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LncRNA KCNQ1OT1 affects cell proliferation, apoptosis and fibrosis through regulating miR-18b-5p/SORBS2 axis and NF-?B pathway in diabetic nephropathy

ABSTRACT: Background It has been reported that long non-coding RNAs (lncRNAs) play vital roles in diabetic nephropathy (DN). Our study aims to research the function of lncRNA KCNQ1OT1 in DN cells and the molecular mechanism. Methods Human glomerular mesangial cells (HGMCs) and human renal glomerular endothelial cells (HRGECs) were cultured in high glucose (30 mM) condition as models of DN cells. KCNQ1 opposite strand/antisense transcript 1 (KCNQ1OT1) and miR-18b-5p levels were detected by quantitative real-time polymerase chain reaction (qRT-PCR). The mRNA and protein levels of Sorbin and SH3 domain-containing protein 2 (SORBS2), Type IV collagen (Col-4), fibronectin (FN), transcriptional regulatory factor-beta 1 (TGF-?1), Twist, NF-?B and STAT3 were measured by qRT-PCR and western blot. Cell viability was detected by cell counting kit-8 (CCK-8) assay for selecting the proper concentration of glucose treatment. Additionally, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and flow cytometry assay were employed to determine cell proliferation and apoptosis, respectively. The targets of KCNQ1OT1 was predicted by online software and confirmed by dual-luciferase reporter assay. Results KCNQ1OT1 and SORBS2 were elevated in DN. Both knockdown of KCNQ1OT1 and silencing of SORBS2 restrained proliferation and fibrosis and induced apoptosis in DN cells. Besides, Overexpression of SORBS2 restored the KCNQ1OT1 knockdown-mediate effects on proliferation, apoptosis and fibrosis in DN cells. In addition, miR-18b-5p served as a target of KCNQ1OT1 as well as targeted SORBS2. KCNQ1OT1 knockdown repressed NF-?B pathway. Conclusion KCNQ1OT1 regulated DN cells proliferation, apoptosis and fibrosis via KCNQ1OT1/miR-18b-5p/SORBS2 axis and NF-?B pathway.

SUBMITTER: Jie R

PROVIDER: S-EPMC7469295 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundLung cancer, especially lung squamous cell carcinoma (LUSC), is one of the most common malignant tumors worldwide. Currently, radiosensitization research is a vital direction for the improvement of LUSC therapy. Long non-coding RNAs (lncRNAs) can be novel biomarkers due to their multiple functions in cancers. However, the function and mechanism of lncRNA KCNQ1OT1 in the radioresistance of LUSC remain to be elucidated.MethodsThe clonogenic assay was employed to determine the radioresistance of SK-MES-1R and NCI-H226R cells. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot were conducted for the detection of gene expression. Cell proliferation was determined by the methyl thiazolyl tetrazolium (MTT) assay, colony formation assay, and 5-ethynyl-2'-deoxyuridine (EdU) staining, and cell apoptosis was assessed by flow cytometry. The relationships between genes were also evaluated by applying the luciferase reporter and radioimmunoprecipitation (RIP) assays.ResultsRadioresistant LUSC cells (SK-MES-1R and NCI-H226R) had strong resistance to X-ray irradiation, and lncRNA KCNQ1OT1 was highly expressed in SK-MES-1R and NCI-H226R cells. Moreover, knockdown of lncRNA KCNQ1OT1 prominently suppressed proliferation, attenuated radioresistance, and accelerated the apoptosis of SK-MES-1R and NCI-H226R cells. More importantly, we verified that miR-491-5p was a regulatory target of lncRNA KCNQ1OT1, and Xenopus kinesin-like protein 2 (TPX2) and RING finger protein 2 (RNF2) were the target genes of miR-491-5p. The rescue experiment results also demonstrated that miR-491-5p was involved in the inhibition of cell proliferation and the downregulation of TPX2 and RNF2 expression mediated by lncRNA KCNQ1OT1 knockdown in SK-MES-1R and NCI-H226R cells.ConclusionsLncRNA KCNQ1OT1 was associated with the radioresistance of radioresistant LUSC cells, and the lncRNA KCNQ1OT1/miR-491-5p/TPX2-RNF2 axis might be used as a therapeutic target to enhance the radiosensitivity of radioresistant LUSC cells.

Project description:Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death, and radiotherapy is currently one of the main treatments. Long non-coding RNAs (lncRNAs) are associated with the radiosensitivity and tumorigenesis of HCC. However, the role and molecular mechanism of potassium voltage-gated channel subfamily Q member 1 overlapping transcript 1 (KCNQ1OT1) in HCC are still unclear. The relative expression of KCNQ1OT1, microRNA-146a-5p (miR-146a-5p) and alkaline ceramidase 3 (ACER3) was quantified by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay. Clonogenic assay was used to assess the radiosensitivity of cells. Cell apoptosis and metastasis were evaluated by flow cytometry and transwell assays, respectively. The protein levels of apoptosis markers, metastasis markers and ACER3 were detected by western blot (WB) analysis. The relationship between miR-146a-5p and KCNQ1OT1 or ACER3 was determined by dual-luciferase reporter assay. Additionally, animal experiments were carried out to explore the effect of KCNQ1OT1 silencing on HCC tumor growth in vivo. KCNQ1OT1 was highly expressed in HCC, and its knockdown hindered the proliferation and metastasis, while increased the radiosensitivity and apoptosis of HCC cells. MiR-146a-5p could interact with KCNQ1OT1, and its inhibition reversed the effects of silenced-KCNQ1OT1 on the radiosensitivity and tumorigenesis of HCC cells. Besides, ACER3 was a target of miR-146a-5p, and its overexpression inversed the effects of miR-146a-5p mimic on the radiosensitivity and tumorigenesis of HCC cells. The expression of ACER3 was regulated by KCNQ1OT1 and miR-146a-5p. Furthermore, KCNQ1OT1 also could reduce the growth of HCC by regulating the miR-146a-5p/ACER3 axis in vivo. Our study suggested that KCNQ1OT1 improved ACER3 expression to regulate the radiosensitivity and tumorigenesis of HCC through sponging miR-146a-5p, indicating that KCNQ1OT1 might be a new therapeutic target for HCC.

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LncRNA KCNQ1OT1 affects cell proliferation, apoptosis and fibrosis through regulating miR-18b-5p/SORBS2 axis and NF-?B pathway in diabetic nephropathy

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