| Background and Objective: Lung cancer is a serious risk to human health,both mortality and morbidity,ranking first in global cancer.The non-small cell lung cancer(NSCLC)is a common subtype of lung cancer,consisting of nearly 80% patient.although the emergence of EGFR/ALK rearrangement kinase inhibitors,monoclonal antibody and new chemotherapy drugs,progression of radiotherapy technology,improvement of surgical operation have made great advances in the treatment of lung malignance,the 5-year survival rate was not significantly improved,about 17%.Two key reasons are responsible for it.firstly,there is rarely effective method to identify the early-stage patients.secondly,a shortage of effective treatment for advanced-stage patient.Hense,to reveal the pathogenesis of lung cancer and find a new therapeutic target is urgent,which is of great significance to improve the prognosis of lung cancer patients.RFPL3 in NSCLC cell lines and tumor histopathological is overexpression,which is mainly distributed in the nucleus of NSCLC,And it is closely related to the lymph node metastasis of lung cancer.When RFPL3 is acetylated by CBP,the two co-bind with hTERT promoter region to coordinate telomerase activity and activate hTERT promoter,leading to promoting the proliferation and tumor development of lung cancer cells.These studies suggest that Up-regulation and nuclear translocation of RFPL3 play an important role in the development and progression of NSCLC.It is expected to be a new target for lung cancer diagnosis and treatment intervention.However,there was few studies on study of stimulation to the expression and sub-distribution of RFPL3 and the effect of RFPL3’ change to cell function.The study is to learn the expression and distribution of RFPL3 among lung normal cell lines(HLF,16HBE)and lung adenocarcinoma cell lines(A549、H1299).Then,investigate the effects of EGF on RFPL3.Next,explore the effect of RFPL3 protein expression and distribution,which is accomplished by trasnfecting RFPL3 gene,on apoptosis.With above results,It is hopeful to offer new data to deeply explore the meachnism of NSCLC and guide clinical treatment.As The computed tomography(CT)is being used more and more widely,the discovery of ground-grass nodules(GGN)is greatly increasing,as well as the recongnition of early lung cancer as a GGN.But there are few reports about the elderly GGN.Therefore,analysising GGN detected by physical examination of lung CT between the elderly and young,including both its imaging characteristics and pathological features,provides reference for diagnosis and treatment for the elderly with GGN founding by clinical examination.Methods: Stimulation of different concentration(0,5,10,20 ng / ml)and different time(24h,48 h,72h)EGF were used to detect the expression of RFPL3 in normal lung cells(HLF,16HBE)and lung gland cells(A549,H1299),and to screene the optimal concentration and time of RFPL3 expression in different cells.The cell lines were divided into EFG-stimulated experimental group and no EGF-stimulated observation group.Western blot and immunofluorescence technique were used to detect the expression of RFPL3 protein in normal lung cells and lung adenocarcinoma Expression and sub-distribution in cells.The RFPL3 gene recombinant plasmid was transfected into A549 cells by transient transfection technique.The transfection efficiency was detected by fluorescence microscopy.The expression level of total protein and nuclear protein in RFPL3 was detected by western blot.The rate of apoptosis between A549 cells was detected by Flow cytometry.A retrospective analysis of 56 cases come from physical examination of lung CT found in the glassy glass nodules and achieved pathological results of the case,anlysising the difference between the elderly and the Middle-young people,ranging from GGNs’ imaging characteristics to nodular pathologyResults:(1)The concentration of RFPL3 protein in lung adenocarcinoma cell line(A549 、H1299)was dose-dependent,and the optimal stimulating concentration was 20 ng / ml.The lung normal cells(HLF,16HBE)RFPL3 protein expression and EGF concentration was ST-T curve,EGF concentration of 10 ng / ml when the maximum expression of RFPL3 protein.The optimal time for EGF to express RFPL3 protein in lung cells(HLF,16 HBE,A549,H1299)was 48 h.(2)(1)The expression of RFPL3 protein in lung cells(HLF,16 HBE,A549 and H1299)was significantly higher than that in normal lung cells(P <0.05).The difference is Statistically significant.(2)The results of western blot showed that the total protein and nuclear protein of control group RFPL3 in the same lung cells(HLF,16 HBE,A549,H1299)were significantly higher than those in the control group(P <0.05).Among experiment group,the expression of RFPL3 in lung adenocarcinoma cells was significantly higher than that in the control group(P <0.05)The expression of nuclear protein was higher than that of normal lung cells,the difference was statistically significant.(3)In the control group,the results of IF show that the RFPL3 protein in lung adenocarcinoma cells(A549,H1299)was mainly distributed in the cytoplasm and the nuclei were also distributed a little.The RFPL3 protein in lung normal cells(HLF,16HBE)was all distributed in the cytoplasm.(4)In the experiment group,The results of IF showed that RFPL3 protein was mainly distributed in the nucleus after stimulation with EGF in lung cells(HLF,16 HBE,A549,H1299).(3)Fluorescence microscopy showed that the transfection efficiency was over 90%.The results of western blot showed that the expression of total protein and nuclear protein in RFPL3 group was significantly higher than blank control group and negative control group,the difference was statistically significant.there was no significant difference between blank control group and negative control group.The results of flow cytometry showed that the apoptotic rate of the transfected group was significantly lower than that of the blank control group and the negative control group.There was significant difference between the transfection group and the blank control group and the negative control group.The blank control group and the negative control group The difference was not statistically significant.3)There was no significant difference in the size,location,edge(lobes,burr,smooth),vacuolate sign,adjacent structural changes(vascular bundle sign,pleural indentation)between the elderly and young-middle aged.Among the elderly’ GGN,it was more commonly to be mGGNs,irregular shape,blurred border,solid.The young and middle-aged GGNs mostly are pGGN,the shape was often round or oval,more clear boundaries,the internal often contain bronchial signs.The malignant rate of GGNs in the elderly was higher than young-middle aged.The difference was statistically significant.Conclusion:(1)EGF significantly upregulated the expression of RFPL3 protein in lung normal and NSCLC cells,and induced RFPL3 protein into the nucleus from cytoplasm.Overexpression and nuclear translocation of RFPL3 protein,inhibite the apoptosis of lung adenocarcinoma A549 cell.RFPL3 is expected to be a new target for NSCLC gene therapy.(2)Among the elderly,the rate of GGN malignancy is higher than the young-middle.to strengthen the elderly lung CT screening and management,vigilance for the elderly GGN is beneficial to early detection of lung cancer and improvement of prognosis. |
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