Hsa-miR-93-5p对喉鳞癌Hep-2细胞增殖和侵袭的影响

发布时间：2018-03-04 13:43

本文选题：喉肿瘤　切入点：鳞状细胞癌　出处：《山西医科大学》2014年硕士论文　论文类型：学位论文

【摘要】：【目的】从前期应用microRNA基因芯片技术检测喉鳞癌癌组织及癌旁组织差异性表达microRNA谱中，通过qRT-PCR技术发现喉鳞状细胞癌石蜡包埋组织中has-mir-93-5p的表达较对应癌旁切缘组织明显上调。拟利用体外转染hsa-miR-93-5p inbibitor，探讨其对人喉鳞癌细胞株Hep-2细胞细胞功能的影响。【方法】 1、取对数生长期的体外培养人喉鳞状细胞癌Hep-2细胞株进行试验。 2、将hsa-miR-93-5p Inhibitor序列（即hsa-miR-93-5p mimisc反向互补序列）转染至Hep-2细胞中作为Inhibitor转染组，以未经任何转染处理的Hep-2细胞作为空白对照组。 3、检测hsa-miR-93-5p Inhibitor转染组和空白对照组Hep-2细胞增殖能力，采用MTS法、平板克隆形成实验。 4、采用Edu法检测细胞分裂增殖能力。 5、采用细胞迁移实验，Transwell侵袭实验检测Inhibitor转染组和空白对照组细胞迁移侵袭能力。 6、采用PI染料法检测细胞周期。 7、采用Annexin V-FITC/PI双染法检测细胞凋亡。 8、采用SPSS13.0软件对所得数据进行统计学分析，，计量资料采用均值±标准差表示，对结果进行t检验，P0.05认为差异有统计学意义。【结果】 1、细胞转染48h后，荧光显微镜检测Hep-2细胞中miR-93转染效率。随机计数100个细胞，其中有荧光的细胞约占90%，符合继续实验的标准。 2、MTS法检测显示hsa-miR-93-5p Inhibitor转染的Hep-2细胞抑制率较空白对照组抑制率明显升高，并且随着转染后培养时间的延长，细胞抑制率更加明显(P0.05)，但增值率相比变化不明显。 3、不同处理的Hep-2细胞Edu法检测细胞增殖，显示Inhibitor转染组较空白对照组明显降低，细胞生长活力得到明显抑制。 4、克隆形成实验结果示Inhibitor转染组的存活分数明显低于空白对照组(P0.05)。 5、流式细胞术检测细胞周期结果，hsa-miR-93-5p抑制物转染的Hep-2细胞处于G2期的细胞数量（9.62±0.71）明显高于空白对照组（0.04±0.06），差异具有统计学意义（P0.05）。 6、流式细胞术检测细胞凋亡结果，Hep-2细胞Inhibitor转染组总凋亡率（1.97±0.11）明显高于空白对照组（1.49±0.11），差异具有统计学意义（P0.05）。 7、细胞迁移实验结果，Hep-2细胞Inhibitor转染组细胞迁移数均明显低于Hep-2细胞空白对照组，差异具有统计学意义（P0.05）。 8、Transwell侵袭实验检测结果，Hep-2细胞Inhibitor转染组细胞侵袭能力（101.5±11.84）明显低于空白对照组（158.67±20.82），差异具有统计学意义（P0.05）。【结论】 1、转染hsa-miR-93-5p Inhibitor序列的喉鳞癌Hep-2细胞的增殖与迁移侵袭能力明显低于未经任何处理的喉鳞癌Hep-2细胞，提示hsa-miR-93-5p可能是喉鳞癌的发病机制中一个重要分子 2、转染hsa-miR-93-5p Inhibitor序列可以抑制喉鳞癌Hep-2细胞的生长，使细胞阻滞于G2期（P0.05）。提示hsa-miR-93-5p可能通过调控喉鳞癌细胞的细胞周期进而发挥其生物学功能。
[Abstract]:[objective] to detect the differential expression of microRNA in laryngeal squamous cell carcinoma (LSCC) and adjacent tissues by using microRNA gene chip technique. The expression of has-mir-93-5p in paraffin embedded laryngeal squamous cell carcinoma (LSCC) was found to be significantly up-regulated than that in adjacent incisor tissue by qRT-PCR technique. The effect of hsa-miR-93-5p inbibitor transfection on the function of Hep-2 cell line was studied by in vitro transfection of hsa-miR-93-5p into human laryngeal squamous cell carcinoma (LSCC) cell line. [methods]. 1. Human laryngeal squamous cell carcinoma (Hep-2) cell line was cultured in logarithmic growth period. 2. Hsa-miR-93-5p Inhibitor sequence (hsa-miR-93-5p mimisc reverse complementary sequence) was transfected into Hep-2 cells as Inhibitor transfection group, and Hep-2 cells without any transfection were used as blank control group. 3. The proliferative ability of Hep-2 cells in hsa-miR-93-5p Inhibitor transfection group and blank control group was detected by MTS assay. 4. The ability of cell division and proliferation was detected by Edu assay. 5. Transwell invasion assay was used to detect the ability of cell migration and invasion in Inhibitor transfection group and blank control group. 6. Pi dye method was used to detect cell cycle. 7. Apoptosis was detected by Annexin V-FITC / Pi double staining. 8. SPSS13.0 software was used to analyze the data. The measurement data was expressed by mean 卤standard deviation, and the difference was statistically significant by t test (P0.05). [results]. 1. After 48 hours of transfection, the transfection efficiency of miR-93 in Hep-2 cells was detected by fluorescence microscope. 2the inhibition rate of Hep-2 cells transfected with hsa-miR-93-5p Inhibitor was significantly higher than that of the control group, and with the extension of culture time after transfection, the inhibition rate of Hep-2 cells was more obvious than that of the control group, but there was no significant change in the proliferation rate. 3. The proliferation of Hep-2 cells was detected by Edu assay with different treatments. The results showed that the Inhibitor transfection group was significantly lower than the blank control group, and the cell growth activity was significantly inhibited. 4. The results of clone formation test showed that the survival fraction of Inhibitor transfected group was significantly lower than that of control group (P 0.05). 5. The cell cycle of Hep-2 cells transfected with hsa-miR-93-5p inhibitor was significantly higher than that of control group (9.62 卤0.71), which was significantly higher than that of control group (0.04 卤0.06), and the difference was statistically significant (P 0.05). 6. The apoptotic rate of Hep-2 cells in Inhibitor transfection group was 1.97 卤0.11, which was significantly higher than that in control group (1.49 卤0.11), and the difference was statistically significant (P 0.05). 7. The results of cell migration assay showed that the number of cell migration in Inhibitor transfected group was significantly lower than that in Hep-2 cell blank control group, and the difference was statistically significant (P 0.05). The invasive ability of Hep-2 cells transfected with Inhibitor was 101.5 卤11.84, which was significantly lower than that of control group (158.67 卤20.82), and the difference was statistically significant (P 0.05). [conclusion]. 1. The proliferation and migration ability of Hep-2 cells transfected with hsa-miR-93-5p Inhibitor sequence was significantly lower than that of untreated Hep-2 cells, suggesting that hsa-miR-93-5p might be an important molecule in the pathogenesis of laryngeal squamous cell carcinoma. 2. Transfection of hsa-miR-93-5p Inhibitor sequence can inhibit the growth of laryngeal squamous cell carcinoma Hep-2 cells and block the cells at G2 phase P0.05, suggesting that hsa-miR-93-5p may play its biological function by regulating the cell cycle of laryngeal squamous carcinoma cells.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R739.65

【参考文献】