RacGAP1 Promotes Malignant Progression In Cervical Cancer By Regulating AP-1 Via Mir-192 And P-JNK

Cervical cancer is one of the most common tumors of the female reproductive system.As reported in GLOBOCAN 2020,cervical cancer(CC)was the fourth most frequently diagnosed cancer and the fourth leading cause of cancer death in women worldwide.Persistent high-risk human papillomavirus(HPV)infection is the main cause of cervical cancer,but the exact molecular mechanism is still unclear.Therefore,looking for molecules related to the occurrence and development of cervical cancer,exploring new prognostic factors and therapeutic targets,and further improving the survival rate of patients are problems that we urgently need to solve.Rac GTPase Activating Protein 1(RacGAP1)is a member of the GTPase-activating protein(RhoGAP)family.It reacts with the GTP-restricted small G protein of the Rho family and then activates the hydrolysis of GTP,restores Rho protein to an inactive state of binding to GDP,regulates Racl and CDC42 proteins to drive tumor growth.Several authors reported that RacGAP1 was highly expressed in a variety of tumors,such as breast cancer,and is associated with poor prognosis.However,its role in cervical cancer is still unclear.This study aims to study the expression of RacGAPl in cervical cancer and its influence on the biological behavior of cervical cancer cells,and to explore the possible mechanism,in the hope that it can provide new ideas and targets for the treatment of cervical cancer.Part Ⅰ Exploration of the hub gene for cervical cancer progression and the expression of RacGAPl in human cervical cancerObjectTo explore key candidate genes for cervical cancer progression through bioinformatics analyses.To detect the expression of RacGAP1 in cervical cancer and to explore the correlation between the expression of RacGAPl and the clinicopathological characteristics and prognosis of patients.MethodsThe key genes of cervical cancer were screened by bioinformatics method.The expression and localization of RacGAPl in fresh cervical cancer tissues and cell lines was detected by qRT-PCR,Western blot and immunofluorescence staining.The expression of RacGAPl and Ki-67 in cervical cancer tissues was detected by immunohistochemistry,and the relationship between the expression of RacGAPl and the clinicopathological features and prognosis of cervical cancer was analyzed.Results1.RacGAP1 and CDK1 are the common DEGs of 10 hub genes in the PPI network and 5 overlapping genes in the data sets.2.The expression of RacGAPl is overexpressed in CC,and it is expressed in both the nucleus and cytoplasm.3.The expression of RacGAPl was associated with the histological grade of cervical cancer(p<0.001).The expression of Ki-67 was positively correlated with RacGAP1.4.Overall survival(OS)and progression-free survival(PFS)were significantly longer in patients with low RacGAP1 expression compared with patients with high RacGAP1 expression.There was no correlation between Ki-67 expression and patients’ OS or PFS.RacGAP1 expression was an independent factor in evaluating the prognosis of cervical cancer(p<0.05).Brief Summary1.RacGAP1 may be a hub gene for the occurrence and development of cervical cancer.2.RacGAP1 is highly expressed in cervical cancer tissues and could be an independent factor for the prognosis of cervical cancer.Part Ⅱ RacGAPl affects the biological behavior of human cervical cancer cell via miR-192ObjectTo explore the effects of RacGAPl on the proliferation,migration,invasion and the cell cycle distribution of human cervical cancer cell lines.To screen the downstream miRNAs of RacGAP1.MethodsActive RhoA was tested using RhoA activation assay Kit.CCK-8 and clone formation assay were used to detect the proliferation ability of CaSki,HeLa and SiHa cell lines.Cervical cancer cell lines with stable up-regulation or down-regulation of RacGAP1 were constructed by lentivirus infection.The effects of RacGAP1 on proliferation,invasion and migration of cervical cancer cell lines in vitro were detected by cell function assay.Flow cytometry was used to detect the effect of RacGAP1 on cell cycle distribution.Xenograft tumor assay in nude mice was used to detect the effect of RacGAPl expression on proliferation of cervical cancer cell lines in vivo.TCGA database was used to screen the downstream differentially expressed miRNAs of RacGAP1.qRT-PCR,Western blot and RIP were used to investigate the relationship between RacGAP1 and miR-192.The effect of miR-192 on biological behavior of cervical cancer was detected by cell function assays.The effect of miR-192 on RacGAPl modified cell function was determined by rescue assay.Results1.The proliferation ability was also decreased sequentially in HeLa,CaSki and SiHa cells.2.Knocking down RacGAP1 could decrease the active RhoA expression.Overexpressing RacGAP1 could enhanced the active RhoA expression.3.Knocking down RacGAP1 could reduce the proliferation function of cervical cancer cell lines and block cell cycle progression(p<0.05).The overexpression of RacGAP1 had the opposite effect(p<0.05).Xenograft tumor assay in nude mice showed that the tumor-forming ability of cells decreased significantly after RacGAP1 knockdown.4.Down-regulated RacGAP1 expression significantly reduced the invasion and migration ability of cervical cancer cells(p<0.05),while overexpression of RacGAP1 promoted the invasion and migration ability of cervical cancer cells(p<0.05).5.The expression of miR-192 was negatively correlated with RacGAP1 expression(R=-0.26,p=5.7e-6).Patients with high miR-192 expression had a good prognosis(p=0.028,log-rank test).Upregulation of miR-192 inhibited cell proliferation,migration and invasion(p<0.05),while knocking down miR-192 got the opposite results(p<0.05).6.The results of rescue experiments showed that the reduced growth,invasion or migration capacity of cervical cancer cells caused by RacGAP1 down-regulation could be partially recovered by down-regulating miR-192(p<0.05).On the contrary,up-regulation of RacGAPl followed by up-regulation of miR-192 attenuated the enhanced growth,invasion or migration capacity of cervical cancer cells due to RacGAP1 up-regulating(p<0.05).Brief Summary1.RacGAP1 promotes the proliferation,cell cycle progression,invasion and migration of cervical cancer cell lines.2.RacGAP1 promotes the malignant progression of cervical cancer by inhibiting miR-192.Part Ⅲ RacGAP1 regulates AP-1 via miR-192 and p-JNKObjectTo detect the possible mechanism of RacGAP1 in cervical cancer combining bioinformatics analysis and molecular biology experiments.MethodsThe downstream genes of RacGAP1 were screened by microarray analysis,and the pathways were screened combining with GSE108422 data set.Western blot and immunohistochemistry were used to verify the changes of related proteins.KEGG enrichment analysis was performed for the target genes of miR-192 and the differential genes in the GSE69990 dataset.Western blot was used to detect the effect of altered miR192 expression on downstream gene expression.The changes of related proteins in the rescue experiment were detected by Western blot.The cells were treated with SP600125,and the effects of SP600125 on cell proliferation were detected by clone formation assay.The effect of SP600125 on RacGAP1 cell proliferation was detected by rescue assay.The changes of related proteins were detected by Western blot.Results1.Bioinformatics analysis and Western blot results showed that RacGAP1 regulates the phosphorylation of c-Jun through the MKK/JNK pathway,thereby affecting the changes of downstream genes.2.Bioinformatics analysis and Western blot results showed that miR-192 affected the expression of c-Jun,and then affected the changes of downstream genes.miR-192 partially reversed downstream protein changes caused by RacGAP1 changes.3.SP600125 inhibits cell proliferation.qRT-PCR and Western Blot analysis showed that after SP600125 treatment,the expressions of miR-192,RacGAP1 and c-Jun did not change significantly,while the expressions of p-c-Jun,c-Myc and c-Met were downregulated(p<0.05).Brief Summary1.RacGAP1 can affect the biological behavior of cervical cancer cells through MKK/JNK signaling pathway.2.RacGAP1 can affect the expression of c-Jun through miR-192 and the phosphorylation of c-Jun through phospho-JNK,thus affecting the formation and stability of AP-1 and changing the expression of downstream genes.Conclusion1.RacGAP1 was high expressed in cervical cancer tissues and was associated with poor prognosis of cervical cancer.2.RacGAP1 promotes the proliferation,cell cycle progression,invasion and migration of cervical cancer cell lines.3.RacGAP1 affected the expression of c-Jun through miR-192 and the phosphorylation of c-Jun through phospho-JNK,thus affecting the formation and stability of AP-1 and changing the expression of downstream genes.In turn,the malignant progression of cervical cancer was promoted.