本文選題：SOX18　切入點(diǎn)：HCC　出處：《山西醫(yī)科大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：目的:肝癌(HCC)是世界上最常見(jiàn)的惡性腫瘤之一。SOX家族是由一類(lèi)含有高度保守的HMG DNA結(jié)合結(jié)構(gòu)域的轉(zhuǎn)錄因子。研究發(fā)現(xiàn)SOX基因與癌癥存在聯(lián)系,SOX7和SOX17屬于SOX家族F組,它們可以通過(guò)抑制Wnt信號(hào)通路來(lái)抑制肝癌細(xì)胞增殖和遷移等。SOX18是F組的另一成員,它在胃癌、卵巢癌、非小細(xì)胞肺癌和乳腺癌中表達(dá)上調(diào),但在肝癌細(xì)胞中SOX18的表達(dá)情況和功能目前還沒(méi)有報(bào)道。因此,本文將通過(guò)檢測(cè)肝癌組織中SOX18表達(dá)和在細(xì)胞水平上改變SOX18的表達(dá)來(lái)探討SOX18與肝癌的關(guān)系。方法:從山西醫(yī)科大學(xué)第一醫(yī)院募集肝癌病例75例,收集患者手術(shù)切除的肝癌組織和癌旁組織,通過(guò)熒光定量PCR檢測(cè)組織中SOX18的相對(duì)表達(dá)量,于手術(shù)結(jié)束開(kāi)始隨訪記錄患者總生存期,總生存期定義為從手術(shù)到死亡的時(shí)間或從手術(shù)至健在病人最后一次觀察的時(shí)間;選擇SOX18表達(dá)水平高的細(xì)胞株MHCC-97H和HepG2進(jìn)行后續(xù)實(shí)驗(yàn),轉(zhuǎn)染SOX18-siRNA敲低這兩個(gè)細(xì)胞中SOX18的表達(dá)水平,觀察敲低SOX18后對(duì)MHCC-97H和HepG2肝癌細(xì)胞的增殖、細(xì)胞周期、細(xì)胞凋亡、細(xì)胞的侵襲和遷移能力的影響;利用癌癥基因組圖譜項(xiàng)目(TCGA)中肝癌細(xì)胞隊(duì)列的高通量RNA測(cè)序結(jié)果進(jìn)行基因集富集分析(GSEA)探討SOX18可能調(diào)控的信號(hào)通路,再比較SOX18表達(dá)下調(diào)后該信號(hào)通路是否受到影響。結(jié)果:熒光定量PCR檢測(cè)75例肝癌患者肝癌組織及其癌旁組織中的SOX18 mRNA水平顯示肝癌組織中SOX18 mRNA表達(dá)顯著高于癌旁組織(P0.001),TCGA肝癌隊(duì)列的高通量RNA測(cè)序數(shù)據(jù)也發(fā)現(xiàn)肝癌組織中SOX18表達(dá)顯著高于正常組織,SOX18高表達(dá)的腫瘤患者的生存期與SOX18表達(dá)較低的患者相比明顯縮短(P0.01);Western blot和實(shí)時(shí)定量PCR檢測(cè)顯示MHCC-97H和HepG2細(xì)胞中SOX18的表達(dá)顯著高于其它細(xì)胞,SOX18 siRNA可以有效下調(diào)MHCC-97H和HepG2兩種細(xì)胞中SOX18的表達(dá);CCK-8法檢測(cè)顯示轉(zhuǎn)染SOX18 siRNA后MHCC-97H和HepG2的增殖能力與對(duì)照組(WT)和未轉(zhuǎn)染組(NC)相比增殖能力明顯減弱;細(xì)胞周期檢測(cè)顯示SOX18下調(diào)后MHCC-97H和HepG2停滯在G1期的細(xì)胞數(shù)增加,而且處于S期和G2/M期的細(xì)胞數(shù)目減少;敲低MHCC-97H和HepG2中SOX18后細(xì)胞遷移和侵襲能力明顯降低;GSEA分析HCC數(shù)據(jù)資料顯示粘附途徑和趨化因子信號(hào)轉(zhuǎn)導(dǎo)途徑與SOX18表達(dá)顯著相關(guān);實(shí)時(shí)定量PCR和Western blot檢測(cè)顯示敲低SOX18能夠降低粘附通路基因(RhoA、PDGF和IGF1R)和趨化因子的信號(hào)通路基因(CCL2,CCL3、CCL5)mRNA和蛋白質(zhì)表達(dá)水平。結(jié)論:本研究首次證明SOX18在肝癌細(xì)胞的增殖、遷移和侵襲中起著關(guān)鍵的作用,該作用可能是通過(guò)影響?zhàn)じ胶挖吇蜃拥男盘?hào)通路來(lái)實(shí)現(xiàn)的。臨床研究顯示SOX18表達(dá)水平與患者的低生存率有關(guān),抑制腫瘤組織中SOX18的表達(dá)可能為肝癌治療提供一個(gè)新的治療策略。
[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.
【學(xué)位授予單位】：山西醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：R735.7

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