miR-193a-3p在食管癌放射抵抗及化疗耐受作用的初步研究

发布时间：2018-05-31 13:04

本文选题：miR-193a-3p + 食管癌　；参考：《安徽医科大学》2016年硕士论文

【摘要】：目的：探讨miR-193a-3p在食管鳞癌放射抵抗及化疗耐受机制中的作用。方法：通过6MV-X射线对四株食管癌细胞进行照射,采用克隆形成法检测出相对敏感株及耐受株细胞；利用MTT方法检测四种化疗药物对四株食管鳞癌细胞的生存率影响；茎环引物实时定量PCR法检测miR-193a-3p在敏感株及耐受株细胞中的表达,分别合成并转染miR-193a-3p的mimic(3PM)或antagomiR(3PA)序列或小干扰RNA(si-PSEN1)以提高或抑制其在细胞中的表达水平,克隆形成法和流式细胞分析术检测miR-193a-3p及其下游基因PSEN1对于放射耐受性及化疗耐受性的影响,Western blot检测r-H2AX、 PSEN1蛋白的表达；细胞增殖实验检测miR-193a-3p与PSEN1对细胞生长的影响。结果：筛选出相对放射敏感细胞株(KYSE150)及耐受细胞株(KYSE410);同时对四种药物最敏感的细胞株为KYSE150,最耐受的细胞株为KYSE410,故将这两株细胞作为后续研究的标本。miR-193a-3p在两株细胞中的表达水平差异显著(1.00：192)；KYSE410细胞中转染miR-193a-3pantagomiR,抑制其在细胞中的表达水平,与对照组相比,其放射敏感性及化疗敏感性均增加,细胞凋亡比例显著增加3.63%,差异有统计学意义(P0.05),同时r-H2AX蛋白表达增加(1.00：1.66)。KYSE510细胞中转染miR-193a-3pmimic,提高其表达水平,与对照组相比,其放射敏感性及化疗敏感性均减小,细胞凋亡比例显著下降11.10%,差异有统计学意义(P0.05),同时r-H2AX蛋白表达减少(1.00：0.85)；在KYSE150中沉默PSEN1表达,与对照组相比,其放射敏感性降低,细胞凋亡比例下降7.07%,差异有统计学意义(P0.05)；在KYSE150中沉默PSEN1及上调miR-193a-3p表达后,与对照组相比,细胞增殖力增加,差异有统计学意义(P0.05)。结论：miR-193a-3p通过调控基因PSEN1促进食管癌细胞放射抵抗性及化疗耐受性,miR-193a-3p和PSEN1可能成为食管癌放化疗耐受的一个潜在标志物。
[Abstract]:Objective: to investigate the role of miR-193a-3p in the mechanism of radiation resistance and chemotherapy tolerance in esophageal squamous cell carcinoma. Methods: four esophageal cancer cell lines were irradiated by 6MV-X rays, the relative sensitive and tolerant cell lines were detected by clone forming method, and the survival rates of four chemotherapeutic drugs on four esophageal squamous carcinoma cells were detected by MTT method. Stem loop primer real-time quantitative PCR assay was used to detect the expression of miR-193a-3p in sensitive and tolerant cell lines, and to synthesize and transfect miR-193a-3p mimico-3PM3 or antagomiRN3PAA sequences or small interfering RNA-PSEN1, respectively, in order to increase or inhibit the expression level of miR-193a-3p in the cells. The effects of miR-193a-3p and its downstream gene PSEN1 on radiation tolerance and chemotherapeutic tolerance were detected by clone formation and flow cytometry. The expression of r-H2AX, PSEN1 protein was detected by Western blot, and the effects of miR-193a-3p and PSEN1 on cell growth were detected by cell proliferation assay. Results: the relative radiosensitive cell line (KYSE150) and the resistant cell line (KYSE410P) were screened, and the most sensitive cell line to the four drugs was KYSE150, and the most tolerant cell strain was KYSE410. therefore, these two cell lines were used as the specimen for further study. MiR-193a-3p was used in the two cell lines. The expression level of miR-19a-3pantagomiR was inhibited by transfection of miR-193a-3pantagomiR in KYSE410 cells. Compared with the control group, the radiosensitivity and chemosensitivity of the cells were increased, and the percentage of apoptosis was increased significantly (P 0.05). Meanwhile, the expression of r-H2AX protein was increased by 1.00: 1.66N. KYSE510 cells transfected with miR-193a-3 pmimicl, and the expression level of miR-193a-3 pmimiccells was increased compared with the control group. Its radiosensitivity and chemosensitivity decreased, the percentage of apoptosis decreased significantly (P 0.05), and the expression of r-H2AX protein decreased by 1.00: 0.85. In KYSE150, the expression of PSEN1 was silenced, compared with the control group, the radiosensitivity was decreased. After silencing PSEN1 and upregulating the expression of miR-193a-3p in KYSE150, the cell proliferation was increased and the difference was statistically significant compared with the control group (P 0.05). ConclusionMiR-193a-3p may be a potential marker of radiotherapy and chemotherapeutic tolerance of esophageal cancer cells by promoting radioresistance and chemotherapeutic tolerance of esophageal cancer cells by regulating gene PSEN1.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

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