ANRIL, H19 and TUG1: a review about critical long non-coding RNAs in cardiovascular diseases

Cardiovascular diseases are the leading cause of death worldwide. They are non-transmissible diseases that affect the cardiovascular system and have different etiologies such as smoking, lipid disorders, diabetes, stress, sedentary lifestyle and genetic factors. To date, lncRNAs have been associated with increased susceptibility to the development of cardiovascular diseases such as hypertension, acute myocardial infarction, stroke, angina and heart failure. In this way, lncRNAs are becoming a very promising point for the prevention and diagnosis of cardiovascular diseases. Therefore, this review highlights the most important and recent discoveries about the mechanisms of action of the lncRNAs ANRIL, H19 and TUG1 and their clinical relevance in these pathologies. This may contribute to early detection of cardiovascular diseases in order to prevent the pathological phenotype from becoming established.

ANRIL, H19 and TUG1: a review about critical long non-coding RNAs in cardiovascular diseases.

Cardiovascular diseases are the leading cause of death worldwide. They are non-transmissible diseases that affect the cardiovascular system and have different etiologies such as smoking, lipid disorders, diabetes, stress, sedentary lifestyle and genetic factors. To date, lncRNAs have been associated with increased susceptibility to the development of cardiovascular diseases such as hypertension, acute myocardial infarction, stroke, angina and heart failure. In this way, lncRNAs are becoming a very promising point for the prevention and diagnosis of cardiovascular diseases. Therefore, this review highlights the most important and recent discoveries about the mechanisms of action of the lncRNAs ANRIL, H19 and TUG1 and their clinical relevance in these pathologies. This may contribute to early detection of cardiovascular diseases in order to prevent the pathological phenotype from becoming established.

Long Non-Coding RNA ANRIL Regulates Inflammatory Factor Expression in Ulcerative Colitis Via the miR-191-5p/SATB1 Axis.

Ulcerative colitis, an inflammatory bowel disease, manifests with symptoms such as abdominal pain, diarrhea, and mucopurulent feces. The long non-coding RNA (lncRNA) ANRIL exhibits significantly reduced expression in UC, yet its specific mechanism is unknown. This study revealed that ANRIL is involved in the progression of UC by inhibiting IL-6 and TNF-α via miR-191-5P/SATB1 axis. We found that in patients with UC, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were significantly overexpressed in inflamed colon sites, whereas ANRIL was significantly under-expressed and associated with disease severity. The downregulation of ANRIL resulted in the increased expression of IL-6 and TNF-α in LPS-treated FHCs. ANRIL directly targeted miR-191-5p, thereby inhibiting its expression and augmenting SATB1 expression. Moreover, overexpression of miR-191-5p abolished ANRIL-mediated inhibition of IL-6 and TNF-α production. Dual luciferase reporter assays revealed the specific binding of miR-191-5p to ANRIL and SATB1. Furthermore, the downregulation of ANRIL promoted DSS-induced colitis in mice. Together, we provide evidence that ANRIL plays a critical role in regulating IL-6 and TNF-α expression in UC by modulating the miR-191-5p/SATB1 axis. Our study provides novel insights into progression and molecular therapeutic strategies in UC.

Expression profile of LncRNA ANRIL, miR-186, miR-181a, and MTMR-3 in patients with preeclampsia.

Preeclampsia (PE) is a leading cause of maternal and neonatal morbidity and mortality worldwide. Several studies demonstrated the role of lncRNAs and miRNAs in the pathogenesis of preeclampsia; the aim was to detect the expression profiles of serum LncRNA ANRIL, miR-186, miR-181a, and MTMR-3 in patients with preeclampsia. The study included 160 subjects divided into 80 subjects considered as a control group, 80 patients with preeclampsia. We found that there was a significant difference between the preeclampsia and control groups with up-regulation of miR-186 median (IQR) = 4, 29 (1.35-7.73) (P < 0.0001), miR-181a median (IQR) = 2.45 (0.83-6.52) (P = 0.028), and downregulation of lncRNA ANRIL median (IQR) = 0.35(0.28-0.528) (P < 0.0001), MTMR median (IQR) = 0.32(0.155-1.11), (P < 0.0001). ROC curve of lncRNA ANRIL, miR-186, miR-181a, and MTMR-3 in preeclampsia patients showing the roles of these markers in the diagnosis of preeclampsia. In conclusion, serum LncRNA ANRIL, miR-186, miR-181a, and MTMR-3 could be promising biomarkers in the diagnosis of preeclampsia.

Panax notoginseng saponins inhibits NLRP3 inflammasome-mediated pyroptosis by downregulating lncRNA-ANRIL in cardiorenal syndrome type 4.

OBJECTIVE: Cardiorenal syndrome type 4 (CRS4) is a complication of chronic kidney disease. Panax notoginseng saponins (PNS) have been confirmed to be efficient in cardiovascular diseases. Our study aimed to explore the therapeutic role and mechanism of PNS in CRS4. METHODS: CRS4 model rats and hypoxia-induced cardiomyocytes were treated with PNS, with and without pyroptosis inhibitor VX765 and ANRIL overexpression plasmids. Cardiac function and cardiorenal function biomarkers levels were measured by echocardiography and ELISA, respectively. Cardiac fibrosis was detected by Masson staining. Cell viability was determined by cell counting kit-8 and flow cytometry. Expression of fibrosis-related genes (COL-I, COL-III, TGF-ß, α-SMA) and ANRIL was examined using RT-qPCR. Pyroptosis-related protein levels of NLRP3, ASC, IL-1ß, TGF-ß1, GSDMD-N, and caspase-1 were measured by western blotting or immunofluorescence staining. RESULTS: PNS improved cardiac function, and inhibited cardiac fibrosis and pyroptosis in a dose-dependent manner in model rats and injured H9c2 cells (p < 0.01). The expression of fibrosis-related genes (COL-I, COL-III, TGF-ß, α-SMA) and pyroptosis-related proteins (NLRP3, ASC, IL-1ß, TGF-ß1, GSDMD-N, and caspase-1) was inhibited by PNS in injured cardiac tissues and cells (p < 0.01). Additionally, ANRIL was upregulated in model rats and injured cells, but PNS reduced its expression in a dose-dependent manner (p < 0.05). Additionally, the inhibitory effect of PNS on pyroptosis in injured H9c2 cells was enhanced by VX765 and reversed by ANRIL overexpression, respectively (p < 0.05). CONCLUSION: PNS inhibits pyroptosis by downregulating lncRNA-ANRIL in CRS4.

Long noncoding RNA antisense noncoding RNA in the INK4 locus inhibition alleviates airway remodeling in asthma through the regulation of the microRNA-7-5p/early growth response factor 3 axis.

Asthma, a chronic inflammatory disease of the airways, clinically manifests as airway remodeling. The purpose of this study was to probe the potential role of long noncoding RNA (lncRNA) antisense noncoding RNA in the INK4 locus (lncRNA ANRIL) in the proliferation and migration of airway smooth muscle cell (ASMC) and to explore its potential mechanisms in asthma. Serum samples were obtained from 30 healthy volunteers and 30 patients with asthma. Additionally, platelet-derived growth factor-BB (PDGF-BB) was used to induce airway remodeling in ASMCs. The level of lncRNA ANRIL and microRNA (miR)-7-5p in serum samples were measured by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). TargetScan predicted the binding site of miR-7-5p to early growth response factor 3 (EGR3) and validated the results using a dual-luciferase reporter assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and Transwell assays were used to detect cellular proliferation and migration, respectively. Subsequently, changes in proliferation- and migration-related genes were verified using western blot analysis and qRT-PCR. These results indicate that lncRNA ANRIL was upregulated in the serum and PDGF-BB-induced ASMCs of patients with asthma, whereas miR-7-5p expression was reduced. EGR3 was a direct target of miR-7-5p. LncRNA ANRIL silencing inhibited the proliferation or migration of ASMCs induced by PDGF-BB through miR-7-5p upregulation. Mechanistic studies indicated that miR-7-5p inhibits the proliferation or migration of PDGF-BB-induced ASMCs by decreasing EGR3 expression. EGR3 upregulation reverses the role of miR-7-5p in airway remodeling. Thus, downregulation of lncRNA ANRIL inhibits airway remodeling through inhibiting the proliferation and migration of PDGF-BB-induced ASMCs by regulating miR-7-5p/EGR3 signaling.

Babao Dan inhibits lymphangiogenesis of gastric cancer in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

Gastric cancer (GC) is one of the most common gastrointestinal malignancies in the world. Growing evidence emphasizes the critical role of long non-coding RNA (lncRNA) in GC tumorigenesis. The aim of the research was to elucidate the effect and mechanism of Babao Dan (BBD) on lymphangiogenesis of GC in vitro and in vivo via lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis. The present study investigated BBD significantly decreased the expression of lncRNA-ANRIL and VEGF-C in GC cells (AGS, BGC823, and MGC80-3) by using real-time quantitative polymerasechain reaction (RT-qPCR) and the secretion and expression of VEGF-C by (enzyme linked immunosorbent assay) ELISA and western blot (WB). BBD significantly inhibited the tumor xenograft of GC growth and the expression of lncRNA-ANRIL, VEGF-C, VEGFR-3 and LYVE-1 in vivo. BBD reduced serum VEGF-C level. In vitro, BBD inhibited the tube formation and decreased the cell viability, proliferation and migration of HLECs by using tube formation, MTT, Hoechst and Transwell assays. In addition, WB assay found that BBD decreased the expression levels of VEGF-C, VEGFR-3, matrix metallopeptidase 2 (MMP-2) and matrix metallopeptidase 9 (MMP-9), and RT-qPCR assay found that the mRNA expression levels of lncRNA-ANRIL, VEGF-C, VEGFR-3, MMP-2, MMP-9, CDK4, Cyclin D1, and Bcl-2 were down-regulated, and the expression of p21 and Bax were increased. Taken together, these results demonstrated that BBD inhibited lymphangiogenesis of GC in vitro and in vivo via the lncRNA-ANRIL/VEGF-C/VEGFR-3 signaling axis.

Significance of the lncRNAs MALAT1 and ANRIL in occurrence and development of glaucoma.

BACKGROUND: Primary open-angle glaucoma (POAG) is the commonest form of glaucoma which is estimated to cause bilaterally blind within 11.1 million people by 2020. Therefore, the primary objectives of this study were to investigate the clinical significance of single-nucleotide polymorphisms (SNPs) in the lncRNAs MALAT1 and ANRIL in a Chinese Han POAG cohort. METHODS: Three hundred and forty-six glaucoma patients and 263 healthy controls were recruited, and totally 14 SNPs in MALAT1 and ANRIL were genotyped between the two populations. RESULTS: The MALAT1 SNPs rs619586 (A>G), rs3200401 (C>T), and rs664589 (C>G) were associated with POAG risk, and the ANRIL SNPs rs2383207 (A>G), rs564398 (A>G), rs2157719 (A>G), rs7865618 (G>A), and rs4977574 (A>G) were associated with POAG (p < 0.05). The MALAT1 haplotypes ACG and ATC, comprised rs619586, rs3200401, and rs664589, increased POAG risk, and the ANRIL haplotype AAGAA, made up of rs2383207, rs7865618, rs4977574, rs564398, and rs2157719, show a significantly increased risk of POAG. In addition, rs619586 (A>G) of MALAT1 and rs564398/rs2157719 of ANRIL were associated with a smaller vertical cup-to-disc ratio, while rs619586 of MALAT1 and rs2383207/rs4977574 of ANRIL were associated with higher intraocular pressure in the POAG population. CONCLUSION: Single-nucleotide polymorphisms and haplotypes in ANRIL and MALAT1 were associated with POAG onset in our study population, which provide more possibilities to POAG diagnosis and treatment.

Long non-coding RNA ANRIL interacts with microRNA-34a and microRNA-125a, and they all correlate with disease risk and severity of Parkinson's disease.

BACKGROUND: This study aimed to investigate the correlation of long non-coding RNA antisense non-coding RNA in the INK4 locus (lncRNA ANRIL) and its target microRNAs (microRNA-34a (miR-34a) and microRNA-125a (miR-125a)) with disease risk and severity of Parkinson's disease (PD). METHODS: Seventy-eight PD patients and 78 age-/gender-matched controls were consecutively enrolled. Their peripheral blood mononuclear cell samples were collected and proposed for the reverse-transcription quantitative polymerase chain reaction to complete lncRNA ANRIL, miR-34a, and miR-125a measurements. RESULTS: LncRNA ANRIL was upregulated, while miR-34a and miR-125a were downregulated in PD patients compared to controls (all p < 0.001). Further, they all showed certain values for PD risk identification by ROC curve analyses, among which lncRNA ANRIL showed the highest AUC (AUC: 0.879, 95% CI: 0.824-0.934). Furthermore, lncRNA ANRIL negatively correlated with miR-34a (p = 0.016) and miR-125a (p = 0.005) in PD patients, but not in controls. In addition, lncRNA ANRIL was observed to positively associate with UPDRS-I score (p = 0.029), UPDRS-III score (p = 0.006), and UPDRS-IV score (p = 0.033), while negatively correlated with MMSE score (p = 0.003). These associations were less distinct as to miR-34a and miR-125a. CONCLUSION: LncRNA ANRIL interacts with miR-34a and miR-125a in PD patients, and they all correlate with disease risk and severity of PD.

Identification and validation of key long non-coding RNAs using co-expression network analysis in Crohn's disease.

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disease of the digestive tract. The underlying molecular mechanism of CD remains unclear. The aim of this study was to investigate the differentially expressed long non-coding RNA (lncRNA) in CD and its possible mechanism, and to verify the expression of lncRNA. METHODS: Microarray GSE67106 and GSE83448 were downloaded from the Gene Expression Omnibus (GEO) database. Differentially expressed lncRNAs (DELs) and messenger RNAs (mRNAs, DEGs), when normalized through the betaqn package in the R, were determined via the limma package. Gene Ontology (GO) and Kyoto Encyclopedia of genes and genomes (KEGG) pathways were studied using the database for the annotation, visualization and integrated discovery (DAVID) version 6.7, along with Gene Set Enrichment Analysis (GSEA) version 3.0. The co-expression of lncRNAs-mRNAs were determined using weighted gene co expression network analysis (WGCNA). The micro RNAs (miRNAs) related to the DELs and DEGs were forecast. A competing endogenous RNA (ceRNA) network was established. RESULTS: There were 42 DEGs and 551 DEGs identified in total among the samples of the CD and normal control, respectively. These DEGs were enriched in such pathways as retinol metabolism, renin angiotensin system, and maturation-related signaling pathways. A lncRNA-mRNA co-expression network was constructed by WGCNA, with CDKN2B-AS (ANRIL), CTC-210G5.1.1, RP11-467L20.10.1, RP11-325F22.5.1, and RP11-59E19.1.1 as hub DELs. Together with miRNAs, a ceRNA network was constructed and functional analysis showed that the cell brush border and plasma membrane, synthesis and transport of lipoprotein, and angiotensin maturation, metabolism, and regulation of blood pressure were involved in the progression of CD. We successfully validated 1 lncRNA ANRIL, in our clinical specimens, ANRIL, which can feature prominently in CD. However, the exact mechanism of lncRNA ANRIL in CD prediction and diagnosis requires further exploration. CONCLUSIONS: This study showed that lncRNA ANRIL has a certain predictive effect on CD occurrence and development and could be a new potential treatment target.

Serum lncRNA-ANRIL and SOX9 expression levels in glioma patients and their relationship with poor prognosis.

BACKGROUND: lncRNA-CDKN2B antisense RNA 1 (ANRIL) and SRY-box transcription factor 9 (SOX9) has abnormal expression in many tumors including glioma, but the underlying molecular mechanism is unclear. This study set out to investigate the serum lncRNA-ANRIL and SOX9 levels in glioma patients and their effects on prognosis. METHODS: We enrolled 142 glioma patients admitted to our hospital from May 2014 to May 2016 into the research group (RG) and 120 healthy subjects receiving concurrent physical examinations into the control group (CG). Fasting peripheral blood (4 mL each) was sampled from subjects from the two groups. Using the quantitative real-time polymerase chain reaction (qRT-PCR), lncRNA-ANRIL and SOX9 were measured to explore their values in the early diagnosis of glioma. Patients from RG were followed up for 3 years to analyze the influence of lncRNA-ANRIL and SOX9 on patient prognosis. We purchased glioma cell lines U251 and U87 and grouped them according to the transfection of different plasmids. We conducted CCK8 assay to test cell proliferation, Transwell assay to test cell invasion, the flow cytometry to test cell apoptosis, and Western Blot assay to measure bcl-2 and bax protein levels. RESULTS: ANRIL and SOX9 were evidently higher in RG than in CG (P<0.01). The receiver operating characteristic (ROC) curve revealed that the diagnostic sensitivity of ANRIL combined with SOX9 for glioma was 81.62%, and the specificity was 90.83% (P<0.01). ANRIL and SOX9 were closely related to tumor grade, tumor diameter, distant metastasis, and family history of glioma (P<0.01). In total, 135 patients were successfully followed up (95.07%). Patients with high levels of ANRIL and SOX9 had a markedly poorer prognosis than those with low levels (P<0.05). ANRIL and SOX9 were markedly higher in glioma cell lines (U251 and U87) than in normal brain cells (P<0.01). The proliferation and invasion of U251 cells were notably reduced after the transfection of ANRIL and SOX9 inhibitory sequences (P<0.01), but the apoptosis was notably increased (P<0.01). Bcl-2 expression was markedly increased in lncRNA-ANRIL-inhibitor and SOX9-inhibitor (P<0.01), while bax expression was markedly reduced in lncRNA-ANRIL-inhibitor and SOX9-inhibitor (P<0.01). CONCLUSION: lncRNA-ANRIL and SOX9 levels were higher in glioma patients than in healthy people. High-lncRNA-ANRIL and SOX9 levels were strongly associated with unfavorable prognosis of patients. The testing of biological behaviors revealed that lncRNA-ANRIL and SOX9 worked as tumor-promoting genes in glioma.

lncRNA ANRIL aggravates the chemoresistance of pancreatic cancer cells to gemcitabine by targeting inhibition of miR-181a and targeting HMGB1-induced autophagy.

Recent studies focus on long noncoding RNAs (lncRNA) as crucial regulators of cancer biology that contribute to essential cancer cell functions such as cell proliferation, apoptosis, and metastasis. In pancreatic cancer, several lncRNAs have been mentioned as important actors in tumorigenesis. However, the function of lncRNA ANRIL (named as ANRIL as follows) in pancreatic cancer has not been elucidated. In the present study, we show that ANRIL was up-regulated while miR-181a was down-regulated in pancreatic cancer tissues and HMGB1 was highly expressed. Knockdown of ANRIL in pancreatic cancer repressed cellular proliferation, invasion, migration, and reduced chemotherapy resistance to gemcitabine. ANRIL was negatively correlated with miR-181a, while overexpression of miR-181a could reverse the effect. For further mechanism research, we found that miR-181a aimed to HMGB1 which activated cell autophagy. Taken together, our results implicate that the ANRIL, by targeting miR-181a, activates the HMGB1-induced cell autophagy, which is thought to be critical for oncogenesis.

Differential non-coding RNAs expression profiles of invasive and non-invasive pituitary adenomas.

BACKGROUND: Pituitary adenoma (PA) accounts for 10-15% of all intracranial neoplasms. Despite their benign nature, PA often shows invasive growth. MicroRNAs (miRNAs) and long non-coding RNAs (lncRNAs) are a class of non-coding RNAs that play important roles in PA initiation and progression. AIM: The aim of this study was to find specific profiles of miR-200a and long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) in PA based on a comparative study using Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analyses of tumor tissue and plasma. METHODS: Plasma and PA tissue samples were obtained from two groups of included patients (15 invasive and 15 non-invasive PA). In addition, plasma samples from patients with invasive PA have collected pre- and post-operation. Plasma and tissue samples subjected to qRT-PCR analyses for the expression levels of miR-200a and lncRNA ANRIL. RESULTS: The expression levels of miR-200a and lncRNA ANRIL were increased in tissue samples patients with invasive PA than in the patients with non-invasive PA. In addition, the expression levels of circulating miR-200a and lncRNA ANRIL were increased in patients with invasive PA than in patients with non-invasive PA in the pre-operation period. However, the expression level of plasma circulating miR-200a and lncRNA ANRIL was decreased in patients with invasive PA in the post-operation period. Our results depicted a miR-200a and lncRNA ANRIL expression in tissue and plasma samples in the patients with invasive PA. In addition, Receiver Operating Characteristic (ROC) curve was used to evaluate the diagnostic value of these circulating miR-200a and lncRNA ANRIL. CONCLUSION: The expression of these tumor-associated ncRNAs has been elevated in the PAs. Therefore, miR-200a and lncRNA ANRIL represents as biomarkers for diagnosis and potential targets for novel invasive PA treatment strategies.

The lncRNA ANRIL Gene rs2151280 GG Genotype is Associated with Increased Susceptibility to Recurrent Miscarriage in a Southern Chinese Population.

BACKGROUND: Genetic factors may play an important role in susceptibility to recurrent miscarriage. Some cardiovascular disease-related candidate genes have been shown to be associated with recurrent miscarriage. Long noncoding RNA ANRIL has been confirmed to be associated with susceptibility to various diseases, such as cardiovascular disease. However, it remains unclear whether the ANRIL gene polymorphism is related to recurrent miscarriage susceptibility. METHODS: Three ANRIL gene polymorphisms (rs2151280, rs1063192 and rs564398) were genotyped in 819 controls and 610 recurrent miscarriage patients through TaqMan real-time polymerase chain reaction. The odds ratios and 95% confidence intervals (CIs) were used to assess the strength of each association. RESULTS: Our results showed that the ANRIL rs2151280 GG genotype was associated with increased susceptibility to recurrent miscarriage (GG vs AA: adjusted OR=1.527, 95% CI=1.051-2.218, p=0.0262; GG vs AG/AA adjusted OR=1.460, 95% CI=1.021-2.089, p=0.0381). By combining the analysis of the risk genotypes in the three SNPs, we found that individuals with 2-3 risk genotypes had a significantly increased risk of recurrent miscarriage compared with those with a 0-1 risk genotype (adjusted OR=1.728, 95% CI=1.112-2.683, p=0.0149). This risk was more significant in subgroups of women less than 35-40 years of age and women with 2-3 miscarriages. CONCLUSION: These results suggested that a specific SNP in the ANRIL gene may be associated with increased susceptibility to recurrent miscarriage in a southern Chinese population.

The role of lncRNA ANRIL in the progression of hepatocellular carcinoma.

OBJECTIVES: The aim of the current study was to identify the long noncoding RNAs (lncRNAs) ANRIL function and molecular pathways underlying hepatocellular carcinoma progression. METHODS: ANRIL knockdown with specific siRNA, and transfected into HepG2 cells according to the protocol of Lipofectamine 2000. Cell proliferation, apoptosis, migration and metastasis were assessed with MTT assay, flow cytometry and wound healing assay, respectively. Moreover, the expression level of ANRIL, apoptosis-related genes, and the Wnt pathway-associated genes were assessed by real time-PCR and Western blot assay. KEY FINDINGS: Knocking down of ANRIL led to alleviated cell growth and increased cell apoptosis of HepG2 cells through markedly increased expression levels of Bax and Bad. In contrast, dramatically diminished the expressions of anti-apoptotic factors including Bid and Bcl-2 in comparison to the scrambled control group (si-NC). Furthermore, ANRIL silencing resulted in an inactivated Wnt/ß-catenin pathway by suppressing key genes associated with this pathway. CONCLUSIONS: Taken together, these findings imply new insights into the regulatory network of the Wnt pathway through lncRNA ANRIL that indicate ANRIL may be a therapeutic factor potential for hepatocellular carcinoma.

Puerarin protects cardiomyocytes from ischemia-reperfusion injury by upregulating LncRNA ANRIL and inhibiting autophagy.

This study analyzed the roles of puerarin and LncRNA ANRIL in myocardial ischemia-reperfusion injury. Hypoxia/reperfusion (H/R) model was established with H9C2 cells. Effects of puerarin of gradient concentrations on cardiomyocytes at different time points of hypoxia and reoxygenation were detected by quantitative real-time polymerase chain reaction (qRT-PCR), cell counting kit-8 (CCK-8), and microscope observation. Effects of puerarin on cardiomyocyte viability, ANRIL expression, contents of lactate dehydrogenase (LDH) and malondialdehyde (MDA), apoptosis, and expressions of autophagy-related genes after H/R injury were determined by CCK-8, quantitative real-time polymerase chain reaction (qRT-PCR), ELISA, flow cytometry, and Western blot, respectively. After cell transfection, the effects of overexpressed and knockdown of ANRIL on cardiomyocytes and H/R-injured cardiomyocytes were examined by rescue experiments. The ischemia-reperfusion (I/R)-injured rat model was established to examine the protective effect of puerarin in vivo. Prolonged hypoxia downregulated ANRIL expression in cardiomyocytes and reduced cardiomyocyte viability. Prolonged reoxygenation increased apoptosis. Both cardiomyocyte viability and ANRIL expression showed a dose-dependent relationship with puerarin. Puerarin reversed the effects of H/R injury on cardiomyocyte viability, ANRIL expression, contents of LDH and MDA, apoptosis, and expressions of autophagy-related genes. Overexpression and knockdown of ANRIL regulated the functions of cardiomyocytes and the expressions of autophagy-related genes. Puerarin reversed the effects of knockdown of ANRIL on H/R-injured cells. The results of In vivo experiments confirmed that puerarin protected myocardial tissues by up-regulating ANRIL and inhibiting autophagy.

Upregulating the Expression of LncRNA ANRIL Promotes Osteogenesis via the miR-7-5p/IGF-1R Axis in the Inflamed Periodontal Ligament Stem Cells.

BACKGROUND: Long non-coding RNA (lncRNA) antisense non-coding RNA in the INK4 locus (ANRIL) is a base length of about 3.8 kb lncRNA, which plays an important role in several biological functions including cell proliferation, migration, and senescence. This study ascertained the role of lncRNA ANRIL in the senescence and osteogenic differentiation of inflamed periodontal ligament stem cells (iPDLSCs). METHODS: Healthy periodontal ligament stem cells (hPDLSCs) and iPDLSCs were isolated from healthy/inflamed periodontal ligament tissues, respectively. The proliferation abilities were determined by CCK-8, EdU assay, and flow cytometry (FCM). The methods of Western blot assay (WB), quantitative real-time polymerase chain reaction (qRT-PCR), alizarin red staining, alkaline phosphatase (ALP) staining, ALP activity detection, and immunofluorescence staining were described to determine the biological influences of lncRNA ANRIL on iPDLSCs. Senescence-associated (SA)-ß-galactosidase (gal) staining, Western blot analysis, and qRT-PCR were performed to determine cell senescence. Dual-luciferase reporter assays were conducted to confirm the binding of lncRNA ANRIL and miR-7-5-p, as well as miR-7-5p and insulin-like growth factor receptor (IGF-1R). RESULTS: HPDLSCs and iPDLSCs were isolated and cultured successfully. LncRNA ANRIL and IGF-1R were declined, while miR-7-5p was upregulated in iPDLSCs compared with hPDLSCs. Overexpression of ANRIL enhanced the osteogenic protein expressions of OSX, RUNX2, ALP, and knocked down the aging protein expressions of p16, p21, p53. LncRNA ANRIL could promote the committed differentiation of iPDLSCs by sponging miR-7-5p. Upregulating miR-7-5p inhibited the osteogenic differentiation of iPDLSCs. Further analysis identified IGF-1R as a direct target of miR-7-5p. The direct binding of lncRNA ANRIL and miR-7-5p, miR-7-5p and the 3'-UTR of IGF-1R were verified by dual-luciferase reporter assay. Besides, rescue experiments showed that knockdown of miR-7-5p reversed the inhibitory effect of lncRNA ANRIL deficiency on osteogenesis of iPDLSCs. CONCLUSION: This study disclosed that lncRNA ANRIL promotes osteogenic differentiation of iPDLSCs by regulating the miR-7-5p/IGF-1R axis.

LncRNA ANRIL negatively regulated chitooligosaccharide-induced radiosensitivity in colon cancer cells by sponging miR-181a-5p.

BACKGROUND: The radiosensitivity of colon cancer cells can be regulated by noncoding RNAs. OBJECTIVES: In this study, the lncRNA antisense non-coding RNA in the INK4 locus (ANRIL) was selected to analyze its regulatory role in chitooligosaccharides (COS)-related radiosensitivity in colon cancer cells. MATERIAL AND METHODS: The ANRIL expression in colon cancer cell lines was examined using real-time quantitative polymerase chain reaction (RT-qPCR), based on which we selected the cell line that presented the highest expression of ANRIL for radiosensitivity research. The cells were exposed to X-rays (0 Gy, 2 Gy, 4 Gy, and 6 Gy) and evaluated for changes in ANRIL and miR-181a-5p expression using RT-qPCR. Cell viability was evaluated using the CCK8 method, while apoptosis was detected with flow cytometry assays. Dual luciferase assays validated the binding between ANRIL and miR-181a-5p. The cell survival rates after differential COS treatments (0 mg/mL, 1.0 mg/mL, 2.0 mg/mL, 3.0 mg/mL, 4.0 mg/mL, and 5.0 mg/mL) were rated using CCK8 assay. The cells with the strongest dosage of COS (5.0 mg/mL) were selected to further investigate the role of ANRIL/miR-181a-5p in modulating the radiosensitivity observed with CCK-8 and flow cytometry assays. RESULTS: The ANRIL was highly expressed in colon cancer cells lines, especially in the SW480 cell line. Irradiation significantly decreased cell viability and ANRIL expression in a dose-dependent manner. The overexpression of ANRIL reduced the cell apoptosis rate after irradiation. MiR-181a-5p directly bound to ANRIL and was upregulated by irradiation in a dose-dependent manner. The suppression of miR-181a-5p decreased cell apoptosis. The COS treatment notably downregulated cell survival and promoted apoptosis in cells exposed to irradiation. The overexpression of ANRIL partially reversed COS-induced apoptosis and the inhibition rate; the upregulation of miR-181a-5p could counteract the impact of ANRIL regulation in cells. CONCLUSIONS: The ANRIL negatively regulated radiosensitivity induced by COS in colon cancer cells by sponging miR-181a-5p.