KRAS4AG12C和KRAS4BG12C对人肺上皮细胞生长和运动的作用差异及机制研究

doi:10.3969/j.issn.1674-8115.2022.08.006

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (8): 1016-1023.doi: 10.3969/j.issn.1674-8115.2022.08.006

• 论著 · 基础研究 • 上一篇

KRAS4A^G12C和KRAS4B^G12C对人肺上皮细胞生长和运动的作用差异及机制研究

邹菁华¹^,²(), 宫淼淼¹^,², 沈瑛¹^,²()

^1.上海交通大学基础医学院药理学与化学生物学系，上海 200025
^2.上海市转化医学协同创新中心，上海 200025

收稿日期:2022-01-10 接受日期:2022-03-08 出版日期:2022-04-26 发布日期:2022-04-26
通讯作者: 沈瑛 E-mail:zouzou@sjtu.edu.cn;yshen0510@sjtu.edu.cn
作者简介:邹菁华（1997—），女，硕士生；电子信箱：zouzou@sjtu.edu.cn。
基金资助:
国家自然科学基金(82073868);上海市科学技术委员会基金(20S11900100);上海市高水平地方高校建设项目——药学学科(PT21010)

Effects and mechanisms of KRAS4A^G12C and KRAS4B^G12C in the proliferation and migration of human pulmonary epithelial cells

ZOU Jinghua¹^,²(), GONG Miaomiao¹^,², SHEN Ying¹^,²()

^1.Department of Pharmacology and Chemical Biology, Shanghai Jiao Tong University College of Basic Medical Sciences, Shanghai 200025, China
^2.Shanghai Collaborative Innovation Center for Translational Medicine, Shanghai 200025, China

Received:2022-01-10 Accepted:2022-03-08 Online:2022-04-26 Published:2022-04-26
Contact: SHEN Ying E-mail:zouzou@sjtu.edu.cn;yshen0510@sjtu.edu.cn
Supported by:
National Natural Science Foundation of China(82073868);Fund of Shanghai Science and Technology Commission(20S11900100);Construction Project of High Level Local Universities in Shanghai—Pharmacy(PT21010)

摘要/Abstract

摘要：

目的·探究含有致癌突变KRAS^G12C 的2种剪接体KRAS4A^G12C和KRAS4B^G12C促进人正常肺支气管上皮细胞生长和运动的差异，并初步探讨其分子机制。方法·在人正常肺支气管上皮细胞BEAS-2B中通过慢病毒包装和感染构建KRAS4A^G12C和KRAS4B^G12C稳定过表达细胞株，蛋白质印迹法（Western blotting）检测模型是否构建成功，倒置相差显微镜观察细胞形态，实时动态细胞成像分析系统观察细胞增殖变化，细胞划痕及细胞迁移实验观察细胞运动的变化。机制探究上，使用RNA高通量测序方法（RNA-seq）对细胞全转录组水平进行测序，对测序结果进行京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）通路富集分析及基因集富集分析（Gene Set Enrichment Analysis，GSEA），寻找差异基因及信号通路变化，实时荧光定量PCR进一步验证。2组间比较采用student's t检验，P<0.05时认为差异具有统计学意义。结果·KRAS4A^G12C和KRAS4B^G12C过表达均引起BEAS-2B细胞形态变化，有伪足状结构和细胞连接增加；BEAS-2B KRAS4B^G12C细胞和BEAS-2B KRAS4A^G12C细胞增殖能力均显著强于亲本BEAS-2B细胞；细胞划痕实验结果表明BEAS-2B KRAS4B^G12C细胞伤痕愈合能力显著强于BEAS-2B KRAS4A^G12C细胞（P=0.006），且显著强于BEAS-2B亲本细胞（P=0.000）；Transwell实验结果表明BEAS-2B KRAS4B^G12C细胞迁移能力显著强于BEAS-2B KRAS4A^G12C细胞（P=0.048），且显著强于BEAS-2B亲本细胞（P=0.033）；与BEAS-2B KRAS4A^G12C细胞相比，BEAS-2B KRAS4B^G12C细胞中细胞黏附分子相关信号通路显著上调（P=0.002）；BEAS-2B KRAS4B^G12C细胞紧密连接蛋白1（claudin 1，CLDN1）的mRNA水平显著高于 BEAS-2B KRAS4A^G12C细胞（P=0.000）；BEAS-2B KRAS4B^G12C细胞黏附分子3（cell adhesion molecule 3，CADM3）的mRNA水平显著高于BEAS-2B KRAS4A^G12C细胞（P=0.000）。结论·该文首次报道了含有致癌突变KRAS^G12C 的2种剪接体KRAS4A^G12C和KRAS4B^G12C促进BEAS-2B细胞生长和运动能力的差异以及潜在的分子机制，即KRAS4A^G12C和KRAS4B^G12C均可促进人肺支气管上皮细胞BEAS-2B增殖，而且KRAS4B^G12C促进肺上皮细胞BEAS-2B运动能力更强，可能与黏附相关基因CLDN1和CADM3表达水平更高相关，为KRAS特异性靶向抑制剂的研究提供了实验依据和理论基础。

关键词: KRAS蛋白，人, 蛋白异构体, 细胞黏附, 细胞运动, 肺癌

Abstract:

Objective ·To explore how the two splice variants of oncogenic mutant KRAS^G12C, KRAS4A^G12Cand KRAS4B^G12C, promote the proliferation and migration of human pulmonary epithelial bronchial BEAS-2B cells. Methods ·The two splice variants of oncogenic mutant KRAS^G12C, KRAS4A^G12Cand KRAS4B^G12C, were stably overexpressed in human normal pulmonary epithelial bronchial BEAS-2B cells by lentivirus packaging and infection system. Western blotting was used to verify whether the model was constructed successfully. Cell morphology was investigated by inverted phase-contrast microscopy. Cell proliferation was observed by the Incucyte Live Cell Analysis System. Cell migration was characterized by scratch wound assay and transwell assay. To investigate the mechanisms, RNA-sequencing (RNA-seq) was used to provide insight into the transcriptome of the indicated cells, Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Set Enrichment Analysis (GSEA) were performed to identify differential genes and signaling pathways enriched in ranked gene lists, and quantitative real-time PCR (qPCR) was used to further verify different genes. Student's t test (t test) was used for comparison between the two groups. Statistical significance was accepted at a value of P<0.05. Results ·Both overexpression of KRAS4A^G12C and KRAS4B^G12C induced morphological changes, including increased invasive pseudopodia structures and cell junctions of BEAS-2B cells. Compared with BEAS-2B cells, the cell proliferation was significantly enhanced in BEAS-2B KRAS4A^G12C and BEAS-2B KRAS4B^G12C cells. In addition, overexpression of KRAS4B^G12C effectively promoted wound healing of BEAS-2B cells (P=0.000) and BEAS-2B KRAS4A^G12C cells (P=0.006), as scratch wound assay characterized. And overexpression of KRAS4B^G12C effectively promoted cell migration of BEAS-2B cells (P=0.033) and BEAS-2B KRAS4A^G12C cells (P=0.048), as transwell assay characterized. Mechanically, compared with BEAS-2B KRAS4A^G12C cells, cell adhesion molecules gene set was enriched and the mRNA levels of claudin 1 (CLDN1) (P=0.000) and cell adhesion molecule 3 (CADM3) (P=0.000) were up-regulated in BEAS-2B KRAS4B^G12C cells. Conclusion ·This research first reports the differences between KRAS4A^G12C and KRAS4B^G12C in promoting BEAS-2B cells proliferation and migration, and the underlying mechanism. The oncogenic mutant KRAS4B^G12C drives cell migration more significantly than KRAS4A^G12C does, which may be related to the expression level of CLDN1 and CADM3. Our study provides insights for the design of KRAS-specific targeting inhibitors and individualized therapy for non-small cell lung carcinoma patients harboring KRAS4A^G12Cand KRAS4B^G12C.

Key words: KRAS protein, human, protein isoform, cell adhesion, cell movement, lung neoplasm

中图分类号:

R730.2

邹菁华, 宫淼淼, 沈瑛. KRAS4A^G12C和KRAS4B^G12C对人肺上皮细胞生长和运动的作用差异及机制研究[J]. 上海交通大学学报（医学版）, 2022, 42(8): 1016-1023.

ZOU Jinghua, GONG Miaomiao, SHEN Ying. Effects and mechanisms of KRAS4A^G12C and KRAS4B^G12C in the proliferation and migration of human pulmonary epithelial cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(8): 1016-1023.

图/表 4

表1 qPCR所用的引物序列

Tab 1 Sequences of primers for qPCR

Gene

Primer sequence（5'→3'）

CLDN1

F：TCTTGCAGGTCTGGCTATTTTA

R：TTGGGTAAGAGGTTGTTTTTCG

CADM3

F： AGAGTGCCCTGATCTTCCCTTTCC

R： GTGGTAGGTGCTGGAGGAGGAG

β-actin

F： CGGGAAATCGTGCGTGAC

R： TGGAAGGTGGACAGCGAGG

图1 KRAS4AG12C 和KRAS4BG12C 过表达对肺上皮细胞形态及增殖的影响Note： A. Western blotting analysis of Flag-tagged KRAS4AG12C stably overexpressed in BEAS-2B cells. B. Western blotting analysis of KRAS4BG12C stably overexpressed in BEAS-2B cells. C. Proliferation rate of BEAS-2B cells increased after stable overexpression of KRAS4AG12C or KRAS4BG12C. D. Morphological changes of BEAS-2B cells. Ku86—Tubulin, as loading control; Con=BEAS-2B; 4A=BEAS-2B KRAS4AG12C; 4B=BEAS-2B KRAS4BG12C.

Fig 1 Effects of KRAS4AG12C or KRAS4BG12C overexpression on pulmonary epithelial cells morphology and proliferation

图2 KRAS4AG12C 和KRAS4BG12C 过表达对肺上皮细胞运动的影响Note： A/B. Mobility increasing of BEAS-2B cells induced by stably overexpressed KRAS4AG12C or KRAS4BG12C (real-time observation by Incucyte, scale bar=300 μm). C/D. Migration increasing of BEAS-2B cells induced by stably overexpressed KRAS4AG12C or KRAS4BG12C. Con=BEAS-2B; 4A= BEAS-2B KRAS4AG12C; 4B=BEAS-2B KRAS4BG12C. ①P=0.000, compared with the control group; ②P=0.006, compared with the 4A group; ③P=0.033, compared with the control group; ④P=0.048, compared with the 4A group.

Fig 2 Effects of KRAS4AG12C or KRAS4BG12C overexpression on pulmonary epithelial cells migration

图3 KRAS4AG12C 和KRAS4BG12C 过表达对黏附相关基因 CLDN1 和 CADM3 表达的影响Note： A/B. KEGG (A) and GSEA (B) analysis of the significantly differential genes between BEAS-2B KRAS4AG12C cells and BEAS-2B KRAS4BG12C cells. C. mRNA levels of CLDN1 in BEAS-2B KRAS4AG12C cells and BEAS-2B KRAS4BG12C cells. D. mRNA levels of CADM3 in BEAS-2B KRAS4AG12C cells and BEAS-2B KRAS4BG12C cells. 4A=BEAS-2B KRAS4AG12C; 4B=BEAS-2B KRAS4BG12C. ①P=0.000, compared with the 4A group; ②P=0.000, compared with the 4A group.

Fig 3 Effects of KRAS4AG12C or KRAS4BG12C overexpression on the mRNA levels of CLDN1 and CADM3

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KRAS4A^G12C和KRAS4B^G12C对人肺上皮细胞生长和运动的作用差异及机制研究

Effects and mechanisms of KRAS4A^G12C and KRAS4B^G12C in the proliferation and migration of human pulmonary epithelial cells

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