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Down-regulation of Xist and Mir-7a-5p improves LPS-induced myocardial injury

ABSTRACT: Background: X-inactive specific transcript (Xist) is a lncRNA, which plays a significant role in X-chromosome inactivation, regulates cell proliferation in tumor cells, and inhibits apoptosis in acute myocardial infarction. On the other hand, miR-7a-5p is involved in cardiomyocytes injury in myocardial ischemia/reperfusion. However, their roles in LPS-induced damage remain unclear. Objectives: This study aimed at using siRNA transfection and lentivirus infection to regulate the expression of xist and miR-7a-5p, and to evaluate their effects on LPS-induced myocardial damage. Method: Mice cardiomyocytes (MCM) cells were divided into six groups, namely the control group, the LPS group, the LPS + lncRNA- group, the LPS + lncRNA+ group, the LPS + miRNA- group, and the LPS + miRNA+ group. Quantitative real-time PCR (qRT-PCR) was performed to assay for the RNA expressions of xist, miR-7a-5p, peroxisome proliferator-activated receptor-? coactivator-1? (PGC-1?), and recombinant mitochondrial transcription factor A (Tfam) in all the groups. The ATP level was determined using the adenosine triphosphate (ATP) assay kit according to the manufacturer's instructions. Flow cytometry was performed to estimate the level of apoptosis and proliferation in cells in each group. Results: The level of xist in the myocardial cells was markedly higher in the LPS group compared with the control group; however, it was reduced in the LPS+ lncRNA- group. There was no significant difference in the expression of xist among the LPS+miRNA-, LPS+miRNA+, and LPS groups. Moreover, the expression of mir-7a-5p was significantly reduced in myocardial cells in the LPS group, and moderately reduced in the LPS+ miRNA- group, but remarkably elevated in the LPS+ miRNA+ group (P<0.05). The expression of mir-7a-5p was comparably similar in the LPS+ lncRNA- group, LPS+ lncRNA+ group, and LPS groups. Further, the levels of PGC-1a, and Tfam were determined. In the LPS group, the expression of PGC-1? was significantly reduced but elevated in the LPS+lncRNA- and LPS+ miRNA- groups (P<0.05). There was no significant difference in the level of PGC-1? among the LPS, LPS+ lncRNA+, and LPS+ miRNA+ groups. The expression of Tfam was markedly reduced in the LPS group (P < 0.05), but elevated after the suppression of xist and mir-7a-5p. The expression of Tfam was not significantly different among the LPS group, LPS+ lncRNA+ and LPS+ miRNA+ groups. Notably, overexpression of mir-7a-5p had a mild effect on the expression of Tfam in the LPS+ miRNA+ group compared with the control group. Besides, ATP expression in the LPS group was markedly reduced, but elevated after the inhibition of xist and mir-7a-5p. Suppressing the expression of xist or mir-7a-5p resulted in reduced cell apoptosis and increased cell proliferation. Conclusions: In this study, we established that down-regulation of xist and mir-7a-5p reduces apoptosis in response to LPS.

SUBMITTER: Liang D

PROVIDER: S-EPMC7532474 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Background: Acute kidney injury (AKI) refers to a sudden loss of renal function. This study was performed to identify the key RNAs acting in the mechanism of sepsis-induced AKI. Methods: Microarray dataset GSE94717 (including six sepsis-induced AKI samples and three control samples) was downloaded from Gene Expression Omnibus database. Differentially expressed miRNAs (DE-miRNAs) were identified. The miRNA targets were predicted and enrichment analysis was performed. Protein-protein interaction (PPI) and competing endogenous RNA (ceRNA) regulatory networks were constructed. Mouse podocytes were treated with lipopolysaccharide (LPS), following by cell viability and PCR analysis. Cellular apoptosis and the ceRNA network were validated. Results: Thirty-one common DE-miRNAs (two up-regulated and 29 down-regulated) by AKI versus control and male AKI versus control were identified. We found the targets of miR-15a-5p, miR-15b-5p, and miR-16-5p were involved in mTOR signaling pathway, and those of miR-29b-3p and miR-16-5p were enriched in PI3K-Akt signaling pathway. RNAs including miR-15b-5p, miR-15a-5p, miR-107, XIST, miR-16-5p, and cullin 3 gene (CUL3) were included in the ceRNA regulatory network. The downregulation of miR-15a-5p and miR-15b-5p and the upregulation of lncRNA XIST and CUL3 gene were validated using qPCR. The miR-15a-5p-XIST-CUL3 regulatory axis was identified and was validated. We confirmed that LPS inhibited the growth of mouse podocytes and seven of the ten miRNAs, but upregulated XIST and CUL3. Transfection analysis showed XIST siRNA enhanced LPS-induced MPC5 cell apoptosis and miR-15a-5p inhibitor reserved it, so did as CUL3 overexpression for miR-15a-5p mimics. Conclusion: The miR-15a-5p-XIST-CUL3 regulatory axis was related to the pathogenesis of sepsis-induced AKI. Highlights Totally, 31 miRNAs were dysregulated between disease and control groups. MiR-15a-5p, miR-15b-5p, and miR-16-5p were involved in mTOR signaling pathway. MiR-16-5p and miR-29b-3p were implicated in PI3K-Akt signaling pathway. The miR-15a-5p-XIST-CUL3 axis was critical for sepsis-induced AKI.

Project description:The increasing rates of morbidity and mortality caused by ischemic heart disease pose a serious threat to human health. Long non‑coding (lnc)RNA small nucleolar RNA host gene 1 (SNHG1) has a protective effect on the myocardium. In the present study, the role of lncRNA SNHG1 in myocardial ischemia reperfusion injury (MIRI) and the underlying mechanisms were investigated. After hypoxia/reoxygenation (H/R) induction, the expression levels of lncRNA SNHG1 were detected using reverse transcription‑quantitative PCR. After lncRNA SNHG1 overexpression via cell transfection, cell viability was detected using an MTT assay, apoptotic rates were detected using TUNEL staining, apoptosis‑related protein expression levels were detected using western blotting and respective kits were used to measure the oxidative stress levels. The Encyclopedia of RNA Interactomes database predicted the presence of binding sites between lncRNA SNHG1 and microRNA (miR)‑450b‑5p, and between miR‑450b‑5p and insulin‑like growth factor 1 (IGF1). These interactions were then verified using luciferase reporter assays. Subsequently, the regulatory mechanism underlying the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis in MIRI was investigated by overexpressing miR‑450b‑5p and knocking down IGF1 expression in H/R‑induced cells. Finally, the expression of PI3K/Akt signaling pathway‑related proteins was detected using western blotting. lncRNA SNHG1 expression was significantly downregulated in H/R‑induced AC16 cells. lncRNA SNHG1 overexpression significantly inhibited apoptosis and decreased oxidative stress levels in H/R‑induced AC16 cells, which was mediated via regulation of the miR‑450b‑5p/IGF1 axis and activation of the PI3K/Akt signaling pathway. Therefore, the present study suggested that activation of the PI3K/Akt signaling pathway via the lncRNA SNHG1/miR‑450b‑5p/IGF1 axis inhibited the apoptosis and oxidative stress levels of H/R‑induced AC16 cells.

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Project description:Background: We have hypothesized that different factors are involved in the severity of ACD and AD because some but not all patients with atopic dermatitis (AD) present with allergic conjunctival disease (ACD) including severe types such as atopic keratoconjunctivitis (AKC) with/without giant papillae. We previously reported that plasma miR-628-3p was up-regulated in AD with severe ACD, but not in severe AD without severe ACD or in our healthy controls. In this study, to investigate the pathogenesis of AD with and without severe ACD, we performed comprehensive plasma miRNA analysis and studied the function of some miRNAs which were significantly up-regulated in ACD. Methods: Transcriptomics analysis of miRNA was performed using the microarray platform from the plasma of nine individuals (AD, severe ACD, controls: n = 3 each). To confirm up-regulation of the 12 miRNAs of the eight miRNA groups we focused on, we performed quantitative miRNA polymerase chain reaction (PCR) assays using 80 plasma samples (AD: 23, severe ACD: 17, controls: 40). To study the function of the eight miRNAs which were significantly up-regulated in ACD, we transfected their mimic to THP-1 cells, a monocyte cell line, and performed comprehensive gene expression analysis of them. The up-regulation of gene expression of interest in transfected THP-1 cells with the hsa-let-7a-5p miRNA mimic was confirmed by quantitative RT-qPCR assay. Results: Quantitative miRNA PCR assays showed that hsa-let-7a-5p, hsa-let-7days-3p, hsa-let-7e-5p, and hsa-miR-151a-5p were significantly up-regulated in both AD-ACD + and AD-ACD - as were hsa-miR-130a-3p, hsa-miR-151a-3p, has-miR-27b-3p, and hsa-miR-146a-5p in AD-ACD + but not in AD-ACD - . The functions of each miRNA were investigated by comprehensive gene expression analysis of THP-1 cells transfected with each miRNA mimic. Of the eight miRNAs, hsa-let-7a-5p, hsa-let-7e-5p, has-miR-27b-3p, and hsa-miR-146a-5p mimic-transfected THP-1 cells showed the up-regulation of CXCL10 (IP-10; interferon gamma-induced protein 10), which might be one of the innate immune-related genes. Quantitative RT-qPCR assays of transfected THP-1 cells with the hsa-let-7a-5p miRNA mimic showed that the 17 genes up-regulated more than 10-fold in the comprehensive gene expression analysis, and TLR3, RIG-I, and MDA5, important innate immune-related genes, were significantly up-regulated. TNFSF13B, AIM2, USP41, STAP1, GBP4, CCL8, and IFI27, reportedly down-regulated by the hsa-miR-628-3p mimic, were also significantly up-regulated in the transfected cells. Conclusion: Hsa-let-7a-5p, which was significantly up-regulated in AD-ACD + and AD-ACD - , could positively regulate the important innate immune-related genes such as TLR3, RIG-I, and MDA5. It is possible that in an allergic disease such as atopic keratoconjunctivitis and/or dermatitis, innate immune responses might be positively regulated by hsa-let-7a-5p in the plasma.

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Down-regulation of Xist and Mir-7a-5p improves LPS-induced myocardial injury

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