刘林,孟涛,雷程,杨新辉,王海江
LIU Lin,MENG Tao,LEI Cheng,YANG Xinhui,WANG Haijiang

• 1:新疆医科大学附属肿瘤医院胃肠外科

摘要(Abstract)：

目的:研究结肠癌细胞中miR-30a与Beclin1诱导的自噬之间的关系。方法:用携带miR-30a基因的病毒感染人结肠癌细胞株HCT116细胞,实验设置空白对照组CON(不转染)、阴性对照组NC(转染空载片段)、miR-30a组(转染miR-30a),分别用5μmol/L奥沙利铂注射液药物处理、或不处理,采用实时荧光定量PCR(q RT-PCR)检测HCT116细胞中miR-30a的表达水平,MTT法检测细胞增殖情况,Transwell实验检测HCT116细胞迁移能力,流式细胞术检测细胞凋亡率;将野生型Beclin1或3'UTR突变Beclin1与miR-30a或阴性对照共转染至HCT116细胞,采用双荧光素法检测各组细胞的荧光强度,并结合q RT-PCR及Western blot实验检测miR-30a与Beclin1表达水平之间的关系。结果:奥沙利铂注射液处理前后,与对照组相比,miR-30a组细胞增殖能力和迁移能力均受抑制,凋亡比例增高(P <0.05);双荧光素酶报告系统显示,与空白对照组和NC组相比,野生型Beclin1和miR-30a共转染后双荧光素酶活性降低(P <0.05),而Beclin1突变体和miR-30a共转染不影响双荧光素酶的活性(P> 0.05); q RT-PCR及Western blot实验表明miR-30a组细胞中Beclin1基因(m RNA)及蛋白表达水平低于对照组(P <0.05)。结论:miR-30a可通过靶向抑制Beclin1的表达,调节结肠癌细胞的自噬过程,进而影响结肠癌的发展。
Objective: To investigate the relationship between miR-30 a and Beclin1 induced autophagy in colorectal cancer( CRC). Methods: Virus carried with miR-30 a gene were transfected into human HCT116 cell lines. Cells were divided into blank control group( not transfected,CON),negative control group( transfected with empty vector,NC) and miR-30 a group( transfected with miR-30 a). Each group was treated with or without 5 μmol/L oxaliplatin injection. And then the expression level of miR-30 a which in HCT116 cells was tested by qRT-PCR. The condition of cell proliferation was examined by MTT assay,and then the migration ability of HCT116 cells was detected by Transwell and apoptosis rate was tested by Flow Cytometry. Wild-type Beclin1 or 3'UTR mutant Beclin1 were cotransfected with miR-30 a or negative control into HCT116 cells. Then the fluorescence intensity of each group was detected by dual-luciferase reporter assay,and the relationship between miR-30 a and Beclin1 was detected by qRT-PCR and Western blot and combined with dual-luciferase reporter assay.Results: Compared with the control group,the proliferation and migration of miR-30 a group cells were significantly inhibited with or without oxaliplatin treatment and the apoptosis rate was significantly increased( P < 0. 05). Dual-luciferase report assay showed that compared with the CON group and NC group,luciferase activity decreased significantly in cells co-transfected with wild type Beclin1 and miR-30 a( P < 0. 05),while cells co-transfected with Beclin1 mutant and miR-30 a didn 't affect the activity of luciferase( P > 0. 05). q RT-PCR and Western blot results showed that the expression level of Beclin1( m RNA) and albumen in cells of miR-30 a group was significantly lower than control group( P < 0. 05). Conclusion: miR-30 a can regulate the autophagy process of colon cancer cells by inhibiting the expression of Beclin1,thus effecting the development of colon cancer.

关键词(KeyWords)： 结肠肿瘤;自噬;细胞凋亡;细胞增殖;miR-30a;Beclin1
colonic neoplasms;autophagy;apoptosis;cell proliferation;miR-30a;Beclin1

Abstract:

Keywords:

基金项目(Foundation): 新疆维吾尔自治区自然科学基金(2016D01C360)

作者(Author): 刘林,孟涛,雷程,杨新辉,王海江
LIU Lin,MENG Tao,LEI Cheng,YANG Xinhui,WANG Haijiang

DOI: 10.19367/j.cnki.2096-8388.2020.08.010

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