SOX18基因对肝癌细胞增殖、迁移及侵袭作用的研究

发布时间：2018-03-08 05:22

本文选题：SOX18　切入点：HCC　出处：《山西医科大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:肝癌(HCC)是世界上最常见的恶性肿瘤之一。SOX家族是由一类含有高度保守的HMG DNA结合结构域的转录因子。研究发现SOX基因与癌症存在联系,SOX7和SOX17属于SOX家族F组,它们可以通过抑制Wnt信号通路来抑制肝癌细胞增殖和迁移等。SOX18是F组的另一成员,它在胃癌、卵巢癌、非小细胞肺癌和乳腺癌中表达上调,但在肝癌细胞中SOX18的表达情况和功能目前还没有报道。因此,本文将通过检测肝癌组织中SOX18表达和在细胞水平上改变SOX18的表达来探讨SOX18与肝癌的关系。方法:从山西医科大学第一医院募集肝癌病例75例,收集患者手术切除的肝癌组织和癌旁组织,通过荧光定量PCR检测组织中SOX18的相对表达量,于手术结束开始随访记录患者总生存期,总生存期定义为从手术到死亡的时间或从手术至健在病人最后一次观察的时间;选择SOX18表达水平高的细胞株MHCC-97H和HepG2进行后续实验,转染SOX18-siRNA敲低这两个细胞中SOX18的表达水平,观察敲低SOX18后对MHCC-97H和HepG2肝癌细胞的增殖、细胞周期、细胞凋亡、细胞的侵袭和迁移能力的影响;利用癌症基因组图谱项目(TCGA)中肝癌细胞队列的高通量RNA测序结果进行基因集富集分析(GSEA)探讨SOX18可能调控的信号通路,再比较SOX18表达下调后该信号通路是否受到影响。结果:荧光定量PCR检测75例肝癌患者肝癌组织及其癌旁组织中的SOX18 mRNA水平显示肝癌组织中SOX18 mRNA表达显著高于癌旁组织(P0.001),TCGA肝癌队列的高通量RNA测序数据也发现肝癌组织中SOX18表达显著高于正常组织,SOX18高表达的肿瘤患者的生存期与SOX18表达较低的患者相比明显缩短(P0.01);Western blot和实时定量PCR检测显示MHCC-97H和HepG2细胞中SOX18的表达显著高于其它细胞,SOX18 siRNA可以有效下调MHCC-97H和HepG2两种细胞中SOX18的表达;CCK-8法检测显示转染SOX18 siRNA后MHCC-97H和HepG2的增殖能力与对照组(WT)和未转染组(NC)相比增殖能力明显减弱;细胞周期检测显示SOX18下调后MHCC-97H和HepG2停滞在G1期的细胞数增加,而且处于S期和G2/M期的细胞数目减少;敲低MHCC-97H和HepG2中SOX18后细胞迁移和侵袭能力明显降低;GSEA分析HCC数据资料显示粘附途径和趋化因子信号转导途径与SOX18表达显著相关;实时定量PCR和Western blot检测显示敲低SOX18能够降低粘附通路基因(RhoA、PDGF和IGF1R)和趋化因子的信号通路基因(CCL2,CCL3、CCL5)mRNA和蛋白质表达水平。结论:本研究首次证明SOX18在肝癌细胞的增殖、迁移和侵袭中起着关键的作用,该作用可能是通过影响黏附和趋化因子的信号通路来实现的。临床研究显示SOX18表达水平与患者的低生存率有关,抑制肿瘤组织中SOX18的表达可能为肝癌治疗提供一个新的治疗策略。
[Abstract]:Objective: hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world. The Sox family is a class of transcription factors containing highly conserved HMG DNA binding domain. They inhibit the proliferation and migration of hepatoma cells by inhibiting the Wnt signaling pathway. SOX18 is another member of group F. it is up-regulated in gastric cancer, ovarian cancer, non-small cell lung cancer and breast cancer. However, the expression and function of SOX18 in HCC cells have not been reported. The relationship between SOX18 and liver cancer was studied by detecting the expression of SOX18 and changing the expression of SOX18 at the cellular level. Methods: 75 cases of liver cancer were recruited from the first Hospital of Shanxi Medical University. The liver cancer tissues and adjacent tissues were collected. The relative expression of SOX18 was detected by fluorescence quantitative PCR, and the total survival time was recorded at the beginning of operation. The total survival time was defined as the time from operation to death or from surgery to the last observation of the patient, and the cell lines MHCC-97H and HepG2, which had high SOX18 expression level, were selected to carry out follow-up experiments, and the expression of SOX18 in the two cells was reduced by transfection of SOX18-siRNA knockout. The effects of SOX18 knockout on the proliferation, cell cycle, apoptosis, invasion and migration of MHCC-97H and HepG2 hepatoma cells were observed. The high throughput RNA sequencing results of HCC cell cohort in cancer genome mapping project (TCGA) were used to study the possible signal pathways regulated by SOX18 by gene set enrichment analysis. Results: the expression of SOX18 mRNA in hepatocellular carcinoma tissues and its adjacent tissues was significantly higher than that of adjacent tissues in 75 patients with hepatocellular carcinoma by fluorescence quantitative PCR. The high throughput RNA sequencing data of P0.001TCGA HCC cohort also showed that the survival time of tumor patients with high expression of SOX18 in HCC tissue was significantly higher than that in patients with high expression of SOX18, compared with patients with lower SOX18 expression. It was also found that the expression of SOX18 in HCC tissues was significantly shorter than that in patients with lower SOX18 expression. PCR analysis showed that the expression of SOX18 in MHCC-97H and HepG2 cells was significantly higher than that in other cells, SOX18 siRNA could effectively down-regulate the expression of SOX18 in MHCC-97H and HepG2 cells. CCK-8 assay showed that the proliferation ability of MHCC-97H and HepG2 after SOX18 siRNA transfection was significantly higher than that of control group. The proliferative ability of the infected group was significantly lower than that of the control group. Cell cycle analysis showed that after SOX18 down-regulation, the number of MHCC-97H and HepG2 stagnated in G1 phase increased, and the number of cells in S phase and G _ 2 / M phase decreased. The ability of cell migration and invasion after knockout of SOX18 in MHCC-97H and HepG2 decreased significantly. The results of HCC data analysis showed that adhesion pathway and chemokine signal transduction pathway were significantly correlated with SOX18 expression. Real-time quantitative PCR and Western blot analysis showed that low SOX18 knockout could decrease the expression of mRNA and protein in adhesion pathway gene (Rho PDGF and IGF1R) and chemokine. Conclusion: this study is the first to prove the proliferation of SOX18 in hepatocellular carcinoma cells. Migration and invasion play a key role, which may be achieved through signaling pathways that affect adhesion and chemokines. Clinical studies have shown that SOX18 expression levels are associated with low survival rates in patients. Inhibition of SOX18 expression in tumor tissues may provide a new therapeutic strategy for liver cancer therapy.
【学位授予单位】：山西医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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