基于肿瘤干细胞miRNA调控研究片仔癀抑制肝癌细胞生长的作用机制

发布时间：2018-01-08 20:08

本文关键词：基于肿瘤干细胞miRNA调控研究片仔癀抑制肝癌细胞生长的作用机制　出处：《福建中医药大学》2017年博士论文　论文类型：学位论文

【摘要】：目的:研究片仔癀干预对肝癌细胞生长的影响及调控机制;建立肝癌干细胞富集体系,筛选肝癌干细胞差异表达miRNA;从肝癌干细胞miRNA调控进一步明确片仔癀对肝癌干细胞生长的干预作用和调控机制。方法:1、培养肝癌HepG2和BEL-7402细胞,并给予不同浓度的片仔癀(0-0.75 mg/mL)干预,通过倒置显微镜观察细胞形态和密度,采用MTT实验检测细胞活力,应用集落形成实验检测细胞存活能力,通过PI染色和流式细胞仪检测细胞周期改变,通过Hoechst染色观察细胞凋亡情况,采用Western-blot检测片仔癀干预对肝癌细胞Bax、Bcl-2、CyclinD1、CDK4、OCT4 和 SOX2 的影响。2、分别在完全培养基常规培养和无血清肿瘤干细胞培养基悬浮培养条件下培养HepG2细胞,采用倒置显微镜观察克隆球和亲本细胞生长情况,通过RT-PCR检测OCT4表达和流式细胞仪检测CD133、CD90阳性细胞比例,进一步采用miRNA芯片筛选克隆球和亲本细胞miRNA差异表达,并通过Q-PCR实验对差异表达进行验证。3、采用无血清肿瘤干细胞培养基悬浮培养HepG2细胞富集肝癌干细胞,并给予片仔癀干预,通过台盼蓝染色计数检测细胞生长情况,通过PI染色和流式细胞仪检测细胞周期改变,通过Hoechst染色观察细胞凋亡情况;采用高内涵观察细胞克隆球形成能力改变,通过皮下移植瘤模型观察细胞体内致瘤能力变化;采用Western-blot检测Bax、Bcl-2、CyclinD1、CDK4、SOX2、OCT4、p21 的表达,通过 Q-PCR、Western-blot 检测miR-483-5p及其靶基因CDKN1A(p21)的表达。结果:1、不同浓度片仔癀干预HepG2和BEL-7402细胞后,显著抑制肝癌细胞生长和降低细胞活力,明显抑制细胞存活能力和阻滞细胞周期从G0/G1到S期的转换的过程,显著诱导细胞凋亡;显著降低Bcl-2/Bax比例,下调细胞周期调控蛋白CyclinD1、CDK4的表达;明显下调肝癌干细胞标志物OCT4和SOX2的表达。2、采用无血清肿瘤干细胞培养基悬浮培养HepG2细胞,成功获得HepG2克隆球细胞,且所获得的克隆球细胞高表达干性基因OCT4,干细胞标记物CD133和CD90双阳性细胞比例明显升高,证实了无血清肿瘤干细胞培养基悬浮培养具有富集肝癌干细胞的作用;miRNA芯片检测结果证实与亲本细胞相比,HegG2克隆球中有234个miRNA表达显著上调,有218个miRNA表达显著下调(cut off1.5倍,P0.05),其中miR-483-5p、miR-582-5p在HegG2克隆球中的表达显著上调(P0.05),Q-PCR检测进一步验证了二者在HepG2克隆球表达上调的结果。3、片仔癀干预对HepG2克隆球细胞具有抑制生长和降低细胞活力的作用,具有抑制细胞从G0/G1期到S期转换的过程和诱导细胞凋亡的作用;能够显著抑制细胞体外克隆球形成能力和体内致瘤能力及生长的作用;片仔癀干预对HepG2克隆球细胞具有显著降低Bcl-2/Bax比例,下调细胞周期调控蛋白CyclinD1、CDK4的表达;明显下调肝癌干细胞标志物OCT4和SOX2的表达;此外可显著下调miR-483-5p的表达,同时上调其靶基因CDKN1A/p21在mRNA和蛋白水平的表达。结论:片仔癀通过调控细胞增殖凋亡相关调控因子Bcl-2/Bax比例、CyclinD1和CDK4的表达具有显著抑制肝癌细胞生长的作用,且具有下调干性基因SOX2和OCT4表达的作用;成功复制无血清肿瘤干细胞培养基悬浮培养富集肝癌干细胞体系,证实miR-483-5p、miR-582-5p等多个miRNA在肝癌干细胞中具有显著调控效应;片仔癀干预显著抑制肝癌干细胞生长和体内外致瘤能力,且通过调控细胞增殖、凋亡相关蛋白Bcl-2/Bax比例、CyclinD1、CDK4和miR-483-5p及其靶基因CDKN1A(p21)表达进而抑制肝癌干细胞生长和诱导细胞凋亡可能是其抑制肝癌细胞生长的重要机制之一。
[Abstract]:Objective: To study pientzehuang intervention on the growth of liver cancer cells and regulation mechanism; establish system of enrichment of liver cancer stem cells, the expression of miRNA in liver cancer stem cells were screened from liver cancer stem cells; miRNA regulation to further clarify the Pianzihuang on hepatocarcinoma stem cells the intervention and control mechanism. Methods: 1. Cultured HepG2 and BEL-7402 cells, and with different concentrations of pientzehuang (0-0.75 mg/mL) intervention by inverted microscope. Cell morphology and density were observed, the cell viability by MTT assay and colony formation assay, using cell viability, by PI staining and flow cytometry cell cycle change and apoptosis was observed by Hoechst staining, detected by Western-blot intervention on pientzehuang Bax cells, Bcl-2, CyclinD1, CDK4, OCT4 and SOX2.2, respectively, in complete medium and serum-free cultured tumor stem Cells cultured HepG2 cells under the condition of medium, observe the clones and parental cell growth by inverted microscope, and flow cytometry to detect the expression of OCT4 CD133 by RT-PCR, the proportion of CD90 positive cells, further using miRNA chip and the expression of miRNA cell clones screening differences, and verified by Q-PCR.3 experiments on the difference the expression of cell culture medium in suspension culture of HepG2 cell enrichment of liver stem cells in serum tumor stem, and give pientzehuang intervention by trypan blue staining to detect the growth of cells, by PI staining and flow cytometry cell cycle change and apoptosis was observed by Hoechst staining; the high content observed cell clone ball formation the ability to change, tumorigenicity changes through subcutaneous transplanted tumor cells in vivo models were detected by Western-blot; Bax, Bcl-2, CyclinD1, CD K4, SOX2, OCT4, p21 expression by Q-PCR, detection of CDKN1A miR-483-5p and its target gene Western-blot (p21) expression. Results: 1 different concentrations of HepG2 and BEL-7402 cells after the intervention of pianzaihuang, significantly inhibited the growth and cell viability of hepatoma cells, inhibit the conversion process of cell survival and cell cycle arrest from G0/G1 to the S phase, significantly induced cell apoptosis; significantly reduced the proportion of Bcl-2/Bax, downregulation of cell cycle regulatory protein CyclinD1, CDK4 expression was down regulated expression of.2; liver cancer stem cell marker OCT4 and SOX2, the cell culture medium in suspension culture of HepG2 cell tumor stem cells, successful acquisition of HepG2, high expression of dry OCT4 gene cloning and cells obtained by double positive cells were stem cell marker CD133 and CD90 increased significantly, confirming no serum tumor stem cell culture has rich nutrient medium In the role of cancer stem cells; miRNA chip test results confirmed that compared with the parent cells, 234 miRNA upregulated the expression of HegG2 in 218 clones, miRNA expression was significantly reduced (cut off1.5 times, P0.05), where miR-483-5p, miR-582-5p expression was significantly up-regulated in HegG2 clones in (P0.05), further Q-PCR test to verify the two results in the upregulation of the expression of.3 HepG2 clones, pientzehuang intervention has reduced cell viability and growth inhibition effect on HepG2 cells, inhibit the cells from G0/G1 phase to S phase transition process and induces cell apoptosis; can inhibit the in vitro clone forming ability and tumorigenicity in vivo and the growth of the intervention significantly reduced the proportion of Bcl-2/Bax; pientzehuang with HepG2 cells, downregulation of cell cycle regulatory protein CyclinD1, CDK4 expression was significantly down regulated in liver cancer stem cells; The expression of OCT4 and SOX2; in addition significantly downregulated the expression of miR-483-5p and its target gene CDKN1A/p21 expression upregulation in mRNA and protein level. Conclusion: pientzehuang through the regulation of cell proliferation and apoptosis related regulation factors Bcl-2/Bax ratio significantly inhibit the growth of hepatocellular carcinoma cells the expression of CyclinD1 and CDK4, and down regulate the expression of stemness genes SOX2 and OCT4; successful replication without cell culture medium suspension culture enrich liver cancer stem cell system, confirmed that miR-483-5p serum tumor stem, miR-582-5p and other miRNA has significant effects on liver cancer stem cells; inhibit cell growth and pientzehuang intervention in liver cancer stem outside the tumorigenic ability, and through the regulation of cell proliferation, apoptosis protein ratio of Bcl-2/Bax, CyclinD1, CDK4 and miR-483-5p and its target gene CDKN1A (p21) expression and inhibit the growth and induce liver cancer stem cells Apoptosis may be one of the important mechanisms to inhibit the growth of hepatoma cells.

【学位授予单位】：福建中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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