Journal of Shanghai Jiao Tong University (Medical Science) >

2022 , Vol. 42 >Issue 8: 1016 - 1023

DOI: https://doi.org/10.3969/j.issn.1674-8115.2022.08.006

Basic research

Effects and mechanisms of KRAS4AG12C and KRAS4BG12C in the proliferation and migration of human pulmonary epithelial cells

  • Jinghua ZOU ,
  • Miaomiao GONG ,
  • Ying SHEN
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  • 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
SHEN Ying, E-mail: yshen0510@sjtu.edu.cn.

Received date: 2022-01-10

  Accepted date: 2022-03-08

  Online published: 2022-04-26

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)

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Abstract

Objective ·To explore how the two splice variants of oncogenic mutant KRASG12C, KRAS4AG12C and KRAS4BG12C, promote the proliferation and migration of human pulmonary epithelial bronchial BEAS-2B cells. Methods ·The two splice variants of oncogenic mutant KRASG12C, KRAS4AG12C and KRAS4BG12C, 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 KRAS4AG12C and KRAS4BG12C 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 KRAS4AG12C and BEAS-2B KRAS4BG12C cells. In addition, overexpression of KRAS4BG12C effectively promoted wound healing of BEAS-2B cells (P=0.000) and BEAS-2B KRAS4AG12C cells (P=0.006), as scratch wound assay characterized. And overexpression of KRAS4BG12C effectively promoted cell migration of BEAS-2B cells (P=0.033) and BEAS-2B KRAS4AG12C cells (P=0.048), as transwell assay characterized. Mechanically, compared with BEAS-2B KRAS4AG12C 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 KRAS4BG12C cells. Conclusion ·This research first reports the differences between KRAS4AG12C and KRAS4BG12C in promoting BEAS-2B cells proliferation and migration, and the underlying mechanism. The oncogenic mutant KRAS4BG12C drives cell migration more significantly than KRAS4AG12C 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 KRAS4AG12C and KRAS4BG12C.

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

Cite this article

Jinghua ZOU , Miaomiao GONG , Ying SHEN . Effects and mechanisms of KRAS4AG12C and KRAS4BG12C in the proliferation and migration of human pulmonary epithelial cells[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022 , 42(8) : 1016 -1023 . DOI: 10.3969/j.issn.1674-8115.2022.08.006

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