miR-217负调控MTDH抑制肝癌细胞增殖和侵袭等作用机制的研究

发布时间：2018-09-05 18:39

【摘要】：背景肝细胞癌(肝癌)在全球恶性肿瘤中发病率居第五位,死亡率居第三位。由于早期无特异表现、治疗后易复发,肝癌病人整体预后较差,是威胁人类健康的重大医疗问题。因此,寻找用于精准早期诊断的的生物标志物和有效的治疗靶点是肝癌研究的重点。近年来发现miR-217已被证实在胃癌、前列腺癌等恶性肿瘤中异常表达,但是在肝癌中miR-217的作用机制仍不明确。异黏蛋白(MTDH)是一种含582个氨基酸的单跨膜蛋白,其在多种肿瘤中均有异常高表达,且其表达水平与肿瘤的进展正相关。MTDH基因的表达还与癌症进展的阶段、分期、以及预后相关。MTDH基因通过调控癌症过程中的多个信号通路参与癌症进程,因此MTDH是一个潜在的治疗靶点。目前对于MTDH在肝癌中的表达调控机制研究还比较少。miRNA能通过间接或者直接调控MTDH的的表达,在结直肠癌中是miRNA间接调控MTDH,在乳腺癌、人头颈部鳞状细胞癌中都是miRNA直接调控MTDH的实例,然而对于miR-217与MTDH的关系尚不明确。我们通过对患者肝癌组织和HepG2细胞的研究,探索miRNA-217介导MTDH在肝癌发生发展中的相关研究及作用机制。目的1.检测miR-217和MTDH在肝癌和癌旁组织中的差异性表达,探讨其相关性;2.验证miR-217与MTDH基因的靶向作用关系;3.检测miR-217对肝癌细胞增殖、凋亡、迁移和侵袭等生物学行为的影响。方法1.自南方医科大学南方医院病理科收集20例肝癌和癌旁组织,分别采用实时荧光定量PCR及Western Blot检测其中miR-217、MTDH mRNA和蛋白水平的表达;2.通过TargetScan预测miR-217与MTDH的结合位点,分别构建MTDH基因3'-UTR区的野生型和突变型荧光素酶报告质粒,与miR-217 mimics共转染HepG2细胞,检测其荧光表达变化。在HepG2细胞中过表达miR-217,通过实时荧光定量PCR、Western Blot及免疫荧光分别检测细胞中MTDH的mRNA和蛋白的表达变化,验证miR-217对MTDH的靶向作用;3.在HepG2细胞中过表达miR-217,通过MTT、流式细胞术、Transwell实验检测miR-217对肝癌细胞增殖、凋亡、迁移和侵袭等生物学特性的影响。结果1.在20例肝癌和癌旁组织中,miR-217在肝癌中的表达量较癌旁组织明显降低(40%),而MTDH mRNA在肝癌中较癌旁组织表达高近一倍,MTDH蛋白水平的表达mRNA水平一致。2.通过转染miR-217 mimics,成功在HepG2细胞中过表达miR-217,miR-217显著抑制野生型MTDH 3'-UTR载体的荧光强度,对突变型MTDH 3'-UTR载体无明显作用。过表达miR-217明显抑制HepG2细胞中MTDH mRNA和蛋白的表达,证明了 miR-217直接靶向于MTDH基因的3'-UTR区,从而抑制MTDH基因的表达。3.在HepG2细胞中过表达miR-217,通过CCK-8实验和流式细胞周期检测发现:较之空白及阴性对照组,过表达miR-217能显著抑制HepG2细胞的增殖,并增加处于G1期、减少S期的HepG2细胞;同时,过表达miR-217,促进HepG2细胞的凋亡;Transwell实验结果表明,过表达miR-217后,转移到下室中的细胞明显减少,证明miR-217显著抑制HepG2细胞的迁移和侵袭。结论1.在肝癌组织中miR-217表达下调,MTDH表达上调。2.miR-217靶向作用于MTDH,抑制其表达。3.miR-217能够显著抑制HepG2细胞的增殖、侵袭和迁移并促进HepG2细胞的凋亡。
[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.
【学位授予单位】：南方医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.7

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