[1]王希方a,郑 伟b,马东瑞c,等.ALKBH5 调控GEFT 的m6A 修饰对胆管癌转移及EMT 的实验研究[J].现代检验医学杂志,2023,38(04):1-7+21.[doi:10.3969/j.issn.1671-7414.2023.04.001]
　WANG Xifanga,ZHENG Weib,MA Dongruic,et al.Experimental Study of GEFT m6A Modification Regulated by ALKBH5 on Cholangiocarcinoma Metastasis and EMT[J].Journal of Modern Laboratory Medicine,2023,38(04):1-7+21.[doi:10.3969/j.issn.1671-7414.2023.04.001]
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ALKBH5 调控GEFT 的m6A 修饰对胆管癌转移及EMT 的实验研究()
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《现代检验医学杂志》[ISSN:/CN:]

卷:
第38卷
期数:
2023年04期
页码:
1-7+21
栏目:
论著
出版日期:
2023-07-15

文章信息/Info

Title:
Experimental Study of GEFT m6A Modification Regulated by ALKBH5 on Cholangiocarcinoma Metastasis and EMT
文章编号:
1671-7414（2023）04-001-08
作者:
王希方1a; 郑 伟1b; 马东瑞1c; 何 莉1a; 孙晶莹1d; 孙 杨1e; 孟 莲2; 孙 超1a
（1．陕西省人民医院a. 肿瘤内科；b. 肝胆外科；c. 教学处；d. 中心实验室；e. 统计室，西安 710068；2．石河子大学医学院第一附属医院病理科，新疆石河子832008）
Author(s):
WANG Xifang1a;  ZHENG Wei1b;  MA Dongrui1c;  HE Li1a;  SUN Jingying1d;  SUN Yang1e;  MENG Lian2;  SUN Chao1a
（1a. Department of Medical Oncology; b. Department of Hepatobiliary Surgery; c.Teaching Office; d. Central Laboratory; e. Statistical Office, Shaanxi Provincial People’s Hospital, Xi’an 710068, China; 2.Department of Pathology, the First Affiliated Hospital of Shihezi University Medical College, Xinjiang Shihezi 832008, China）
关键词:
胆管癌; m6A 甲基化; alKB 同源蛋白; 鸟嘌呤核苷酸交换因子; 上皮间充质转化
分类号:
R735.8；R730.43
DOI:
10.3969/j.issn.1671-7414.2023.04.001
文献标志码:
A
摘要:
目的　探究alkB 同源蛋白5（ALKBH5）调控鸟嘌呤核苷酸交换因子T（guanine nucleotide exchange factorsT, GEFT）的m6A 修饰对胆管癌转移和上皮间充质转化（epithelial-mesenchymal transition, EMT）的影响。方法　将HuCCT1 细胞按照转染类别分为Control 组，NC-sh 组、ALKBH5-sh 组、NC-LV 组、ALKBH5-LV 组、ALKBH5-LV+NC-sh 组和ALKBH5-LV+GEFT-sh 组。Control 组细胞不进行转染，使用Lipofectamine 2000 对其他组细胞分别转染相应的慢病毒。采用MTT 法检测细胞增殖水平，流式细胞仪检测细胞凋亡水平，Transwell 实验检测细胞迁移和侵袭水平。采用RT-qPCR 分析HuCCT1 细胞中的ALKBH5，GEFT，Bax，Bcl-2，MMP2，MMP9，E-cadherin，N-cadherin 和Vimentin 的mRNA 水平。采用Western blot 分析HuCCT1 细胞中的ALKBH5 和GEFT 蛋白水平。采用MeRIP-qPCR 分析HuCCT1 细胞中GEFT m6A 甲基化水平。结果　与Control 组和NC-sh 组比较，ALKBH5-sh 组ALKBH5 和GEFT 的mRNA 和蛋白水平降低，相对细胞活力降低，细胞凋亡率和Bax mRNA 水平升高，Bcl-2 mRNA 水平降低，迁移和侵袭细胞数量降低，MMP2 和MMP9 mRNA 水平降低，E-cadherin mRNA 水平升高，N-cadherin 和Vimentin 水平降低，GEFT m6A 甲基化水平升高，差异具有统计学意义（F=43.347~1 995.868，均P<0.001）。与Control 组和NC-LV 组比较，ALKBH5-LV 组ALKBH5 和GEFT 的mRNA 和蛋白水平升高，相对细胞活力升高，细胞凋亡率和Bax mRNA 水平降低，Bcl-2 mRNA 水平升高，迁移和侵袭细胞数量升高，MMP2 和MMP9 mRNA 水平升高，E-cadherin mRNA 水平降低，N-cadherin 和Vimentin 水平升高，GEFT m6A 甲基化水平降低，差异具有统计学意义（F=42.421~720.275，均P<0.001）。与ALKBH5-LV+NC-sh 组比较，ALKBH5-LV+GEFT-sh 组GEFT 的mRNA 和蛋白水平降低，相对细胞活力降低，细胞凋亡率和Bax mRNA 水平升高，Bcl-2 mRNA 水平降低，迁移和侵袭细胞数量降低，MMP2 和MMP9mRNA 水平降低，E-cadherin mRNA 水平升高，N-cadherin 和Vimentin 水平降低，差异具有统计学意义（t=7.175~77.872，均P<0.001）。结论　ALKBH5 和GEFT 均促进胆管癌细胞的生长和转移，ALKBH5 可能通过调控GEFT m6A 甲基化修饰来影响胆管癌的转移及EMT。
Abstract:
Objective　 To explore the effect of m6A modification of guanine nucleotide exchange factor T (GEFT) regulated by alkB homolog 5 (ALKBH5) on metastasis and epithelial-mesenchymal transformation (EMT) of cholangiocarcinoma. Methods　 HuCCT1 cells were divided into control group, NC-sh group, ALKBH5-sh group, NC-LV group, ALKBH5-LV group,ALKBH5-LV+NC-sh group and ALKBH5-LV+GEFT-sh group according to the type of transfection. The cells in control group were not transfected, while the cells in other groups were transfected with lentivirus with Lipofectamine 2000. Cell proliferation was detected by MTT method, apoptosis was detected by flow cytometry, and cell migration and invasion were detected by Transwell test. The mRNA levels of ALKBH5, GEFT, Bax, Bcl-2, MMP2, MMP9, E-cadherin, N-cadherin and Vimentin in cells were detected by RT-qPCR. The protein levels of ALKBH5 and GEFT in cells were detected by Western blot. The methylation level of GEFT m6A in cells was detected by MeRIP-qPCR. Results　Compared with control group and NC-sh group, ALKBH5 and GEFT mRNA and protein levels decreased, relative cell viability decreased, apoptosis rate and Bax mRNA level increased, Bcl-2 mRNA level decreased, the number of migratory and invasive cells decreased, MMP2 and MMP9 mRNA levels decreased, E-cadherin mRNA level increased, N-cadherin and vimentin levels decreased, GEFT m6A methylation level increased in ALKBH5-sh group, the differences were statistically significant (F=43.347~1 995.868, all P<0.001). Compared with control group and NC-LV group, ALKBH5 and GEFT mRNA and protein levels increased, relative cell viability increased, apoptosis rate and Bax mRNA level decreased, Bcl-2 mRNA level increased, the number of migratory and invasive cells increased, MMP2 and MMP9 mRNA levels increased, E-cadherin mRNA level decreased, N-cadherin and vimentin levels increased, GEFT m6A methylation level decreased in ALKBH5-sh group, the differences were statistically significant (F=42.421~720.275, all P<0.001). Compared with ALKBH5-LV+NC-sh group, GEFT mRNA and protein levels decreased, relative cell viability decreased, apoptosis rate and Bax mRNA level increased, Bcl-2 mRNA level decreased, the number of migratory and invasive cells decreased, MMP2 and MMP9 mRNA levels decreased, E-cadherin mRNA levels increased, N-cadherin and Vimentin levels decreased in ALKBH5-LV+GEFT-sh group, and the differences were statistically significant (t=7.175~77.872, all P<0.001). Conclusion　Both ALKBH5 and GEFT promoted the growth and metastasis of cholangiocarcinoma cells. ALKBH5 may affect the metastasis and EMT of cholangiocarcinoma by regulating the methylation of GEFT m6A.

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相似文献/References:

[1]肖小平,郭 玲a,张 熊b.胆管癌组织LOXL2 mRNA与Tenascinm-C mRNA表达的临床应用研究[J].现代检验医学杂志,2017,32(03):79.[doi:10.3969/j.issn.1671-7414.2017.03.021]
　XIAO Xiao-ping,GUO Linga,ZHANG Xiongb.Clinical Application of the Expression of LOXL2 mRNA and Tenascin-C mRNA in Tissues of the Bile Duct Cancer[J].Journal of Modern Laboratory Medicine,2017,32(04):79.[doi:10.3969/j.issn.1671-7414.2017.03.021]
[2]遆振宇a,高小鹏,千东维b,等.血清 sPDL1水平和外周血 NLR在判断晚期胆管癌患者生存预后中的意义[J].现代检验医学杂志,2019,34(06):41.[doi:10.3969 ／ j.issn.1671-7414.2019.06.010]
　TI Zhen-yua,GAO Xiao-peng,QIAN Dong-wei,et al.Soluble Programmed Death-Ligand 1 (sPDL1) and Neutrophilto-Lymphocyte Ratio (NLR) Predicts Prognostic Survival in Advanced Biliary Tract Cancer Patients Treated with Palliative Chemotherapy[J].Journal of Modern Laboratory Medicine,2019,34(04):41.[doi:10.3969 ／ j.issn.1671-7414.2019.06.010]

备注/Memo

备注/Memo:
基金项目： 国家自然科学基金（82060487）：GEFT 调控Rac1/CDC42/β-catenin 信号通路影响横纹肌肉瘤干细胞生物学特性促进横纹肌肉瘤进展的机制研究；陕西省自然科学基础研究计划项目（2022JQ-983）：hsa-circ-0027308 促进GEFT 表达参与胆管癌发生发展的机制研究；西安市科技计划项目（22YXYJ0133）：去RNA 甲基化酶ALKBH5 上调GEFT 表达促进上皮间质转化参与胆管癌的发生发展。
作者简介：王希方（1982-），男，硕士，主治医师，研究方向：肿瘤转移机制研究，E-mail：1yyxwxf205@163.com。
通讯作者： 孙超（1988-），女，硕士，主治医师，研究方向：肿瘤发生发展的分子机制研究，E-mail：sunchaoSX2016@126.com。
更新日期/Last Update: 2023-07-15