Rap1GAP affects proliferation, invasion and migration of cervical cancer cells through AMPK signaling pathway

  Background  Rapid proliferation of tumor cells and uncontrolled pelvic lymph node metastasis are the main causes of death in patients with advanced cervical cancer. Low expression of Rap1 GTPase-activating protein (Rap1GAP) is associated with tumor cell proliferation, invasion and poor prognosis.

  Objective  To investigate the effect of Rap1GAP on the proliferation, invasion and migration of cervical cancer cells SiHa and C33a and its potential molecular mechanism.

  Methods  The standardized pan-cancer dataset were downloaded from UCSC (https://xenabrowser.net/) database. The expression data of ENSG00000076864 (Rap1GAP) gene in cervical cancer samples were extracted from TCGA TARGET GTEx (PANCAN, N=19131, G=60499), and log2 (x + 0.001) transformation was performed for each expression value. Rap1GAP gene expression was obtained. Western blotting was used to detect the expression of RAP1GAP in cervical cancer cells SiHa and C33a and normal cervical endothelial cells H8. The expression of Rap1GAP was up-regulated and down-regulated by lentivirus transfection in SiHa and C33a cells, respectively. According to different treatments, the cells were divided into normal control group (SiHa and C33a cells), no-load group (NC-Rap1GAP), OE-Rap1GAP group (overexpression of Rap1GAP) and sh-Rap1GAP group (low expression of Rap1GAP), and the transfection efficiency was verified. Cell proliferation, invasion and migration were detected by plate cloning assay and Transwell assay, respectively. Western blotting and qRT-PCR were used to detect the protein and mRNA expressions of Rap1GAP, E-cadherin and N-cadherin in each group after up-regulating and down-regulating Rap1GAP expression. Phosphorylated AMPK (p-AMPK) and AMPK protein expression were detected by Western blotting.

  Results  The expression of Rap1GAP in cervical cancer tissues was significantly higher than that in normal cervical tissues (P<0.05). Western blotting showed that compared with H8 cells, the expression of Rap1GAP protein in C33a cells increased, while the expression of Rap1GAP protein in SiHa cells decreased (P<0.05). Compared with SiHa group, the ability of colony formation, invasion and migration of cells in OE-Rap1GAP group were decreased. Compared with C33a group, the colony formation ability, invasion and migration ability of sh-Rap1GAP group were enhanced. The results of Western blotting and qRT-PCR showed that the protein and mRNA expressions of Rap1GAP and E-cadherin in the OE-Rap1GAP group increased compared with those in the SiHa group. The protein and mRNA expressions of N-cadherin and p-AMPK were significantly decreased (P<0.01). Compared with C33a group, the protein and mRNA expressions of Rap1GAP and E-cadherin in sh-Rap1GAP group were decreased, while the protein and mRNA expressions of N-cadherin and p-AMPK were increased (P<0.01).

  Conclusion  Rap1GAP inhibits the proliferation, invasion and migration of cervical cancer cells, which may be related to AMPK signaling pathway.