【摘要】：背景肝細(xì)胞癌(肝癌)在全球惡性腫瘤中發(fā)病率居第五位,死亡率居第三位。由于早期無(wú)特異表現(xiàn)、治療后易復(fù)發(fā),肝癌病人整體預(yù)后較差,是威脅人類健康的重大醫(yī)療問(wèn)題。因此,尋找用于精準(zhǔn)早期診斷的的生物標(biāo)志物和有效的治療靶點(diǎn)是肝癌研究的重點(diǎn)。近年來(lái)發(fā)現(xiàn)miR-217已被證實(shí)在胃癌、前列腺癌等惡性腫瘤中異常表達(dá),但是在肝癌中miR-217的作用機(jī)制仍不明確。異黏蛋白(MTDH)是一種含582個(gè)氨基酸的單跨膜蛋白,其在多種腫瘤中均有異常高表達(dá),且其表達(dá)水平與腫瘤的進(jìn)展正相關(guān)。MTDH基因的表達(dá)還與癌癥進(jìn)展的階段、分期、以及預(yù)后相關(guān)。MTDH基因通過(guò)調(diào)控癌癥過(guò)程中的多個(gè)信號(hào)通路參與癌癥進(jìn)程,因此MTDH是一個(gè)潛在的治療靶點(diǎn)。目前對(duì)于MTDH在肝癌中的表達(dá)調(diào)控機(jī)制研究還比較少。miRNA能通過(guò)間接或者直接調(diào)控MTDH的的表達(dá),在結(jié)直腸癌中是miRNA間接調(diào)控MTDH,在乳腺癌、人頭頸部鱗狀細(xì)胞癌中都是miRNA直接調(diào)控MTDH的實(shí)例,然而對(duì)于miR-217與MTDH的關(guān)系尚不明確。我們通過(guò)對(duì)患者肝癌組織和HepG2細(xì)胞的研究,探索miRNA-217介導(dǎo)MTDH在肝癌發(fā)生發(fā)展中的相關(guān)研究及作用機(jī)制。目的1.檢測(cè)miR-217和MTDH在肝癌和癌旁組織中的差異性表達(dá),探討其相關(guān)性;2.驗(yàn)證miR-217與MTDH基因的靶向作用關(guān)系;3.檢測(cè)miR-217對(duì)肝癌細(xì)胞增殖、凋亡、遷移和侵襲等生物學(xué)行為的影響。方法1.自南方醫(yī)科大學(xué)南方醫(yī)院病理科收集20例肝癌和癌旁組織,分別采用實(shí)時(shí)熒光定量PCR及Western Blot檢測(cè)其中miR-217、MTDH mRNA和蛋白水平的表達(dá);2.通過(guò)TargetScan預(yù)測(cè)miR-217與MTDH的結(jié)合位點(diǎn),分別構(gòu)建MTDH基因3'-UTR區(qū)的野生型和突變型熒光素酶報(bào)告質(zhì)粒,與miR-217 mimics共轉(zhuǎn)染HepG2細(xì)胞,檢測(cè)其熒光表達(dá)變化。在HepG2細(xì)胞中過(guò)表達(dá)miR-217,通過(guò)實(shí)時(shí)熒光定量PCR、Western Blot及免疫熒光分別檢測(cè)細(xì)胞中MTDH的mRNA和蛋白的表達(dá)變化,驗(yàn)證miR-217對(duì)MTDH的靶向作用;3.在HepG2細(xì)胞中過(guò)表達(dá)miR-217,通過(guò)MTT、流式細(xì)胞術(shù)、Transwell實(shí)驗(yàn)檢測(cè)miR-217對(duì)肝癌細(xì)胞增殖、凋亡、遷移和侵襲等生物學(xué)特性的影響。結(jié)果1.在20例肝癌和癌旁組織中,miR-217在肝癌中的表達(dá)量較癌旁組織明顯降低(40%),而MTDH mRNA在肝癌中較癌旁組織表達(dá)高近一倍,MTDH蛋白水平的表達(dá)mRNA水平一致。2.通過(guò)轉(zhuǎn)染miR-217 mimics,成功在HepG2細(xì)胞中過(guò)表達(dá)miR-217,miR-217顯著抑制野生型MTDH 3'-UTR載體的熒光強(qiáng)度,對(duì)突變型MTDH 3'-UTR載體無(wú)明顯作用。過(guò)表達(dá)miR-217明顯抑制HepG2細(xì)胞中MTDH mRNA和蛋白的表達(dá),證明了 miR-217直接靶向于MTDH基因的3'-UTR區(qū),從而抑制MTDH基因的表達(dá)。3.在HepG2細(xì)胞中過(guò)表達(dá)miR-217,通過(guò)CCK-8實(shí)驗(yàn)和流式細(xì)胞周期檢測(cè)發(fā)現(xiàn):較之空白及陰性對(duì)照組,過(guò)表達(dá)miR-217能顯著抑制HepG2細(xì)胞的增殖,并增加處于G1期、減少S期的HepG2細(xì)胞;同時(shí),過(guò)表達(dá)miR-217,促進(jìn)HepG2細(xì)胞的凋亡;Transwell實(shí)驗(yàn)結(jié)果表明,過(guò)表達(dá)miR-217后,轉(zhuǎn)移到下室中的細(xì)胞明顯減少,證明miR-217顯著抑制HepG2細(xì)胞的遷移和侵襲。結(jié)論1.在肝癌組織中miR-217表達(dá)下調(diào),MTDH表達(dá)上調(diào)。2.miR-217靶向作用于MTDH,抑制其表達(dá)。3.miR-217能夠顯著抑制HepG2細(xì)胞的增殖、侵襲和遷移并促進(jìn)HepG2細(xì)胞的凋亡。
[Abstract]:BACKGROUND Hepatocellular carcinoma (HCC) ranks fifth in the incidence of malignant tumors worldwide, and its mortality ranks third. Because of its early non-specific manifestations, easy recurrence after treatment and poor overall prognosis of HCC patients, it is a major medical problem threatening human health. In recent years, it has been found that the expression of microRNAs-217 is abnormal in gastric cancer, prostate cancer and other malignant tumors. However, the mechanism of action of microRNAs-217 is still unclear in hepatocellular carcinoma. The expression of MTDH gene is also associated with the stage, stage and prognosis of cancer. MTDH gene is involved in the cancer process by regulating multiple signaling pathways in the cancer process, so MTDH is a potential therapeutic target. Or directly regulate the expression of MTDH, in colorectal cancer is the indirect regulation of MTDH by microRNAs, in breast cancer, human head and neck squamous cell carcinoma are all examples of direct regulation of MTDH by microRNAs. However, the relationship between microRNAs-217 and MTDH is still unclear. Objective 1. To investigate the differential expression of microRNAs-217 and MTDH in hepatocellular carcinoma and adjacent tissues, 2. To verify the targeting effect of microRNAs-217 on MTDH gene, 3. To investigate the effects of microRNAs-217 on the proliferation, apoptosis, migration and invasion of hepatocellular carcinoma cells. Twenty cases of hepatocellular carcinoma and adjacent tissues were collected from the Department of Pathology, Southern Hospital of University, and real-time fluorescence quantitative PCR and Western Blot were used to detect the expression of microRNAs-217, MTDH mRNA and protein levels. 2. Wild-type and mutant luciferase reporter plasmids in the 3'-UTR region of MTDH gene were constructed by predicting the binding sites of microRNAs-217 and MTDH by TargetScan. Mi-217 mimics were co-transfected into HepG2 cells to detect their fluorescent expression. Mi-217 was overexpressed in HepG2 cells. The expression of MTDH mRNA and protein was detected by real-time fluorescence quantitative PCR, Western Blot and immunofluorescence, respectively, to verify the targeting effect of Mi-217 on MTDH. 3. Mi-217 was overexpressed in HepG2 cells, and was detected by MTT, flow cytometry. Results 1. In 20 cases of hepatocellular carcinoma and adjacent tissues, the expression of microRNAs-217 in hepatocellular carcinoma was significantly lower than that in adjacent tissues (40%), while the expression of MTDH mRNA in hepatocellular carcinoma was nearly twice as high as that in adjacent tissues, and the expression of MTDH protein was almost twice as high as that in adjacent tissues. RNA level was consistent. 2. Mi-217 was successfully overexpressed in HepG2 cells by transfecting Mi-217 mimics. Mi-217 significantly inhibited the fluorescence intensity of wild-type MTDH-3'-UTR vectors and had no significant effect on mutant MTDH-3'-UTR vectors. Overexpression of Mi-217 significantly inhibited the expression of MTDH mRNA and protein in HepG2 cells, which proved that Mi-217 directly targeted to MTDH. Overexpression of Mi-217 in HepG2 cells was detected by CCK-8 assay and flow cytometry. Compared with blank and negative control group, overexpression of Mi-217 significantly inhibited the proliferation of HepG2 cells, increased the G1 phase and decreased the S phase of HepG2 cells. At the same time, overexpression of Mi-217 promoted the proliferation of HepG2 cells. Transwell assay showed that the number of cells transferred to the lower chamber after overexpression of microRNAs-217 was significantly reduced, indicating that microRNAs-217 significantly inhibited the migration and invasion of He pG2 cells. Conclusion 1. In hepatocellular carcinoma tissues, the expression of microRNAs-217 was down-regulated and the expression of MTDH was up-regulated. 2. MicroRNAs-217 targeted at MTDH and inhibited its expression. 3. MicroRNAs-217 significantly inhibited the migration and invasion of He pG2 cells. PG2 cell proliferation, invasion and migration, and promote the apoptosis of HepG2 cells.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.7

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8 李鳳霞;Myrothecine A通過(guò)miR-221調(diào)控肝癌細(xì)胞增殖及樹(shù)突狀細(xì)胞成熟機(jī)制的研究[D];揚(yáng)州大學(xué);2015年

9 肖澤林;乙肝病毒X蛋白通過(guò)SPHK1促進(jìn)肝癌細(xì)胞增殖的作用及其分子機(jī)制的研究[D];南開(kāi)大學(xué);2015年

10 武承鳳;MIF通過(guò)ERK信號(hào)通路對(duì)肝癌細(xì)胞增殖的實(shí)驗(yàn)研究[D];甘肅中醫(yī)藥大學(xué);2016年

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