Study Of The Rac3 Regulated Autophagy And Apoptotic Functional Molecular Mechanism In Non-small Cell Lung Cancer Cells

Background:Lung cancer is currently the most common malignant tumor in the world.In China,there are 733,300 new cases of lung cancer each year,accounting for 17.08%of the total number of new cancer cases,and 610,200 deaths,accounting for 21.68%of the total number of cancer deaths.At present,the overall situation of lung cancer treatment is still unsatisfactory,and various studies are yet to be carried out.Rac3 is a small G protein with molecular weight of 21k D and belongs to the Rac family of the Rho family.This family is considered to be an important regulator of the cytoskeleton and plays an important role in tumor and neuronal development.Rac3 is highly expressed in normal neuronal tissues.In addition,it is highly expressed in a variety of tumor cells,such as glioma,breast cancer,lung adenocarcinoma and prostate cancer.However,the mechanism of Rac3 in the occurrence and development of lung cancer is still unclear.In the early stage of the research,we used Rac3 gene-edited cells to perform RNA sequencing technology and found that Rac3 might regulate melanoma antigen A6（Melanoma antigen A6,MAGEA6,also known as MAGE3B,MAGE6）.MAGEA6 is a member of the melanoma antigen family,which is conserved in eukaryotes and is often abnormally activated in tumors.MAGEA6 regulates m TORC1 by inhibiting AMPKα,regulates autophagy,and promotes tumor survival.Whether Rac3 regulates MAGEA6 and whether it is related to the occurrence and development of lung cancer has not been reported yet,and it needs to be further verified.Autophagy is a highly conservative biological process,which often plays a double-edged role in tumors.On one hand,autophagy can increase the tumor’s ability to resist harsh environments,and on the other hand,autophagy can also directly cause tumor apoptosis.Since MAGEA6 can regulate autophagy-related AMPKα/m TOR pathway,it suggests that Rac3 may regulate autophagy.However,the mechanism of Rac3 regulating autophagy and apoptosis in non-small cell lung cancer is currently less relevant in China and internationally,and more comprehensive and specific exploration is needed.Methods:1.Through TCGA data-mining,compared the expression of Rac3 in cancer and normal tissues,analyzed the correlation between different clinical features and Rac3expression in tumors,and analyzed the relationship between the expression of Rac3 and the overall survival by Kaplan-Meier curves and COX proportional regression.2.sg RNA sequence was designed online,constructed CRISPR/cas9 gene knock-out plasmid,transfected into A549 knocking out Rac3 to construct stable knockout cell line,and constructed stable overexpression cell line by Rac3 overexpression lentivirus infecting A549.3.RNA sequencing was performed on the Rac3 knock-out and overexpression cell lines,searched for differential genes.The genes directly affected by Rac3 knockout and overexpression were obtained through the Venn diagram analysis of differential genes,and low-throughput verification of these genes was performed by real-time fluorescent quantitative PCR.4.Autophagy was induced by EBSS starvation,Western Blot was performed to detect autophagy-related proteins LC3,SQSTM-1/p62,and LC3 dual fluorescent lentiviral infection method was performed to detect autophagy levels.5.After autophagy was induced,Annexin V-FITC/PI double staining flow cytometry was performed to detect the proportion of apoptosis,the JC-1 fluorescent probe method was performed to detect the mitochondrial membrane potential,and Western Blot was performed to detect the cleaved-caspase3.These three experiments verified the occurrence of apoptosis.6.Differential gene MAGEA6 and AMPKαwere detected by Western Blot,and it also performed on ERK1/2 pathway,PI3K-AKT pathway and m TOR pathway which was related to autophagy.7.Transfected the MAGEA6 overexpression plasmid into the Rac3 knockout cell line to rescue the expression of MAGEA6,and detecting LC3,cleaved-caspase3,AMPKαand phosphorylated AMPKαby Western Blot,verifying whether overexpression of MAGEA6could rescue the autophagy,apoptosis and pathway changing that caused by Rac3 knockout.8.Tissue microarray was established,the expression of Rac3,MAGEA6 and GABRA3which was regulated by Rac3 and low abundance in A549 were detected by immunohistochemical staining in vivo,analyzed the relationship between gene expression and clinical characteristics,and performed Kaplan-Meier survival analysis.9.The expression of AMPKαand cleaved-aspase3 were performed by tissue microarray and immunohistochemistry,and the correlation analysis was performed in AMPKα,cleaved-aspase3,Rac3,MAGEA6 and GABRA3,verified that could Rac3 inhibit AMPKαpathway by MAGEA6 and whether GABRA3 was related to AMPKαpathway and cleaved-caspase3 in vivo.Results:1.Data-mining of 1145 cases of TCGA showed that the expression of Rac3 in lung cancer was higher than that in adjacent lung tissues,and the expression in squamous cell carcinoma was higher than that in adenocarcinoma tissues.The difference was statistically significant.2.The correlation between Rac3 expression and age,gender,smoking history,lung lobe where the tumor was located and T2 stage were statistically significant,p<0.05.The correlation between Rac3 expression and central lung cancer,R0 resection,and tumor stage were not statistically significant.3.Kaplan-Meier curve indicated that the high expression of Rac3 in non-small cell lung cancer indicated a poor prognosis,p<0.05,which was statistically significant.In further pathological subtype analysis,it was found that high expression of Rac3 in squamous cell carcinoma could indicate a poor prognosis,p<0.05.But in adenocarcinoma the difference between expression and prognosis was not statistically significant.4.Univariate COX analysis suggested that T,N,M staging and clinicopathological staging,the lobe of the tumor,whether the tumor was completely resected,and the expression of Rac3 were risk factors which affected tumor prognosis.Multivariate COX analysis suggested that expression of Rac3 was the independent risk factors affecting prognosis.5.GSEA enrichment analysis revealed 25 up-regulated pathways of Rac3,including signaling pathways such as RNA polymerization,DNA replication,spliceosome,cell cycle,and basic transcription factors.There was only one down-regulated pathway that aldosterone regulated sodium reabsorption.6.Two sg RNAs were designed online,and Rac3 knock-out cell lines KO1 and KO2 were successfully constructed through CRISPR/cas9 gene-editing technology.The number of missing bases were 2bp and 35bp,respectively.Western Blot detected that Rac3 did not express any more in these two knock-out cell lines.7.The RNA-Seq were performed in Rac3 knock-out and overexpression cell lines.The threshold was set as log₂ fold Change≤-2 and log₂ fold Change≥2 in screening,it indicated that the knockout group had 88 differentially expressed genes compared with the control group,including 10 genes up-regulated and 78 genes down-regulated.Compared with the control group,the overexpression group had 160 different genes,of which 61 were up-regulated genes and 99 were down-regulated genes.The Venn diagram indicated that there were four different genes had intersections,ADAMTS2,CSRNP3,GABRA3 and MAGEA6.Further low-throughput verification by RT-q PCR confirmed that the 4 genes were all low-expressed while Rac3 was knocked out,and high while Rac3 was over-expressed,and the difference was statistically significant.8.Autophagy induced by EBSS.Compared with wild-type A549,the ratio of LC3-II/LC3-I of the Rac3 knock-out cell line increased,the level of SQSTM-1/p62 decreased.And in the Rac3 overexpressing cell line,the ratio of LC3-II/LC3-I decreased,and the difference was statistically significant.The LC3 dual-fluorescence lentivirus was induced autophagy after infection.Compared with the wild type,the number of fluorescence aggregation points were significantly increased after Rac3 knockout,while the number of fluorescence aggregation points in the Rac3 overexpression group decreased.It suggested that Rac3overexpression inhibited autophagy,and Rac3 knock-out promoted autophagy.9.Annexin V-FITC/PI flow cytometry indicated that the proportion of apoptosis increased after induction of autophagy.Compared with wild-type A549,the percentage of apoptosis increased after Rac3 knock-out,while the percentage of apoptosis decreased after Rac3overexpression.The JC-1 fluorescent probe method detected apoptosis.Apoptosis increased significantly after autophagy was induced.Compared with wild-type A549,JC-1 green fluorescence was stronger in knock-out cell lines,while it was weaker in overexpression cell line.After autophagy was induced,the level of cleaved-caspase3 was detected by Western Blot.Compared with wild-type A549,the expression of cleaved-caspase3 was increased after Rac3 knock-out,and decreased after Rac3 overexpression.It is suggested that Rac3 knockout promoted autophagy-induced apoptosis,and Rac3overexpression inhibited autophagy-induced apoptosis.10.MAGEA6 and autophagy-related pathway proteins were detected by Western Blot.After Rac3 knockout,MAGEA6 was down-regulated,AMPKαand phosphorylated AMPKαwere up-regulated,phosphorylated m TOR was down-regulated.When Rac3overexpression MAGEA6 was up-regulated,AMPKαand phosphorylated AMPKαwere down-regulated,the level of phosphorylated m TOR raised.It is suggested that Rac3reduced AMPKαby up-regulating the expression of MAGEA6 and activated the m TOR pathway,which might inhibit autophagy and apoptosis.11.The Rac3 knockout cell line was transfected with MAGEA6 overexpression plasmid.Western Blot were performed with LC3,phosphorylated AMPKαand cleaved-caspase3.It indicated that after overexpression of MAGEA6,the expression changing of LC3 II,cleaved-caspase3 and phosphorylated AMPKαcaused by Rac3 knock-out were rescued.12.The tissue microarray was established,Rac3,MAGEA6 and GABRA3 in non-small cell lung cancer were detected by immunohistochemical staining.Both Rac3 and MAGEA6 were highly expressed in tumors.Rac3 was highly expressed in patients with squamous cell carcinoma,tumors larger than 3 cm,clinical stage II-III and Ki67 positive,p<0.05.MAGEA6 was highly expressed in patients with stageⅡ-Ⅲ,p<0.05.13.The tissue microarray was detected by immunohistochemical staining of AMPKαand cleaved-caspase3 and correlation analysis in Rac3,MAGEA6 and AMPKα.It suggested that Rac3 expression is significantly related to MAGEA6,and MAGEA6 expression was significantly related to AMPKαin vivo.Conclusion:1.Rac3 was highly expressed in non-small cell lung cancer,and the high expression of Rac3 was an independent survival risk factor for the poor prognosis in non-small cell lung cancer.2.Transcriptomics analysis showed that there were 88 differentially expressed genes after Rac3 knockout,of which 10 were up-regulated and 78 were down-regulated;after Rac3overexpression,there were 160 differentially expressed genes,of which 61 were up-regulated and 99 were down-regulated.Among those genes,there were only 4 differential genes that existed both in Rac3 knockout and overexpression,namely ADAMTS2,CSRNP3,GABRA3 and MAGEA6.3.Rac3 inhibited autophagy and autophagy-induced apoptosis in A549.4.Rac3 inhibited autophagy by up-regulating MAGEA6.Up-regulating MAGEA6reduced AMPKαphosphorylation,activated of the m TOR pathway,and inhibited autophagy and autophagy-induced apoptosis.5.Both Rac3 and MAGEA6 were highly expressed in non-small lung cancer tissues in vivo,and the high expression of these two genes were the risk factors for poor prognosis.6.In vivo there was a correlation in Rac3,MAGEA6 and GABRA3,and there was a correlation between MAGEA6 and AMPKα.Rac3 might also regulate AMPKαpathway through MAGEA6,affect autophagy and apoptosis in vivo.