomes, making them ideal candidates for liquid biopsy applications. For example, hsa_circ_0005075 was found to be significantly upregulated in the plasma of patients with CRC¹⁵. Prognostically, a meta-analysis involving 1430 patients with CRC revealed that abnormally expressed circRNAs were correlated with tumor diameter (P = 0.0350), differentiation (P = 0.0038), lymphatic metastasis (P = 0.0119), distant metastasis (P < 0.0001), TNM stage (P = 0.0002), and depth of invasion (P = 0.001) in patients with CRC¹⁶. These advancements underscore the dual utility of circRNAs as diagnostic tools and predictors of therapeutic response.

In this study, we identified the circular RNA hsa_circ_0005571, which is located at genomic coordinates 19:18285849–18286507, utilizing bioinformatics analysis. qRT‒PCR analysis revealed elevated expression levels of hsa_circ_0005571 in CRC tissues, their corresponding adjacent noncancerous tissues, and associated cell lines. We subsequently assessed the role of hsa_circ_0005571 in the proliferation and invasion of CRC cells. Through biological analysis, we found that hsa_circRNA_0005571 binds specifically to miR-520f-3p, which is consistent with the binding site of LRP8 on miR-520f-3p. By experimentally exploring this mechanism, our study demonstrated that hsa_circRNA_0005571 promotes proliferation and migration through the miR-520f-3p/LRP8 axis in CRC. These results suggest that hsa_circ_0005571 has potential as a biomarker for the diagnosis and treatment of CRC.

Materials and methods

Bioinformatics analysis

A total of 42 pairs of CRC tissues and their corresponding adjacent normal tissues were collected from patients undergoing surgery at the Affiliated Hospital of Nantong University. None of the patients had received radiotherapy, chemotherapy, or immunotherapy prior to tumor resection, and informed consent was obtained from each participant. The samples were cryogenically preserved at temperatures below − 80 °C for subsequent analytical procedures.

This study was approved by the Ethics Research Committee of the Affiliated Hospital of Nantong University.

CRC tissue samples

A total of 42 pairs of CRC tissues and their corresponding adjacent normal tissues were collected from patients undergoing surgery at Affiliated Hospital of Nantong University. None of the patients had received radiotherapy, chemotherapy, or immunotherapy prior to tumor resection, and informed consent was obtained from each participant. The specimens were cryogenically preserved at temperatures below − 80 °C for subsequent analytical procedures.

This study was approved by the Ethics Research Committee of Affiliated Hospital of Nantong University.

Cell culture and transfection

The normal human colorectal cell line NCM460, along with the human CRC cell lines SW480 and HCT116 and the MSI-high (LoVo) and MSS (HT29) cell lines, were obtained from the Biological Sample Bank at the Affiliated Hospital of Nantong University. The cells were maintained in culture at 37 °C with 5% CO₂ in a thermostatically controlled incubator. Small interfering RNAs (siRNAs) designed to specifically target hsa_circ_0005571 (designated si-circ_0005571#1: 3′-GCCACTAACAGGCAATCTA-5′ and si-circ_0005571#2: 3′-CACTAACAGGCAATCTATA-5′), along with a corresponding negative control (si-NC), were obtained from Ruibo Company, Guangzhou, China. The transfection procedure was conducted using Lipofectamine RNAiMAX (Invitrogen).

RNA extraction and qRT‒PCR

Total RNA was extracted from CRC tissues and cells using TRIzol reagent (Invitrogen) according to the manufacturer’s protocol. RNA was converted to complementary DNA (cDNA) through reverse transcription using a PrimeScript RT Kit (Invitrogen). qRT‒PCR was performed on a 7500 Real-Time PCR system (Applied Biosystems). The relative expression of hsa_circ_0005571 was calculated using the 2^-ΔΔCt method. The sequences of the primers used were as follows: hsa_circ_0005571, forward: 5′-GGAGAGAAGTCTGCCACTAACAGG-3′, reverse: 5′-CACAGTGCTTCATAGTAGAGGGTCA-3′; miR-520f-3p forward: 5′-GAATTCGCCTCAAGAGAACAAAGTGGAG-3′, reverse: 5′-AGATCTCCCATGGGGGCTCAGCCC CT-3′; LRP8 forward: 5′-CTGTGGCGACATTGATGAGTG-3′; reverse: 5′-TCTTGGTCAGTAGGTCCATCTC-3′; and GAPDH forward: 5′-TGAAGGTCGGAGTCAACGGATTTGGT-3′, reverse: 5′-CATGTGGGCCATGAGGTCCACCAC-3′.

EdU assay

In accordance with the RiboBio EdU kit instructions, the transfected cells were resuspended in a 96-well plate, and 100 µl of diluted EdU working solution was added to a final concentration of 10 µM. Two hours later, 4% paraformaldehyde was added to fix the cells, and then Apollo staining solution was added to the 96-well plate in the dark. Finally, the labeled cells were observed under a fluorescence microscope (Olympus).

Cell proliferation assay

The transfected cells (HCT116 and SW480) were resuspended, mixed, and seeded into a 96-well plate at a density of 2 × 10³ per well. After incubation for the indicated time, the cells were harvested, treated with 10 µl of CCK-8 solution and 90 µl of medium per well, and cultured for an additional 4 h. The OD value of each well was then measured at 450 nm using a microplate reader at the specified time point.

Colony formation assay

CRC cells transfected with siRNAs (2 × 10³ cells/well) were seeded into 6-well plates and gently swirled to ensure even distribution. The cells were cultured for 2–3 weeks to allow colony formation. After the medium was removed, the colonies were washed with PBS, fixed with 4% paraformaldehyde, and stained with 0.1% crystal violet (Beyotime) for 15 min. Excess dye was removed by washing the plates with distilled water. The plates were then air-dried, and the colonies were quantified under an Olympus phase-contrast microscope.

Wound-healing assay

CRC cells were seeded in 6-well plates and cultured until they reached approximately 95% confluence. A sterile 200 µL pipette tip (0.5 mm width) was used to create a scratch wound in the cell monolayer. Images of the scratch were captured using a light microscope 12 h after wounding. For quantitative analysis, scratch closure was measured using automated macro scripts in ImageJ. To ensure objectivity, images were also analyzed independently by two blinded researchers using a MATLAB-based MASK-RCNN algorithm, which demonstrated strong interobserver agreement (> 95% concordance). The assay was performed in three independent replicates.

Transwell invasion assay

Twenty-four-well plates were precoated with Matrigel (Invitrogen). CRC cells were seeded in the upper chamber of the Transwell insert (8-µm pores), and antigen-free complete medium was added to the lower chamber. The Matrigel concentration was optimized through gradient validation experiments. Briefly, inserts were coated with 0.5, 1.0, or 2.0 mg/mL Matrigel diluted in serum-free medium. CRC cells (HCT116/SW480; 2 × 10⁵ cells/well) were then added to the upper chamber and allowed to invade for 24 h. The 1.0 mg/mL coating provided optimal discrimination between invasive and noninvasive cells. Higher concentrations (≥ 2.0 mg/mL) caused overpolymerization, physically blocking cell penetration. Conversely, lower concentrations (≤ 0.5 mg/mL) inadequately mimicked the physiological basement membrane barrier. After 24 h, the cells were fixed with 4% paraformaldehyde for 30 min, stained with 0.1% crystal violet, and rinsed thoroughly with PBS to remove residual dye. Invasion was assessed by light microscopy (Olympus).

In vivo assay

Four-week-old male BALB/c nude mice were purchased from the Animal Experimentation Centre of Nantong University. The HCT116 cells used in our study were treated with si-circ0005571 or si-NC. A total of 5 × 10⁶ treated cells in suspension were administered via subcutaneous injection into the flank of each mouse. The mice were housed in a specific pathogen-free (SPF) environment and were randomly allocated into two groups, with each group consisting of three mice (n = 3 per group). Tumor volume assessments commenced on the seventh day following inoculation and were conducted at weekly intervals thereafter. The volume was calculated using the modified ellipsoid formula: volume (mm3) = a⋅b2/2. After 35 days, the mice were euthanized through cervical dislocation while under 5% isoflurane anesthesia to facilitate subsequent analysis, during which the tumors were excised and weighed. All the measurements were performed by two independent researchers unaware of the group allocations. This study was approved by the Animal Ethics Committee of the Affiliated Hospital of Nantong University. All the experiments were performed in accordance with the relevant guidelines and regulations.

Western blot

Cells transfected with siRNA were harvested and lysed in RIPA lysis buffer (Solarbio, Beijing, China) to extract total protein. The protein concentration for each group was determined using a BCA protein quantification kit (Solarbio) according to the manufacturer’s instructions. Proteins were separated by 10% sodium dodecyl sulfate‒polyacrylamide gel electrophoresis (SDS‒PAGE) and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was blocked with nonfat milk and then incubated overnight with primary antibodies against LRP8 (Sangon Biotech, Shanghai, China). The membrane was subsequently incubated with the appropriate species-matched secondary antibody. GAPDH (Sangon Biotech) served as the loading control.

Statistical analysis

All continuous data are presented as the means ± standard deviations (SDs) and were analyzed with GraphPad Prism 9.0. For categorical and qualitative variables expressed numerically, the χ2 test was employed and analyzed with SPSS 20.0 software. Differences in the expression of hsa_circ_0005571 among clinical samples were evaluated using paired t tests, and one-way ANOVA followed by the least significant difference (LSD) test was used to analyze the differences among three or more groups. The mutual correlations between circ-0005571, miR-520f-3p and LRP8 were explored by Pearson’s correlation analysis. Statistical significance was defined as p < 0.05.

Results

Bioinformatics analysis of circRNAs in CRC

Differentially expressed circRNAs identified by the three comparative methods were intersected, and corresponding gene annotations were retrieved from the circBase database (http://www.circbase.org/). Table 1 summarizes these results, and a Venn diagram was constructed to illustrate the overlap: 21 differentially expressed circRNAs were identified, consisting of 12 downregulated, 7 upregulated, and 2 showing inconsistent regulation (Fig. 1A). A heatmap of intersecting circRNAs was generated using the ggplot package (Fig. 1B). Based on these findings, hsa_circ_0005571 was selected for further investigation.

Table 1.

Common difference circRNA obtained by comparing microarray data in three ways.

Gene	Position (genone browser link)	Strand	Gene symbol	Trend
hsa_circ_0004826	chr6:144808683–144814592	+	UTRN	Change
hsa_circ_0017636	chr10:7285519–7327916	−	SFMBT2	Change
hsa_circ_0000199	chr1:243708811–243736350	−	AKT3	DOWN
hsa_circ_0000437	chr12:109046047–109048186	−	CORO1C	DOWN
hsa_circ_0001326	chr3:111632165–111639266	+	PHLDB2	DOWN
hsa_circ_0003380	chr8:106431371–106456609	+	ZFPM2	DOWN
hsa_circ_0004228	chr4:5664833–5667376	−	EVC2	DOWN
hsa_circ_0005328	chr6:163876310–163899928	+	QKI	DOWN
hsa_circ_0005600	chr17:57430575–57430887	+	YPEL2	DOWN
hsa_circ_0005830	chr4:905460–907456	−	GAK	DOWN
hsa_circ_0006867	chr4:151388824–151412187	−	LRBA	DOWN
hsa_circ_0008016	chr8:38287199–38287466	−	FGFR1	DOWN
hsa_circ_0008732	chr9:16727794–16738483	−	BNC2	DOWN
hsa_circ_0009022	chr18:9583114–9595100	−	PPP4R1	DOWN
hsa_circ_0005571	chr19:18285849–18286507	+	IFI30	UP
hsa_circ_0008002	chr14:50130032–50141145	−	POLE2	UP
hsa_circ_0008197	chr1:51032748–51061888	−	FAF1	UP
hsa_circ_0084927	chr8:95676924–95677424	+	ESRP1	UP
hsa_circ_0085173	chr8:102570646–102571040	+	GRHL2	UP
hsa_circ_0120174	chr2:47600601–47601187	+	EPCAM	UP
hsa_circ_0120479	chr2:58311223–58316858	+	VRK2	UP

Fig. 1.

Abnormal upregulation of circRNAs in CRC. (A) Venn diagram illustrating the abnormal expression of circRNAs in the CRC datasets (GSE138734 and GSE116589). (B) Heatmap in which each cell reflects the expression level of each circRNA in the sample. Expression levels correlate with color intensity: red indicates upregulated circRNA expression, while green indicates downregulated expression.

Identification and characterization of hsa_circ_0005571 in colorectal cancer

The gene structure and circular conformation of hsa_circ_0005571 (Fig. 2A,B, respectively) were obtained from the Cancer-Specific CircRNA Database (CSCD) database (http://gb.whu.edu.cn/). Its spliced sequence and a schematic illustrating its structural relationship with the IFI30 gene (Fig. 2C) were retrieved from the circBase and CircularRNA Interactome databases (https://circinteractome.nia.nih.gov/).

Fig. 2.

Gene information for hsa_circ_0005571 and its gene structure. (A) The gene structure of hsa_circ_0005571. (B) The red triangle marks the microRNA response element (MRE), which acts as a miRNA sponge and serves as the binding site for circRNA and miRNA. The blue cross represents the RNA binding protein (RBP) binding site, which may indicate a role for circRNA in protein regulation. The green strip denotes the open reading frame (ORF) of hsa_circ_0005571, suggesting a potential link to circRNA’s translational function. (C) Schematic diagrams illustrating hsa_circ_0005571 derived from the IFI30 gene.

Expression of Hsa_circ_0005571 in CRC

The relative expression level of hsa_circ_0005571 was significantly elevated in CRC tissues compared with adjacent nontumor tissues (Fig. 3A). Elevated hsa_circ_0005571 levels were correlated with advanced TNM stage and lymph node metastasis (Fig. 3B,C). To investigate its clinical relevance, we analyzed the associations between hsa_circ_0005571 expression and clinicopathological characteristics in patients with CRC (Table 2). Patients were stratified into high- and low-expression groups based on tumor tissue level. Kaplan–Meier survival analysis with log-rank tests revealed significantly reduced survival in the high-expression cohort (Fig. 3F). Furthermore, the expression of hsa_circ_0005571 was markedly greater in CRC cell lines (HCT116, SW480, LoVo, and HT29) than in normal colorectal epithelial cells (NCM460) (Fig. 3D). These findings suggest that hsa_circ_0005571 overexpression may promote CRC progression. To functionally validate this observation, we performed knockdown experiments in which hsa_circ_0005571 was targeted in HCT116 and SW480 cells (Fig. 3E).

Fig. 3.

Hsa_circ_0005571 is upregulated in CRC. (A) Relative expression of hsa_circ_0005571 in CRC tissue samples. (B) TNM stage. (C) Lymph node metastasis. (D) Upregulation of hsa_circ_0005571 expression in CRC cell lines. (E) Knockdown of hsa_circ_0005571 in CRC cells. (F) Kaplan-Meyer survival analysis was performed in patients with high versus low expression of CRC. Data were expressed as mean ± SD. *p < 0.05, **p < 0.01,***p < 0.001.

Table 2.

Associations between hsa_circ_0005571 expression and clinicopathological features of CRC patients.

Factor	Quantities	hsa_circ_0005571 expression		χ2	P value
		High	Low
Aggregate	42	25	17
Gender				0.038	0.845
Male		13	9
Female		11	8
Age				0.032	0.859
< 60		8	5
≥ 60		17	12
Tumor size				4.356	0.037*
< 5 cm		11	13
≥ 5 cm		14	4
Site of tumor				0.514	0.474
Colon		16	9
Rectum		9	8
Depth of infiltration				8.576	0.003**
T1-T2		5	11
T3-T4		20	6
Lymph node metastasis				4.748	0.029*
Yes		22	10
No		3	7
TNM stage				7.412	0.006**
Ⅰ–Ⅱ		10	14
Ⅲ–Ⅳ		15	3

Data were expressed as mean ± SD. *p < 0.05, **p < 0.01.

Effects of Hsa_circ_0005571 on the proliferation of CRC cells

The findings from the colony formation and CCK-8 assays demonstrated that, compared with the negative control condition, diminished expression of hsa_circ_0005571 resulted in a substantial reduction in CRC cell colony numbers and inhibited the proliferation of HCT116 and SW480 cells (Fig. 4A,B). Additionally, the results of the EdU assays revealed that, compared with si-NC, downregulation of hsa_circ_0005571 markedly reduced the proliferative capacity of CRC cells (Fig. 4C).

Fig. 4.

Hsa_circ_0005571 promotes proliferation of CRC cells. (A) Cell proliferation was analyzed by CCK-8 assays. (B) Silencing hsa_circ_0005571 expression reduced colony-forming ability of CRC cells. (C) Silencing hsa_circ_0005571 expression suppressed CRC cell proliferation. Data were expressed as mean ± SD. *p < 0.05.

Effects of Hsa_circ_0005571 on the migration of CRC cells

To evaluate the influence of hsa_circ_0005571 on cell metastasis, wound healing and Transwell migration assays were performed. The results demonstrated that the knockdown of hsa_circ_0005571 significantly reduced the metastatic capacity of HCT116 and SW480 cells in vitro compared with that of the control group (si-NC) (Fig. 5A,B).

Fig. 5.

Hsa_circ_0005571 promotes invasion of CRC cells. (A) Wound-healing assay showing reduced cell migration following transfection with si-hsa_circRNA_0005571. (B) Transwell migration assay demonstrating reduced migration of cells transfected with si-hsa_circRNA_0005571 compared to control. Data were expressed as mean ± SD. *p < 0.05.

Effects of Hsa_circ_0005571 on in vivo action in CRC cells

To investigate the in vivo effects of hsa_circ_0005571, an in vivo tumorigenicity assay was performed. The knockdown of hsa_circ_0005571 resulted in significant reductions in tumor volume and weight, suggesting that the silencing of this circRNA hinders the progression of CRC in vivo (Fig. 6). Overall, these findings suggest that downregulation of hsa_circ_0005571 mitigates CRC cell growth.

Fig. 6.

Silencing hsa_circ_0005571 inhibited the growth of CRC cells in vivo. (A) Inhibiting hsa_circ_0005571 reduced the size of tumors. (B) Inhibiting hsa_circ_0005571 reduced the volume of tumors. (C) Inhibiting hsa_circ_0005571 reduced the weight of tumors. Data were expressed as mean ± SD. *p < 0.05.

The mechanism of hsa_circ_0005571 in CRC

Using the starBase (https://rnasysu.com/encori/) and CircBank (https://www.circbank.cn/) prediction tools, we determined that hsa_circ_0005571 binds to miR-520f-3p. Target sequence prediction for LRP8 and miR-520f-3p was performed using TargetScan Human 7.2 (https://www.targetscan.org/vert_72/). This analysis revealed that hsa_circ_0005571 and LRP8 share a common binding site on miR-520f-3p (Fig. 7A). The expression of miR-520f-3p was significantly lower in CRC cell lines (HCT116, SW480, LoVo, and HT29) than in normal colorectal epithelial cells (NCM460) (Fig. 7B). Consistent with its tumor-suppressive role, CCK-8 assays demonstrated that knocking down miR-520f-3p using siRNAs promoted cell proliferation (Fig. 7C). Analysis via the GEPIA database (http://gepia.cancer-pku.cn/) indicated that LRP8 is highly expressed in CRC tissues (Fig. 7D), and elevated LRP8 expression was also confirmed in CRC cell lines (Fig. 7E). Western blot analysis of LRP8 protein expression revealed that miR-520f-3p knockdown increased LRP8 levels, whereas hsa_circ_0005571 knockdown reversed this effect (Fig. 7F).

Fig. 7.

Hsa_circ_0005571 acts as a suppressor via regulation of miR-520f-3P/LRP8 axis. (A) The binding sites of miR-520f-3P in hsa_circRNA_0005571 and LRP8. (B) The expression of miR-520f-3P was measured by qRT-PCR in the CRC cells. (C) Cell proliferation was measured by CCK-8 analysis in the CRC cell. (D) The expression of LRP8 in READ and COAD was analyzed by GEPIA website. (E) The expression of LRP8 in CRC cell lines. (F) The expression of LRP8 was detected by western blotting assay. Data were expressed as mean ± SD. *p < 0.05,**p < 0.01,***p < 0.001.

Discussion

CRC is a widely prevalent malignancy characterized by its ability to manifest throughout the colorectal region. It is one of the most prevalent cancers worldwide and frequently develops as a result of epithelial proliferation driven by chronic inflammation. Risk factors include smoking, alcohol consumption, obesity, a family history of the disease, and diets that are high in fat and low in fiber¹⁷. In 2020, CRC accounted for more than 1.9 million newly diagnosed cases and approximately 935,000 deaths, representing 10% of the global cancer incidence and mortality rates¹⁸. CRC cells demonstrate attributes of unchecked proliferation, invasion, and metastasis, which contribute to accelerated disease progression and a high incidence of recurrence. The increasing incidence, especially among younger populations, highlights the urgent need for the development of effective biomarkers and personalized treatment strategies.

CircRNAs are increasingly recognized as pivotal regulators in the field of cancer biology. CircRNAs are generated through a process known as back-splicing, resulting in a stable covalently closed loop structure that lacks the 5′ cap and 3′ poly-A tail characteristic of mRNAs. This unique configuration imparts resistance to exonuclease-mediated degradation^19,20. Owing to their distinctive structural configuration, circRNAs serve as stable mediators of tumor progression, frequently functioning as microRNA (miRNA) sponges or interacting with RNA-binding proteins (RBPs)²¹. CircRNAs have been recognized as potential biomarkers and therapeutic targets because of their role in cancer. For example, circFBXO7 inhibits the progression of non-small cell lung cancer by acting as a sponge for miR-296-3p²², whereas circBCBM1 facilitates breast cancer brain metastasis through the circBCBM1/miR-125a/BRD4 axis²³. Research indicates that hsa_circ_0006357 facilitates proliferation and metastasis in CRC, whereas hsa_circ_0000392 and hsa_circ_101277 are significantly overexpressed in CRC, thereby increasing cellular growth and invasion^24,25,26. We conducted a comprehensive analysis of the GSE138734 and GSE116589 microarray datasets to identify hsa_circ_0005571, which exhibited significant differential expression. Subsequent validation demonstrated that hsa_circ_0005571 is markedly upregulated in CRC tissues and cell lines, indicating its potential role as an oncogenic factor in CRC.

Our functional analyses confirmed the tumor-promoting role of hsa_circ_0005571 in CRC. Knockdown of hsa_circ_0005571 in CRC cell lines (HCT116 and SW480) significantly suppressed cell proliferation, colony formation, migration, and in vivo tumor growth. Although these findings are robust, larger-sample studies are warranted to further validate these results due to current sample size limitations. To explore the underlying molecular mechanisms, we used bioinformatics to predict potential miRNA binding sites for hsa_circ_0005571. Notably, both hsa_circ_0005571 and LRP8 share binding sites for miR-520f-3p. We therefore propose that hsa_circ_0005571 functions as a competing endogenous RNA (ceRNA), sequestering miR-520f-3p to derepress LRP8 expression, thereby regulating CRC progression.

We observed significantly lower expression of miR-520f-3p in CRC tissues than in matched adjacent normal tissues. Conversely, LRP8 expression was markedly elevated in CRC. The knockdown of miR-520f-3p led to the upregulation of both hsa_circ_0005571 and LRP8 mRNA. Subsequently, knocking down hsa_circ_0005571 reversed the pro-proliferative effect induced by miR-520f-3p knockdown in CRC cells. Consistently, Western blot analysis confirmed that LRP8 protein expression was dependent on the levels of hsa_circ_0005571 and miR-520f-3p.

In conclusion, hsa_circ_0005571 is highly expressed in CRC tissues and cell lines and significantly enhances CRC cell proliferation and migration. Hsa_circRNA_0005571 acts as an oncogene and may be involved in the biological process of CRC through the hsa_circRNA_0005571/miR-520f-3P/LRP8 axis. In the future, we can expand the cohort validation (multicenter and prospective), explore the application of liquid biopsy, and study its combination with other markers in diagnostic models. We can also evaluate its role in treatment resistance and conduct preliminary drug-adherence studies (e.g., stability, toxicity). Despite significant challenges, this study provides an important foundation for a deeper understanding of the biological role and translational potential of hsa_circ_0005571 in colorectal cancer. Overcoming these challenges will be a key direction for future research.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Haipeng Ge and Jun Qin conceived and designed research. Haipeng Ge, Jiaxuan Zhang, Ran Tao, Li Ding, Song Jiang, Jiale Shen performed the research. Haipeng Ge, Jiaxuan Zhang, Song Jiang analyzed the data. Haipeng Ge, Jiaxuan Zhang, Longjun Bian edited the manuscript. All authors approved final version of manuscript.

Funding

This study was funded by the General project of National Natural Science Foundation of China (82173270), and Nantong Municipal Health Commission Scientific Research Project (MA2021005).

Data availability

The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Competing interests

The authors declare no competing interests.