| Cancer has become a serious threat to human life and health of the number one killer,the world's new cancer patients increase year by year,according to the relevant data shows that the number one cancer in the world is lung cancer,followed by gastric cancer and breast cancer.Cancer has seriously affected people's life and quality of life,the incidence and mortality are increasing year by year,how to prevent and treat cancer has become the number one problem in the field of oncology.Gastric cancer(GC),the most common malignant tumor occurring in the Gastric epithelium worldwide and in China,accounts for the largest number of gastrointestinal tumors and the second largest number of all malignant tumors.Although the incidence of gastric cancer in recent decades in the world has a significant trend of decline,but the death rate of gastric cancer in China is still the first of all kinds of tumors.In 2017,the state of the epidemiology of gastric cancer in China released by the national cancer center reported that 951,000 new cases of gastric cancer and 723,000 deaths were reported worldwide in 2012.China accounts for nearly half of all cases and deaths worldwide.At present,the clinical treatment of gastric cancer include general treatment,surgical treatment,radiotherapy,chemotherapy and immunotherapy,mediated cisplatin chemotherapy combined surgery and/or radiation therapy has obtained the good curative effect and prognosis,fluorouracil or clinical commonly used cisplatin plus capecitabine as the standard therapy for gastric cancer chemotherapy.Cisplatin is the first metal complex with anticancer activity discovered.It is a non-specific drug of the cell cycle and has cytotoxicity.It can inhibit the DNA replication process of cancer cells and damage the membrane structure of cancer cells.Clinically,it is mainly used for the treatment of Such as gastric cancer,ovarian cancer,testicular cancer and lung cancer and so on.In the course of chemotherapy for gastric cancer,fluorouracil or capecitabine are commonly used as first-line drugs.In vitro studies revealed that cisplatin with different strain after gastric cancer cells,will be accompanied by obvious changes in gene and protein level,and regulation of cell proliferation,apoptosis,cell cycle and cell migration and invasion,and other functions,to achieve the purpose of killing tumor cells,and in order to obtain cisplatin potential targets for the treatment of gastric cancer.However,with the rapid development of high-throughput technology,large-scale studies on the changes in the expression levels of small RNA after cisplatin treatment in gastric cancer cells have not been reported,especially the changes in the expression profile of differential mi RNAs.Therefore,it is urgent to reveal the differential mi RNAs expression profiles after cisplatin treatment of gastric cancer cells,and to develop new combined small molecule targets of cisplatin for the treatment of gastric cancer,so as to provide an important new intervention method for the clinical treatment of gastric cancer.Based on it,the specific work to be carried out in this study is as follows.Objective Based on small RNA high-throughput sequencing technology,the changes of mi RNAs expression profile after cisplatin treatment of gastric cancer cells NCI-N87 were studied,and the potential regulatory targets of differentially expressed mi RNAs were studied in combination with the dual luciferase reporter gene system and cell function experiments,so as to reveal its potential mechanism.Methods First,cisplatin with gradient concentration was used to treat human gastric cancer cells NCI-N87.Cell images were collected and cell proliferation activity was measured by MTT method to screen the best cisplatin concentration of cisplatin induced injury of gastric cancer cells NCI-N87.Secondly,gastric cancer cells were treated based on the optimal cisplatin concentration as the screened above.After the cells were collected,small RNA high-throughput sequencing and bioinformatics analysis were conducted to obtain differentially expressed mi RNAs,and heatmap and volcano analysis were conducted.Based on the above differences,5 significantly up-regulated mi RNAs and 5significantly down-regulated mi RNAs were screened for real-time fluorescence quantitative PCR verification.The target gene prediction software Target Scan and mi RDB was used for target gene prediction for the mi RNAs that have been verified and high-throughput sequencing,and Venn diagram was drawn with the R language package.The GO and KEGG cluster analysis was performed for the cross target genes obtained by the above two software,and the cross regulatory network was constructed by Cytoscape software.The 3?-UTR region of the target gene regulated by mi RNAs was obtained by screening,and the dual luciferase reporter gene vector was constructed to measure the activity of firefly luciferase and renilla luciferase,and the in vitro activity of the target gene regulated by mi NRAs was obtained by taking the ratio of the two.In vitro,mi RNAs mimics and inhibitors verified systematically by double luciferase reporter genes were synthesized.Gastric cancer cells NCI-N87 treated with cisplatin were transfected.Cell proliferation activity was determined by MTT assay,cell cycle and apoptosis were determined by flow cytometry,and cell migration and invasion were determined by Transwell assay.Results 1)When cisplatin concentration reaches 15?g/m L,the damage of NCI-N87 to gastric cancer cells is close to 50%.It can be seen from the cell pictures collected that,at this concentration,certain damage was caused to the gastric cancer cell NCI-N87,but more than 50% of the cells were still in good condition.Therefore,the optimal concentration of cisplatin induced injury of NCI-N87 in gastric cancer cells was determined to be 15?g/m L.2)Compared with the control group,a total of 49 significantly differentially expressed mi RNAs were obtained in cisplatin treated gastric cancer cells NCI-N87,including33 significantly up-regulated mi RNAs and 16 significantly down-regulated mi RNAs.3)Compared with the control group,the expression level of hsa-mir-1246 was significantly up-regulated and the expression level of hsa-mir-892 b was significantly down-regulated after cisplatin treatment.4)Based on Target Scan and mi RDB database target gene prediction analysis,it was found that there were 196 cross-regulatory genes in hsa-mir-1246 and 323cross-regulatory genes in hsa-mir-892 b in the two databases.Further,according to the cross regulation genes to GO function clustering and KEGG pathway clustering analysis found that hsa-mi R-1246 control target genes were involved in 30 cells and molecules containing a biological process and the process of three KEGG signaling pathways,has-mi R-892 b regulating target genes were involved in 29 cells and molecules containing a biological process function and the process of the 11 KEGG signaling pathways,both with complex cross regulation network.5)Compared with NC group and Mut+Mimic group,the ratio of firefly luciferase and renilla luciferase in WT+Mimic group was not changed,indicating that hsa-mir-1246 had no regulatory activity in ATP2B2 3?-UTR region of target gene.Similarly,compared with NC group and Mut+Mimic group,the ratio of firefly luciferase and renilla luciferase in WT+Mimic group were significantly lower(**:p<0.01),suggesting that hsa-mir-892 b regulates target gene activity by downregulating the target gene IL1 A 3?-UTR region.6)Hsa-mir-892 b Inhibitor can synergistically inhibit the proliferation activity of cisplatin-treated gastric cancer cells NCI-N87,increase the G0/G1 phase of gastric cancer cells NCI-N87,inhibit the S phase of gastric cancer cells NCI-N87,and synergistically increase the apoptosis level,migration and invasion ability of cisplatin-treated gastric cancer cells NCI-N87.7)After hsa-mir-892 b Mimic treatment of gastric cancer cells of NCI-N87,when compared to Cisplatin and Cisplatin+Inhibitor groups,the protein and m RNA expression level of IL1 a was significantly decreased,and had a statistically significant.Conclusion In this study,differential mi RNAs expression profiles of cisplatin treated gastric cancer cells were obtained based on small RNA high-throughput sequencing technology,and the regulatory activity of hsa-mir-892 b on its target gene IL1 A was verified by the dual luciferase reporter gene system.Cell function experiments showed that hsa-mir-892 b had the potential function to inhibit the activity of gastric cancer cell NCI-N87,and could act together with cisplatin on tumor cells,which has a potential clinical development and application prospect. |
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