[1]时晓晓a,董 翔a,于若卉b,等.miR-203a-3p 靶向GLS1 调控HCC 细胞增殖、迁移、侵袭的机制研究[J].现代检验医学杂志,2022,37(05):86-92+122.[doi:10.3969/j.issn.1671-7414.2022.05.018]
　SHI Xiao-xiaoa,DONG Xianga,YU Ruo-huib,et al.Mechanism of miR-203a-3p Targeting GLS1 to Regulate the Proliferation, Migration and Invasion of HCC Cells[J].Journal of Modern Laboratory Medicine,2022,37(05):86-92+122.[doi:10.3969/j.issn.1671-7414.2022.05.018]
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miR-203a-3p 靶向GLS1 调控HCC 细胞增殖、迁移、侵袭的机制研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第37卷
期数:
2022年05期
页码:
86-92+122
栏目:
论著
出版日期:
2022-09-15

文章信息/Info

Title:
Mechanism of miR-203a-3p Targeting GLS1 to Regulate the Proliferation, Migration and Invasion of HCC Cells
文章编号:
1671-7414（2022）05-086-08
作者:
时晓晓a; 董 翔a; 于若卉b; 苏君君a; 齐东东a; 白 杨a; 董 猛ab
河北省沧州中西医结合医院a. 肝胆外二科；b. 耳鼻咽喉科，河北沧州 061000
Author(s):
SHI Xiao-xiaoa; DONG Xianga; YU Ruo-huib; SU Jun-juna; QI Dong-donga; BAI Yanga; DONG Mengab
a.Department of Hepatobiliary Surgery；b.Department of Otorhinolaryngology，Hebei Cangzhou Hospital of Integrated Traditional Chinese and Western Medicine，Hebei Cangzhou 061000, China
关键词:
肝细胞癌; 微小核糖核酸-203a-3p; 肾型谷氨酰胺酶; 增殖; 迁移; 侵袭; 谷氨酰胺代谢; Wnt/β-catenin通路
分类号:
R735.7；R730.43
DOI:
10.3969/j.issn.1671-7414.2022.05.018
文献标志码:
A
摘要:
目的　探究微小核糖核酸（microRNAs, miRNA, miR）-203a-3p 对肝细胞癌（hepatocellular carcinoma，HCC）细胞增殖、迁移、侵袭的影响及其潜在分子机制。方法　采用实时荧光定量PCR（quantitative real-time PCR，qRTPCR）检测HCC 细胞、人正常肝细胞以及临床HCC 组织中miR-203a-3p 相对表达；采用细胞增殖实验、细胞划痕实验和Transwell 实验分别检测miR-203a-3p 对HCC 细胞增殖、迁移、侵袭的影响；生物信息学网站预测miR-203a-3p 的潜在靶基因（GLS1），双荧光素酶实验进行验证；探究HCC 细胞对谷氨酰胺的依赖性及抑制谷氨酰胺酶1(glutaminase 1，GLS1) 对HCC 细胞增殖、迁移、侵袭的影响；蛋白免疫印迹（Western blot）实验检测Wnt/β-catenin 信号通路关键蛋白β-catenin，p-GSK-3β 和c-Myc 表达。结果　HCC 癌组织中miR-203a-3p（0.32±0.07）表达明显低于癌旁正常组织（1.02±0.03），差异有统计学意义（t ＝ 41.105，P ＜ 0.001）；HCC 细胞HepG2（0.34±0.05），HCCLM3（0.58±0.06），Huh7（0.43±0.05），Hep3B（0.29±0.04）中miR-203a-3p 相对表达水平明显低于人正常肝细胞LO2（1.01±0.02）中miR-203a-3p 表达，差异有统计学意义（F ＝ 119.080，P ＜ 0.001）。与Blank 组相比，miR-203a-3p 过表达组HCC细胞增殖（0.61±0.05 vs 1.24±0.06）， 迁移率（21.43%±2.01% vs 60.22%±3.14%） 及侵袭能力（76.54±13.56 vs221.06±16.54）均明显降低，差异有统计学意义（t ＝ 14.849，13.900，10.562，均P ＜ 0.001）。GLS1 是miR-203a-3p的靶基因，miR-203a-3p 靶向负调控GLS1 表达。HCC 细胞中GLS1 高表达呈现高酶活性，HCC 细胞对谷氨酰胺存在明显依赖性。GLS1 抑制组α-KG，谷氨酸水平均较Blank 组和siRNA-NC 组明显降低，差异有统计学意义（F ＝ 64.754，35.627，均P ＜ 0.001）。GLS1 抑制组细胞增殖（0.59±0.04）、迁移率（30.15%±1.02%）和侵袭能力（69.59±15.74）较Blank 组明显降低（1.29±0.07，59.67%±1.45%，202.14±13.52），差异均有统计学意义（t ＝ 16.499，16.278，11.215，均P ＜ 0.001）。miR-203a-3p 过表达和GLS1 表达抑制明显抑制了Wnt/β-catenin 信号通路关键蛋白β-catenin，p-GSK-3β 和c-Myc 的表达，差异均有统计学意义（t ＝ 11.129 ～ 28.213，均P ＜ 0.001）。转染GLS1 可逆转miR-203a-3p 对HCC 细胞生物学行为及Wnt/β-catenin 信号通路关键蛋白表达的抑制作用。结论　HCC 中miR-203a-3p 显著低表达，其过表达能够抑制HCC 细胞的增殖、迁移和侵袭，可能与GLS1 调控谷氨酰胺代谢及miR-203a-3p 靶向GLS1调控Wnt/β-catenin 信号通路活性有关。
Abstract:
Objective　To explore the effect of micro ribonucleic acid (miRNA) -203a-3p on proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells and its potential molecular mechanism. Methods　Quantitative real-time PCR (qRTPCR) was used to detect the relative expression of miR-203a-3p in HCC cells, human normal liver cells LO2 and clinical HCC tissues. Cell proliferation assay, cell scratch assay and Transwell assay were used to detect the effects of miR-203a-3p on the proliferation, migration and invasion of HCC cells. The potential target gene (GLS1) of miR-203a-3p was predicted by bioinformatics website and verified by double luciferase assay. The dependence of HCC cells on glutamine was detected to explore the effects of GLS1 expression inhibition on the proliferation, migration and invasion of HCC cells. The expression of key proteins β-catenin, P-GSK-3 β and c-Myc in Wnt/β-catenin signaling pathway was detected by Western blot. Results　 The expression of miR-203a-3p in HCC cancer tissues was significantly lower than that in adjacent normal tissues (1.02±0.03 vs 0.32±0.07), and the difference was statistically significant (t=41.105, P ＜ 0.001). The relative expression level of miR-203a-3p in HCC cells HepG2 (0.34±0.05), HCCLM3 (0.58±0.06), Huh7 (0.43±0.05), Hep3B (0.29±0.04) was significantly lower than that of LO2 (1.01±0.02) in human normal liver cells,and the difference was statistically significant (F=119.080, P ＜ 0.001). Compared with the control group, HCC cell proliferation in miR-203A-3p overexpression group (0.61±0.05 vs 1.24±0.06 ), the migration rate(21.43%±2.01% vs 60.22%±3.14%) and invasion ability (76.54±13.56 vs 221.06±16.54) were significantly decreased, and the difference was statistically significant (t=14.849,13.900,10.562, all P ＜ 0.001).Conversely, the opposite results were obtained after miR-203a-3p expression was down-regulated. GLS1 was the target gene of miR-203a-3p, and miR- 203a-3p negatively regulates GLS1 expression. HCC cells showed high expression of GLS1 and high enzyme activity, and HCC cells showed significant dependence on glutamine.The levels of α-KG and glutamate in GLS1 inhibited group were significantly lower than those in Blank group and siRNA-NC group, and the differences were statistically significant (F=64.754, 35.627, all P ＜ 0.001). GLS1 inhibited cell proliferation (20.59±0.04), the migration rate (30.15%±1.02%), invasion ability (69.59±15.74) were significantly lower than blank control group（1.29±0.07，59.67%±1.45%，202.14±13.52）, the differences were statistically significant (t=16.499,16.278,11.215, all P ＜ 0.001). MiR-203a-3p overexpression and GLS1 expression inhibition significantly inhibited the expression of key proteins β-catenin, P-GSK-3β and c-Myc in the Wnt/β-catenin signaling pathway, with statistically significant differences (t=11.129 ～ 28.213, all P ＜ 0.001).Transfection with GLS1 reversed the inhibition of miR-203a-3p on the biological behavior of HCC cells and the expression of key proteins in the Wnt/β-catenin signaling pathway. Conclusion　MiR-203a-3p was significantly underexpressed in HCC, and its overexpression could inhibit the proliferation, migration and invasion of HCC cells, which may be related to GLS1 regulation of glutamine metabolism and miR- 203a-3p targeting GLS1 regulation of Wnt/β-catenin signaling pathway activity.

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备注/Memo

备注/Memo:
基金项目：沧州市重点研发计划指导项目（204106065）。
作者简介：时晓晓（1986-），男，硕士研究生，主治医师，研究方向：普通外科，肝胆胰外科，E-mail：shanqiu0819@163.com。
通讯作者：董猛（1980-），男，副主任医师，研究方向：普通外科，肝胆胰外科。
更新日期/Last Update: 2022-09-15