Molecular Mechanism Of ARHGEF3 Regulating ACLY Protein Homeostasis And Promoting Proliferation Of Non-small Cell Lung Cancer

Background:Lung cancer is one of the most common malignant tumors and a major disease that endangers human life and health.Lung cancer includes small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC).NSCLC consists of lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC)and large cell carcinoma.According to statistics,non-small cell lung cancer(NSCLC)is the most common pathological type in clinical lung cancer,and its incidence accounts for about 85%of all primary lung cancer.At present,a lot of studies have been conducted on the pathogenesis of non-small cell lung cancer,but there are still many key scientific issues that have not been clarified.Although some progress has been made in the treatment of non-small cell lung cancer,its therapeutic effect,especially in advanced patients,is still very limited.Therefore,it is of great theoretical significance and application value to explore the pathogenesis of non-small cell lung cancer and find new targets related to its diagnosis and treatment.Rho GTPase is involved in the regulation of cell growth,apoptosis,cell cycle progression and cytoskeleton formation and many other life activities.The abnormal activation of Rho GTPase is closely related to the occurrence and development of breast cancer,liver cancer,gastric cancer,colon cancer and other cancers.Guanine nucleotide exchange factors(GEF s),as upstream activators of Rho GTPase,show high abundance and activity in human tumors.ARHGEF3 is a member of the guanine nucleotide exchange factor family and plays a key role in the activation of Rho GTPase.In acute myeloid leukemia,ARHGEF3 regulates cancer cell differentiation by specifically activating RhoA-Rock-JNK signaling pathway.In nasopharyngeal carcinoma,ARHGEF3 promotes the proliferation of cancer cells by regulating the expression of apoptotic proteins BIRC8 and caspase-3.At present,there are few studies on ARHGEF3 in tumor,and its role and mechanism in the occurrence and development of non-small cell lung cancer have not been reported.Therefore,exploring the role and molecular mechanism of ARHGEF3 in the occurrence and development of NSCLC is helpful to clarify the pathogenesis of tumors and provide a theoretical basis for the discovery of new therapeutic targets for NSCLC.Materials and methods:1.A clinical cohort analysis for ARHGEF3 expression(1)Clinical samples of NSCLC patients were collected,and the protein and mRNA expression levels of ARHGEF3 in clinical NSCLC tissue samples and corresponding adjacent normal tissue samples were analyzed by western blot and real-time quantitative PCR(Q-PCR).(2)Non-small cell lung cancer cell lines were collected,and the protein and mRNA expression levels of ARHGEF3 in normal lung epithelial cells and non-small cell lung cancer cells were analyzed by western blotting and real-time PCR.2.Explore the biological function of ARHGEF3 in the malignant proliferation of nonsmall cell lung cancer cells(1)The biological function of ARHGEF3 in non-small cell lung cancer(NSCLC)was investigated by crystal violet,colony formation and flow cytometry.(2)The stable cell line of non-small cell lung cancer that interferes with ARHGEF3 expression was constructed by specific shRNA,and the effect of ARHGEF3 on the proliferation of non-small cell lung cancer cells was verified by constructing subcutaneous xenograft tumor model in nude mice.3.Investigate the molecular mechanism of ARHGEF3 in regulating proliferation of NSCLC cells(1)ARHGEF3 was overexpressed in non-small cell lung cancer cells,and the target protein interacting with ARHGEF3 was found by mass spectrometry.(2)In non-small cell lung cancer cells,the interaction between ARHGEF3 and citrate lyase(ACLY)was verified by immunoprecipitation(Co-IP)and immunofluorescence(IF)experiments.(3)In non-small cell lung cancer cells,the expression of ARHGEF3 was overexpressed or interfered with.The changes of ACLY protein level and mRNA level were detected by western blotting and real-time PCR to clarify the expression regulation relationship between ARHGEF3 and ACLY(4)To explore the specific mechanism of ARHGEF3 regulating ACLY acetylation through SIRT2 by molecular clonal,immunoblotting and co-precipitation.(5)By constructing mutant with defective ACLY acetylation function,we elucidated that K17 and K86 are the key sites of ARHGEF3 regulating ACLY expression.4.Detect the effect of ARHGEF3 on fatty acid synthesis by regulating the expression of ACLYThe expression of ARHGEF3 was overexpressed or interfered with in non-small cell lung cancer cells,and the content changes of acetyl-CoA and free fatty acid were detected by using acetyl-CoA content assay kit and free fatty acid content assay kit.5.Determine whether the function of ARHGEF3 in promoting proliferation of NSCLC cells depends on the activity of guanylate exchange factor(GEF)(1)The GEF inactivated mutant plasmids L269E and W440L were constructed by molecular cloning.(2)The effects of L269E and W440L on ACLY acetylation were detected by western blot,immunoprecipitation,etc.6.Verify the role of ARHGEF3 in promoting the proliferation of non-small cell lung cancer by regulating the expression of ACLY through SIRT2 in animal level(1)A stable cell line with ARHGEF3 knockdown and ACLY overexpression was constructed.The growth rate of the tumor was detected by subcutaneous tumorigenicity assay in mice,and whether ARHGEF3 promoted the proliferation of non-small cell lung cancer by regulating the expression of ACLY was verified.(2)The mutant K17R and K86R with defective ACLY acetylation function were constructed,and the tumorigenic effect of the mutant and wild-type ACLY cell lines was compared by subcutaneous tumorigenesis assay in mice.The K17 and K86 sites were identified as the key sites of ARHGEF3 regulating ACLY expression.Results:1.Clinical cohort analysis revealed high expression of ARHGEF3 in NSCLC(1)The expression of ARHGEF3 in non-small cell lung cancer tissues was higher than that in adjacent tissues.(2)The expression of ARHGEF3 in non-small cell lung cancer cells was higher than that in normal lung epithelial cells.2.ARHGEF3 promotes the proliferation of NSCLC cells(1)Using specific siRNA knockdown of ARHGEF3 expression,it was found that the growth and colony formation ability of NSCLC cells were significantly inhibited,and the cell cycle was arrested in the G0/G1 phase.(2)Stable cell lines with low expression of ARHGEF3 were constructed by shRNA.In vivo and in vitro experiments showed that the proliferation of NSCLC cells and the growth of subcutaneous xenografts were significantly inhibited.3.ARHGEF3 regulates SIRT2-mediated ACLY acetylation to affect ACLY protein expression(1)ARHGEF3 binds to ACLY(2)Overexpression of ARHGEF3 enhanced the interaction between SIRT2 and ACLY and reduced the acetylation level of ACLY;Knockdown of ARHGEF3 expression could weaken the interaction between SIRT2 and ACLY and increase the acetylation level of ACLY.(3)ARHGEF3 had no interaction with K17R and K86R,and had no effect on the acetylation of K17R and K86R,indicating that K17 and K86 are important sites for ARHGEF3 to regulate ACLY acetylation.(4)Knockdown of ACLY expression inhibited the proliferation of non-small cell lung cancer cells,and overexpression of ARHGEF3 could not attenuate the proliferation inhibition induced by ACLY knockdown.On the contrary,interference with the expression of ARHGEF3 could inhibit the proliferation of NSCLC cells,and overexpression of ACLY could attenuate the proliferation inhibition induced by ARHGEF3.4.ARHGEF3 promotes fatty acid synthesis by regulating ACLYIn non-small cell lung cancer cells,overexpression of ARHGEF3 could increase the protein level of ACLY and the content of acetyl-CoA and free fatty acids.Conversely,knockdown of ARHGEF3 expression reduced the protein level of ACLY,acetyl-CoA and free fatty acid content.5.ARHGEF3 promotes proliferation of NSCLC cells independently of its GEF activity In non-small cell lung cancer cells,overexpression of mutants L269E and W440L,which lack GEF activity,still reduced ACLY acetylation as well as wild-type ACLY.6.ARHGEF3 promotes the proliferation and tumorigenesis of NSCLC cells by regulating the expression of ACLY(1)The results of subcutaneous tumorigenicity in mice showed that interfering with the expression of ARHGEF3 significantly inhibited tumor growth;Overexpression of ACLY attenuated the tumor growth inhibition induced by ARHGEF3 knockdown.(2)The results of subcutaneous tumor formation in mice showed that the tumor weight and volume of K17R and K86R mutant stable cell lines with defective ACLY acetylation function were significantly higher than those of wild-type stable ACLY cell lines.Conclusions:1.ARHGEF3 is abnormally high expressed in non-small cell lung cancer and promotes the malignant proliferation of non-small cell lung cancer cells.2.ARHGEF3 interacts with ACLY.3.ARHGEF3 regulates ACLY acetylation through SIRT2 and thus affects ACLY expression.4.K17 and K86 are the key sites of ARHGEF3 in regulating ACLY expression.5.ARHGEF3 regulates ACLY acetylation independent of its GEF activity.6.ARHGEF3 promotes proliferation and tumorigenesis of non-small cell lung cancer cells by regulating ACLY expression.