The Research Of Correlation Between FAM136A Expression And Clinical Prognosis Of Lung Cancer And Biological Function

Objective(s):China has the highest incidence and mortality of lung cancer globally,especially in Xuan Wei of Yunnan.Although the project has reduced morbidity and mortality,it is still the highest incidence than in other regions.The purpose of this study is to discover the essential pathogenic genes related to the survival and prognosis of lung cancer in this region,to study it's in vitro biological effects and biological signal transduction mechanism,and to provide a theoretical basis for proper clinical treatment.Methods:One.Bioinformatics analysis and Screening of differentially expressed genes in regional lung cancer.A total of 9 representative lung cancer cases in the Xuanwei area and 6 cases in the non-Xuanwei area confirmed by pathology after thoracic surgery in the Third Affiliated Hospital of Kunming Medical University were collected.The Agilent mRNA expression microarray sequencing results were used to filter the differentially expressed genes(DEGs).Visualization and Integrated Discovery(DAVID)tools were used for gene ontology(GO)functional enrichment analysis and KEGG pathway enrichment analysis.Protein-protein interaction network analysis was performed using the STRING database.Cytoscape software was used for visual analysis,and CytoHubba,a plugin built into the software,was used to screen out Hub genes.Finally,Kaplan-Meier Plotter software was used for survival analysis prediction.FAM136A,a candidate oncogene with experimental research value and potential clinical significance,was chosen.Two.Correlation analysis between clinical expression and prognosis of lung cancer patients of FAM136A,the candidate oncogene.1.To expand the sample size,177 lung cancer tissues(72 cases in Xuanwei group and 105 cases in the non-Xuanwei group)were selected,and the corresponding paracancer beside normal lung tissues were used as the control group.Immunohistochemical SP method was used to detect the expression intensity and distribution area of FAM136A in 177 lung cancer tissues and control tissue.The correlation between its expression and clinicopathological features,the correlation between survival and prognosis were analyzed.2.Seventy-five cases of Xuan Wei lung cancer and 102 cases of non-Xuan Wei lung cancer were selected.The expression intensity and distribution area of FAM136A in 177 cases of lung cancer were detected by immunohistochemical SP method.The correlation between its expression and the clinicopathological characteristics of the patients was analyzed.Also,the survival prognosis was analyzed.Three.In vitro cell biological function experiment1.First,six lung adenocarcinoma cells,A549,H1975,H2342,H522,XLA-07,PC-9,and BEAS-2B(The virus-transformed human bronchial epithelial cell line),16HBE(transformed human bronchial epithelial-like cell line)were selected to detect the basal expression level of FAM13 6A protein by Western blot.Then,two lung adenocarcinoma cells,A549,and pc-9 were chosen for subsequent experiments in cells in vitro and experiments in nude mice in vivo.2.To synthesis specific knock-down small interfering RNA,namely siRNA-FAM136A,further synthesis and package of the virus LV-siRNA-faml36A,and subsequent functional experiments were carried out after instantaneous infection of cell lines A549 and PC-9.The scramble one was used as control.3.The effect of FAM136A knockdown on cell proliferation was detected by CCK8.4.After targeted synthesis and package knockout to slow down the virus LV-shFAM136A and infect A549 and pc-9 cells,the Wound Healing experiment was used to detect the effect of FAM13 6A knockdown on cell migration.5.The impact of knockout on cell cycle distribution and apoptosis was detected by flow cytometry.6.ATP detection kit was used to detect the effect of FAM136A reduction on cellular ATP metabolism levels.7.LDH detection kit was used to detect the impact of FAM136A on the metabolic status of LDH cells;8.The morphological effects of FAM136A on autophagy were observed by transmission electron microscopy.9.Western blot was used to detect FAM136A knockdown on the essential nodal proteins in the cell proliferation signaling pathway,the effects on critical proteins of the cell cycle,the changes in the expression levels of autophagy-related core proteins.Four.Zoological experiments in vivo1.The cells of A549 were amplified after infecting with lentivirus LV-shFAM136A.The model of transplanted lung cancer was established by injection of infected cells into the armpit of nude mice at the appropriate age of week.The length and short diameters of xenografts in nude mice were measured daily.The bodyweight of nude mice was weighed,and the growth status of nude mice was observed.After feeding for a proper time,the nude mice were sacrificed with a broken neck and dislocation.The xenografts were dissected;the long and short length of the tumor were measured.The net weight of the xenografts was weighed for statistical analysis.Results:One.We first screened 15 pairs of differentially expressed genes(9 cases of representative lung cancer in the Xuanwei area and 6 cases of the non-Xuanwei site)from the mRNA expression profile microarray of lung cancer tissues and their paracancer normal lung tissues,among which 1440 up-regulated genes and 2120 down-regulated genes were expressed in Xuan Wei lung cancer group.In the non-Xuanwei lung cancer group,1182 genes were up-regulated,and 1661 genes were down-regulated.Group for the Screening of Xuan Wei differentially expressed gene chip results through bioinformatics analysis,using the DAVID tool to GO after enrichment degree.KEGG pathway enrichment analysis found the differentially expressed gene biology function mainly related to angiogenesis,cell adhesion,ring an amp response,extracellular matrix,drug response,blood clotting,clearance outside the cell,cell's outer membrane and extracellular region,cytoplasmic membrane,the lateral aspect of the cytoplasmic membrane,cellular connection,calcium channel,direct protein phosphorylation,related gene,and cell proliferation,cell cycle regulation,complement,and coagulation system-related regulation pathways.The protein-protein interaction network was analyzed using the STRING database,and Cytoscape software was further used to screen out core genes(Hub)genes.The top ten core genes for the connectivity degree of the protein interaction network structure relationship were FAM136A,LPAR3,ACTN2,GNAI1,PBP,JUN,ANXA1,EGF,PIK3R1,and AGTR1.Finally,online survival analysis and prediction were conducted using Kaplan-Meier Plotter software.The prediction results showed that FAM136A was near related to patients' overall survival and disease-free progression.This provides a new entry point for follow-up research.Two.The screening sample size was further expanded to collect the lung cancer sample tissues of 177 cases(75 cases in the Xuan Wei group and 102 cases in the non-Xuanwei group)with complete follow-up information of patients.The expression intensity and distribution of FAM136A were detected by the immunohistochemical SP method.The correlation between the clinicopathological characteristics of patients with FAM136A expression was statistically analyzed.Unfortunately,the results showed that there was no statistical difference between the expression of FAM136A in Xuan Wei and non-Xuanwei cancer tissues.Still,there were statistically significant differences between the expression of cancer tissues and normal tissues in the Xuan Wei group and the non-Xuanwei group.Comprehensive statistics showed that the positive expression of FAM136A could be detected in 79 cases(44.6%),and the positive expression of FAM136A was correlated with TNM stage and lymph node metastasis.More importantly,we found that the overall survival of FAM136A positive expression patients was shortened and independently associated with patients with lymph node metastatic lung cancer,which could be an independent prognostic factor for the poor prognosis of these patients.Three.Results of experiments in vitroThe following experiments were carried out successively after A549,and PC-9 cells were infected with lentivirus LV-shFAM136A.1.The proliferation activity of A549 and pc-9 lung cancer cells was inhibited after transfection with LV-shFAM136A by CCK8.2.After infection with lentivirus LV-shFAM136A,the migration ability of lung cancer cells in A549 and pc-9 detected by the Wound Healing test was significantly reduced.3.Flow cytometry detection found that A549 and pc-9 lung cancer cell apoptosis increased,in which A549 cells were more significant,and the cell cycle distribution changes were mainly increased in the G1 phase decreased in the S phase.4.ATP detection kit detected that the ATP synthesis level increased after FAM136A reduction.5.The detection of the LDH detection kit found that after the decrease of FAM136A,the metabolic status of LDH in cells was reduced.6.It was discovered by transmission electron microscopy that FAM136A reduction had no obvious morphological effect on autophagy.7.The expression level of c-Jun,a key node protein in the cell proliferation signaling pathway,was reduced by Western blot.The expression level of cell cycle key protein CDK4/CDK6 decreased.Four.The animal experiment in vivoWith the extension of time,the xenograft volume of the nude mice in the experimental group and control group showed a gradually increasing trend.Still,the tumor proliferation rate of the transplanted tumor of lentivirus LV-shFAM136A infected cell line was significantly slower than that of the unrelated lentivirus infected group.At the appropriate age of the week,the nude mice were sacrificed with their necks broken,the tumor body was dissected,and the length and length of the tumor were measured.Statistical analysis showed that the tumor volume in the experimental group was significantly smaller than that in control group.No abnormal changes were observed in the growth status of nude mice,and no statistically significant difference was observed in the weight change of nude mice.Conclusion(s):One.In this study,bioinformatics analysis showed that the mRNA expression spectrum of Xuanwei lung cancer tissues was different to some extent,but not specific.Compared with other lung cancer databases,the different gene functions mainly focus on angiogenesis,cell adhesion,adenosine cyclic monophosphate response,extracellular matrix,drug response,etc.It is mainly involved in the regulation of cell proliferation,cell cycle,complement,and coagulation system.Two.We screened different expression of candidate genes FAM136A by mRNA expression profile chip and combined with bioinformatics analysis for lung cancer in Xuan Wei group.Confusingly,immunohistochemical results showed no difference between Xuanwei and non-Xuanwei groups.This reminds us that chip results were not consistent with the differences in clinical practice.However,there were significant differences between the two groups in comparing cancer tissue and paracancer tissue.More importantly,we have proved that FAM136A is indeed related to the survival prognosis of lung cancer patients from two aspects of bioinformatics prediction and clinical evidence.It can be used as a new candidate indicator to judge the prognosis of patients.Crucially,we found that the expression of FAM136A was independently related to the overall survival prognosis of patients with lymph node metastasis,and could be used as an independent prognostic factor to determine the survival of patients with lymph node metastasis,which was worthy of further study.Three.FAM136A was associated with increased proliferation and cell migration of lung cancer cell lines A549 and pc-9.FAM136A can affect the cell cycle distribution of lung cancer cells A549 and pc-9 and play a role in the biological effect of inhibiting apoptosis.FAM136A plays a role in ATP metabolism and LDH metabolism of lung cancer cells A549 and pc-9.The role of FAM136A in autophagy needs further study.FAM136A can regulate the expression of A549 and pc-9 proliferating proteins in lung cancer cells and promote the proliferation of A549 and pc-9 lung cancer cells.FAM136A can regulate cell cycle distribution and promote cell proliferation by promoting the expression of cell line A549 and PC-9 proliferation protein C-Jun,and by increasing the expression level of cell line A549 and PC-9 cyclin CDK4/CDK6.The MYC-Faml36A-CDK4/CDK6 regulatory axis may be a potential proliferative signaling pathway.However,its exact and more detailed mechanism needs to be clarified in further experimental design.Four.The volume of xenografts increased slowly in nude mice after FAM136A was knocked out,while that of the control group increased rapidly after transplantation.There were significant differences between the two groups,indicating that FAM136A could promote the proliferation of lung cancer cells in nude mice.Simultaneously,there was no significant difference in body weight and growth status in nude mice.