全反式维甲酸调控miRNA200s逆转肝癌细胞上皮间质转化的研究

发布时间：2018-04-28 09:03

本文选题：肝癌细胞 + 全反式维甲酸　；参考：《重庆医科大学》2017年硕士论文

【摘要】：目的:体外探讨全反式维甲酸(all-trans-retinoic acid,ATRA)是否通过调控micro RNA 200家族(mi R200s)逆转肝癌细胞的上皮细胞间充质转化(epithelial mesenchymal transition,EMT),从而抑制肿瘤细胞的增殖迁徙侵袭,促进其成熟分化,并寻找相关下游靶基因,探讨可能的分子生物学机制,为ATRA用于肝癌的临床治疗提供新的思路。方法:以小鼠肝癌细胞Hepa1-6为研究对象,首先体外给予0、0.1、1.0和10.0μmol/L不同终浓度的ATRA处理,台盘蓝拒染实验计数细胞,绘制细胞生长曲线,克隆形成实验、结晶紫染色检测细胞增殖及克隆形成能力;Hoechst检测细胞凋亡,划痕实验检测细胞的平行迁徙能力,Transwell实验检测纵向迁徙及侵袭能力。荧光定量PCR(real-time PCR)法检测肝前体细胞标志甲胎蛋白(alpha fetoprotein,AFP)及成熟肝细胞标志白蛋白(albumin,ALB)和细胞角蛋白18(cytokeratin 18,CK18),酪氨酸转氨酶(tyrosine aminotransferase,TAT),载脂蛋白B(Apo lipoprotein B,Apo B)的m RNA水平表达;Western Blot及免疫荧光检测AFP、ALB、CK18的蛋白水平表达。吲哚菁绿(Indocyanine green,ICG)摄取释放实验及过碘酸希夫(Periodic acid-Schiff,PAS)染色检测细胞的代谢解毒及糖原合成功能。Real-time PCR检测上皮标志蛋白-上皮细胞钙粘蛋白(epithelia cadherin,E-cadherin),以及间质标志蛋白:神经型钙粘蛋白(nerve cadherin,N-cadherin)、锌指蛋白转录因子snail、波形蛋白Vimentin和上皮-间质转化因子twist的m RNA表达。初步筛选出对Hepa1-6细胞作用最佳的ATRA浓度。其次,体外用最佳浓度的ATRA处理Hepa1-6细胞,real-time PCR检测mi R200s的表达情况,筛选出表达变化具有显著差异的mi R200成员mi R200a-3p、mi R200c-3p、mi R141-3p。采用抑制mi RNA200功能的三种特异性mi RNA antagomir处理Hepa1-6细胞,再用最佳浓度的ATRA继续培养细胞,分为:a.空白对照组;b.ATRA组;c.mi RNA antagomir Negative Control(NC)+ATRA组;d.mi R-200s antagomir+ATRA共三组。MTT分析法检测细胞增殖情况,克隆形成实验检测细胞克隆形成,Annexin V-FITC/PI双染法流式细胞术检测细胞凋亡,划痕实验检测细胞的平行迁徙能力,Transwell实验检测纵向迁徙能力及侵袭能力,Real-time PCR检测成熟肝细胞标志蛋白ALB、CK18的表达,ICG及PAS染色检测细胞的成熟分化作用。转录因子芯片检测ATRA处理前后345种转录因子活性,获得差异因子,生物信息学分析可能受mi R-200分子调控的差异因子靶基因,并进行real-time PCR法及western blot验证。结果:第一部分:0.1、1.0和10.0μmol/L ATRA处理后Hepa1-6细胞的增殖、迁徙、侵袭能力较control组明显下降(p0.05),凋亡率增加,ICG摄取及PAS染色阳性细胞数显著增多(p0.05),上皮标志蛋白E-cadherin,CK18及成熟肝细胞标志ALB、CK18、TAT、Apo B的表达明显上调,肝肿瘤细胞标志AFP下降,间质标志蛋白N-cadherin,Vimentin、Snail、Twist的表达明显下调(p0.05),这些调节作用呈现出对ATRA的剂量依赖性,10.0μmol/L ATRA组的作用最强。第二部分:1)Real-time PCR结果显示10.0μmol/LATRA诱导后Hepa1-6细胞中mi RNA200a-3p、200c-3p、141-3p的m RNA表达明显上调(p0.05),而其他7个mi R200家族分子的表达与对照组相比无统计学差异(p0.05)。2)ATRA处理后Hepa1-6细胞的增殖、凋亡、迁移、侵袭以及成熟分化作用较对照组出现与第一部分相同的作用趋势。NC+ATRA组与ATRA诱导组Hepa1-6细胞的增殖、凋亡率、迁移、侵袭能力、成熟肝细胞标志表达及分化能力均无差异(p0.05)。而与NC+ATRA组相比,mi R-200a/200c/141-3p antagomir+ATRA三组Hepa1-6细胞的增殖率、划痕愈合率及侵袭细胞均增多(p0.05),其中mi R-200a/200c-3p antagomir+ATRA两组增多更明显,甚至达到control组水平,但三组克隆形成率均较NC+ATRA组无差异;同时三组凋亡率均较NC+ATRA组均明显下降(p0.05)。只有mi R-200a/200c-3p antagomir+ATRA两组与NC+ATRA组相比,transwell纵向迁移能力增强,ALB、CK18的m RNA表达下调,PAS及ICG阳性细胞数减少(p0.05),mi R-141-3p antagomir不影响ATRA对肝癌细胞的纵向迁移和促进分化成熟作用。3)转录因子芯片结果显示ATRA处理后有8个转录因子活性上调,35个转录因子活性下调,生物信息学结果分析mi R-200a/200c/141-3p与klf12、zeb1、Pou4f1、Ets1、Jun基因相关,相对应于AP2、AREB1、Brn-3、Ets1/PEA3及Fra-1/JUN转录因子,而其他7个mi R-200亚型与ATRA调控的43个差异因子均不相关。ATRA诱导后转录因子Jun、Fra-1、Pouf1、Etv4、zeb1的m RNA表达显著下调,klf12表达上调(p0.05)。并且mi R-200a/200c/141-3p antagomira特异性抑制了mi R-200a/200c/141-3p的生物学作用后,三组较mi RNA Negative control组Jun、Fra-1、Pouf1、Etv4、zeb1的蛋白表达显著上调(p0.05),Klf12的蛋白表达未见明显改变。结论:ATRA呈浓度依赖性逆转小鼠肝癌细胞Hepa1-6的上皮间质转化,抑制细胞的增殖、迁移及侵袭能力,促进细胞凋亡及成熟分化,并具有成熟肝细胞的合成代谢功能。ATRA可能通过调控mi R-200a-3p、mi R-200c-3p、mi R-141-3p逆转肝癌细胞上皮间质转化,其中mi R-200a-3p、mi R-200c-3p、mi R-141-3p均参与介导ATRA抑制肝癌细胞增殖、平行迁移、侵袭的作用及促凋亡作用;同时mi R-200a-3p、mi R-200c-3p还介导了ATRA抑制肝癌细胞纵向迁移及促进分化成熟的作用,并且mi R-200s调控肝癌EMT的发生可能与Jun、Fra-1、Pouf1、Etv4、zeb1相关。
[Abstract]:Objective: To investigate in vitro whether all-trans-retinoic acid (ATRA) can reverse the epithelial mesenchymal transition (epithelial mesenchymal transition, EMT) by regulating the micro RNA 200 family (MI R200s), thus inhibiting the proliferation and invasion of tumor cells, promoting its maturation and differentiation, and searching for the related downstream targets. To explore the possible molecular biological mechanism to provide new ideas for the clinical treatment of liver cancer by ATRA. Methods: the mouse hepatoma cell Hepa1-6 was used as the research object. First, the ATRA treatment of the different final concentrations of 0,0.1,1.0 and 10 mu mol/L was given in vitro, the cell blue stain experiment was used to count the fine cell, the cell growth curve was plotted, the cloning experiment was made and the crystallization was crystallized. Cell proliferation and clone formation ability were detected by purple staining. Hoechst was used to detect cell apoptosis, scratch test to detect the parallel migration ability of cells. Transwell test was used to detect vertical migration and invasion ability. Fluorescence quantitative PCR (real-time PCR) method was used to detect alpha fetoprotein (alpha fetoprotein, AFP) and mature hepatocyte marker albumin (real-time PCR) method. Albumin, ALB) and cytokeratin 18 (cytokeratin 18, CK18), tyrosine aminotransferase (tyrosine aminotransferase, TAT), apolipoprotein B (Apo lipoprotein B, Apo) level expression. Periodic acid-Schiff (PAS) staining was used to detect the metabolic detoxification of cells and the function of glycogen synthesis by.Real-time PCR to detect epithelial marker protein, epithelia cadherin, E-cadherin, and interstitial marker protein: neuronal cadherin (nerve cadherin, N-cadherin), zinc finger protein transcription factor Snail, vimentin M RNA expression of tin and epithelial mesenchymal transition factor twist. The optimum concentration of ATRA for Hepa1-6 cells was screened. Secondly, Hepa1-6 cells were treated with the best concentration of ATRA in vitro. Real-time PCR was used to detect the expression of MI R200s. Hepa1-6 cells were treated with three specific mi RNA antagomir, which inhibit the function of MI RNA200, and then the cells were continued to be cultured with the best concentration of ATRA, divided into a. blank control group, b.ATRA group and c.mi RNA antagomir group. Cell clones were formed, cell apoptosis was detected by Annexin V-FITC/PI double staining flow cytometry, parallel migration ability was detected by scratch test, vertical migration ability and invasion ability were detected by Transwell test, Real-time PCR was used to detect the mature hepatocyte marker protein ALB, CK18 surface expression, ICG and PAS staining to detect the maturation and differentiation of cells. The activity of 345 transcription factors before and after ATRA treatment was detected by the recording factor chip, and the difference factor was obtained. The bioinformatics analysis could be regulated by the MI R-200 molecule, and the real-time PCR method and Western blot were used to verify. The results were as follows: the first part: the proliferation, migration, and invasion energy of Hepa1-6 cells after the treatment of 0.1,1.0 and 10 micron ATRA. Compared with control group (P0.05), the apoptosis rate increased, the number of ICG uptake and PAS staining positive cells increased significantly (P0.05), the epithelial marker protein E-cadherin, CK18 and mature hepatocyte marker ALB, CK18, TAT, Apo B, the expression of liver tumor cell markers decreased. P0.05, which showed a dose dependence on ATRA, the 10 micron mol/L ATRA group had the strongest effect. The second part: 1) Real-time PCR results showed mi RNA200a-3p in Hepa1-6 cells induced by 10 mu mol/LATRA, 200c-3p, and the expression and control of the other 7 family members. Compared with the control group, the proliferation, apoptosis, migration, invasion and mature differentiation of Hepa1-6 cells were similar to those of the control group after ATRA treatment. The proliferation, apoptosis, migration, invasiveness, the expression and differentiation of mature hepatocyte markers were no more than that of the control group. The proliferation, apoptosis, migration, invasion and maturation of Hepa1-6 cells were compared with those of the control group after ATRA treatment. The difference (P0.05). Compared with the NC+ATRA group, the proliferation rate of Hepa1-6 cells in the MI R-200a/200c/141-3p antagomir+ATRA three groups, the scar healing rate and the invasive cells increased (P0.05), and the increase of MI R-200a/200c-3p antagomir+ATRA two group was more obvious, even reached the control group, but the three groups had no difference compared with those of the NC+ATRA group. The apoptosis rate of the three groups were all significantly lower than those in the NC+ATRA group (P0.05). Only the MI R-200a/200c-3p antagomir+ATRA two groups were compared with the NC+ATRA group, and the vertical migration ability of Transwell was enhanced, the m RNA expression of ALB, CK18 decreased and the number of PAS and positive cells decreased. The transcriptional factor.3) transcriptional factor chip results showed that 8 transcriptional factors were up-regulated after ATRA treatment, and the activity of 35 transcriptional factors was down. The bioinformatics results showed that MI R-200a/200c/141-3p was associated with klf12, ZEB1, Pou4f1, Ets1, and Jun genes, and corresponded to AP2, AREB1, Brn-3, and transcription factors, and the other 7 The 43 difference factors regulated by ATRA are not related to.ATRA induced transcription factor Jun, Fra-1, Pouf1, Etv4, the RNA expression of M is down significantly, and klf12 expression is up regulated (P0.05). The protein expression of TV4, ZEB1 increased significantly (P0.05), and the expression of Klf12 protein was not obviously changed. Conclusion: ATRA has a concentration dependent reversal of the epithelial transformation of Hepa1-6 in mouse hepatoma cells, inhibiting cell proliferation, migration and invasion, promoting cell apoptosis and maturation, and having the metabolic function.ATRA of mature liver cells. By regulating mi R-200a-3p, MI R-200c-3p and MI R-141-3p to reverse the epithelial mesenchymal transition of hepatoma cells, MI R-200a-3p, MI R-200c-3p and MI R-141-3p are involved in inhibiting the proliferation, parallel migration, invasion and apoptosis of hepatoma cells. And promote the role of differentiation and maturation, and MI R-200s regulation of liver cancer EMT may be related to Jun, Fra-1, Pouf1, Etv4, ZEB1.

【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7

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