| Background: Gastric Cancer(Gastric,GC)is one of the most common malignant tumors in the world,which is often characterized by high incidence and high mortality.At present,the application of traditional histopathological classification standard for gastric cancer have been unable to fully meet the clinical needs,in 2014 the Cancer Genome Atlas planning group(TCGA),GC will be divided into 4 subtypes,including AT binding domains of 1A gene(the AT-rich interactive domain 1A,ARID1A)in 2 groups(subtype EB virus type GC,microsatellite instability type GC)in high frequency mutation,and scholars put forward the ARID1 A mutation as one of the factors of GC.As the ARID1 A chromatin remodeling complex is an important one,can repair the damage of DNA synthesis,regulation of gene transcription after DNA in embryonic development,tissue repair,cell cycle,apoptosis and tumor formation plays an important role in the.Research shows that ARID1 A mutations in a variety of tumors,including gastric cancer and the mutation rate is 8-27%,the gene mutation can often lead to protein loss or decline,is one of the factors in this type of cancer prognosis prediction.At present,the comprehensive treatment of gastric cancer is still based on chemotherapy,but many patients in the treatment process often due to drug sensitivity or drug resistance,leading to disease progression,metastasis.Lyu et al.Found that the sensitivity of ovarian clear cell carcinoma to cisplatin decreased after ARID1 A.At present,the combination of platinum based chemotherapy is one of the best options for adjuvant chemotherapy and palliative chemotherapy in gastric cancer.In this study,we used sh RNA lentivirus to transfect gastric cancer cells and silenced the ARID1 A gene,and to investigate the effect of ARID1 A on the biological characteristics of gastric cancer cells and the sensitivity to oxaliplatin.Objective: The lentiviral transfected into gastric cancer cell line stably transfected ARID1 A gene,construct the gastric cancer cell line model is silent,to explore ARID1 A gene silencing on the biological behavior of gastric cancer cells,and the effect of oxaliplatin sensitivity of human gastric cancer cells,and to explore the possible mechanism of gastric cancer cells to oxaliplatin platinum resistance.Methods: 1.The transcription and translation of ARID1 A gene in gastric cancer cells were detected by q PCR and Western blot.2.The gastric cancer cell lines MGC803 and AGS were screened by lentivirus(sh RNA and green fluorescent protein gene specific to human ARID1 A gene).The gene was silenced and the gastric cancer cell lines were screened by puromycin.The effect of ARID1 A gene silencing was verified by fluorescence microscopy and Western blot.3.Flow cytometry and Transwell method were used to detect the biological behavior of gastric cancer cell apoptosis,cell cycle and cell invasion after silencing ARID1 A gene.4.CCK8 was used to detect ARID1 A gene silencing group and negative plasmid of gastric cancer cells to oxaliplatin sensitivity;using flow cytometry,the same oxaliplatin concentration,apoptosis ARID1 A gene silencing vector group and negative rate of gastric cancer cells.5.Application of Western blot detection method,before and after the addition of oxaliplatin,the expression of Akt protein in gastric cancer and the same concentrations of oxaliplatin 48 h,negative control group and ARID1 A group gene silencing the expression of Akt protein in gastric cancer cells.Results: 1.ARID1 A expression in gastric cancer cell line MGC803,AGS.2.Successfully constructed ARID1 A silencing stable gastric cancer cell line.Fluorescence microscopy showed that the expression of ARID1 A in 72 h after transfection,cell fluorescence silencing of MGC803 and AGS group of gastric cancer cell line Western blot in more than 80%;compared with the empty vector group and gene silencing group of gastric carcinoma cells ARID1 A protein expression in gastric cancer cells MGC803 protein expression(by 46%,AGS protein expression in gastric cancer cells by 65%)(P<0.01). 3.The results of CCK8 showed that in the cell plate after the early(within 24h),changes of gastric cancer cell proliferation activity between ARID1 A gene silencing group and negative control groups were not significant(P>0.05);in the cell plate after 24h-72 h,ARID1A gene silencing group of gastric carcinoma cell proliferation activity is higher than negative empty vector control group(P<0.05).Flow cytometry showed that ARID1 A gene silencing group of gastric carcinoma cells apoptosis cells compared with the negative control groups decreased(5.12 + 1.09 VS 8.76 + 1.02)(P<0.05);cell cycle compared with the empty vector group,ARID1 A gene silencing group in gastric cancer cell cycle distribution table is more cells in S phase and the decrease of G1 phase cells(P<0.05).Transwell invasion test showed that compared with the control group,the invasion ability of ARID1 A gene silencing group was significantly increased(P<0.01).4.The results of CCK8 showed that in the concentration of oxaliplatin(4 g/ml,8 g/ml,12 g/ml,16 g/ml)when compared with the negative control groups,the inhibition of ARID1 A gene silencing group of gastric cancer cell rate decreased(P<0.01);in oxaliplatin concentration(1 g/ml,2 g/ml)when compared with the negative control groups,the inhibition of ARID1 A gene silencing group of gastric cancer cell rate did not change significantly,the difference was not statistically significant(P>0.05).The results of flow cytometry showed that the apoptosis rate of ARID1 A gene silencing group was lower than that of the negative control group(10.63 + 1.15 VS + + 19.05 + + 2.45).5.Western blot results showed that in AGS and MGC803 cell lines,in the addition of oxaliplatin 48 h,oxaliplatin group of gastric carcinoma cells than the drug group the down-regulation of Akt protein expression;at the same concentrations of oxaliplatin 48 h,ARID1A gene silencing group of gastric carcinoma cells Akt protein expression compared to negative expression vector group(P<0.01).Conclusions: 1.After ARID1 A gene silencing,the proliferation activity of gastric cancer cells was increased,the apoptosis was decreased,more cells entered the S phase,and the cell invasion ability increased. 2.After ARID1 A gene silencing,the sensitivity of gastric cancer cells to oxaliplatin was decreased.3.After ARID1 A gene silencing,the sensitivity of gastric cancer cells to oxaliplatin was decreased,which may be related to the changes of Akt signaling pathway. |
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