Decoding the regulatory roles of non-coding RNAs in cellular metabolism and disease

Abstract

Non-coding RNAs, including long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and circular RNAs (circRNAs), are being studied extensively in a variety of fields. Their roles in metabolism have received increasing attention in recent years but are not yet clear. The regulation of glucose, fatty acid, and amino acid metabolism is an imperative physiological process that occurs in living organisms and takes part in cancer and cardiovascular diseases. Here, we summarize the important roles played by non-coding RNAs in glucose metabolism, fatty acid metabolism, and amino acid metabolism, as well as the mechanisms involved. We also summarize the therapeutic advances for non-coding RNAs in diseases such as obesity, cardiovascular disease, and some metabolic diseases. Overall, non-coding RNAs are indispensable factors in metabolism and have a significant role in the three major metabolisms, which may be exploited as therapeutic targets in the future.

Graphical abstract

Dr. Lin and colleagues summarize the functional roles of ncRNAs in the occurrence and development of cancer and vascular diseases, review the regulatory mechanisms of ncRNAs in the physiological processes of glucose, lipid, and amino acid metabolism, and highlight the importance of ncRNA-based strategies in the diagnosis and therapy of diseases.

Introduction

Over the past three decades, non-coding RNAs (ncRNAs) of the genome have been likened to the “dark matter” of the universe.^1,2 Although most of the genome is transcribed into RNAs, only 1%–2% of the human genome has the potential to encode proteins or peptides³; the majority of RNAs are without coding potential and referred to as ncRNAs.⁴ The complexity of an organism is correlated to the amount of ncRNAs, which suggests the functional importance of ncRNAs for normal development, physiology, and disease.^5,6,7 The functional discovery of ncRNA has been expanded with the development of next-generation deep sequencing technologies as well as novel molecular technologies.^8,9 Currently, ncRNAs could be classified as housekeeping RNAs (such as rRNAs and tRNAs) and regulatory RNAs (such as microRNAs, small nucleolar RNAs [snoRNAs], and long non-coding RNAs [lncRNAs]).^10,11,12,13 Functional ncRNA molecules regulate the target genes at transcriptional, posttranscriptional, translational, and posttranslational levels.^14,15 Their potential roles in gene regulation make ncRNAs prospective targets for drug development and epigenetic therapeutics.¹⁶

ncRNAs are classified into lncRNAs (>200 nt), snoRNAs (60–300 nt), and small RNAs (<200 nt) based on the number of their ncRNA nucleotides.^17,18,19 ncRNAs participate in numerous cellular processes and signaling transduction pathways, and the specific molecular mechanisms of their functional outputs are accurately connected to their subcellular localization and interactions with molecular partners.²⁰ ncRNAs have also been reported to be involved in the pathogenesis of various cardiovascular diseases and cancers via the regulation of glucose metabolism, lipid metabolism, and amino acid metabolism.^21,22,23,24

Although genetic factors play important roles in the pathogenesis of diseases, the role of epigenetic regulation has received more and more attention.^25,26 Metabolism is the critical pathway, connecting the environment to the pathogenesis of cardiovascular diseases and cancers.²⁷ A potential interventional strategy to alter the metabolism in diseases begins with clarifying the regulatory mechanism of ncRNAs in the cell metabolism in pathology and then establishing effective prevention approaches targeting metabolic pathways.^28,29

The mechanisms of non-coding RNA metabolic pathways

Metabolism is an indispensable biological process that occurs throughout the whole life cycle of each cell, either utilizing energy to make macromolecules and biomolecular polymers, which is referred to as anabolism, or releasing energy when breaking down the macromolecules into simpler molecules, which is referred to as catabolism.³⁰ Metabolism is a continuous exchange of matter and energy in an organism to maintain cellular and whole-body function.³¹ Regulation of glucose, fatty acid (FA), and amino acid metabolism is an imperative physiological process that occurs in living organisms and an important pathological process that takes part in cancer and cardiovascular diseases.^32,33,34 As such, metabolic reprogramming has been widely recognized as a hallmark of cancer malignancy and cardiac adaptation.^35,36 Glucose is central to all of metabolism and is the main source of energy involved in cellular energy production.³⁷ Carbohydrates, lipids, and proteins all eventually decompose, and energy is stored in glucose, which then serves as the primary metabolic fuel of mammals.³⁸ Glucose is the final substrate at the cellular level that enters the cell and converts to adenosine triphosphate (ATP).³⁹ Fatty acids serve as cellular signaling molecules that play an important role in the etiology of metabolic syndrome.⁴⁰ The metabolism of fatty acids is highly regulated and is currently considered an attractive target to regulate the human pathological process of diseases like obesity, diabetes, cancer, and cardiovascular complications.⁴¹ The metabolism of amino acids is aberrantly regulated in many cancers and cardiovascular diseases that display a dependence on particular amino acids.³³ Amino acids are involved in the survival and proliferation of cells under genotoxic, oxidative, and nutritional stress. Thus, targeting amino acid metabolism is becoming a potential therapeutic strategy for cancer and cardiovascular patients (Figure 1).⁴²

Figure 1.

ncRNAs contribute to regulating metabolic rearrangement by targeting metabolism-related signals

Changes in metabolism are currently considered defects in the human pathological process of diseases like obesity, diabetes, cancer, and cardiovascular complications. ncRNAs and their molecular partners or genomic targets are committed to directly regulating the expression or function of metabolic enzymes. A deeper understanding of the interplay between ncRNAs and metabolism renders a better knowledge of the intimate mechanisms that alter the metabolism of glucose, glutamine, lipids, or hormones and other proteins and offers a strong basis for establishing effective preventive approaches targeting metabolic pathways.

Non-coding RNAs in glucose metabolism

Cellular glucose metabolism, including the processes of glycogenesis, glycolysis, the pentose phosphate pathway (PPP), and gluconeogenesis, produces ATP for energetic support or provides the precursors for the synthesis of biological macromolecules.⁴³

The entry of glucose into cells via glucose transporters (GLUTs) is the first step in glycolysis. GLUT1 is encoded by the antisense strand of Solute carrier 2A (SLC2A).⁴⁴ GLUT families are a wide group of membrane proteins that play essential roles in the uptake and storage of glucose.^45,46 Based on amino acid sequence similarity and substrate affinity, GLUT proteins can be classified into three classes.^47,48 Clearly, the class I GLUTs (GLUTs 1–4 and 14) catalyze the transport of glucose and other hexoses into and out of cells, but not fructose transport. However, class II (GLUTs 5, 7, 9, and 11) and class III GLUT (GLUTs 6, 8, 10, 12, and HMIT) proteins do not necessarily have a primary function in facilitating the uptake of glucose. In addition, GLUT1 and GLUT3 are the major GLUTs in human islets, and GLUT2 is expressed in rodent pancreatic β cells.^49,50

GLUT1 was the first GLUT to be identified and is expressed in varying degrees in all tissues of the body.⁵¹ Several ncRNAs, including lncRNAs, microRNAs, and circRNAs, are known to regulate GLUT1 levels and possibly GLUT1 catalytic activity (Figure 2 and Table S1). The neighbor of BRCA1 gene 2 (NBR2) is an lncRNA that promotes the expression of GLUT1, which is proposed to be induced by 2-DG (2-Deoxy-D-glucose), phenformin treatment, or glucose starvation. NBR2 knockdown by two independent NBR2 short hairpin RNAs (shRNAs) inhibits phenformin-induced GLUT1 expression and glucose uptake, rendering cancer cells more susceptible to phenformin treatment. In addition, using NBR2 status as a biomarker to predict response to biguanide therapy in cancer patients is possible.⁵² lncRNA GAS6-AS1 (GAS6 antisense RNA1) suppresses the E2F transcription factor 1 (E2F1)-mediated transcription of GLUT1. An overexpression plasmid transfected with GAS6-AS1 prevents the progression of lung adenocarcinoma (LUAD) and inhibits glucose metabolism reprogramming.⁵³ lncRNA SLC2A1-AS1 (SLC2A1 antisense RNA 1) inhibits glycolysis by negatively regulating GLUT1 expression via the inhibition of Forkhead box M1 (FOXM1) binding to the promoter of GLUT1, to restrain hepatocellular carcinoma (HCC) cell proliferation and tumor metastasis.⁵⁴ MicroRNAs miR-140 and miR-143 inhibit cancer cell proliferation and invasion by suppressing the translation of GLUT1 by binding to the 3′ untranslated region (or UTR) of GLUT1 mRNA, which could be attenuated by the competitive binding of TMPO-AS1 (TMPO Antisense RNA 1). Transfection of small interfering RNA (siRNA) targeting TMPO-AS1, or delivering miR-140 and miR-143 mimics, significantly reduced the protein level of GLUT1 in endometrial cancer (EC) cells, which is similar to the therapeutic effect of shRNA targeting GLUT1.⁵⁵ In addition, hsa-miR-144-5p and hsa-miR-186-3p have been implicated in binding to the 3′ UTR of GLUT-1’s mRNA and consequently inhibiting its translation into protein.⁵⁶ circRNF13 (circular RNA RNF13) promotes GLUT1 protein degradation by upregulating the protein levels of SUMO2 (Small Ubiquitin-like Modifier 2) to inhibit the cell proliferation and metastasis of nasopharyngeal carcinoma cells (NPCs).⁵⁷ circRNA_100290 promotes glycolysis and cell proliferation in oral squamous cell carcinoma (OSCC) by counteracting miR-378A-mediated GLUT1 inhibition, which is suggested to be attenuated by si-circRNA_100290 (si-circRNA).⁵⁸

Figure 2.

ncRNAs regulate the molecules involved in glucose metabolism

ncRNAs regulate glucose uptake and glycolytic flux by modulating GLUTs and glycolic enzymes. The major metabolites, the main metabolic enzymes, and the main metabolic pathways are indicated. →, promotion; ┤, inhibition. GLUT, glucose transporter; HK2, hexokinase 2; PFKFB2, fructose-2,6-bisphosphatase 2; PKM2, pyruvate kinase isozymes M2.

GLUT3 is the main GLUT in neurons and was found to be highly expressed in brain tumor.⁵⁹ A novel miRNA, miR-3189 is recruited to the 3′ UTR of GLUT3 to inhibit GLUT3 expression, thereby working as a tumor suppressor to inhibit tumorigenesis. The expression of miR-3189 can be increased by knocking down HDAC2 using the lentiviral system of an shRNA library or by delivering miR-3189 mimics in vitro.⁶⁰ circMYLK, circRNA derived from the MYLK gene, promotes the expression of GLUT3 as a molecular sponge of miR-195-5p, resulting in increased glycolysis and proliferation of non-small cell lung cancer (NSCLC) cells (Figure 2 and Table S1).⁶¹

GLUT4 is present in the cytoplasm of vesicles, which are translocated to the plasma membrane under insulin stimulation.⁶² lncRNA CRNDE (colorectal neoplasia differentially expressed) positively regulates GLUT4 transcription and glucose intake in colorectal tumors, which are repressed by insulin and insulin-like growth factor (IGF) treatment.^63,64 lncRNA DREH (downregulated expression by hepatitis B virus X) is the first lncRNA observed to promote glucose transport by increasing the expression of GLUT4 in the plasma membrane of adipocytes and skeletal muscle cells.^65,66 Glucose levels in adipocytes can be reduced by repressing the expression level of GLUT4 using miR-27a, which can be attenuated by transfecting antagomir-27a.⁶⁷ In addition, overexpression of miR-106b with lentivirus significantly decreased glucose uptake and GLUT4 translocation, whereas miR-106b sponge delivered via lentiviruses diminished this effect (Figure 2 and Table S1).⁶⁸

Previous studies suggest that ncRNAs participate in the regulation of three key rate-limiting enzymes of glycolysis: hexokinases (HKs), phosphofructokinase 1 (PFK1), and pyruvate kinases (PKs). HKs prime glucose for intracellular utilization, and HK2 is the most active isozyme among the five major isoforms of the family.⁶⁹ HK2 is mainly expressed in insulin-sensitive tissues, such as the heart, skeletal muscle, and adipose tissue, and in a wide range of tumors. HK2 catalyzes the first rate-limiting step of glucose metabolism, producing glucose-6-phosphate.^70,71 PFK1 catalyzes the phosphorylation of fructose 6-phosphate (F6P) to fructose 1,6-bisphosphate (F1,6BP) and is critical in determining the rate of glycolytic flux.⁷² PKs are also critical enzymes that catalyze the final reaction of glycolysis, irreversibly converting ADP and phosphoenolpyruvate to ATP and pyruvate.⁷³ Four isoenzymes of PK have been identified so far: M, K, L, and R types. PKM1 and PKM2 promote the tricarboxylic acid (TCA) cycle and glycolysis, respectively. Allosteric, as well as covalent, modifications can affect PK activity.⁷⁴ A discussion of RNA involvement in the regulation of HKs, PFK1, and PKs follows.

HK2 levels are regulated through a number of mechanisms (Figure 2 and Table S1). lncRNA UCA1 (urothelial carcinoma-associated 1) upregulates HK2 expression at the mRNA level and increases glucose consumption and lactate production in bladder cancer cells, which is dependent on the activation of STAT3 and repression of miR-143. Rapamycin or STAT3 siRNA reduces HK2 at the transcriptional level, whereas lncRNA SARCC (suppressing androgen receptor in renal cell carcinoma) and miR-143 mimics reduce HK2 at the posttranscriptional level, respectively.^75,76 lncRNA PVT1 (plasmacytoma variant translocation 1) increases the mRNA level of HK2 and promotes cell proliferation and invasion in osteosarcoma. Cells transfected with si-PVT1 exhibit significantly suppressed glucose consumption and lactate production.⁷⁷ Overexpression of lncRNA loc285194 plasmid significantly inhibited laryngeal squamous cell carcinoma cell proliferation by repressing HK2 expression, thereby inhibiting tumor growth.⁷⁸ Zinc-finger antisense 1 (ZFAS1) is an lncRNA highly expressed in glioma tissues and cells. ZFAS1 binds to the 3′ UTR of HK2 mRNA to compete with miR-1271-5p, increasing HK2 expression levels and resulting in glioma cell proliferation and migration. Treatment with si-ZFAS1 or miR-1271-5p mimics downregulates HK2 expression and blocks glioma cell proliferation, migration, and invasion.⁷⁹ miRNAs and circRNAs play important roles in the regulation of HK2 expression or activity in glycolysis.⁸⁰ miRNAs, including miR-9-1, miR-216b, miR-125a-5p, miR-615, miR-202, miR-487a, and miR-653, interact with the 3′ UTR of HK2 mRNA, weakening HK2 expression at the posttranscriptional level and acting as tumor suppressors in various cancer types, like breast cancer, osteosarcoma (OS), and pancreatic cancer.^{81,82,83,84,85} circRNAs, including circCDKN2B-AS1, circRNF20, and circ_0061140, work as the sponge RNA of HK2, binding microRNAs, upregulating the expression levels of HK2, and promoting cancer cell proliferation and tumorigenesis. Delivery of miRNA mimics or sh-circRNA in vivo and in vitro to weaken HK2 expression levels may serve as a potential therapeutic strategy to decrease cancer cell proliferation and diminish the Warburg effect (aerobic glycolysis).^86,87,88

Allosteric regulation of PFK1 by 6-phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2; PFKFB) alters PFK1 efficiency.⁸⁹ The human body has four isozymes of PFKFB, namely PFKFB1, PFKFB2, PFKFB3, and PFKFB4, which display tissue-specific expression patterns and different kinase-to-phosphatase activities.⁹⁰ lncRNA LINC00538 (YIYA), related to the cytosolic cyclin-dependent kinase CDK6, promotes CDK6-dependent PFKFB3 phosphorylation, leading to the catalysis of glucose 6-phosphate (G6P) to fructose 2,6-bisphosphate/fructose 1,6-bisphosphate (F2,6BP/FBP).⁹¹ LINC00092 promotes ovarian cancer progression by binding with PFKFB2, whereas si-LINC00092 significantly reduces ovarian cancer cell invasive ability and metastatic potential, increasing anoikis rate.⁹² LINC00115 downregulates PFKFB2 by targeting miR-489-3p, thereby aggravating the progression of retinoblastoma (RB). Furthermore, transfection of LINC00115 interference fragment (si-lnc) in vitro reduces the proliferation and migration ability of RB cells.⁹³ miR-210-5p mimic inhibits the expression of PFKFB2, represses the process of glycolysis, and induces energy stress in pancreatic cancer.⁹⁴ miR-613 inhibits the expression of PFKFB2 by binding to the 3′ UTR of PFKFB2, thus significantly inhibiting gastric cancer cell growth, inhibiting colony-forming ability and cell invasion (Figure 2 and Table S1).⁹⁵

PK is the last rate-limiting enzyme in glycolysis. PKM2 deficiency in cardiomyocytes accelerates the development of pressure overload-induced heart failure, and the aberrant expression of PKM2 is correlated with tumor progression.^96,97,98 The enzymatic activities and downstream effect of PKM2 are regulated by ncRNAs through multiple mechansims. Downregulation of lncRNA-p21 (also called as TRP53COR1 (P53 Pathway Corepressor 1 protein tumor)) in human prostate cancer correlated with an increase in PKM2 levels, leading to tumor formation and growth, whereas the shPKM2, 3-bromopyruvate (3-Brpa) or rapamycin treatment, largely reduced tumor burden.⁹⁹ FEZF1-AS1 (FEZF1 Antisense RNA 1) is an lncRNA upregulated in more than 60% of colorectal cancer tissues, promoting colorectal cancer cell proliferation and metastasis. FEZF1-AS1 increases PKM2 levels by promoting gene transcription as well as increasing protein stability. Conversely, shFEZF1-AS1 significantly decreased total and nuclear PKM2 levels, and si-PKM2 blocked FEZF1-AS1 induction of colorectal cancer cell growth.¹⁰⁰ SNHG3 (Small Nucleolar RNA Host Gene 3) functions as an miR-330-5p sponge to positively regulate PKM1 and PKM2 expression, increasing glycolysis carboxylation and enhancing breast tumor cell proliferation. Similarly, antagomir (anti-miR-330) treatment also promotes glycolytic metabolism and cell proliferation. Transfection with si-SNHG3 or miR-330-5p mimic significantly inhibits cancer cell proliferation, decreases lactate production, reduces oxygen consumption rate (OCR), and reduces extracellular acidification rate.¹⁰¹ miR-124 suppresses the Warburg effect via the interaction with RNA binding protien PTBP1 (polypyrimidine tract binding protein 1) and downstream PKM axis. miR-124 mimic treatment significantly reverses the growth promotion caused by antagomir-124 and induces apoptosis and/or autophagy as well as a significant decrease in lactate production.¹⁰² The downregulation of miR-214 achieved by injection of adenoviral vectors (AdP14ARF) containing miR-214 inhibitor upregulated PKM2 expression, which contributed to facilitating mitochondrial fusion and further alleviating brain injury after general anesthesia.¹⁰³ miR-489-3p targets the 3′ UTR of PKM2 and inhibits glycolysis of pancreatic cancer cells and pancreatic cancer progression. Treatment with anti-miR-489-3p inhibitors increased pancreatic cancer cell growth ability and promoted pancreatic cancer cell migration and invasion.¹⁰⁴ Under hypoxia, circMAT2B (circular RNA MAT2B) upregulates the expression level of PKM2 by sponging miR-338-3p, thereby enhancing glycolysis and promoting the progression of HCC.¹⁰⁵ Recent studies also suggested that circular RNA, circSMOC1, plays a role in pulmonary arterial hypertension by regulating pulmonary vascular remodeling through pulmonary artery smooth muscle cells. circSMOC1 inhibits specific splicing of PKM prophase RNA by binding directly to PTBP1, leading to the upregulation of PKM2 to enhance glycolysis (Figure 2 and Table S1).¹⁰⁶

The TCA cycle works as the central hub of cellular metabolism, bioenergetics, and redox homeostasis and is the most critical metabolic cycle in physiology.¹⁰⁷ Current evidence indicates that ncRNAs contribute to the regulation of the TCA cycle both directly and indirectly (Figure 3 and Table S1). Pyruvate dehydrogenase (PDH) acts as the gatekeeper when acetyl-CoA enters the TCA cycle, by converting pyruvate to acetyl-CoA. Increasing miR-195 levels leads to increased total and specific lysine acetylation of PDH in the failing myocardium. This effect reduces PDH activity and inhibits cardiac energy metabolism.¹⁰⁸ The activity of PDH is primarily regulated via dephosphorylation by PDH kinases 1 through 4 (PDK1–4).¹⁰⁹ miR-23a directly binds to the 3′ UTR of PDK4 mRNA and subsequently inhibits PDK4 expression, thereby promoting PDH activation and oxidative phosphorylation to generate sufficient ATP for human colorectal cancer cell lines and tissue cell proliferation. Moreover, miR-23a inhibitor reduced PDH activity, acetyl-CoA, OCR, and ATP production, which were abolished by miR-23a mimic or shPDK4.¹¹⁰ The first key enzyme in the TCA cycle is citrate synthase (CS), which catalyzes the condensation of acetyl-CoA with oxaloacetate to form citric acid. Growth arrest-specific transcript 5 (GAS5) is a mitochondrial lncRNA. The overexpression of GAS5 simulates the signal of glucose deficiency, inhibits the formation of CS metabolon, causes abnormal levels of citric acid and an imbalance of mitochondrial energy metabolism, and inhibits cell growth and proliferation in breast cancer.¹¹¹ The second key enzyme in the TCA cycle is isocitrate dehydrogenase (IDH), which catalyzes the oxidative decarboxylation of isocitrate to form α-ketoglutarate (α-KG). IDH3 is the principal subtype of the three IDHs, and the loss of function of IDH3 might be detrimental to cell growth due to the disruption of the TCA cycle.¹¹² No related ncRNA was reported to correlate with the expression of the activity of IDH3. IDH1 and IDH2 subtypes catalyze the NADP⁺-dependent oxidative decarboxylation of isocitrate to α-KG (alpha-ketoglutarate) to generate carbon dioxide (CO₂) and nicotinamide adenine dinucleotide phosphate (NADPH).¹¹³ The introduction of IDH1-AS1 (psin-IDH1-AS1) enhances the activity of IDH1 by promoting the homodimerization of IDH1, reducing cell proliferation, and increasing cell apoptosis, thus inhibiting glioblastoma tumor growth both in vivo and in vitro.¹¹⁴ Furthermore, c-Myc downregulates IDH1 and promotes lactate production by inhibiting the transcription of the lncRNA IDH1-AS1.^115,116 IDH1 mutation occurring at codon 132 (R132) in prostate cancer (PCa) increases the expression of circRNA-51217 to sponge miRNA-646, thereby inducing the TGF-β1 (transforming growth factor-beta) and phosphorylation of SMAD Family Member 2/3 (SMAD2/3) signaling pathway to increase PCa cell invasion. This provides the rationale and evidence for the use of Tibsovo (ivosidenib) for the treatment of adult patients with relapsed or refractory AML (acute myeloid leukemia ) with IDH1 mutations.¹¹⁷ Upregulation of miR-183 inhibits IDH2 expression and induces HIF-1α (hypoxia inducible factor 1 subunit alpha) expression in malignant gliomas, which in turn may be effective for glioma formation or survival. Transfection with anti-miR-183 (mirVana inhibitor) resulted in increased IDH2 mRNA expression and downregulated HIF-1α in glioma cells.¹¹⁸

Figure 3.

ncRNAs regulate the molecules involved in the tricarboxylic acid (TCA) cycle

ncRNAs regulate the steps of the TCA cycle in cardiac energy metabolism, tumor cell growth, proliferation, and invasion. PDH, pyruvate dehydrogenase; PDK4, pyruvate dehydrogenase kinase 4; CS, citrate synthase; IDH, isocitrate dehydrogenase.

The rate-limiting enzyme in glucose metabolism is glucose-6-phosphate dehydrogenase (G6PD). This enzyme converts glucose metabolism from glycolysis into the PPP, which is vital for the production of reduced NADPH.¹¹⁹ lncRNA PDIA3P (protein disulfide isomerase family A member 3 pseudogene 1) interacts with c-Myc to enhance its binding affinity to the G6PD promoter, stimulating G6PD expression and PPP flux, which promotes the proliferation and drug resistance of multiple myeloma (MM) cells. As a potential therapeutic target for MM patients, the administration of shRNA targeting PDIA3P sensitized MM cells to bortezomib treatment, which restrains MM progression.¹²⁰ LINC00242 (Long Intergenic Non-Protein Coding RNA 242) sponges miR-1-3p expression, upregulating G6PD expression in gastric cancer tissues and cells. As a result, the shRNA of LINC00242 or miR-1-3p mimics in gastric cancer cells significantly inhibits glucose utilization, lactate concentration, and ATP production in vitro and attenuates gastric cancer tumorigenesis in vivo.¹²¹ lncRNA SNHG14 (small nucleolar RNA host gene 14) and lncRNA OR3A4 (olfactory receptor family 3 subfamily A member 4) participate in the progression of NSCLC by sponging the miR-206/G6PD axis or miR-1207-5P/G6PD axis, respectively.^122,123 miR-206 directly binds to the 3′ UTR of G6PD to downregulate the level of G6PD. The mimic of miR-206 induces cell-cycle arrest in G0/G1 phase by inhibiting G6PD expression and inhibits cell proliferation of skeletal muscle cells and cancer cells.^{124,125,126,127} miR-24 negatively regulates the expression of G6PD by binding to the CUGAGCC motif of G6PD, damaging the G6PD-mediated mitochondrial function and inducing oxidative stress in cardiac hypertrophy cells. Overexpression of the G6PD vector in hypertrophic cardiomyocytes alleviates phenylephrine-induced mitochondrial dysfunction and oxidative stress, which is comparable to the treatment of miR-24 inhibitor (Figure 4 and Table S1).¹²⁸

Figure 4.

ncRNAs regulate the molecules involved in the pentose phosphate pathway (PPP)

The PPP flux is generally enhanced in cancer cells by increased activity of G6PD to meet the bioenergy requirement for growth and proliferation. Recent studies have shown that the PPP is regulated by different ncRNA classes. G6PD, glucose-6-phosphate dehydrogenase.

Non-coding RNA in fatty acid metabolism

Fatty acids (FAs) are a class of molecules comprising a long aliphatic hydrocarbon chain with different lengths and saturations. The metabolism of FA involves a series including FA synthesis, transportation, release, and decomposition, playing a significant role in many biological processes.¹²⁹ The synthesis of de novo FAs starts from acetyl-CoA and NADPH, which are synthesized from the TCA cycle and the PPP. Several lines of evidence demonstrate ncRNA-mediated regulation of the lipogenic enzymes, such as ATP-citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FASN) (Figure 5 and Table S2).¹³⁰

Figure 5.

ncRNAs regulate the molecules involved in fatty acid metabolism

ncRNAs regulate lipid metabolism, including during adipocyte differentiation, HDL biogenesis, and cholesterol metabolism. ACC, acetyl-CoA carboxylase; ACLY, ATP-citrate lyase; FASN, fatty acid synthase; SREBPs, sterol regulatory element-binding proteins; CPT1, carnitine palmitoyl transferase 1.

ACLY catalyzes the conversion of the six-carbon citrate molecule to oxaloacetate and two-carbon acetyl-CoA, the precursor for FA synthesis. It has been demonstrated that lncRNA FLJ22763 plasmid significantly suppressed malignancy of gastric cancer cells by decreasing the expression of mRNA and protein levels of ACLY.¹³¹ lncRNA TINCR (terminal differentiation-induced lncRNA) is significantly upregulated in nasopharyngeal carcinoma and positively correlated with poor survival rate. Current mechanistic studies suggest that TINCR binds the A1 domain (amino acids 1–620) of ACLY, which contains the ATP-binding, citrate-binding, and CoA-binding regions. This binding shields it from degradation by ubiquitin, which maintains total cellular levels of acetyl-CoA. Silencing of TINCR by siRNA or shRNA blocks acetylation of histone H3K27 and de novo synthesis of lipids and inhibits nasopharyngeal carcinoma progression and chemical resistance.¹³² lncRNA CASC9 (cancer susceptibility candidate 9) negatively regulates the expression of miR-146b-5p and miR-146b-5p, which degrades ACLY by directly targeting the 3′ UTR of ACLY. In salivary adenoid cystic cancer (SACC) cells, treatment with sh-CASC9 or miR-146b-5p mimics decreases the proliferation, migration, invasion, and apoptosis of SACC cells.¹³³ Yang et al. revealed that liver-specific knockdown of Gm16551 by transfection with a specific shRNA adenovirus upregulated the expression of ACLY and resulted in increased plasma triglyceride levels.¹³⁴ Adipocyte miR-221-3p mimic suppressed the expression of ACLY protein, impaired adipocyte lipid storage, and promoted metabolic disease and cancer progression.¹³⁵ In addition, Liu et al.¹³⁶ reported that miR-22 mimics downregulated ACLY expression and inhibits the growth and metastasis of breast cancer cells. Further, miR-133b mimic inhibits the expression and activation of ACLY and subsequently decreases the proliferation and invasion of gastric cancer cells.¹³⁷ The low level of miR-206 is associated with the upregulated expression levels of ACLY. Transfection of miR-206 mimic reduces breast cancer cell-cycle progression and inhibits mammosphere formation and metastasis (Figure 5 and Table S2).¹³⁸

Acetyl-CoA is generated in mitochondria and is the direct raw material of FA synthesis. The preliminary reaction of FA synthesis is to transport acetyl-CoA from mitochondria to cytosol. The citrate pyruvate cycle is a critical transportation process, helping transfer acetyl-CoA from mitochondria to cytosol. Cytosolic acetyl-CoA reacts with bicarbonate and ATP, forming malonyl-CoA, which is catalyzed by ACC. ACC has two major isoforms, ACC1 and ACC2. These two isoforms are preferentially expressed in different tissues.¹³⁹ ACC1 is the lipogenic isoform, mainly expressed in white adipose tissue, mammary gland, and liver, catalyzing the rate-limiting step in FA synthesis. The activity of ACC is regulated mainly by phosphorylation, and phosphorylation of Serine residues repressed the synthesis activity of ACC.^139,140 lncRNA TUG1 (taurine-upregulated gene 1) attenuates fatty accumulation and reverses diabetes development via downregulation of miR-204, further increasing ACC phosphorylation levels.^139,141 Nuclear paraspeckle assembly transcript 1 (NEAT1) is an lncRNA that sponges the expression of miR-139-5p. miR-139-5p suppresses hepatic lipid accumulation by reducing the expression of ACC at both mRNA and protein levels. Thus, knockdown of NEAT1 expression with si-NEAT1 or sh-NEAT1 restrains fat deposition in non-alcoholic fatty liver disease (NAFLD) by increasing miR-139-5p expression, further reducing the expression of ACC.¹⁴² Two miRNAs expressed in the cotton aphid Aphis gossypii Glover (miR-276 and miR-3016) are correlated with spirotetramat resistance by inverse regulating the gene expression of ACC at the transcriptional level. Treatment with miR-276 and miR-3016 mimics significantly decreased ACC transcript levels and increased the sensitivity of A. gossypii to the spirotetramat.¹⁴³ Sterol regulatory element-binding protein 1 (SREBP-1) is a master transcription factor in lipogenesis and lipid metabolism and regulates the expression of ACC. miR-21 inactivation with anti-miR-21 oligonucleotides decreases SREBP-1 mRNA expression levels and reduces ACC levels in human PCa cells, significantly suppressing cell proliferation and prostate tumorigenesis (Figure 5 and Table S2).¹⁴⁴

FASN is possibly the most studied enzyme related to FA metabolism and cancer. FASN catalyzes consecutive condensation reactions to form long-chain FAs from a molecule of acetyl-CoA and malonyl-CoA, mostly generating 16-carbon palmitate. lncRNA PVT1 was demonstrated to act as a molecular sponge of miR-195 and increase the protein expression of FASN. Silencing of PVT1 with siRNA suppresses OS cell invasion, migration, and proliferation and induces apoptosis and cell-cycle arrest.¹⁴⁵ The HOXD (Homeobox D Cluster) antisense growth-associated lncRNA (HAGLR), whose transcript is a novel lncRNA, may augment the expression of FASN and enhance de novo lipogenesis. HAGLR expression was disrupted by using a lentivirus-mediated shRNA expression system in NSCLC cells, which consequently inhibited cellular free FA content, cell proliferation, and invasion.¹⁴⁶ miR-27 and miR-195 resulted in the downregulation of FASN by targeting the 3′ UTR of the FASN mRNA. The transfection of miR-27 and miR-195 inhibitors restored the cellular level of FASN and slowed down the development of hypoxia-induced cardiomyocyte injury.¹⁴⁷ miR-4310 mimics, which block FASN-mediated lipid synthesis in HCC cells, represses HCC tumor growth and metastasis.¹⁴⁸ Similarly, miR-33a mimic treatment suppressed the expression of FASN in breast cancer cells (Figure 5 and Table S2).¹⁴⁹

FA oxidation (FAO) is a repeat cycle for FA shortening (two carbons per cycle) producing NADH, FADH2, and acetyl-CoA in each cycle.¹⁵⁰ In FAO, the carnitine palmitoyl transferase (CPT) system is essential for the transportation of FAs, especially long-chain FAs previously activated as FA-CoA, from the cytoplasm into the mitochondria, to enter the β-oxidation route. CPT1 catalyzes the rate-limiting step of FAO, converting FA-CoA into carnitine derivatives. AGAP2-AS1 (AGAP2 Antisense RNA 1) directly binds to the CPT1, increasing its expression by improving RNA stability. In addition, AGAP2-AS1 could serve as a ceRNA (competitive endogenous RNA) by sponging miR-15a-5p to increase CPT1 expression at the transcript level.¹⁵¹ lncRNA-HCP5 (lncRNA–HLA complex P5) targeted miR-3619-5p, leading to the transactivation of CPT1 and facilitated FAO, which was reversed by miR-3619-5p mimics (Figure 5 and Table S2).¹⁵²

CPT1A, a subtype within the CPT1 gene family, is expressed highly in many cancer types. The expression of CPT1A is negatively regulated by miR-33. After T cells are activated, CD28 will restrict miR-33 expression, which enables the expression of CPT1A. This expression then remodels the mitochondria crest and leads to the activation of T cell protective memory.¹⁵³ NEAT1 could serve as a ceRNA by sponging miR-107 to upregulate CPT1A expression at the transcript level. In addition, the transfection of sh-NEAT1 or mimics-miR-107 inhibited the progression of breast cancer cells.¹⁵⁴ Another member of the CPT1 family is CPT1C. miR-1291 inhibits pancreatic cancer cell and breast cancer cell proliferation by downregulating the mRNA and protein levels of CPT1C in an ERRα-dependent manner.¹⁵⁵ miR-106a-5p and miR-320a directly inhibit CPT1 expression, which could be diminished by circRNA_0001805 by forming an intermolecular interaction axis of circRNA_0001805/miR-106a-5p/miR-320a. The injection with circRNA_0001805 adenoviruses or mimics of miR-106a-5p and miR-320a rescued high-fat diet (HFD)-induced lipid metabolism disorder and hepatic inflammation in an NAFLD mouse model (Figure 5 and Table S2).¹⁵⁶

The bulk of the energy stored in fat is in FAs. The FAs produced by lipolysis bind to albumin to form very-high-density lipoproteins, which are transported through the bloodstream to energy-hungry tissues. FAs are taken up by tissues through various transporters, including CD36, FATP (fatty acid transport protein), and FABP (fatty acid binding protein). CD36 is fatty acid translocase (FAT), which belongs to the class B scavenger receptors. Transfection of miR-29a mimics can reduce the expression of the FA transposase CD36 in HepG2 cells. Meanwhile, transgenic mice with overexpressed miR-29a showed not only remission of HFD-induced weight gain but also remission of subcutaneous, visceral, intestinal fat accumulation and hepatocyte steatosis compared with control mice.¹⁵⁷ miR-20a-5p inhibits CD36 expression by binding to its 3′ UTR and reduces lipid accumulation.¹⁵⁸ miR-26a binds directly to the 3′ UTR of CD36, and its mimics abrogated particulate matter 2.5 (PM2.5)-induced steatosis in HepG2 cells by inhibiting CD36.¹⁵⁹ miR-101b directly targets and inversely regulates the expression of CD36 at the transcriptional level. miR-101b inhibitor aggravates FAs, including PA (palmitic acid), or OA (OMEGA-3 fatty acids)-induced lipid accumulation (Figure 5 and Table S2).¹⁶⁰

Non-coding RNAs in amino acid metabolism

In humans, amino acids take part in the synthesis of tissue proteins and contribute to energy metabolism, including 10%–15% of ATP generation as a direct source of energy.¹⁶¹ Aminotransferases (ATs) are pyridoxal 5′-phosphate-dependent enzymes that catalyze the transamination reactions between amino acid donors and keto acid acceptor substrates.^162,163 The most important transaminases in the human body are glutamic pyruvic transaminase/alanine AT (GPT/ALT) and glutamic oxaloacetic transaminase/aspartate AT (GOT/AST), which function to catabolize amino acids that enter the citric acid cycle.¹⁶⁴ Their expression levels are the most commonly used biochemical markers of hepatic damage.

Injection of adeno-associated viruses (AAV) carrying lncRNA Gm2199 through the tail vein protects against carbon tetrachloride (CCl)-induced liver injury in mice by increasing hepatocyte proliferation and decreasing serum ALT, AST, and hepatic hydroxyproline.¹⁶⁵ Yangonin (Yan), a bioactive compound extract from kava, improves cell viability and reduces AST, ALT, total cholesterol (TC), and total triglyceride (TG) levels by significantly inhibiting the expression of miR-194 in alcoholic liver disease.¹⁶⁶ Gan-Jiang-Ling-Zhu decoction inhibits miR-138-5p expression and accelerates FA β-oxidation, improves hepatic steatosis and inflammation, reduces liver index and liver TG content, and significantly reduces serum ALT and AST levels in rats (Figure 6 and Table S3).¹⁶⁷

Figure 6.

ncRNAs regulate the molecules involved in amino acid metabolism

ncRNAs regulate the metabolism of various amino acids and glutamine. Amino acid synthesis, utilization, and involvement in other metabolism pathways are usually changed in diseases. ALT, alanine aminotransferase; AST, aspartate aminotransferase; PC, pyruvate carboxylase; GDH, glutamate dehydrogenase.

The roles of circRNAs in regulating serine/glycine metabolism have not been well elucidated. Recent studies suggested that shRNA targeting-circ-MBOAT2 (membrane bound O-acyltransferase domain containing 2) suppressed tumor progression and glutamine catabolism by sponging miR-433-3p.¹⁶⁸ circMYH9, which is significantly upregulated in colorectal cancer tissues, promotes serine/glycine metabolism in a p53-dependent manner and leads to the proliferation of colorectal cancer cells.¹⁶⁹ According to a recent study by Eyob et al., circGRM4 was found to be significantly upregulated in the eyes of cystathionine β-synthase-deficient mice in response to extremely high circulating glutamate concentrations, which can lead to retinal vascular dysfunction.¹⁷⁰ circPSD3 (circRNA Pleckstrin and Sec7 domain-containing 3) acts as a sponge for miR-92b-3p. In vivo overexpression of circPSD3 after injection of AAV8-circPSD3 through the tail vein arrested the deterioration of CCl₄-induced hepatic fibrosis as indicated by reduced serum ALT and AST content and the degree of hepatic fibrogenesis.¹⁷¹ Furthermore, circCREBBP acts as a sponge for hsa-miR-1291 and subsequently alleviates liver fibrosis, with reduced serum ALT and AST contents (Figure 6 and Table S3).¹⁷²

Deamination is the removal of an amino group from a molecule. Enzymes that catalyze this reaction are called deaminases. Deamination can be divided into two categories: oxidative deamination and non-oxidative deamination.¹⁷³ Oxidative deamination refers to a deamination reaction in which amino acids are oxidized through the action of enzymes. The enzymes that catalyze this reaction are called amino acid oxidase or amino acid dehydrogenase, mainly including L-amino acid oxidase, D-amino acid oxidase, and L-glutamate dehydrogenase. Oxidative deamination of L-glutamate is important in the catabolism of amino acids. Because glutamic acid is the only amino acid capable of oxidative deamination in many organisms, it is now believed that, at least in animals, most amino acids are deaminated by the combined action of amino conversion and oxidative deamination of glutamic acid, and some amino acids can also be synthesized by the reverse reaction of this combined action. Glutamate dehydrogenase (GDH) is widely distributed and plays a very important role in the process of amino acid deamination.¹⁷⁴ RICTOR (Rapamycin-insensitive companion of mammalian target of rapamycin) acts as a potential HMGB1 (high mobility group box 1) ceRNA in the early stage of HCC. Indeed, elevated HMGB1 mRNA promoted the expression of RICTOR mRNA by competitively binding with miR-429 and other members of the miR-200 family. The overexpression of HMGB1/RICTOR 3′ UTR downregulated the expression of GDH. When downregulating miR-200 family members with antagomir, the stemness characteristics and tumorigenesis were disturbed in HCC (Figure 6 and Table S3).¹⁷⁵

Ketogenesis produces acetone, acetoacetate, and β-hydroxybutyrate molecules by breaking down FAs. The α-ketoacid generated by the deamination of ketogenic amino acids is first converted to acetoacetic acid, then enters the TCA cycle as acetyl-CoA, becomes oxaloacetic acid, and then generates sugar through glycogen heterogenesis.¹⁷⁶ Pyruvate carboxylase (PC) is a metabolic enzyme involved in the first step of gluconeogenesis.¹⁷⁷ The Zfp36l2-AS overexpression plasmid induced ACC-α (ACACA) dephosphorylation and impaired pyruvate carboxylase protein stability, promoted intramuscular fat deposition, activated the fast-spasticity muscle phenotype, and induced muscle atrophy.¹⁷⁸ The lentivirus vector overexpressing miR-143-3p gene attenuates pyruvate carboxylase expression by binding to the 3′ UTR of the pyruvate carboxylase mRNA and affects the activity of breast cancer cells.¹⁷⁹ The let-7 miRNA negatively regulates the expression of pyruvate carboxylase, and transgenic overexpression of pyruvate carboxylase or CRISPR-Cas9-based knockdown of let-7 leads to inhibition of cell growth in silk gland enlargement, which in turn leads to the increase of silk production (Figure 6 and Table S3).¹⁸⁰

Ketogenic amino acids produced by protein decomposition are deaminated to form ketoacids, which are decomposed into acetoacetic acid in the process of metabolism, and acetoacetic acid can be converted into acetyl-CoA and then synthesized into FAs. Glycogenic amino acids from protein breakdown can be converted to fat via pyruvate to acetyl-CoA. Phenylalanine is catalyzed by phenylalanine hydroxylase (PAH) to form tyrosine, which is also the synthesis pathway of tyrosine. Tyrosine traps ammonia to produce 4-hydroxyphenylpyruvate, which is catalyzed by 4-hydroxyphenylpyruvate dioxygenase (HPD) decarboxylation to produce urine melanic acid; urine melanic acid continues to be metabolized, will oxidize ring opening, and finally lyses into fumaric acid and acetoacetic acid. Fumaric acid enters the TCA cycle and acetoacetic acid enters ketone body metabolism. Phenylketonuria (PKU) is an autosomal recessive disorder of phenylalanine metabolism, mainly caused by mutations in the PAH gene, resulting in elevated levels of phenylalanine (Phe) in the blood and brain, leading to brain dysfunction. Li et al. identified two lncRNAs, HULC (nucleotides 183 to 216) and Pair (PAH-activating lincRNA;, nucleotides 460 to 496), present in humans and mice, respectively, that interact with PAH and regulate its function.¹⁸¹ Galnac-labeled HULC mimics could be enriched in the liver, and together with sapropterin could significantly reduce the concentration of excessive Phe in mice, without detectable effects on liver and kidney function.¹⁸¹ Combining HULC mimics with current dietary restrictions, sapropterin dihydrochloride supplements, and enzyme replacement or replacement therapies may further improve patient outcomes, especially for patients with severe PKU. Perry and Ulitsky pointed out that lncRNA mimics can be extensively modified, which promotes high stability and reduces immunogenicity in vivo compared with therapeutic mRNAs that need to be translated by ribosomes, and can be easily labeled with organ-targeting peptides for tissue-specific distribution.¹⁸² The low conservation between mouse and human lncRNAs hinders the identification of “pathogenic” or “therapeutic” lncRNAs, but some functional elements in this study are likely to be conserved between the two species, thus overcoming the major challenge of antisense oligonucleotides (ASOs). Leucine metabolism belongs to the branched-chain amino acid metabolism pathway, which is catalyzed by branched-chain amino acid aminotransferase (BCAT) and branched-chain α-ketoacid dehydrogenase (BCKD) to transamination and decarboxylation to isovaleryl-CoA.¹⁸³ Mersey et al.¹⁸⁴ represents the first report demonstrating the action of miR-29b, which targets the dihydrothioctinamide branched acyltransferase component of BCKD and prevents translation upon binding within HEK293 cells. snoRNAs play essential roles in rRNA biogenesis to adapt to the changing demands of splicing and protein synthesis during health and disease. Kim et al.¹⁸⁵ demonstrated that snoRNA-activated PARP-1 ADPRylates DDX21 (DExD-box helicase 21) in BRCA1/2-wild-type breast cancer cells to promote enhanced ribosome biogenesis and cell proliferation.

Conclusion

The inability to fully utilize and/or store energy and the disruption of usual metabolic processes characterize metabolic disorders that affect millions of people worldwide. At present, metabolic disorders are recognized as one of the major global challenges. Therefore, the molecular mechanisms underlying the development of metabolic disorders need to be clarified to develop effective therapeutic strategies. In recent years, more and more attention has been paid to ncRNAs as a novel intracellular regulatory mechanism. A growing body of evidence suggests that dysregulation of ncRNAs may be involved in the occurrence and development of metabolic disorders.

The specificity that ncRNAs exert on significant functions in the progress of diseases makes them ideal as biomarkers for metabolic disorders. Various therapeutics of metabolic disorders based on RNA, including ASOs, ASO anti-miRNAs, siRNAs, shRNAs, miRNA mimics, miRNA sponges, therapeutic circular RNAs, and CRISPR-Cas9-based gene editing, have been developed and translated into the clinic.¹⁸⁶ Currently, some RNA-based therapeutics have been approved by the FDA and/or the European Medicines Agency (EMA).¹⁸⁷ To date, some miRNA therapies associated with cardiovascular disease, including heart failure, cardiac fibrosis, neointima formation, atherosclerosis, and lipid metabolism, are already undergoing clinical evaluation.¹⁸⁸ In addition, the development of many new tumor-targeted cancer therapeutics to enhance therapeutic efficacy and reduce side effects based on ncRNAs has been an active area of investigation in recent years.¹⁸⁹

ncRNAs play vital roles in the three major metabolisms. In glucose metabolism, many studies have demonstrated that ncRNAs regulate GLUTs, represented by GLUT1, GLUT2, GLUT3, and GLUT4, thereby affecting glucose metabolism. Also, ncRNAs control several essential enzymes of glycolysis. In FA metabolism, ncRNAs mediate the regulation of lipogenic enzymes, regulate FAO, and also act on FA transport proteins among others. In amino acid metabolism, certain ncRNAs are involved in the regulation of several processes, such as GPT/ALT and GOT/AST in transamination, deamidation, gluconeogenesis, and ketogenic amino acid metabolism. In addition, there have been research advances in ncRNA-based therapies for related metabolic diseases. Hence, recent research advances have demonstrated that ncRNAs are indispensable factors in metabolic pathways, and their potential as therapeutic targets for metabolism-related diseases is substantial.

ncRNAs are the vast majority of RNAs synthesized by the genome, and the categories of ncRNAs remain to be explored. The discovery of ncRNAs has revolutionized the comprehension of the connection between genetics and disease. There is now a growing body of research focusing on the study of regulatory and functional roles of ncRNAs in disease occurrence and development. ncRNA-based strategies could play a key role in the diagnosis and therapy of diseases.