Effects Of HSG Gene Silencing On Proliferation And Apoptosis Of Lung Adenocarcinoma A549 Cells

Research background and purposeLung cancer is currently one of the most morbid and fatal lung malignancies in the world,and is the leading cause of cancer death in China Smoking has been proved to be one of the main pathogenesis factors of lung cancer,with more than 1.5 million lung cancer patients dying every year.It has approximately caused 158000 deaths in the United States in the year 2015.The majority of patients have been treated for local advanced or distant metastasis,so the treatment of lung cancer has been very urgent.The pathological division of clinical lung cancer can be divided into non-small cell lung cancer and small cell lung cancer,about 85%of lung cancer is non-small cell lung cancer,which is including lung adenocarcinoma,squamous cell carcinoma and large cell carcinoma(LCC)and other histological subtypes.Currently,the treatment methods for lung cancer include surgical resection of tumor and regional lymph nodes,radiotherapy and chemotherapy,but the curative effect is limited.Surgery is the best treatment for early lung cancer.With the development and change of society,lung adenocarcinoma has become the most common type of histopathology of lung cancer,accounting for about 50%of the incidence of non-small cell lung cancer(NSCLC).It usually occurs in peripheral lung epithelial cells from type Ⅱ alveolar epithelial cells or bronchoalveolar stem cells.Lung adenocarcinoma is characterized by the formation of glands or ducts and a large amount of bronchial mucosa.It generally originates in peripheral lung tissue.The etiology of lung adenocarcinoma is not fully understood.The major risk factors for lung adenocarcinoma are smoking or passive smoking(passive smokers are at a higher risk comparatively),exposure to cigarette smoke,asbestos,chromium,or arsenic compounds.However,lung adenocarcinoma is often diagnosed in the late stage,and the prognosis of lung adenocarcinoma patients is usually poor,but its metastasis is the main reason for the low survival rate and poor prognosis.lt also tends to develop resistance.Overall,the overall survival rate of patients with lung adenocarcinoma has not been significantly improved by the development of surgery and chemoradiotherapy.The overall 5-year survival rate is about 16%.Therefore,advanced and effective treatment is urgently needed.In recent years,a great deal of research has been done on the genes and proteins associated with pulmonary adenocarcinoma,and many genetic mutations and abnormalities related to pulmonary adenocarcinoma have been discovered.Gene-targeted drug therapy has become an important part of the treatment of lung adenocarcinoma.The occurrence of tumor is related to growth,invasion and metastasis,which is the molecular genetic basis of malignant transformation and tumor progression.However,there has been little research on some new genes and their association with lung adenocarcinoma.Therefore,the identification of key genes or targets related to tumorigenesis can lead us to a new stage of treatment for lung adenocarcinoma.Hyperplasia suppressor gene(HSG),also known as Mitofusin 2(mfn2),localizes to the mitochondrial outer membrane,which contains one GTPase domain in its NH2-terminal region,one coiled-coil domain,two transmembrane spans,and one coiled-coil domain in the carboxy-terminal region.HSG also plays a key role in mitochondrial fusion trafficking,and mitophagy,thus regulating mitochondrial morphology and function.Moreover,HSG is a strong inhibitor of cell proliferation in vitro and in vivo,mediated by cell cycle blocking and inhibiting protein kinase signals activated by mitochondrials.It has been reported that in animal models of vascular stenosis,atherosclerosis and hypertension,cell proliferation,migration and remodelling of cardiovascular disease can be significantly inhibited,and the development of cardiovascular disease can be slowed down.In addition,the pro-apoptotic function of HSG in various malignancies,such as hepatocellular and gastric cancers,was also taken into consideration and researched.Although there are strong evidences that HSG could inhibit tumor development,systematic studies on the effects of HSG on apoptosis and proliferation of lung adenocarcinoma A549 cells and in vivo experimental data are lacking.Therefore,this study intends to investigate the effects of HSG gene silencing on apoptosis and proliferation of lung adenocarcinoma A549 cells by constructing RNAi lentivirus vector to silence HSG gene and constructing xenograft tumor in nude mice in vitro and in vivo.It provides a new direction for the treatment of lung adenocarcinoma in the future.Methods:1.Human lung adenocarcinoma A549 cells were selected to construct the lentiviral vector carrying HSG gene.The lentiviral vector and the packaged plasmid vector were combined to transfect 293T cells to obtain high concentration virus clusters,and the titer of the virus was determined.The infection efficiency was calculated by fluorescence microscopy.The cells were divided into three groups,named the Blank group and the human lung adenocarcinoma A549 cells after lentivirus infection,named si-NC group(negative control group)and si-HSG group(HSG specific interference RNAi group).2.Real-time fluorescence quantitative PCR(RT-PCR)and Western blot were used to detect the expression of apoptosis-related proteins Bax,caspase-3,caspase-8 and bal-2 in the HSG gene silencing group and corresponding control group.3.The cell counting kit(CCK-8)was selected for the assessment of A549 cell survival.Cell cycle was detected by flow cytometry with propidine iodide staining.Apoptosis of A549 cells was detected by flow cytometry(FCM),and cell cycle and apoptosis rate were determined by FCM.Apoptosis rate=early apoptosis rate+late apoptosis rate.4.BALB/C nude mice were selected to establish xenotransplantation models and divided into Blank group,si-NC group and si-RNAi group.After successful modeling,tumor volume was measured every 5 days and growth curve was drawn.HSG gene silencing and corresponding control group cells were subcutaneously injected into nude mice.The expression of HSG protein in tumor tissues of mice in each group was detected by immunohistochemistry,and the tissue was detected by TUNEL method to calculate the apoptosis index.Results:1.HSG silencing reduces mRNA and protein expression of HSG and shows good morphology of lung adenocarcinoma A549 cells in vitroAfter the construction of lentivirus vector for si-HSG,the mRNA and protein expression of HSG in each group were detected by RT-PCR and Western blot analysis.Compared with the blank group,there was no significant difference of HSG mRNA expression which was observed in the si-NC group(P>0.05),but HSG mRNA expression in the si-HSG group decreased by 38%(P<0.05).Western blot analysis further indicated that HSG protein expression was significantly down-regulated by 46%in the si-HSG group as compared with that in the blank group(P<0.05).No significant difference in the mRNA and protein expression of HSG was noted between the si-NC group and the blank group(P>0.05).The morphology of A549 cells after a span of 48-h culturing was observed under an inverted microscope.For the blank and NC groups,cells were stumped to the wall in the shape of oval and fusiform,and they were then connected closely and grew well with optimum bright cytoplasm and fast expansion rate.Compared with the blank and NC groups,the si-HSG group cells proliferated more rapidly.2.The effects of HSG silencing on A549 cells proliferation were detected by CCK-8 assay Compared with the blank group,no significant difference was noted in the si-NC group in terms of the cell proliferation rates at 24,48,and 72 h(all P>0.05).The si-HSG group showed significant increased cell survival rates at 24,48,and 72 h in comparison with those in the blank group and the si-NC group(all P<0.05).Comparison between different time points indicated that cell proliferation rates at the time points of 24,48,and 72 h significantly increased as compared with that in the 0 h(all P<0.05).Compared with the cell proliferation rate at 24 h,the time points of 48 and 72 h showed a significant increase in cell proliferation rates,but There was no significant difference(both P>0.05).Comparison between the time points of 48 and 72 h revealed significantly higher cells proliferation rate at 72h,,but there was no significant difference(P>0.05).2)In order to explore and study the effect of HSG on the cell cycle and apoptosis of lung adenocarcinoma A549 cells,flow cytometry of PI staining and Annexin V-FITC double staining was conducted.Based on the obtained results,the si-HSG group showed significantly lower ratio of cells at G0/G1 phase and increased ratio of cells at G2/M and S phases than the blank group(P<0.05).There was no significant difference between the si-NC group and the blank group in terms of cell ratio at G0/G1,S,and G2/M phases(all P>0.05),indicating that down-regulation of HSG expression could arrest less cells at G0/G1 phase,enhance the ratio of cells at S and G2/M phases,and thus,promote the proliferation of A549 cells.3)The effects of HSG silencing on apoptosis of A549 cells were detected by flow cytometry.There were many early apoptoses and late apoptoses in the blank and si-NC groups.Significant decreases in early apoptosis and late apoptosis of A549 cells were observed in the si-HSG group.Compared with the blank group,the si-HSG group showed significant decrease in cell apoptosis rate(P<0.05).No significant difference between the blank group and the si-NC group was noted in terms of cell apoptosis rate(P>0.05).4)The effects of HSG silencing on the expressions of cell apoptosis-related proteins were detected by carrying out RT-PCR and Western blot analysis.In comparison with the blank group,the si-NC group was noted showing no remarkable difference in terms of the mRNA and protein expressions of Bax,Caspase-3,Caspase-8,and Bcl-2(all P>0.05).The si-HSG group showed significantly decreased mRNA and protein expressions of Bax,Caspase-3,and Caspase-8 but increased Bcl-2 mRNA and protein expression in the si-HSG group as compared with the blank group(all P<0.05).HSG silencing decreases Bax,Caspase-3,and Caspase-8 expressions and increases Bcl-2 expression.3.According to the results,Over time the volume of transplant tumors increased significantly in each group(P<0.05),there was no significant difference in tumor volume at various points in the si-NC group(all P>0.05)compared to the Blank group,and the tumor volume increased significantly at various points in the si-HSG group compared to the Blank group and si-NC group(P<0.05).The results of the 32nd day transplant tumor weighing showed that the tumor quality of si-HSG group was significantly improved compared with that of blank group and si-NC group(P<0.05).2)The protein expression of HSG in tumor tissues of each group was detected by immunohistochemistry.The positive reactant of HSG presented as brown granules in cell plasma and cell nucleus.Based on the mean OD value of each group,no significant difference of HSG protein expression was noted between the blank and si-NC groups(P>0.05).In comparison with the blank group,HSG protein was significantly decreased in the si-HSG group(P<0.05).3)According to the TUNEL staining results,the apoptotic cells mainly presented as clay-bank dense granules in cell nucleus.Few apoptotic cells were observed in the blank and si-NC groups.The si-HSG group showed much fewer apoptotic cells.The statistical analysis indicated that no significant difference of AI was noted between the blank and si-NC groups(P>0.05).When compared with the blank group,the si-HSG group revealed significantly decreased AI(P<0.05).Conclusions:1.HSG silencing reduces mRNA and protein expression of HSG and shows good morphology of lung adenocarcinoma A549 cells in vitro.HSG silencing enhances lung adenocarcinoma A549 cell viability and cycle entry,but inhibits apoptosis in vitro.2.HSG silencing decreases Bax,Caspase-3,and Caspase-8 expressions and increases Bcl-2 expression.3.After HSG silence,lung adenocarcinoma A549 cells proliferate faster in naked mice,and the apoptosis index of the cells decreases,which inhibits the apoptosis of tumor cells.4.HSG gene silencing may be a factor in promoting the progress of lung adenocarcinoma.HSG may be an anti-cancer gene in the progress of lung adenocarcinoma,and it is also a very interesting gene in lung adenocarcinoma research.