miR-338-3p对甲状腺乳头状癌细胞系TPC-1增殖、侵袭、迁移和上皮-间质转化的调控作用

doi:10.3969/j.issn.1673-8640.2022.06.016

摘要/Abstract

摘要：

目的探讨微小RNA-338-3p（miR-338-3p）过表达靶向沉默信息调节因子（SIRT）6对人甲状腺乳头状癌细胞系TPC-1增殖、侵袭、迁移和上皮-间质转化（EMT）的影响。方法采用荧光素酶报告实验分析miR-338-3p与SIRT6的靶向关系。构建SIRT6 pcDNA载体过表达SIRT6,将miR-338-3p mimic与pcDNA-SIRT6单独或联合转染至TPC-1细胞,根据转染质粒的不同分为6组：对照组（不作处理）、mimic-NC组（转染mimic-NC）、miR-338-3p mimic组（转染miR-338-3p mimic）、pcDNA-SIRT6组（转染pcDNA-SIRT6）、mimic-NC+pcDNA-SIRT6组（共转染mimic-NC和pcDNA-SIRT6）和miR-338-3p mimic+pcDNA-SIRT6组（共转染miR-338-3p mimic和pcDNA-SIRT6）。分别检测各组TPC-1细胞的克隆形成率、侵袭细胞数、划痕愈合率及上皮型钙黏蛋白（E-cad）、神经型钙黏蛋白（N-cad）和波形蛋白的表达量。结果 miR-338-3p的靶基因为SIRT6。与对照组比较,miR-338-3p mimic组克隆形成率、侵袭细胞数和划痕愈合率显著降低（P<0.05）,E-cad蛋白表达显著升高（P<0.05）,N-cad、波形蛋白表达显著降低（P<0.05）;pcDNA+SIRT6组克隆形成率、侵袭细胞数和划痕愈合率显著升高,E-cad蛋白表达水平显著降低（P<0.05）,N-cad、波形蛋白表达显著升高（P<0.05）。与pcDNA-SIRT6组比较,miR-338-3p mimic+pcDNA-SIRT6组克隆形成率、侵袭细胞数、划痕愈合率显著降低（P<0.05）,E-cad蛋白表达显著升高（P<0.05）,N-cad、波形蛋白表达显著降低（P<0.05）。结论 miR-338-3p过表达靶向SIRT6可抑制TPC-1细胞的增殖、侵袭、迁移和EMT。

关键词: 微小RNA-338-3p, 沉默信息调节因子6, 甲状腺乳头状癌

Abstract:

Objective To investigate the effect of microRNA（miR）-338-3p overexpression of sirtuin （SIRT）6 on proliferation,invasion,migration and epithelial-mesenchymal transformation（EMT） of human thyroid papillary carcinoma cell line TPC-1. Methods Luciferase reporting assay was used to detect the targeting relationship between miR-338-3p and SIRT6. SIRT6 pcDNA vector was constructed to overexpress SIRT6,and miR-338-3p mimic and pcDNA-SIRT6 were transfected into TPC-1 cells either alone or in combination. The transfected plasmids were classified into 6 groups,including control group（no treatment）,mimic-NC group（transfected mimic-NC） and miR-338-3p mimic group（transfected miR-338-3p mimic）,pcDNA-SIRT6 group（transfected pcDNA-SIRT6）,mimic-NC+pcDNA-SIRT6 group（co-transfected mimic-NC and pcDNA-SIRT6） and miR-338-3p mimic+pcDNA-SIRT6 group（co-transfected with miR-338-3p mimic and pcDNA-SIRT6）. The clone genesis rate,invasive cell number,scratch healing rate of TPC-1 cells and the expression levels of E-cadherin（E-cad）,N-cadherin（N-cad） and Vimentin in epithelial cells were determined. Results The target gene of miR-338-3p was SIRT6. Compared with the control group,clone genesis rate,invasive cell number and scratch healing rate in miR-338-3p mimic group were decreased（P<0.05）. The protein expression of E-cad was increased（P<0.05）,while the protein expressions of N-cad and Vimentin were decreased（P<0.05）. The clone genesis rate,invasive cell number and scratch healing rate were increased in pcDNA+SIRT6 group. The protein expression level of E-cad was decreased（P<0.05）,while the protein expressions of N-cad and Vimentin were increased（P<0.05）. Compared with pcDNA-SIRT6 group,clone genesis rate,invasive cell number and scratch healing rate in miR-338-3p mimic+pcDNA-SIRT6 group were decreased（P<0.05）,while E-cad protein expression was increased（P<0.05）. The protein expressions of N-cad and Vimentin were decreased（P<0.05）. Conclusions The miR-338-3p overexpression targets SIRT6 to inhibit TPC-1 cell proliferation,invasion,migration and EMT.

Key words: MicroRNA-338-3p, Sirtuin 6, Thyroid papillary carcinoma

中图分类号:

R446.7

李涛, 李佳, 潘婧, 梁霄, 于丽娜. miR-338-3p对甲状腺乳头状癌细胞系TPC-1增殖、侵袭、迁移和上皮-间质转化的调控作用[J]. 检验医学, 2022, 37(6): 583-589.

LI Tao, LI Jia, PAN Jing, LIANG Xiao, YU Lina. Regulation of miR-338-3p on proliferation,invasion,migration and epithelial-mesenchymal transformation of thyroid papillary carcinoma cell line TPC-1[J]. Laboratory Medicine, 2022, 37(6): 583-589.

图/表 7

图1 生物信息学网站在线预测SIRT6与miR-338-3p的靶向关系

图2 miR-338-3p与SIRT6的靶向关系注：（a）各组miR-338-3p相对表达量比较;与miR-338-3p mimic组比较,*P<0.05;（b）各组SIRT6相对表达量比较;与miR-338-3p mimic组比较,*P<0.05;（c）荧光素酶报告实验结果;与对照组或SIRT6 Wt组比较,*P<0.05;1为SIRT6 Wt组;2为SIRT6 Wt+miR-338-3p mimic组;3为SIRT6 Mut组;4为SIRT6 Mut+miR-338-3p mimic组;与SIRT6 Wt组比较,*P<0.05。

图3 各组SIRT6 mRNA和蛋白相对表达量比较注：（a）各组SIRT6 mRNA相对表达量比较;（b）SIRT6蛋白表达的免疫印迹图;（c）各组SIRT6蛋白表达量比较;与pcDNA-SIRT6组比较,*P<0.05。

图4 各组细胞克隆形成率的比较注：（a）克隆形成实验（×200）;（b）各组克隆形成率比较;1为对照组;2为mimic-NC组;3为miR-338-3p mimic组;4为pcDNA-SIRT6组;5为mimic-NC+pcDNA-SIRT6组;6为miR-338-3p mimic+pcDNA-SIRT6组;与对照组比较,*P<0.05;与pcDNA-SIRT6组比较,#P<0.05。

图5 各组细胞侵袭数比较注：（a）Transwell小室检测细胞侵袭（×200）;（b）各组侵袭细胞数比较;1为对照组;2为mimic-NC组;3为miR-338-3p mimic组;4为pcDNA-SIRT6组;5为mimic-NC+pcDNA-SIRT6组;6为miR-338-3p mimic+pcDNA-SIRT6组;与对照组比较,*P<0.05;与pcDNA-SIRT6组比较,#P<0.05。

图6 各组划痕愈合率的比较注：（a）划痕愈合实验（×200）;（b）各组划痕愈合率比较;1为对照组;2为mimic-NC组;3为miR-338-3p mimic组;4为pcDNA-SIRT6组;5为mimic-NC+pcDNA-SIRT6组;6为miR-338-3p mimic+pcDNA-SIRT6组;与对照组比较,*P<0.05;与pcDNA-SIRT6组比较,#P<0.05。

图7 miR-338-3p过表达靶向SIRT6对TPC-1细胞E-cad、N-cad、波形蛋白表达的影响注：（a）各组E-cad、N-cad和波形蛋白表达的免疫印迹图;1为对照组;2为mimic-NC组;3为miR-338-3p mimic组;4为pcDNA-SIRT6组;5为mimic-NC+pcDNA-SIRT6组;6为miR-338-3p mimic+pcDNA-SIRT6组;（b）各组E-cad、N-cad和波形蛋白表达量比较; 对照组; mimic-NC组; miR-338-3p mimic组; pcDNA-SIRT6组; mimic-NC+pcDNA-SIRT6组; miR-338-3p mimic+pcDNA-SIRT6组;与对照组相比,*P<0.05;与pcDNA-SIRT6组相比,#P<0.05。

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