大鼠肝再生相关lncRNA对大鼠肝细胞株BRL-3A增殖的作用研究

发布时间：2018-04-08 22:33

本文选题：lncRNA　切入点：大鼠肝细胞BRL-3A　出处：《河南师范大学》2017年硕士论文

【摘要】：近些年来,研究证明lncRNA能够调控多种生理和病理进程,包括对大鼠肝再生的调控。但是lncRNA对肝再生的调控机制还不清楚。为了了解lncRNA是如何调控肝再生,本研究采用高通量测序、RT-PCR检测大鼠2/3肝切除诱导的肝再生3个时间点肝细胞中表达有意义的lncRNA。结果表明在大鼠肝再生3个时间点肝细胞中表达变化有意义的lncRNA有770条,其中差异表达的lncRNA有28条。分析在肝再生中有意义表达的lncRNA的具体表达情况,发现有254条表达发生上调,464条表达下调,52条表达上/下调。差异表达的28条lncRNA中13条表达上调,12条表达下调,3条表达上/下调。为了解这些差异表达的lncRNA对肝再生的调节作用,采用生物信息学方法预测其靶基因,结果表明lncRNA的共表达(TRANS作用)基因中表达差异的465条。lncRNA的调控(CIS作用)基因差异表达的10条。为进一步筛选出待研究的lncRNA及其在肝再生中的作用,用DESeq,结合GO和KEGG信息分析基因功能发现基因在细胞中具有多种作用。用Ingenuity中的Ingenuity Pathway Analysis(IPA)检测大鼠肝再生不同时间点差异表达的基因相应的蛋白质在细胞中作用,结果显示相关基因在细胞生长、增殖、分化、细胞通讯、信号传导、分子运输、细胞增殖、作为信号分子等方面具有一定的调控作用。IPA分析信号通路发现差异表达lncRNA的靶基因主要参与的信号通路有ILK通路、ERK/MAPK通路、SAPK/JNK Signaling通路等。本文从差异表达lncRNA中筛选其中的两个并研究其对肝细胞增殖的调控作用。高通量测序结果表明lncRNA TCONS_00027980和TCONS_00042303在肝再生不同时间点表达水平显著变化,RT-PCR检测不同时间点再生肝组织中两种lncRNA的RNA表达水平,实验结果表明高通量测序结果是可信的。本文首先用干涉技术降低lncRNA在大鼠BRL-3A细胞中的表达水平,MTT法检测BRL-3A细胞的活性,流式细胞术检测BRL-3A增殖情况的变化,RT-PCR和Western-blot用来检测与增殖相关基因的表达情况。检测结果表明MTT法检测表明实验组比对照组细胞活性增加(P0.05);流式细胞术实验检测显示与对照组相比,实验组的S+G2/M期细胞数在一定程度上增加了(P0.05);而real-time PCR(RT-PCR)检测mRNA表达水平,结果表明实验组与对照组相比,细胞增殖相关基因MYC、CCNA2、CCND1、BCL-2表达上调(P0.05),凋亡相关基因的BAX表达下调(P0.05);蛋白质免疫印迹(Western-blot)检测表明,实验组细胞增殖相关基因MYC、CCNA2、CCND1、BCL-2表达上调(P0.05),凋亡相关基因的BAX表达下调(P0.05)。lncRNA TCONS_00027980和TCONS_00042303能够通过降低细胞增殖相关基因的表达水平抑制大鼠肝细胞BRL-3A的细胞增殖。
[Abstract]:In recent years, it has been demonstrated that lncRNA can regulate various physiological and pathological processes, including the regulation of rat liver regeneration.However, the regulatory mechanism of lncRNA on liver regeneration is unclear.In order to understand how lncRNA regulates liver regeneration, we used high-throughput sequencing RT-PCR to detect the expression of LNC in rat hepatocytes induced by 2 / 3 hepatectomy at three time points.The results showed that there were 770 differentially expressed lncRNA in rat liver cells at three time points of liver regeneration, among which 28 were differentially expressed lncRNA.By analyzing the specific expression of lncRNA in liver regeneration, it was found that there were 254 up-regulated, 464 up-regulated and 52 up-/ down-regulated expression of lncRNA.Among 28 differentially expressed lncRNA, 13 were up-regulated, 12 were down-regulated, and 3 were up-/ down-regulated.In order to understand the regulatory effect of differentially expressed lncRNA on liver regeneration, the target genes were predicted by bioinformatics.The results showed that there were 10 differentially expressed genes in the coexpression of lncRNA.In order to screen out the lncRNA and its role in liver regeneration, we used DESeq, go and KEGG information to analyze the gene function and found that the gene has many functions in the cell.Ingenuity Pathway analysis in Ingenuity was used to detect the role of proteins of differentially expressed genes at different time points in rat liver regeneration. The results showed that the related genes were involved in cell growth, proliferation, differentiation, cell communication, signal transduction and molecular transport.IPA analysis showed that the target genes involved in the differential expression of lncRNA were mainly involved in the ILK pathway, ERK / MAPK pathway, SAPKP / JNK Signaling pathway and so on.Two of them were screened from differentially expressed lncRNA and their effects on hepatocyte proliferation were studied.The results of high-throughput sequencing showed that the expression levels of lncRNA TCONS_00027980 and TCONS_00042303 were significantly changed at different time points during liver regeneration. RT-PCR was used to detect the RNA expression levels of two kinds of lncRNA in regenerated liver tissues at different time points. The results showed that the results of high-throughput sequencing were reliable.In this paper, the expression level of lncRNA in rat BRL-3A cells was reduced by interference technique. The activity of BRL-3A cells was detected by MTT assay. The changes of BRL-3A proliferation were detected by flow cytometry. RT-PCR and Western-blot were used to detect the expression of proliferation-related genes.The results showed that the cell activity of the experimental group was higher than that of the control group by MTT, the number of S G _ 2 / M phase cells of the experimental group was increased to some extent than that of the control group, and the expression of mRNA was detected by real-time PCRRT-PCR.The results showed that the expression of MYCnCCNA2CCND1 / BCL-2 was up-regulated, the expression of apoptosis-related gene BAX was down-regulated (P0.05), and the Western blot assay showed that the expression of MYC- CCNA2CCND1- BCL-2 was down-regulated by Western blot (Western blot), and the expression of apoptosis-related gene was down-regulated.In the experimental group, the expression of MYCnCCNA2CCND1 / BCL-2 gene was up-regulated, and the BAX expression of apoptosis-related gene down-regulated P0.05. lncRNA TCONS_00027980 and TCONS_00042303 could inhibit the proliferation of rat hepatocyte BRL-3A by decreasing the expression level of cell proliferation-related genes.
【学位授予单位】：河南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575

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