Effect Of FOXC1 Gene Silencing On The Sensitivity Of A549 Cells To Cisplatin Chemosensitivity

Background Cancer is a predominating public health problem in the world.Among the cancer-related deaths,lung cancer ranks first and consists of more than 50 histomorphological subtypes and has a poor prognosis with a 5-year survival rate less than 15%.Non-small cell lung cancer accounts for about 85% of lung cancer.In clinic,only a small percentage of with non-small cell lung cancer patients have the chance of surgical resection if they have been diagnosed in the early stages?stage ? or ??.While,more than 60% of the patients have been in local progression or metastasis stage?stage ? or ??when be diagnosed,losing the chance of surgery.Therefore,conventional chemotherapy and radiotherapy are still the main treatment strategies for lung cancer patients.Cisplatin is one of the most common drugs for various cancers,such as the non-small cell lung cancer,ovarian cancer,prostate cancer,esophageal cancer,bladder cancer and so on.There are a variety of mechanisms of its anti-cancer action and the chief one is to activate the damage response of DNA to induce mitochondrial apoptosis.With the wide application of cisplatin since the 1980 s,it has been gradually noticed that the efficacy of cisplatin is significant in the early stage of chemotherapy,but will get worse and worse by the continuously improved drug resistance.The rapidly improved drug resistance of cisplatin severely restricts its further application and is the primary cause of chemotherapy failure.Transcription factors are a series of biological factors which could be binding with the specific sequence upstream of a target gene.They can affect the development of the target organ or cells' differentiation,metabolism,apoptosis and other biological processes by regulating the expression of some targeted genes.The transcription factor forkhead box C1?FOXC1?is an important member of the forkhead box?FOX?superfamily.More researches have indicated that FOXC1 could not only be related to the occurrence and development of various tumors,such as nasopharyngeal carcinoma,liver cancer,basal-like breast cancer,melanoma,pancreatic cancer,etc.,but also promote the proliferation,migration,invasion and distant metastasis of cancer cells.In addition,it has also been found the expression of FOXC1 in NSCLC tissues is significantly higher than that in adjacent normal tissues and could correspond to the tumor differentiation,lymph node metastasis and TNM staging.Silencing FOXC1 could help inhibit the epithelial-mesenchymal transition of A549 cells and attenuate the migration and invasion of A549 cells.Some studies have reported that the overexpression of FOXC1 could cause the anthracycline chemotherapy resistance of sporadic triple-negative breast cancer.However,the role of siRNA-FOXC1 in reversing drug resistance of lung adenocarcinoma is still to be studied,especially its effect on the drug resistancerelated proteins.Objective In this study,A549 cells were used to investigate the effect of transfection of siRNA-FOXC1 on the proliferation and apoptosis of cells treated with cisplatin and the variation of apoptosis proteins Bcl-2 and Bax in the process.The study aims to prove whether siRNA-FOXC1 can affect the sensitivity of lung adenocarcinoma A549 to DDP or not by inhibiting the expression of FOXC1.It could be helpful for elucidating the mechanism of complex chemotherapy resistance and finding out some more effective anticancer strategies.Materials and Methods 1.Materials The human lung adenocarcinoma A549 cells?purchased from the Shanghai Institute of Life Sciences,Chinese Academy of Sciences?were selected as experiment objects.All the cells were cultured in RPMI1640 complete medium containing 10% fetal bovine serum and putted in the 37? and 5% CO₂ incubator.2.Methods 2.1 Experiments were divided into two parts.In the first part,the effect of drug concentration and action duration would be investigated.Cells will be treated with cisplatin at different concentrations?empty control,0.5,1.0,2.0,4.0,8.0,16.0,32.0,256.0 nmol/L groups?and with different stimulation time of 24 h and 48 h,respectively.In the second part,the effect of cisplatin on A549 cells would be carried out after silencing the FOXC1.Test groups would be divided into blank,transfection reagent,siRNA-NC,siRNA-NC +cisplatin,siRNA-FOXC1 and siRNA-FOXC1+ cisplatin groups.2.2 Cell transfection: In the logarithmic growth phase of A549 cells,when the cell growth density had reached 60%-70%,siRNA-FOXC1 and its negative contrast?siRNA-NC?would be transfected into cells by liposome transfection.2.3 CCK-8:CCK-8 assay was adopted to detect the proliferation inhibition rate of A549 cells treated with cisplatin before and after siRNA-FOXC1 transfection.2.4 Flow Cytometry: Flow cytometry was used to detect the apoptosis rate of A549 cells treated with cisplatin before and after siRNA-FOXC1 transfection.2.5 q RT-PCR: q RT-PCR was used to detect the expression of FOXC1,Bcl-2 and Bax m RNA in A549 cells treated with cisplatin before and after transfection with siRNA-FOXC1.2.6 Western Blot:Western blot was used to detect the quantity of expression of FOXC1,Bcl-2 and Bax protein in A549 cells treated with cisplatin before and after transfection with siRNA-FOXC1.3.Statistical analysis Statistical analysis of the obtained data using SPSS 24.0 statistical software.All the experimental data were presented with mean and standard deviation values????±s?.Independent sample t-test or adjusted t-test was used to compare the statistical date form cells proliferation inhibition between two groups.The difference of apoptosis and expression of FOXC1,Bcl-2 and Bax m RNA and protein among 6 groups of A549 cells were analyzed in one-way analysis of variance.Pairwise comparison was conducted by LSD-t test with a test level of 0.05.Results 1.The optimal concentration and action duration of cisplatin The concentration of 4.0nmol/L and action duration of 24 h were determined to be the optimal parameters of cisplatin treatment based on the observed inhibition rate of A549 cells in the first part of experiments.2.Effect of cisplatin on the proliferation of A549 cells after transfection of siRNA-FOXC1.The results of CCK8 detection indicated the inhibition rate of siRNAFOXC1+cisplatin group was significantly higher than that of siRNA-NC+ cisplatin group.The difference between them was statistically significant?P<0.05?.3.Effect of cisplatin on apoptosis of A549 cells in vitro after transfection with siRNA-FOXC1.The results of flow cytometry indicated the early apoptotic rate,late apoptotic rate and total apoptotic rate of A549 cells in siRNA-FOXC1+cisplatin group were all significantly higher than those in other groups.The difference was statistically significant?P<0.05?.4.The effect of cisplatin on the expression of FOXC1,Bcl-2 and Bax m RNA and protein in A549 cells after transfection with siRNA-FOXC1.The results of q RT-PCR and Western Blot showed that,the relative expressions of FOXC1,Bcl-2 m RNA and protein of A549 cells in siRNA-FOXC1+cisplatin group were all decreased compared to other groups,while the relative expressions of Bax m RNA and protein were increased.The differences were statistically significant?P<0.05?.Conclusion The chemotherapy sensitivity of A549 cells to cisplatin can be improved through inhibiting the expression of FOXC1 in cells,which could provide some useful information for further exploring the mechanism of lung adenocarcinoma resistance to cisplatin and other chemotherapeutic drugs.