miR-199a调控肝星状细胞增殖和迁移及其分子机制
发布时间：2019-05-05 07:40
【摘要】：目的探讨mi R-199a对肝星状细胞(Hepatic Stellate Cells,HSCs)活化增殖和迁移能力的影响,并确定mi R-199a直接调控的靶基因以及靶基因对HSCs的作用。方法1构建mi R-199a过表达载体pcDNA3/pri-mi R-199a,合成mi R-199a的反义寡聚核苷酸链即ASO-199a,然后通过脂质体转染的方法转染肝星状细胞LX-2,将实验分为四组,pc DNA3空载质粒组(pc DNA3组)、pc DNA3/pri-mi R-199a质粒组(mi R-199a组)、乱序寡聚核苷酸组(ASO-ctrl组)和mi R-199a ASO组(ASO-199a组)。2采用实时荧光定量PCR(q RT-PCR)实验检测过表达mi R-199a或者mi R-199a功能被抑制后,对肝星状细胞LX-2中mi R-199a的m RNA表达水平的影响。3通过CCK-8实验和细胞集落形成实验观察mi R-199a对LX-2细胞增殖能力以及细胞集落形成能力的影响。4运用Transwell实验判断mi R-199a对LX-2细胞迁移能力的影响。5利用计算机生物信息学网站Targetscan、Pictar、mi RDB预测并筛选出SIRT1基因可能是mi R-199a的侯选靶基因。6利用荧光报告载体实验验证SIRT1是mi R-199a的直接靶基因。7通过q RT-PCR实验检测经TGF-β1处理后的LX-2细胞中,mi R-199a、SIRT1、α-SMA和collagen I m RNA表达水平的变化。8采用q RT-PCR实验和Western blot实验分别在m RNA水平和蛋白水平检测过表达mi R-199a或抑制mi R-199a功能后,SIRT1的表达水平的变化。9合成si-SIRT1和si-NC,将LX-2细胞中SIRT1基因表达沉默后再分别通过q RTPCR实验、CCK-8实验和细胞集落形成实验检测LX-2细胞的活化增殖和细胞集落形成能力的改变。结果1 q RT-PCR实验结果显示,在肝星状细胞LX-2中过表达mi R-199a后,与pc DNA3对照组比较,mi R-199a组细胞中mi R-199a m RNA表达水平升高,差异具有统计学意义(P0.01);将细胞内源性的mi R-199a功能封闭后,与ASO-ctrl对照组相比,ASO-199a组细胞中mi R-199a m RNA表达水平降低,差异具有统计学意义(P0.01);2 CCK-8实验结果显示,在LX-2细胞中过表达mi R-199a后,与pc DNA3组比较,mi R-199a组细胞增殖能力增强,差异具有统计学意义(P0.01);将细胞内源性mi R-199a功能封闭后,与ASO-ctrl组比较,ASO-199a组细胞增殖能力下降,差异具有统计学意义(P0.01)。3细胞集落形成实验结果显示,在LX-2细胞中过表达mi R-199a后,与pc DNA3组相比,mi R-199a组细胞集落数增多,差异具有统计学意义(P0.01);细胞内源性mi R-199a的功能被封闭后,与ASO-ctrl组相比,ASO-199a组细胞集落数减少,差异具有统计学意义(P0.01)。4 Transwell实验结果显示,在LX-2细胞中过表达mi R-199a后,与pc DNA3组比较,mi R-199a组细胞迁移数明显增多,差异具有统计学意义(P0.05);细胞内源性mi R-199a的功能被封闭后,与ASO-ctrl组比较,ASO-199a组细胞迁移数明显减少,差异均具有统计学意义(P0.05)。5通过计算机生物信息学网站预测到SIRT1基因可能是mi R-199a的候选靶基因。6 EGFP荧光报告载体实验证明了mi R-199a对SIRT1的直接调控作用。7在TGF-β1处理后的细胞中,α-SMA和collagen I的m RNA表达水平均升高,差异均具有统计学意义(P0.01);而且mi R-199a m RNA表达水平升高,差异具有统计学意义(P0.01);但是SIRT1表达水平下降,差异具有统计学意义(P0.01)。8在LX-2细胞中过表达mi R-199a后,与pc DNA3组比较,mi R-199a组细胞中SIRT1 m RNA表达水平和蛋白表达水平均降低,差异均具有统计学意义(P0.05);封闭细胞内源性mi R-199a的功能后,与ASO-ctrl组比较,ASO-199a组细胞中SIRT1 m RNA和蛋白表达水平均升高,差异均具有统计学意义(P0.05);9将细胞中的SIRT1基因表达沉默后促进了LX-2细胞的活化增殖(P0.05)和细胞集落形成(P0.01),差异均具有统计学意义。结论1 mi R-199a可以促进LX-2细胞的增殖、迁移和活化。2 SIRT1是mi R-199a的直接靶基因。3沉默SIRT1基因的表达能够促进LX-2细胞的活化增殖和细胞集落形成能力,mi R-199a可能通过靶定SIRT1促进了肝星状细胞的活化增殖。
[Abstract]:Objective To study the effects of mi R-199a on proliferation and migration of hepatic stellate cells (HSCs), and to determine the effect of mi R-199a on HSCs. Method 1 The expression vector pcDNA3/ pri-mi R-199a of mi R-199a was constructed, the antisense oligodeoxynucleotide chain of mi R-199a was ASO-199a, then the hepatic stellate cell LX-2 was transfected with the method of liposome transfection, and the experiment was divided into four groups. In this study, the expression of mi R-199a or mi R-199a was detected by real-time fluorescence quantitative PCR (q-RT-PCR). The effect of mi R-199a on the proliferation of LX-2 cells and the formation of the colony-forming ability of the mi R-199a in the hepatic stellate cell LX-2. The effect of mi R-199a on the proliferation of LX-2 cells and the ability of the colony formation was determined by the experiment of CCK-8 and cell-colony formation. SIRT1 gene may be the candidate target gene of mi R-199a. The direct target gene of SIRT1 is mi R-199a was verified by the fluorescence reporter vector experiment. The expression of mi R-199a and SIRT1 in the LX-2 cells treated with TGF-1991a was detected by the q-RT-PCR. The expression level of the RNA-SMA and the collagen I m RNA was changed. The expression level of SIRT1 was changed after the expression of the mi R-199a or the inhibition of the mi R-199a function was detected by the q-RT-PCR and Western blot. After the expression of the SIRT1 gene in the LX-2 cells, the activation and proliferation of the LX-2 cells and the change of the colony-forming ability of the LX-2 cells were detected by a q-RTPCR experiment, a CCK-8 experiment and a cell-colony formation assay, respectively. Results The results of 1-q RT-PCR showed that, after the expression of mi R-199a in the hepatic stellate cell LX-2, the expression of mi R-199a mRNA in the mi R-199a group was significantly higher in the mi R-199a group, and the difference was of statistical significance (P0.01), and the endogenous mi R-199a of the cells was closed and compared with the control group of the ASO-ctrl group. The expression of mi R-199a mRNA in the ASO-199a group was lower and the difference was of statistical significance (P0.01). The results of CCK-8 showed that after the expression of mi R-199a in the LX-2 cells, the proliferation ability of the mi R-199a group was enhanced and the difference was statistically significant (P0.01). After the function of the endogenous mi R-199a of the cells was closed, the cell proliferation ability of the ASO-199a group was decreased, and the difference was of statistical significance (P0.01). The results of the cell colony formation showed that after the expression of the mi R-199a in the LX-2 cells, the number of cell colonies in the mi R-199a group increased, The difference was statistically significant (P0.01); after the function of the endogenous mi R-199a of the cells was closed, the number of cells in the ASO-199a group was reduced, and the difference was of statistical significance (P0.01). The results of the 4 Transwell experiment showed that after the expression of the mi R-199a in the LX-2 cells, the expression of the mi R-199a in the LX-2 cells was compared with the pc-group. The number of cell migration in the group of mi R-199a was significantly increased (P0.05). After the function of the endogenous mi R-199a of the cells was closed, the number of cells in the ASO-199a group was significantly reduced after the function of the endogenous mi R-199a of the cells was closed. The results showed that the direct regulatory effect of mi R-199a on SIRT1 was demonstrated by the computer bioinformatics web site, and the direct regulatory effect of mi R-199a on SIRT1 was demonstrated. The expression of m-RNA of SMA and collagen I was both increased and the difference was of statistical significance (P0.01); and the expression of mi R-199a mRNA was increased, and the difference was of statistical significance (P0.01); however, the expression level of SIRT1 decreased and the difference was of statistical significance (P0.01). After the expression of mi R-199a in LX-2 cells, Compared with the control group, the expression level of SIRT1 mRNA and the level of protein expression in the cells of the mi R-199a group were both decreased and the difference was statistically significant (P0.05); after the function of the endogenous mi R-199a of the closed cells, the levels of SIRT1 m RNA and protein in the ASO-199a group increased, The difference was statistically significant (P0.05); after the expression of the SIRT1 gene in the cells, the activation and proliferation of the LX-2 cells (P0.05) and the colony formation were promoted (P0.01). Conclusion 1 mi R-199a can promote the proliferation, migration and activation of LX-2 cells. SIRT1 is a direct target gene of mi R-199a.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
本文编号：2469384
[Abstract]:Objective To study the effects of mi R-199a on proliferation and migration of hepatic stellate cells (HSCs), and to determine the effect of mi R-199a on HSCs. Method 1 The expression vector pcDNA3/ pri-mi R-199a of mi R-199a was constructed, the antisense oligodeoxynucleotide chain of mi R-199a was ASO-199a, then the hepatic stellate cell LX-2 was transfected with the method of liposome transfection, and the experiment was divided into four groups. In this study, the expression of mi R-199a or mi R-199a was detected by real-time fluorescence quantitative PCR (q-RT-PCR). The effect of mi R-199a on the proliferation of LX-2 cells and the formation of the colony-forming ability of the mi R-199a in the hepatic stellate cell LX-2. The effect of mi R-199a on the proliferation of LX-2 cells and the ability of the colony formation was determined by the experiment of CCK-8 and cell-colony formation. SIRT1 gene may be the candidate target gene of mi R-199a. The direct target gene of SIRT1 is mi R-199a was verified by the fluorescence reporter vector experiment. The expression of mi R-199a and SIRT1 in the LX-2 cells treated with TGF-1991a was detected by the q-RT-PCR. The expression level of the RNA-SMA and the collagen I m RNA was changed. The expression level of SIRT1 was changed after the expression of the mi R-199a or the inhibition of the mi R-199a function was detected by the q-RT-PCR and Western blot. After the expression of the SIRT1 gene in the LX-2 cells, the activation and proliferation of the LX-2 cells and the change of the colony-forming ability of the LX-2 cells were detected by a q-RTPCR experiment, a CCK-8 experiment and a cell-colony formation assay, respectively. Results The results of 1-q RT-PCR showed that, after the expression of mi R-199a in the hepatic stellate cell LX-2, the expression of mi R-199a mRNA in the mi R-199a group was significantly higher in the mi R-199a group, and the difference was of statistical significance (P0.01), and the endogenous mi R-199a of the cells was closed and compared with the control group of the ASO-ctrl group. The expression of mi R-199a mRNA in the ASO-199a group was lower and the difference was of statistical significance (P0.01). The results of CCK-8 showed that after the expression of mi R-199a in the LX-2 cells, the proliferation ability of the mi R-199a group was enhanced and the difference was statistically significant (P0.01). After the function of the endogenous mi R-199a of the cells was closed, the cell proliferation ability of the ASO-199a group was decreased, and the difference was of statistical significance (P0.01). The results of the cell colony formation showed that after the expression of the mi R-199a in the LX-2 cells, the number of cell colonies in the mi R-199a group increased, The difference was statistically significant (P0.01); after the function of the endogenous mi R-199a of the cells was closed, the number of cells in the ASO-199a group was reduced, and the difference was of statistical significance (P0.01). The results of the 4 Transwell experiment showed that after the expression of the mi R-199a in the LX-2 cells, the expression of the mi R-199a in the LX-2 cells was compared with the pc-group. The number of cell migration in the group of mi R-199a was significantly increased (P0.05). After the function of the endogenous mi R-199a of the cells was closed, the number of cells in the ASO-199a group was significantly reduced after the function of the endogenous mi R-199a of the cells was closed. The results showed that the direct regulatory effect of mi R-199a on SIRT1 was demonstrated by the computer bioinformatics web site, and the direct regulatory effect of mi R-199a on SIRT1 was demonstrated. The expression of m-RNA of SMA and collagen I was both increased and the difference was of statistical significance (P0.01); and the expression of mi R-199a mRNA was increased, and the difference was of statistical significance (P0.01); however, the expression level of SIRT1 decreased and the difference was of statistical significance (P0.01). After the expression of mi R-199a in LX-2 cells, Compared with the control group, the expression level of SIRT1 mRNA and the level of protein expression in the cells of the mi R-199a group were both decreased and the difference was statistically significant (P0.05); after the function of the endogenous mi R-199a of the closed cells, the levels of SIRT1 m RNA and protein in the ASO-199a group increased, The difference was statistically significant (P0.05); after the expression of the SIRT1 gene in the cells, the activation and proliferation of the LX-2 cells (P0.05) and the colony formation were promoted (P0.01). Conclusion 1 mi R-199a can promote the proliferation, migration and activation of LX-2 cells. SIRT1 is a direct target gene of mi R-199a.
【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R575
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