miR-199a調控肝星狀細胞增殖和遷移及其分子機制
發(fā)布時間：2019-05-05 07:40
【摘要】：目的探討mi R-199a對肝星狀細胞(Hepatic Stellate Cells,HSCs)活化增殖和遷移能力的影響,并確定mi R-199a直接調控的靶基因以及靶基因對HSCs的作用。方法1構建mi R-199a過表達載體pcDNA3/pri-mi R-199a,合成mi R-199a的反義寡聚核苷酸鏈即ASO-199a,然后通過脂質體轉染的方法轉染肝星狀細胞LX-2,將實驗分為四組,pc DNA3空載質粒組(pc DNA3組)、pc DNA3/pri-mi R-199a質粒組(mi R-199a組)、亂序寡聚核苷酸組(ASO-ctrl組)和mi R-199a ASO組(ASO-199a組)。2采用實時熒光定量PCR(q RT-PCR)實驗檢測過表達mi R-199a或者mi R-199a功能被抑制后,對肝星狀細胞LX-2中mi R-199a的m RNA表達水平的影響。3通過CCK-8實驗和細胞集落形成實驗觀察mi R-199a對LX-2細胞增殖能力以及細胞集落形成能力的影響。4運用Transwell實驗判斷mi R-199a對LX-2細胞遷移能力的影響。5利用計算機生物信息學網站Targetscan、Pictar、mi RDB預測并篩選出SIRT1基因可能是mi R-199a的侯選靶基因。6利用熒光報告載體實驗驗證SIRT1是mi R-199a的直接靶基因。7通過q RT-PCR實驗檢測經TGF-β1處理后的LX-2細胞中,mi R-199a、SIRT1、α-SMA和collagen I m RNA表達水平的變化。8采用q RT-PCR實驗和Western blot實驗分別在m RNA水平和蛋白水平檢測過表達mi R-199a或抑制mi R-199a功能后,SIRT1的表達水平的變化。9合成si-SIRT1和si-NC,將LX-2細胞中SIRT1基因表達沉默后再分別通過q RTPCR實驗、CCK-8實驗和細胞集落形成實驗檢測LX-2細胞的活化增殖和細胞集落形成能力的改變。結果1 q RT-PCR實驗結果顯示,在肝星狀細胞LX-2中過表達mi R-199a后,與pc DNA3對照組比較,mi R-199a組細胞中mi R-199a m RNA表達水平升高,差異具有統(tǒng)計學意義(P0.01);將細胞內源性的mi R-199a功能封閉后,與ASO-ctrl對照組相比,ASO-199a組細胞中mi R-199a m RNA表達水平降低,差異具有統(tǒng)計學意義(P0.01);2 CCK-8實驗結果顯示,在LX-2細胞中過表達mi R-199a后,與pc DNA3組比較,mi R-199a組細胞增殖能力增強,差異具有統(tǒng)計學意義(P0.01);將細胞內源性mi R-199a功能封閉后,與ASO-ctrl組比較,ASO-199a組細胞增殖能力下降,差異具有統(tǒng)計學意義(P0.01)。3細胞集落形成實驗結果顯示,在LX-2細胞中過表達mi R-199a后,與pc DNA3組相比,mi R-199a組細胞集落數(shù)增多,差異具有統(tǒng)計學意義(P0.01);細胞內源性mi R-199a的功能被封閉后,與ASO-ctrl組相比,ASO-199a組細胞集落數(shù)減少,差異具有統(tǒng)計學意義(P0.01)。4 Transwell實驗結果顯示,在LX-2細胞中過表達mi R-199a后,與pc DNA3組比較,mi R-199a組細胞遷移數(shù)明顯增多,差異具有統(tǒng)計學意義(P0.05);細胞內源性mi R-199a的功能被封閉后,與ASO-ctrl組比較,ASO-199a組細胞遷移數(shù)明顯減少,差異均具有統(tǒng)計學意義(P0.05)。5通過計算機生物信息學網站預測到SIRT1基因可能是mi R-199a的候選靶基因。6 EGFP熒光報告載體實驗證明了mi R-199a對SIRT1的直接調控作用。7在TGF-β1處理后的細胞中,α-SMA和collagen I的m RNA表達水平均升高,差異均具有統(tǒng)計學意義(P0.01);而且mi R-199a m RNA表達水平升高,差異具有統(tǒng)計學意義(P0.01);但是SIRT1表達水平下降,差異具有統(tǒng)計學意義(P0.01)。8在LX-2細胞中過表達mi R-199a后,與pc DNA3組比較,mi R-199a組細胞中SIRT1 m RNA表達水平和蛋白表達水平均降低,差異均具有統(tǒng)計學意義(P0.05);封閉細胞內源性mi R-199a的功能后,與ASO-ctrl組比較,ASO-199a組細胞中SIRT1 m RNA和蛋白表達水平均升高,差異均具有統(tǒng)計學意義(P0.05);9將細胞中的SIRT1基因表達沉默后促進了LX-2細胞的活化增殖(P0.05)和細胞集落形成(P0.01),差異均具有統(tǒng)計學意義。結論1 mi R-199a可以促進LX-2細胞的增殖、遷移和活化。2 SIRT1是mi R-199a的直接靶基因。3沉默SIRT1基因的表達能夠促進LX-2細胞的活化增殖和細胞集落形成能力,mi R-199a可能通過靶定SIRT1促進了肝星狀細胞的活化增殖。
[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.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575
本文編號：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.
【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R575
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