| Foreword:Lung cancer is the most dangerous cancer to human health in the world,among which NSCLC accounts for 80%.Although EGFR and ALK gene mutations exist in non-small cell lung cancer,targeted drug therapy can be used.However,it is only suitable for some cases with gene mutations,and the high resistance rate in the early stage limits the current gene targeted therapy effect.Therefore,the discovery of proteins related to proliferation and metastasis of NSCLC cells is of great significance for the early diagnosis of NSCLC and the development of new targeted drugs.Replication factor C(RFC)is a protein complex composed of five unique subunits,RFC1,RFC2,RFC3,RFC4,RFC5,with molecular weight of 140,40,38,37 and 36 k Da,respectively.The extension of primer DNA template by DNA polymerase ? and DNA polymerase ? requires protein proliferating cell nuclear antigen(PCNA)and replication factor C.Replication factor C,also known as activator 1,is a part of the whole enzyme of DNA polymerase in eukaryotic cells.It plays an important role in DNA replication,DNA damage repair and synthesis regulation.DNA replication plays an important role in cancer progression and cell cycle.It is suggested that the abnormal amplification of RFC gene may be related to the proliferation and invasion of cancer cells.Related studies have shown that RFC1 can regulate transcription by acting with deacetylase-1,which is an important factor in regulating NF kappa B pathway activity;RFC2 is highly expressed in nasopharyngeal carcinoma,which is closely related to proto oncogene C-myc;RFC4 is not only highly expressed in liver cancer,but also inhibited the proliferation of cancer cells,increased the number of necrotic cells,and increased the sensitivity to chemotherapy after silencing.In addition,RFC4 also in lung,prostate,colon,stomach and skin malignant tumors,RFC4 was also highly expressed in the immortalized cells of simian vacuolation virus 40(SV40)with pulmonary fibrosis,suggesting that RFC may be the first protein involved in tumor malignant transformation;RFC5was found to be highly expressed in human papillomavirus positive squamous cell carcinoma.RFC3 is a 38 k Da protein among the five subunits,which is necessary for the interaction between the other three subunits and the large one.RFC3 has been reported to be related to the proliferation,invasion and migration of triple negative breast cancer,liver cancer,ovarian cancer,acute myeloid leukemia and esophageal adenocarcinoma cells.However,the role of RFC3 in NSCLC has not been studied.Objective: In this study,we explored the expression of RFC3 in non-small cell lung cancer and adjacent tissues,and its relationship with clinical features and prognosis.In addition,MTT method,flow cytometry analysis,invasion,migration and scratch test were used to evaluate the proliferation, apoptosis and invasion and migration ability of A549 and H1299 cell lines when RFC3 was regulated.Finally,according to the literature review,bioinformatics analysis and Western blot to evaluate the changes of downstream proteins to find the role of RFC3 in two cell lines.Methods:1.165 cases of NSCLC with complete follow-up data were collected from the First Affiliated Hospital of China Medical University.Among them,123 cases were diagnosed as adenocarcinoma and 42 cases as squamous cell carcinoma.The expression of RFC3 gene in nonsmall cell lung cancer(NSCLC)and its adjacent tissues was evaluated by immunohistochemistry.The correlation between RFC3 and clinical indexes and prognosis of NSCLC was studied by chi square test,single factor analysis and multi factor analysis.Kaplan Meier method was used to compare the effect of RFC3 expression on the overall survival of patients.2.Western blot was used to detect the expression of RFC3 in each non-small cell lung cancer cell line.In lung adenocarcinoma cell lines A549 and H1299,RFC3 expression was up-regulated and down regulated by over expression plasmid and RNA silencing fragment si RNA,respectively.The effects of RFC3 on the biological behavior of lung adenocarcinoma cells were verified by MTT assay,flow cytometry,transwell invasion and migration test and scratch test.3.In lung adenocarcinoma cell lines A549 and H1299,RFC3 expression was up-regulated and down regulated by over expression plasmid and RNA silencing fragment si RNA,respectively.Western blot was used to detect Wnt1,?-Catenin,c-myc,the ratio of p-GSK3-?(ser9)to GSK3-?,and the expression of E-cadherin,Ncadherin and vimentin.Results: 1.In the immunohistochemical study,the proportion of high expression of RFC3 in 123 cases of adenocarcinoma and its adjacent tissues was 46.34% and 20.33%,respectively.The difference was statistically significant.However,the high expression rate of RFC3 was 16.67% in42 cases of squamous cell carcinoma and 11.90% in the corresponding adjacent tissues.The expression of RFC3 was correlated with TNM stage,differentiation and lymph node metastasis.Multivariate analysis suggested that RFC3 expression was an independent factor affecting the survival of patients with lung adenocarcinoma.Single factor analysis suggested that the expression of RFC3 had no significant effect on the survival of patients with lung squamous cell carcinoma.Kaplan Meier survival analysis showed that the survival time of patients with low RFC3 expression was significantly longer than that of patients with high RFC3 expression.2.In the non-small cell lung cancer cell lines,except A549 and LK2 cell lines and normal human bronchial epithelial cell lines,the expression level of RFC3 in other non-small cell lung cancer cell lines was significantly higher than that in normal human bronchial epithelial cell lines.The results of flow cytometry showed that the up-regulated or down regulated RFC3 did not affect the proliferation of A549 and H1299 cells.The up regulated RFC3 promoted A549 cells from G0 / G1 phase to S phase,while the down regulated RFC3 resulted in H1299 cells stagnating in G0 / G1 phase.The number of apoptotic cells in RFC3 up-regulated group decreased,but there was no significant difference between the two groups.Paclitaxel and erlotinib were added to A549 and H1299 cell lines to induce apoptosis,and the number of apoptotic cells up-regulated RFC3 was significantly reduced,while the number of apoptotic cells down regulated RFC3 was significantly increased.Transwell invasion and migration test and scratch test indicated that the invasion and migration ability of A549 cells increased after RFC3 up regulation,while H1299 cells decreased after RFC3 down regulation,which indicated that RFC3 had an effect on the invasion and migration ability of lung adenocarcinoma.3.After RFC3 overexpression in A549,the expression of Wnt1,?-Catenin,cmyc,N-cadherin and vimentin increased,p-GSK3-?(SER)/ GSK3-? increased,while E-cadherin decreased.After RFC3 was knocked out in H1299,the expression of Wnt1,?-Catenin,c-myc,Ncadherin and vimentin decreased,p-GSK3-?(SER)/ GSK3-? decreased,while E-cadherin increased.It is suggested that RFC3 may activate Wnt / ?-catenin signaling pathway of A549 and H1299 cells,and promote the epithelial interstitial of lung adenocarcinoma to cause the invasion and metastasis of cancer cells.Conclusion: 1.The expression of RFC3 in lung adenocarcinoma is higher than that in paracancerous tissue.The expression of RFC3 is related to TNM stage,tumor cell differentiation and lymph node metastasis.2.The expression of RFC3 is an independent risk factor for the survival of lung adenocarcinoma.Patients with high expression of RFC3 have short overall and relapse free survival.3.RFC3 has a positive correlation with the invasion and migration ability of lung adenocarcinoma cells.The expression of RFC3 can promote cell cycle progression and inhibit cell apoptosis.4.RFC3 may activate Wnt / ?-catenin signaling pathway of A549 and H1299 cells,and promote the epithelial-mesenchymal transition of lung adenocarcinoma to cause the invasion and metastasis of cancer cells. |
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