【作者】 李俊；

【导师】 徐存拴；

【作者基本信息】 河南师范大学 ， 生物学、细胞生物学， 2017， 硕士

【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 TCONS₀0027980和TCONS₀0042303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（P<0.05）;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（P<0.05）;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（P<0.05）,凋亡相关基因的BAX表达下调（P<0.05）;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（P<0.05）,凋亡相关基因的BAX表达下调（P<0.05）。lncRNA TCONS₀0027980和TCONS₀0042303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。更多还原

【Abstract】 Emerging evidences have revealed that long non-coding RNAs（lnc RNAs）functioned in a wide range of physiological and pathophysiological processes including rat liver regeneration,and could regulate gene expression in the transcriptional and post-transcriptional levels.However,the underlying mechanism for lncRNAs participation in liver regeneration is largely unknown.To define the mechanisms how the lncRNAs regulate LR,we performed bio-chip technology,high-throughput sequencing and RT-PCR to detect the expression of lnc RNAs at 0,2 and 6h during LR after 2/3 hepatectomy（PH）.The results indicated that 770 lncRNAs expressed meaningfully and 28 lncRNAs significantly expressed were involved in LR.To understand more about the lncRNA expressed meaningfully regulation of LR,using the bioinformatic methods to predict the target genes and the result showed 465 genes expressed significantly among the co-expression genes（TRANS）.More clearly,254 genes expression level increased and 464 genes expression level decreased,and 52 genes were positive/negative regulated.And the lncRNA（CIS）expressed significantly were 10.Then we combined the DESeq with the information of the GO and KEGG to analyze the lncRNAs’ function for selecting ones to more research.Ingenuity Pathway Analysis（IPA）was employed to analyze the signaling pathways and physiological activities that regulated by these genes,and the results suggested that these genes were potentially related to ILK,SAPK/JNK and ERK/MAPK signaling pathways,and possibly regulate many important physiological activities in LR in terms of cell proliferation,cell differentiation,cell survival,apoptosis and necrosis.We researched the two selected lnc RNAs’ function mechanism in rat LR.Through High-throughput sequencing the lncRNA TCONS₀0027980 and TCONS₀0042303 was demonstrated that they expressed differentially during the rat LR.To study the effect of the long non-coding RNA,TCONS₀0027980 on the proliferation of the rat liver cell line BRL-3A in vitro for explore influence of TCONS₀0027980 and TCONS₀0042303 on rat liver regeneration.The expressed differentially lncRNAs were screened through High-throughput sequencing and then lnc RNA TCONS₀0027980 was selected.The expression of TCONS₀0027980 and TCONS₀0042303 were knocked down by RNA interference,then MTT method,Flow cytometry and RT-PCR were used to detect the effect of them on the cell viability,cell cycle and cell proliferation of rat cell line BRL-3A.The results of MTT assay showed thecell viability of the interference group（siRNA）was significantly higher than the negative group（NC）at 48 h after transfection（P<0.05）.Meanwhile,Flow cytometry found the cell number of S+G2/M period in the cell cycle of the interference group was increased（P<0.05）compared with the negative group.Moreover,RT-PCR and the Western-blot asserted that the mRNA and protein expression level of the cell proliferation-related genes MYC,CCNA2,CCND1,BCL2 were significantly increased respectively（P<0.05）,and the apoptosis-related gene BAX was significantly decreased（P<0.05）.lncRNA TCONS₀0027980 and TCONS₀0042303 may inhibit the cell proliferation of rat cell line BRL-3A via lowering the expression of cell proliferation-related genes in vitro.更多还原