天津医药

天津医药 ›› 2021, Vol. 49 ›› Issue (12): 1255-1260.doi: 10.11958/20202783

• 细胞与分子生物学 • 上一篇    下一篇

m6A修饰调控的COL6A5对肺腺癌细胞侵袭能力的影响

王馨，黄嘉星，潘辉林，冯正富，汤锐明，邱惠思△   

  1. 王馨（1983），女，硕士，副主任医师，主要从事肺癌发生发展的机制研究。E-mail：16208918@163.com
  • 收稿日期:2020-10-10 修回日期:2021-07-19 出版日期:2021-12-15 发布日期:2021-12-27
  • 通讯作者: △通信作者 E-mail：huisi22@126.com E-mail:huisi22@126.com
  • 作者简介:王馨（1983），女，硕士，副主任医师，主要从事肺癌发生发展的机制研究。E-mail：16208918@163.com

The effect of COL6A5 downregulated by m6A modification on lung adenocarcinoma cell invasion

WANG Xin, HUANG Jia-xing, PAN Hui-lin, FENG Zheng-fu, TANG Rui-ming, QIU Hui-si△   

  • Received:2020-10-10 Revised:2021-07-19 Published:2021-12-15 Online:2021-12-27

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摘要： 目的 明确6型胶原蛋白α5（COL6A5）在肺腺癌中的表达及意义。方法 通过在线数据库及实时荧光定 量PCR（qPCR）法检测COL6A5在肺腺癌细胞和组织中的表达。采用GEPIA数据库分析COL6A5的表达量与肺腺癌 患者预后的相关性。生物信息学预测结合 RNA 甲基化免疫沉淀-实时荧光定量 PCR（MeRIP-qPCR）实验检测 COL6A5转录本上的m6A位点。qPCR法检测RNA去甲基酶ALKBH5在肺腺癌组织中的表达量，并分析ALKBH5与 COL6A5在27例肺腺癌组织中表达的相关性。qPCR和Western blot检测ALKBH5对COL6A5的抑制作用。生物信息 学预测结合Transwell实验和Western blot验证COL6A5在肺腺癌中的作用。结果 在线数据库Oncomine分析显示， COL6A5 在肺癌中低表达，GEPIA 数据库分析显示肺腺癌组织中 COL6A5 mRNA 表达水平显著低于正常组织（P＜ 0.05）。qPCR 结果显示，COL6A5 mRNA 在 27 例肺腺癌组织中的表达量低于癌旁组织；与正常肺上皮细胞相比， COL6A5 mRNA在肺腺癌细胞中的表达量明显受到抑制（P＜0.05）。生物信息学预测结合MeRIP-qPCR 实验证实， COL6A5 mRNA上存在m6A位点。肺腺癌组织中ALKBH5 mRNA的表达水平高于癌旁正常组织，且与COL6A5 mRNA 表达呈负相关（r=-0.599，P＜0.01）。在H1299细胞中敲低ALKBH5，可显著上调COL6A5的表达。分析与COL6A5共 表达的基因，结果显示 COL6A5 可能参与调控肺腺癌细胞上皮间质转换、止血和细胞表面相互作用等生命活动。 Transwell和Western blot证实，过表达COL6A5可显著抑制H1299细胞的侵袭转移能力。结论 ALKBH5在肺腺癌中 下调COL6A5，过表达COL6A5可明显抑制肺腺癌细胞的侵袭转移能力。

关键词: 肺肿瘤, 腺癌, 肿瘤侵润, 胶原Ⅵ型, AlkB同源蛋白5, RNA脱甲基酶, 计算生物学, 6型胶原蛋白α5, m6A

Abstract: Objective To elucidate the role and expression pattern of COL6A5 in lung adenocarcinoma (LUAD). Methods The expression pattern of COL6A5 in LUAD was detected through Oncomine database. The relationship between COL6A5 and the prognosis of LUAD patients was analyzed by GEPIA database. The qPCR assay was used to detect the expression of RNA demethylase ALKBH5 in lung adenocarcinoma tissue. The m6A sites on COL6A5 was confirmed by bioinformatics analysis and MeRIP-qPCR experiment. The inhibitory effect of ALKBH5 on COL6A5 was detected by qPCR and Western blot assy. Bioinformatics analysis and Transwell assay were performed to determine the role of COL6A5 in LUAD. Results Oncomine analysis of the online database showed that the expression of COL6A5 was significantly reduced in LUAD. GEPIA database analysis showed that the level of COL6A5 mRNA expression was significantly lower in LUAD tissue than that of normal tissue (P＜0.05). qPCR result showed that COL6A5 mRNA expression was lower in LUAD tissues than that of adjacent normal tissues in 27 LUAD patients. Compared to normal pulmonary epithelial cells, COL6A5 mRNA was significantly inhibited in LUAD cells (P＜0.05). Furthermore, bioinformatics analysis and MeRIP-qPCR assay confirmed that there were m6A sites on COL6A5 mRNA. The expression level of ALKBH5 mRNA was higher in LUAD than that of adjacent normal tissues, and it was negatively correlated with COL6A5 mRNA expression (r=-0.599, P＜0.01). Knocking down ALKBH5 significantly enhanced COL6A5 expression in H1299 cells. After the analysis of co-expressed genes with COL6A5, it was found that COL6A5 may be involved in the regulation of life activities including epithelial transition, hemostasis, and cell surface interaction in LUAD cells. Results of Transwell assay and Western blot assay confirmed that the over-expression of COL6A5 could inhibit the invasion of H1299 cells. Conclusion The down regulated COL6A5 by ALKBH5 can inhibit the invasion metastasis of LUAD cells .

Key words: lung neoplasms, adenocarcinoma, neoplasm invasiveness, collagen type Ⅵ, AlkB homolog 5, RNA demethylas, computational biology, type Ⅵ collagen alpha-5 chain (COL6A5), m6A

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