Study On The Effect And Related Pathway Of RNA Interference-mediated AQP5 Expression Silencing In Nonsmall-cell Lung Cancer

Background:Lung cancer is one of the most common malignancies with high morbidity and mortality worldwide.Non-small-cell lung cancer(NSCLC)is the most common type,accounting for approximately 80% of all lung cancer cases..Conventional treatment methods for lung cancer include surgical treatment,radiation therapy,and chemotherapy.With the rapid development of molecular biology,targeted gene therapies are gradually becoming one of the main treatment methods for lung cancer,and certain effects have achieved.Researchers have discovered many oncogenes and cancer-associated proteins that play a decisive role in the development and progression of NSCLC,providing new research and treatment ideas for the treatment of NSCLC.Therefore,the search for key genes and related signaling pathways in the pathogenesis of NSCLC,and reveal the molecular mechanisms,are essential for targeted therapy.Aquaporin 5(AQP5)is an aquaporin transmembrane that regulates intracellular water balance.Recent studies have shown that AQP5 is upregulated in a variety of tumor cells and can promote the proliferation of malignant tumor cells,and gene silencing can inhibit tumor growth.However,the correlation between AQP5 and NSCLC remains to be explored.Purpose:Based on the research at home and abroad,the expression of AQP5 gene in non-small cell lung cancer cells was silenced by genetic engineering recombination technology,and the effects of AQP5 on tumor cell proliferation,apoptosis,migration,and invasion were explored.Further study on the effect of AQP5 gene silencing on related functional proteins and ERK1/2 signal transduction pathways,and explore the role of AQP5 and NSCLC in the development and the involved signal pathways and molecular mechanisms,provide a new direction for the targeted treatment of NSCLC.Method:Western Blot analysis was used to detect the expression of AQP5 in normal human lung cells MRC-5 and human lung cancer cell lines HCC827,A549,H1299,and H358.AQP5 high expression cell line A549 was selected.The specific eukaryotic expression plasmids AQP5-p GCH1/Neo(sh AQP5)and NC-p GCH1/Neo(sh NC)were designed and constructed.The liposomes were transfected into A549 cells and stable transfected cell lines were obtained by screening.Real-time PCR and Western Blot were used to detect the gene and protein expression levels of AQP5 in the untreated cell group,blank control group,and gene silencing group.2.Use cell colony formation assays to test the proliferation of the three groups of cells.MTT assay was used to detect the cell proliferation activity.Flow cytometry was used to investigate the cell cycle distribution and apoptosis.The expression of apoptosis-related proteins Bcl-2,Cleaved caspased-3 and Bax was detected by Western Blot.3.Cell wound scratch assay to observe the migration ability of the three groups of cells,Transwell invasion experiment to observe the invasive ability of each group of cells,Western Blot assay to detect cell migration and invasion-related proteins,MMP-2,MMP-9 and Vimentin expression levels.Western Blot was used to detect the expression of p-ERK,p-CREB and c-fos related proteins in ERK1/2 signaling pathway.Explore the signaling pathways that AQP5 may affect.On the basis of the above in vitro experiments,three kinds of cells were inoculated subcutaneously in nude mice to establish a xenotransplantation model in vivo,and the tumor growth curve was observed and recorded.Tumor tissue was removed 30 days after inoculation and weighed to compare proliferative capacity.Three sections of tumor tissue were taken for Tunel to detect the apoptosis of tumor cells.Result:1.AQP5-p GCH1/Neo plasmid containing AQP5 sh RNA and control plasmid NC-p GCH1/Neo were successfully constructed and stably transfected into A549 cells;AQP5 m RNA and protein expression levels were significantly reduced in A549 cells after transfection.2.Cell colony experiments showed that colony formation ability of AQP5 sh RNA cells decreased significantly;MTT assay showed that the absorbance of AQP5 sh RNA cells decreased at each time point,and the longer the time,the more significant the difference.The cell cycle was detected by flow cytometry.The results showed that AQP5 sh RNA cells were arrested in G0/G1 phase,while the number of cells in S phase and G2/M phase was significantly decreased.Flow cytometry revealed significant apoptosis in AQP5 sh RNA cells.Apoptosis-related protein assays showed that Bcl-2 expression was down-regulated and Cleaved caspased-3 and Bax were up-regulated,indicating that AQP5 silencing inhibited tumor cell proliferation,inhibited tumor cell division,and promoted apoptosis of A549 cells by affecting apoptosis-related protein expression..3.Cell migration experiments showed that the migration ability of AQP5 sh RNA cells was decreased.Transwell cell invasion assays showed that the AQP5 sh RNA group entered the lower chamber cells and the invasive ability decreased.Western Blot results showed that the expression of MMP-2 and MMP-9 as key factors of migration and Vimentin expression of mesenchymal transition protein decreased,indicating that AQP5 protein down-regulates tumor cell migration and invasion,and inhibits epithelial-mesenchymal process of tumor cells.4.The expression of p-ERK,p-CREB and c-fos in AQP5 sh RNA group was significantly reduced.This indicates that the down-regulation of AQP5 can reduce the phosphorylation of ERK1/2 signaling pathway and inhibit the ERK1/2 signaling pathway.5.The tumor volume and weight of mice in AQP5 sh RNA group were significantly smaller than those in the control group,and the tumor growth rate was significantly decreased.Tunel assay detected tumor apoptosis results showed that there were more apoptotic cells in tumor tissue of AQP5 sh RNA group mice.It shows that AQP5 gene silencing can inhibit tumor proliferation and promote tumor apoptosis in vivo.Conclusion:1.AQP5 gene silencing cell line of non-small cell lung cancer A549 cells was established using RNA interference technology.2.It was confirmed that AQP5 is closely related to the biological function of non-small cell lung cancer A549.The down-regulated expression of AQP5 inhibits the proliferation,migration and invasion of tumor cells,promotes the apoptosis of tumor cells,and regulates the expression level of related functional proteins,and inhibits the ERK1/2 signaling pathway.3.Provide new potential markers and targets for the diagnosis and treatment of non-small cell lung cancer.4.Provide experimental data for RNA interference treatment of tumor diseases.