目的：研究长链非编码RNA mir-100-let-7a-2-mir-125b-1簇宿主基因（long non-coding RNA mir-100-let-7a-2-mir-125b-1 cluster host gene,LncRNA MIR100HG）调控miR-142-5p/ SRY相关的高迁移率族盒蛋白5（SRY-related high mobility group box 5,SOX5）轴对口腔鳞癌（oral squamous cell carcinoma,OSCC）细胞增殖、侵袭能力的影响。方法：采用实时荧光定量PCR和免疫印迹检测体外培养的人口腔上皮细胞和人OSCC细胞CAL27、SAS、SCC-25中LncRNA MIR100HG、miR-142-5p与SOX5表达。体外培养CAL27细胞,随机分为对照组、LncRNA MIR100HG敲低组（转染LncRNA MIR100HG siRNA质粒）、miR-142-5p mimics组（转染miR-142-5p mimics）、共转染阴性对照组（转染空载质粒和miR-142-5p阴性对照）、LncRNA MIR100HG敲低+miR-142-5p inhibitor组（转染LncRNA MIR100HG siRNA质粒和miR-142-5p inhibitor）,分组转染后,采用实时荧光定量PCR和免疫印迹检测各组细胞LncRNA MIR100HG、miR-142-5p及SOX5表达;采用3-（4,5-二甲基噻唑-2）-2,5-二苯基四氮唑溴盐法、5-乙炔基-2'-脱氧尿苷染色及集落形成实验检测各组细胞增殖;采用Transwell侵袭实验检测各组CAL27细胞侵袭;采用免疫印迹检测各组CAL27细胞上皮间质转化相关蛋白表达。将各组细胞接种在裸鼠背部右侧腋窝附近皮下构建OSCC移植瘤模型,饲养3周后检测各组移植瘤裸鼠肿瘤体积及重量。采用双荧光素酶报告实验检测CAL27细胞LncRNA MIR100HG对miR-142-5p、miR-142-5p对SOX5的靶向调控。结果：与人口腔上皮细胞相比,CAL27、SAS、SCC-25中LncRNA MIR100HG、SOX5蛋白与mRNA表达升高,miR-142-5p表达降低。与对照组相比,LncRNA MIR100HG敲低组、miR-142-5p mimics组细胞SOX5蛋白与mRNA表达、细胞活力、增殖率、集落形成比例、移植瘤裸鼠肿瘤体积及重量、侵袭数、N-cadherin蛋白表达降低,miR-142-5p表达、E-cadherin蛋白表达升高。下调miR-142-5p可减弱LncRNA MIR100HG敲低对CAL27细胞各指标的作用。CAL27细胞中LncRNA MIR100HG靶向下调miR-142-5p、miR-142-5p靶向下调SOX5。结论：敲低LncRNA MIR100HG可通过调控miR-142-5p/SOX5轴而抑制OSCC细胞增殖、上皮间质转化及侵袭,并可延缓其体内肿瘤生长。
Abstract
Objective To investigate the effect of the long non-coding RNA mir-100-let-7a-2-ir-125b-1 cluster host gene (LncRNA MIR100HG) regulating miR-14-2-5p / SRY related high mobility box protein 5(SOX 5) axis on the proliferation and invasion capacity of oral squamous cell carcinoma (OSCC) cells. Methods Quantitative real-time PCR and immunoblot were performed to detect the expression of LncRNA MIR100HG, miR-142-5p and SOX 5 in CAL 27, SAS, and SCC-25 in vitro cultured human oral epithelial cells and human OSCC-derived cells. When CAL 27 cells were cultured in vitro, They were randomly divided into control group, LncRNA MIR100HG knockdown group (transfection with LncRNA MIR100HG siRNA plasmid), miR-142-5p mimics group (transfected with miR-142-5p mimics), cotransfection negative control group (transfection with empty plasmid and miR-142-5p negative control), LncRNA MIR100HG knockdown + miR-142-5p inhibitor group (transfected with LncRNA MIR100HG The siRNA plasmid and miR-142-5p inhibitor), After the group-transfection, The expression of LncRNA MIR100HG, miR-142-5p and SOX 5 were analyzed by real-time PCR and immunoblotting; Using the method 3- (4, 5-dimethylthiazole-2) -2, 5-diphenyl tetrazolium bromine salt (MTT) method, 5-ethylene-2-2 ′ -deoxyuridine (Edu) staining and colony formation assay to detect cell proliferation in each group; CAL 27 cell invasion in each group was detected by Transwell invasion assay; Immunoblotting was used to detect protein expression associated with epithelial stromal transformation in each group of CAL 27 cells. OSCC tumor model was constructed in the area near the right armpit of the back of nude mice. The tumor volume and weight of nude mice were detected after 3 weeks of feeding. A dual luciferase reporter assay was used to detect the targeted regulation of miR-142-5p and SOX 5 by miR-142-5p. Results Compared with human oral epithelial cells, LncRNA MIR100HG, SOX 5 protein and mRNA were increased in CAL 27, SAS, SCC-25 and miR-142-5p decreased. In LncRNA MIR100HG knockdown, SOX 5 protein and mRNA expression, cell viability, proliferation rate, colony formation ratio, tumor volume and weight, invasion number, N-cadherin protein expression, miR-142-5p expression than control group. Downregulation of miR-142-5p can weaken the effect of LncRNA MIR100HG knockdown on various indicators of CAL27 cells. Targeted downregulation of miR-142-5p by LncRNA MIR100HG and miR-142-5p and SOX 5 in CAL 27 cells. Conclusion Knockdown of LncRNA MIR100HG can inhibit OSCC cells proliferation, epithelial stromal transformation and invasion by regulating the miR-142-5p/SOX5 axis, and can delay tumor growth in vivo.
关键词
LncRNA MIR100HG /
miR-142-5p/SOX5 /
口腔鳞癌 /
增殖 /
侵袭
Key words
LncRNA MIR100HG /
miR-142-5p/SOX5 /
oral squamous cell carcinoma /
proliferation /
invasion
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基金
河北省保定市科技计划支持项目（2241ZF180）
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