| Gastric cancer,as a common malignant tumor of upper digestive tract,has a high mortality.According to statistics,the death rate of Chinese stomach cancer ranks the third among malignant tumors in 2020.In recent years,although the incidence and mortality of gastric cancer have been decreasing,the detection methods of early gastric cancer are often asymptomatic and signs,and most patients are not easily accepted by invasive endoscopy,which leads to that some patients have missed the best treatment opportunity when diagnosed.Therefore,in order to improve the early diagnosis rate of gastric cancer and improve the prognosis of patients with gastric cancer,the search for effective molecular therapeutic targets in gastric cancer and the molecular mechanism causing the occurrence and development of gastric cancer have become the current research hotspot.Our previous results showed that Insulin gene enhancer binding protein 1(ISL1)is abnormally highly expressed in gastric cancer and can significantly promote gastric cancer cell proliferation,tumor growth,cell migration and invasion.In tumor,the dynamic balance of cell proliferation and apoptosis is broken,resulting in the disorder of cell proliferation and apoptosis.Cisplatin,as a commonly used chemotherapy drug,promotes apoptosis of tumor cells by causing DNA damage,and cisplatin resistance in gastric cancer is closely related to the disorder of apoptosis.Therefore,the study of the effect of ISL1 on apoptosis and cisplatin sensitivity in gastric cancer as well as the specific molecular regulatory mechanism will provide new ideas for the diagnosis and treatment of gastric cancer,help to improve the early clinical diagnosis rate of gastric cancer and improve the prognosis of patients with gastric cancer.Objective:The objective of this study was to investigate the specific molecular regulation mechanism of ISL1 on apoptosis and cisplatin sensitivity in gastric cancer.To further improve the mechanism of ISL1,a classical transcription factor,in gastric cancer,it also provides a theoretical basis for improving the early diagnosis rate of gastric cancer and improving the prognosis of patients with gastric cancer.Methods:1.ISL1 protein expression levels in gastric cancer cell lines BGC823,MKN45,SGC7901,AGS,MGC803 and normal gastric mucosal epithelial cell line GES1 were detected by Western blot assay,and two cell lines AGS with low expression of ISL1 and MGC803 with high expression of ISL1 were selected as the follow-up research system.2.The AGS with the lowest expression of ISL1 were transfected with lentivirus overexpression of ISL1,and were used as the ISL1 overexpression group.MGC803 with the highest expression of ISL1 was transfected with lentivirus knockdown ISL1 and used as the ISL1 knockdown group.The lentivirus transfection was determined by Western blot assay,and the regulation effect of ISL1 expression on the expression levels of anti-apoptotic factor Bcl-2 and pro-apoptotic factor Bax of AGS or MGC803 was detected.The effect of ISL1 expression on the apoptosis rate of gastric cancer cells was detected by flow cytometry.3.On the basis of overexpression or knockdown ISL1 of AGS or MGC803 lentivirus transfection,after cisplatin treatment,CCK-8 cell proliferation-toxicity assay and flow cytometry apoptosis assay were performed respectively: The effect of ISL1 expression on cisplatin IC50 value of gastric cancer cells and the effect of ISL1 expression on cisplatin treated gastric cancer cells were detected by CCK-8 cell proliferation-toxicity assay.The effect of ISL1 expression on the apoptosis rate of gastric cancer cells was detected by flow cytometry.4.Online databases PITA,Target Scan,Pic Tar,micro T,mi Rmap,and mi RDB were used to find upstream miRNA molecules that may regulate ISL1.miRNA molecules in cells with high expression of ISL1 and cells with relatively low expression of ISL1 were detected by RT-q PCR assay,and miRNA molecules that may regulate ISL1 were preliminarily screened.Transient overexpression of miRNA219a-5p,miRNA29a-3p and miRNA371b-5p in gastric cancer cells MGC803 was performed by mimics of miRNA molecule overexpression sequence.Western blot assay was used to detect the effect of overexpression of miRNA219a-5p,miRNA29a-3p and miRNA371b-5p on ISL1 protein expression in gastric cancer cells MGC803.5.Based on the transient overexpression of miRNA29a-3p in gastric cancer cells MGC803,Western blot assay was performed to detect the effect of miRNA29a-3p expression on the expression levels of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in gastric cancer cells MGC803.Flow cytometry was used to detect the effect of miRNA29a-3p expression on the apoptosis rate of gastric cancer cells MGC803.Results:1.Western blot assay showed that compared with normal gastric mucosal epithelial cell line GES1,the expression level of ISL1 protein in AGS gastric cancer cell line BGC823,MKN45,SGC7901,AGS and MGC803 was the lowest.MGC803 gastric cancer cell line had the highest expression level of ISL1 protein,so AGS and MGC803 were selected as the study system for abnormally low expression of ISL1,and Mg C803 as the study system for abnormally high expression of ISL1.Lentiviral transfection of AGS or MGC803 was performed to overexpress or knockdown ISL1.2.Western blot assay showed that the expression level of anti-apoptotic protein Bcl-2 was up-regulated and that of pro-apoptotic protein Bax was down in AGS cells after overexpression of ISL1 compared with NC in the control group.Contrarily,Compared with the control group sh NC,the expression level of anti-apoptotic protein Bcl-2 was decreased and the expression level of pro-apoptotic protein Bax was up-regulated in MGC803 cells after ISL1 knockdown.Flow cytometry showed that the apoptosis rate of AGS cells decreased after overexpression of ISL1 compared with the control group.Contrarily,Compared with the control group sh NC,the apoptosis rate of MGC803 was up-regulated after ISL1 knockdown.3.The inhibitory effect of cisplatin on the proliferation of gastric cancer cells was determined by the proliferation-toxicity assay of CCK-8 cells.However,compared with the control group,the IC50 value of cisplatin was up-regulated after overexpression of ISL1,and the inhibitory effect of cisplatin on the proliferation of gastric cancer cells was reversed.Contrarily,Compared with the control group sh NC,the cisplatin IC50 value of MGC803 decreased after ISL1 knockdown,and the inhibitory effect of cisplatin on the proliferation of MGC803 was enhanced.Similarly,flow cytometry showed that cisplatin promoted the apoptosis of gastric cancer cells,but overexpression of ISL1 reversed the promoting effect of cisplatin on AGS cell apoptosis compared with NC in the control group.Contrarily,Compared with the control group sh NC,ISL1 knockdown enhanced the promotion effect of cisplatin on apoptosis of gastric cancer cells MGC803.4.We preliminarily screened out 17 miRNA molecules that may be involved in regulating ISL1 expression through online database prediction.The expression levels of miRNA219a-5p,miRNA29a-3p and miRNA371b-5p were significantly decreased in the gastric cancer cell line MGC803 with abnormally high ISL1 expression,while significantly up-regulated in the gastric cancer cell line AGS with abnormally low ISL1 expression.Western blot assay showed that the ISL1 protein level was not significant after transient overexpression of miRNA219a-5p and miRNA371b-5p in gastric cancer cells MGC803 compared with con in the control group.After transient overexpression of miRNA29a-3p,ISL1 protein levels were significantly reduced.5.Western blot assay showed that,compared with con in the control group,after transient overexpression of miRNA29a-3p in gastric cancer cells MGC803,the level of ISL1 protein was decreased,the level of anti-apoptotic protein Bcl-2 was decreased,and the level of pro-apoptotic protein Bax was up-regulated.Flow cytometry showed that the apoptosis rate of MGC803 increased after transient overexpression of miRNA29a-3p compared with con in the control group.Conclusions:1.ISL1 inhibited the apoptosis of gastric cancer cells by promoting the expression of anti-apoptotic factor Bcl-2 and antagonizing the expression of pro-apoptotic factor Bax at the protein level.2.ISL1 can increase the IC50 value of gastric cancer cells to cisplatin and inhibit cisplatin sensitivity of gastric cancer cells.This effect is caused by ISL1 inhibiting apoptosis of gastric cancer cells.3.miRNA29a-3p negatively regulated ISL1.miRNA29a-3p promotes apoptosis of gastric cancer cells,which is contrary to the antagonistic effect of ISL1 on apoptosis of gastric cancer cells,suggesting that the inhibition of ISL1 on apoptosis of gastric cancer cells may be negatively regulated by miRNA29a-3p upstream. |
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