PLCE1基因对食管鳞癌细胞自噬的作用及初步分子机制研究

发布时间：2018-08-08 12:27

【摘要】：目的:探讨PLCE1对食管鳞癌自噬水平的影响;研究下调食管鳞癌中沉默PLCE1所诱发的自噬水平对癌症细胞凋亡的影响;初步研究PLCE1调控食管鳞癌自噬水平的分子机制。方法:(1)用PLCE1 si RNA转染食管鳞癌细胞系Eca-109、TE-1,应用MDC染色、AO染色及细胞免疫荧光检测方法,检测下调PLCE1表达对肿瘤细胞内自噬水平的影响;(2)运用自噬特异性抑制剂3-MA或Beclin-1 si RNA,抑制由沉默PLCE1表达所诱发的自噬,运用MDC染色检测食管鳞癌细胞内的自噬水平,通过MTT方法、流式细胞仪检测凋亡方法检测肿瘤细胞的凋亡和增殖水平受到的影响,应用Western Blot方法检测自噬相关分子Beclin-1和LC3的表达水平,以及凋亡相关分子cleaved-PARP、Bax、Bcl-2、cleaved-caspase-3的蛋白表达水平;(3)根据课题组前期研究结果,运用Western Blot实验检测mi R-106b-5p的宿主基因MCM7的表达,结合Target Scan,mi Randa,DIANAm T,mi RDB,mi RWalk等靶基因预测软件分析,运用荧光素酶报告试验检测mi R-106b-5p与自噬相关基因Beclin-1之间的靶向关系;(4)在食管鳞癌细胞ECA109内共转染mi R-106b-5p模拟物与PLCE1 si RNA,通过MDC染色和AO染色,检测肿瘤细胞内的自噬水平,通过Western Blot方法检测食管癌细胞内自噬基因Beclin-1的表达水平。结果:(1)使用PLCE1 si RNA转染食管鳞癌细胞系,MDC染色和AO染色的结果显示,沉默PLCE1的表达后,细胞内的自噬囊泡数量明显增多,细胞免疫荧光检测提示LC3蛋白的表达水平明显升高;(2)MDC染色结果显示沉默PLCE1后所诱发的自噬上调可被3-MA抑制;MTT实验结果显示,PLCE1 si RNA和3-MA共同处理的ECA109细胞系和TE-1细胞系的增殖水平与PLCE1 si RNA单独处理组相比有明显升高,Beclin-1 si RNA与PLCE1 si RNA共处理组的细胞增殖水平与对照组及PLCE1 si RNA处理组相比有明显升高;流式细胞术结果显示,PLCE1 si RNA处理组细胞的凋亡水平,在转染3-MA或Beclin-1 si RNA后明显降低;Western Blot结果显示,下调PLCE1蛋白表达后,Beclin-1蛋白及LC3蛋白的表达水平明显升高,3-MA或Beclin-1 si RNA与PLCE1 si RNA共处理组中,cleaved-caspase-3、Bax及cleaved-PARP蛋白的表达水平与PLCE1 si RNA单独处理组相比,表达水平有明显降低,而Bcl-2蛋白的表达水平升高;(3)Western Blot实验结果显示,mi R-106b-5p的宿主基因MCM7在沉默PLCE1后表达明显降低;Target Scan,mi Randa,DIANAm T,mi RDB,mi RWalk等靶基因预测软件分析结果显示,mi R-106b-5p可能与自噬基因Beclin-1存在靶向关系,荧光素酶报告试验结果显示,mi R-106b-5p通过靶向结合3’UTR调控下游基因Beclin-1;Western Blot结果显示,与对照组相比,mi R-106b-5p模拟物组的Beclin-1蛋白表达量明显降低;(4)在Eca-109细胞系和TE-1细胞系中转染mi R-106b-5p模拟物及PLCE1 si RNA,通过MDC染色和AO染色后发现,共转染mi R-106b-5p模拟物及PLCE1 si RNA后,肿瘤细胞内的自噬囊泡数量比PLCE1 si RNA单独处理组也明显降低,Western Blot结果显示,共转染mi RNA模拟物及PLCE1 si RNA后,Beclin-1的表达水平与单独转染PLCE1 si RNA的细胞组相比明显降低。结论:(1)PLCE1的过表达可以下调食管鳞癌细胞内的自噬和凋亡水平,从而促进食管细胞的恶性转化;(2)PLCE1对调控食管鳞癌细胞自噬的分子机制可能是通过上调mi R-106b-5p,靶向抑制Beclin-1的表达,进而下调肿瘤细胞内的自噬过程;(3)本次研究提示通过靶向干预PLCE1的表达,诱导食管鳞癌细胞的自噬,能够成为食管癌治疗的新策略。
[Abstract]:Objective: To investigate the effect of PLCE1 on the autophagy level of esophageal squamous cell carcinoma, to study the effect of autophagy induced by PLCE1 in esophageal squamous cell carcinoma and to investigate the molecular mechanism of autophagy by PLCE1 to regulate the autophagy level of esophageal squamous cell carcinoma. Methods: (1) transfection of Eca-109, TE-1, MDC staining, AO staining with PLCE1 Si RNA The effect of down regulated PLCE1 expression on the autophagy level in tumor cells was detected by color and cell immunofluorescence. (2) autophagy, 3-MA or Beclin-1 Si RNA, was used to inhibit autophagy induced by silent PLCE1 expression. Autophagy was detected by MDC staining in esophageal squamous cell carcinoma cells, using MTT method and flow cytometry to detect the loss of autophagy. The apoptosis and proliferation level of tumor cells were detected by the method of death. Western Blot method was used to detect the expression level of autophagy related molecules Beclin-1 and LC3, as well as the protein expression level of cleaved-PARP, Bax, Bcl-2, cleaved-caspase-3 of apoptosis related molecules. (3) according to the results of earlier study of the group, Western Blot test was used to detect M The expression of the host gene MCM7 of I R-106b-5p, combined with Target Scan, MI Randa, DIANAm T, MI RDB, MI targets and other target gene prediction software analysis, and the use of luciferase report test to detect the target relationship between the autophagy related genes and those of the autophagy related genes. (4) the simulants were co transfected in the squamous carcinoma cells of the esophagus. The autophagy level in tumor cells was detected by MDC staining and AO staining. The expression level of autophagic gene Beclin-1 in esophageal cancer cells was detected by Western Blot method. Results: (1) transfection of esophageal squamous cell carcinoma cell lines using PLCE1 Si RNA. The results of MDC staining and AO staining showed that the number of autophagic vesicles in the cells was clear after the expression of silent PLCE1. The expression of LC3 protein was significantly increased by cell immunofluorescence, and (2) MDC staining showed that the up regulation of autophagy induced by silent PLCE1 could be suppressed by 3-MA. The MTT experiment showed that the proliferation of ECA109 cell lines and TE-1 cell lines, which were treated by PLCE1 Si RNA and 3-MA, was compared with that of PLCE1 alone. The cell proliferation level of Beclin-1 Si RNA and PLCE1 Si RNA co processing group was significantly higher than that of the control group and PLCE1 Si RNA treatment group. The results of flow cytometry showed that the apoptosis level of PLCE1 Si RNA treatment group was significantly lower than that of the PLCE1 Si Group. The expression level of Beclin-1 protein and LC3 protein increased obviously. The expression level of cleaved-caspase-3, Bax and cleaved-PARP protein in 3-MA or Beclin-1 Si RNA and PLCE1 Si RNA co processing group was significantly lower than that of the single treatment group. The expression of the host gene MCM7 of MI R-106b-5p decreased markedly after the silence of PLCE1, and Target Scan, MI Randa, DIANAm T, MI RDB, and other target gene prediction software analysis showed that there might be a targeting relationship with autophagic gene. Control downstream gene Beclin-1; Western Blot results showed that the expression of Beclin-1 protein in the MI R-106b-5p simulation group decreased significantly compared with the control group; (4) the MI R-106b-5p analog and PLCE1 Si were transfected in the Eca-109 cell line and TE-1 cell line. The number of autophagic vesicles in the tumor cells was significantly lower than that in the PLCE1 Si RNA treatment group. The Western Blot results showed that the expression level of the Beclin-1 was significantly lower than that of the MI RNA mimics and PLCE1 Si RNA. Conclusion: (1) the overexpression of the esophageal squamous cell carcinoma cells can be downregulated in the cells of the squamous cell carcinoma of the esophagus. The level of macrophage and apoptosis promotes malignant transformation of esophageal cells; (2) the molecular mechanism of PLCE1 to regulate autophagy in esophageal squamous cell carcinoma may be by up regulation of MI R-106b-5p, targeting the expression of Beclin-1 and down regulation of autophagy in the tumor cells; (3) this study suggested that the expression of PLCE1 was targeted by target intervention to induce the squamous cell carcinoma of the esophagus. Cell autophagy can become a new strategy for the treatment of esophageal cancer.
【学位授予单位】：石河子大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R735.1

【相似文献】