| The contents of this project are as follows:Objective:To investigate the effect and its mechanism of RORαin the growth and metastasis of human NSCLC cells.Methods:1.The m RNA level of RORαin normal lung epithelial cells and NSCLC cells were detected by Real-time PCR,and the protein level of RORαin normal lung epithelial cells and NSCLC cells were detected by western blot(WB).The RNAi interference were transiently transfected into human A549 cells in vitro.The expression of RORαwere detected by Real-time PCR after transfection.The clony ability of cells were tested by clony formation assay.The cells migration ability were detected by scratch assay and transwell migration assay.The expression of RORαwere detected by immunohistochemical(IHC)staining in cancer tissues and paracancerous tissues on human lung adenocarcinoma(LUAD)tissue chips,and the expression of RORαnucleus and cytoplasm were performed by immunohistochemical score(H-Score),and After the K-S normality test,they were divided into RORαnucleus and cytoplasmic of high expression group and low expression group.The expressions of RORαcytoplasm,nucleus and related clinical indicators were performed by correlation analysis and chi-square test,and the five-year survival rate of patients were analyzed by Kaplan-Meier method;the total factors affecting the five-year survival of LUAD patients were analyzed by Cox multivariate regression analysis;The expression of RORαin normal lung epithelial cells and NSCLC cells of the nucleus and cytoplasm were detected by WB and laser confocal assay.2.The nuclear localization sequence(NLS)of RORαwere predicted on the NLS Mapper website and construct the full length of RORαeukaryotic expression vector(pc-RORα)and the RORαeukaryotic expression vector with NLS removed(pc-△RORα)and the control eukaryotic expression vector(pc-NC)were transiently transfected into human NSCLC cells in vitro,the RORαexpression and localization in the nucleus and cytoplasm of NSCLC cells were detected by WB and laser confocal,the cells proliferation were detected by CCK-8 technology after transfect 72h,the cell cloning ability were detected clone formation test,the cell migration ability were detected by Scratch test in vitro,the cell migration ability were detected by Transwell migration test,the cell invasion ability were detected by Transwell invasion test,the cell cycle in vitro were detected by flow cytometry(FCM).Finally,the changes in related signal pathways including AKT and Erk were detected by WB in each group of cells.3.A mouse LUAD tumor models were established by using 6×10~6 A549cells/right axillary subcutaneous tumor-bearing nude mice,and the vector were injected into the tumor in situ after 10 days.The tumor growths were recorded every two days and the mice were sacrificed on the 15 days.The morphological change of tumor tissues were observed by HE staining.The expression of RORαwere verified by WB and laser confocal.The change of related signal pathways including AKT and Erk were verified by WB in tumor tissues.Results1.The Real-time PCR results showed that the level of RORαm RNA in human NSCLC cell lines A549 and 95D cells was higher than that in normal lung epithelial BEAS-2B cells(P<0.05).WB results showed that RORαlevels in A549 and 95D cells was higher than those in BEAS-2B(P<0.05).After transfection with RORα-RNAi for48h,the level of RORαin A549 cells was significantly decreased.The results of the clone formation experiment showed that compared with the control group(RORα-NC),the cell colony formation ability of RORα-RNAi group was significantly decreased(P<0.05).The results of the scratch experiment showed that compared with RORα-NC,the non-directed migration ability of RORα-RNAi cells was significantly decreased(P<0.05).The results of Transwell migration assay showed that compared with RORα-NC,the directed migration ability of RORα-RNAi cells was significantly decreased(P<0.05).The immunohistochemistry(Immunohistochemistry,IHC)was stained of the tissue chip,and the statistical analysis of the clinical data showed that compared with the adjacent tissues,RORαwas highly expressed in the cytoplasm of cancer tissues(P<0.05),and low expression in the nucleus of cancer tissue(P<0.05),and the higher the expression of RORαin the cytoplasm,the lower the 5-year survival rate(P<0.05).The expression of RORαin the nucleus is no correlated with the 5-year survival time(P>0.05).The WB results showed that compared with BEAS-2b cells,RORαprotein was higher in the nucleus of A549 and 95D cells(P<0.05),and was higher in the cytoplasm of A549,95D and H1299 cells(P<0.05).The laser confocal results showed that compared with BEAS-2b cells,the RORαprotein content was higher in the nucleus of A549 and 95D cells,and lower in the nucleus of H1299 cells(P<0.05).2.The results of double enzyme digestion experiment and sequencing showed that p EGFP-N1 basic-RORαeukaryotic expression vector(named:pc-RORα)and the p EGFP-N1 basic-RORαeukaryotic expression vector without NLS(named:pc-ΔRORα)was successfully constructed.After cells was transfected with control plasmid(named pc-NC),pc-RORαplasmid and pc-ΔRORαplasmid,Real-Time PCR results showed that compared with the pc-NC group,the RORαm RNA level in the pc-RORαgroup was significantly increased(P<0.05),there was no difference in RORαm RNA level in pc-ΔRORαgroup compared with pc-RORαgroup(P>0.05).WB results showed that compared with pc-NC group,the RORαprotein level in the pc-RORαgroup was increased(P<0.05),the RORαprotein level in the pc-ΔRORαgroup was not different from that in the pc-RORαgroup(P>0.05).WB results showed that compared with pc-NC group,the cytoplasm and nucleus of RORαprotein levels in pc-RORαgroup were increased(P<0.05),compared with the pc-RORαgroup,the RORαprotein level in the cytoplasm of the pc-ΔRORαgroup increased(P<0.05),and the RORαprotein level in the nucleus decreased(P<0.05).The laser confocal results showed that compared with the pc-NC group,the RORαprotein levels in the cytoplasm and nucleus of the pc-RORαgroup was increased(P<0.05).Compared with the pc-RORαgroup,the RORαprotein level in the cytoplasm of the pc-RORαgroup was increased(P<0.05).CCK-8 results showed that the cell proliferation leve of pc-RORαgroup was significantly higher than that of pc-NC group(P<0.01),and the cell proliferation level of pc-ΔRORαgroup was significantly higher than that of pc-RORαgroup(P<0.05).The clone formation results showed that the colony forming ability of the pc-RORαgroup was significantly higher than that of the pc-NC group(P<0.05),and the colony forming ability of the pc-ΔRORαgroup was significantly higher than that of the pc-RORαgroup(P<0.05).The results of the scratch experiment showed that the non-directional migration ability of the pc-RORαgroup was significantly higher than that of the pc-NC group(P<0.05).Compared with the pc-RORαgroup,the non-directional migration ability of the pc-ΔRORαgroup was significantly increased(P<0.01).The results of the Transwell migration experiment showed that compared with the pc-NC group,the directional migration ability of the pc-RORαgroup was significantly increased(P<0.05).Compared with the pc-RORαgroup,the in vitro migration ability of the pc-ΔRORαgroup was obvious Increased(P<0.05).The results of transwell invasion experiment showed that the invasion ability of cells in vitro did not change(P>0.05).Real-time PCR results showed that the m RNA levels of MMP-2,MMP-3,MMP-9 did not change(P>0.05).The results of the A549 cell cycle experiment showed that compared with the pc-NC group,the proportion of S phase in the cell cycle of the pc-RORαgroup was increased(P>0.05),compared with the A549 cells in the pc-RORαgroup,the ratio of G2/M phase in the cell cycle of the pc-△RORαgroup was significantly decreased(P>0.05),and the ratio of S phase was decreased(P>0.05).The results of 95D cell cycle experiment showed that compared with pc-NC group,the ratio of pc-RORαgroup cell in S phase was significantly decreased,G2/M phase ratio was increased(P>0.05);compared with pc-RORαgroup 95D cells,pc-△RORαgroup cell cycle G2/M phase ratio was decreased(P>0.05).The WB test results of the cells showed that compared with the pc-NC group,the p-AKT,p-Erk,N-Cadherin,and Vimentin protein levels in the pc-RORαgroup were significantly increased(P<0.05);compared with the pc-RORαgroup,the protein levels of p-AKT,p-ERK,N-Cadherin and Vimentin in the pc-ΔRORαgroup were significantly increased(P<0.05).3.The nude mouse Xenograft model showed that the tumor volume of the pc-RORαgroup was significantly higher than that of the pc-NC group from the 11th day(P<0.05),the tumor volume of mice in the pc-ΔRORαgroup was significantly higher than that in the pc-RORαgroup from day 5(P<0.05).Mouse tumor weighing results showed that the tumor weight of the pc-RORαgroup was significantly higher than that of the pc-NC group(P<0.01),and the tumor weight of the pc-ΔRORαgroup was significantly higher than that of the pc-RORαgroup(P<0.05).The results of HE staining of tumor tissues showed that compared with the pc-NC group,the pc-RORαand pc-ΔRORαgroups had more cells and irregular shapes.Compared with pc-RORαgroup,pc-ΔRORαis composed of fewer fibroblasts.The results of H&E staining of lung tissue showed that compared with the pc-NC group,the pc-RORαgroup had more tumor cells and thickened lung septum;compared with the pc-RORαgroup,the number of tumor cells in the pc-ΔRORαgroup further increased,and the lung septum was further thickened.The results of the WB experiment showed that compared with the pc-NC group,the RORαprotein level in the pc-RORαgroup was significantly increased(P<0.05);compared with pc-RORα,the RORαprotein level in the pc-ΔRORαgroup did not change significantly(P>0.05).The laser confocal results showed that compared with the pc-NC group,the RORαprotein levels in the cytoplasm and nucleus of the pc-RORαgroup were increased(P<0.05),compared with the pc-RORαgroup,the level of RORαprotein of the pc-ΔRORαgroup in the cytoplasm was increased(P<0.05).The WB test results of tumor tissues showed that compared with the pc-NC group,the AKT,p-AKT,p-Erk,N-Cadherin and Vimentin protein levels in the pc-RORαgroup were significantly increased(P<0.05),and the JNK protein level was significantly reduced(P<0.05);Compared with pc-RORα,the protein levels of p-AKT,p-Erk,N-Cadherin and Vimentin in 95D cells in the pc-ΔRORαgroup were significantly increased(P<0.05),and the Erk protein level was significantly reduced(P<0.05).Conclusions1.The high expression of RORαin the cytoplasm is positively related to the 5-year survival rate of NSCLC patients.2.The high expression of RORαin the cytoplasm promotes the growth of NSCLC cells and tumors,which may be ralate AKT and Erk pathways. |
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