HIF-1α基因沉默对人脉络膜黑色素瘤细胞上皮—间质转化的影响

发布时间：2018-06-28 13:21

本文选题：脉络膜黑色素瘤 + 缺氧诱导因子-1α　；参考：《青岛大学》2014年硕士论文

【摘要】：目的：以特异性小干扰RNA (siRNA)沉默缺氧培养的人眼脉络膜黑色瘤细胞(OCM-1)中缺氧诱导因子-1α (HIF-1α)基因,观察其对细胞骨架分子波形蛋白(Vimentin)表达的影响,以初步探讨HIF-1α对人眼脉络膜黑色瘤上皮-间质转化(EMT)的调控作用。方法：在正常培养基中加入100μmo1/L的氯化钴(CoCl2)以模拟肿瘤内部缺氧微环境,构建靶向HIF-1α的siRNA干扰载体,同时设立β-actin阳性对照、无义寡核苷酸阴性对照及空载脂质体对照组,以LipofectamineTM2000为载体介导siRNA转染缺氧培养的OCM-1细胞,以RT-PCR和Western blot方法检测缺氧培养前后及转染前后HIF-1α Vimentin mRNA和蛋白的表达情况。结果:OCM-1细胞经缺氧培养后可由上皮细胞样形态转变成间质细胞样形态。与常氧组相比,单纯缺氧组HIF-1α mRNA表达无明显差异(P0.05),蛋白表达显著升高(P0.01),而Vimentin mRNA和蛋白的表达均显著升高(P0.01)。缺氧培养的各组之间相比,阳性对照组β-actin mRNA表达明显下降(P0.01),说明转染成功；HIF-1a干扰组HIF-1α和Vimentin mRNA和蛋白表达均明显下降(P0.01)；无义寡核苷酸阴性对照组及空载脂质体对照组各检测因素均无明显差别(P0.05)。结论： 1.缺氧培养可使OCM-1细胞由上皮细胞样形态转变成间质细胞样形态,提示缺氧微环境可促使OCM-1细胞发生EMT。 2.缺氧可在蛋白水平上调OCM-1细胞中HIF-1α的表达,提示缺氧对HIF-1α的调控主要发生在转录后水平。 3.HIF-1α可通过转录激活Vimentin,使其表达上调,提示HIF-1α可能参与调控OCM-1细胞的EMT,进而影响其侵袭、转移能力。 4.可成功利用LipofectamineTM2000为载体将siRNA转入OCM-1细胞内。 5.HIF-lα-siRNA转染OCM-1细胞可成功下调HIF-1α及Vimentin的表达,进一步说明缺氧条件下Vimentin的表达、EMT的发生受HIF-1α的调控；从分子水平抑制HIF-1α的表达,可能抑制肿瘤侵袭、转移,从而可为肿瘤治疗提供新方向。
[Abstract]:Aim: to study the effect of hypoxia inducible factor 1 伪 (HIF-1 伪) gene on cytoskeleton vimentin (vimentin) expression in cultured human eye choroidal melanoma cells (OCM-1) by silencing hypoxia with specific small interfering RNA (siRNA). To investigate the effect of HIF-1 伪 on epithelial-interstitial transformation (EMT) of human choroidal melanoma. Methods: to construct siRNA interference vector targeting HIF-1 伪 by adding 100 渭 mol / L Cobalt chloride (CoCl2) into normal medium to simulate the anoxic microenvironment of tumor, and to set up 尾 -actin positive control, negative control group and no-load liposome control group. The expression of HIF-1 伪 Vimentin mRNA and protein was detected by RT-PCR and Western blot before and after hypoxia culture. Results the cells of OCM-1 could be transformed from epithelial cells to interstitial cells by hypoxia. Compared with normoxic group, the expression of HIF-1 伪 mRNA in hypoxia group was not significantly different (P0.05), but the expression of Vimentin mRNA and protein was significantly increased (P0.01). The expression of 尾 -actin mRNA in positive control group was significantly decreased (P0.01), which indicated that the expression of HIF-1 伪 and Vimentin mRNA and protein in HIF-1a interference group decreased significantly (P0.01). There was no significant difference between negative control group and no-load liposome control group (P0.05). Conclusion: 1. Hypoxia can up-regulate the expression of HIF-1 伪 in OCM-1 cells at the protein level, suggesting that the regulation of HIF-1 伪 by hypoxia mainly occurs at the post-transcriptional level. 3. HIF-1 伪 can activate Vimentinthrough transcription and up-regulate the expression of HIF-1 伪. These results suggest that HIF-1 伪 may be involved in the regulation of EMTs of OCM-1 cells, thus affecting their invasion and metastasis ability. 4. The expression of HIF-1 伪 and Vimentin in OCM-1 cells was successfully down-regulated by transfection of siRNA with LipofectamineTM2000. 5. HIF-l 伪 -siRNA transfected OCM-1 cells could down-regulate the expression of HIF-1 伪 and Vimentin, which further indicated that the expression of Vimentin was regulated by HIF-1 伪 and inhibited the expression of HIF-1 伪 at molecular level. It may inhibit tumor invasion and metastasis, thus providing a new direction for tumor therapy.
【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.7

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