| BackgroundAbout one million people are diagnosed with gastric cancer worldwide each year.Among them,east Asia is the region with the highest incidence of gastric cancer,and about 738,000 people die of gastric cancer every year.All these made gastric cancer the fourth in tumor morbidity and second in mortality.After 2000,several large controlled studies showed that adjuvant chemotherapy has a beneficial effect on survival in patients with gastric cancer.These studies indicated that patients who receive adjuvant therapy after gastrectomy have a higher survival rate than those who receive simply undergo surgery.Many subsequent studies have also found beneficial effects of adjuvant chemotherapy in patients with gastric cancer.Therefore,adjuvant chemotherapy for gastric cancer has received research and clinical attention and has become one of the standard treatment options for advanced gastric cancer after resection in recent 20 years.For advanced gastric cancer,platinum-containing chemotherapy is the standard treatment for advanced gastric cancer,but drug resistance may occur in some patients.At present,the mechanism of platinum resistance in gastric cancer is not yet clear.The chemotherapy-induced expression of tumor stem cells is one of the reasons for the failure of platinum therapy.Therefore,it is one of the important directions of gastric cancer research to strengthen the research on the mechanism of gastric cancer and to find a practical preventive and therapeutic approach.MiRNAs are a group of non-coding single-stranded small molecules with a length of about 18-24 nucleotides that are widely found in eukaryotes.MiRNAs are a group of short sequences of RNA that are not involved in protein coding.They inhibit their translation or induce their degradation by binding to the 3 ’UTR of target gene mRNA,thus playing a regulatory role in protein translation.So far,more than 2,500 miRNAs(miRbase database)have been identified,whose heterotopic expression is associated with tumor proliferation,invasion,metastasis,growth and apoptosis.More and more studies subsequently found that onco-miRNA(onco-miRNA)and tumor suppressor miRNA(suppressor miRNA)play an important role in the progress of gastric cancer.Previous studies have found that miR-492 promotes proliferation,metastasis,drug resistance and association with tumor stem cells in a variety of tumors.The main purpose of this study was to clarify the expression of miR-492 in gastric cancer,as well as the effect of miR-492 on proliferation,apoptosis,migration,invasion and platinum-type treatment of gastric cancer cells,to furtherclarify the mechanism of miR-492,and to provide a new perspective for the occurrence,development and drug resistance of gastric cancer.Methods1.Detection of miR-492 expression in gastric cancer tissues and gastric cancer cell lines,as well as the relationship between miR-492 expression in gastric cancer tissues and prognosis.The expression of miR-492 in gastric cancer tissues and gastric cancer cell lines(SNU-5,MGC-803,AGS and SGC-7901)and gastric mucosal epithelial cell lines(GES-1)was detected and quantitatively analyzed by RT-PCR.Kaplan-Meier curves were used to analyze the correlation between miR-492 expression and OS in patients.2.Effect of miR-492 on proliferation,metastasis and apoptosis of gastric cancer cell lines and its mechanism.CCK-8,flow apoptosis detection,and Transwell was used to detect cell proliferation and metastasis after transfection with miR-492 mimic and inhibitor,respectively.RT-PCR was used to verify the effect of miR-492 on stem cell markers and DNMT3 B.RT-PCR was used to detect the expression of miR-492 and DNMT3 B in gastric cancer tissues and analyze the correlation between them.3.Mechanism of miR-492 regulating the stemness of gastric cancer cells through DNMT3 B.Lentivirus was used to interfere DNMT3 B and WB was used forverification.CCK-8,flow apoptosis detection,and Transwell was used to detect the effect of DNMT3 B on proliferation and metastasis of gastric cancer cells.WB and RT-PCR were used to verify the effect of miR-492 on stemness markers and analyze the stemness of gastric cancer cell lines by pellet-forming ability.4.Effect of miR-492 on drug resistance of gastric cancer through DNMT3 B.Subcutaneous tumor grafts were constructed by subcutaneous injection of gastric cancer cells from nude mice and gastric cancer cells transfected with inhibitor.The tumor size was measured to draw the growth curve,and the expression of CD133 was detected by flow classification.Immunohistochemistry was used to detect the proliferation index.5.Statistical methodsStatistical analysis was performed using SPSS 22.0.Count data is expressed by M±SD,measurement data is expressed by median and range,t test is used for comparison between the two groups,Kaplan-Meier,Log rank is used for survival analysis,P<0.05 is considered statistically significant.Results1.MiR-492 was highly expressed in tumor tissues and tumor cell lines(P<0.05).Further analysis of the relationship between the level of miR-492 expression and the patient’s prognosis found that the highexpression of miR-492 was associated with poor prognosis(P=0.0287).2.MiR-492 promoted the proliferation,metastasis and anti-apoptosis by increasing the stemness of gastric cancer cells;it was found that miR-492 has a regulatory effect on the expression of DNMT3 B and its mechanism was that the combination of miR-492 and DNMT3 B transcription promoter that inhibited the transcription of DNMT3 B.3.Demonstrate that DNMT3 B can inhibit the expression of CD133,Nanog,Oct3/4 and BMI-1 in gastric cancer,reduce the number of gastric cancer cells,and prove that miR-492 is involved in the regulation of CD133,Nanog,Oct3/4 and BMI-1 by inhibiting DNMT3 B.MiR-492 binding to the promoter of DNMT3 B inhibited the transcription of DNMT3 B.The down-regulation of DNMT3 B resulted in the down-regulation of the methylation of the promoter regions of CD133,Nanog,Oct3/4 and BMI-1,thereby increasing the transcription of CD133,Nanog,Oct3/4 and BMI-1.4.MiR-492 promoted tumor growth.After miR-492 is inhibited,tumor growth is slowed(P<0.05),but this inhibition is partially reversed after inhibiting the expression of DNMT3 B.Conclusion1.MiR-492 is highly expressed in gastric cancer tissues and gastric cancer cell lines.2.MiR-492 increase the proliferation,migration and anti-apoptosisability by regulating the stemness transformation of gastric cancer cells.3.MiR-492 negatively regulates DNMT3 B,reducing the methylation level of the latter to CD133,Nanog,Oct3/4 and BMI-1 promoter,resulting in increased tumor cell stemness,thus increasing the proliferation,metastasis and anti-apoptosis ability of gastric cancer cells.4.MiR-492 regulates the stemness of gastric cancer cells through DNMT3 B and participates in the resistance to cisplatin. |
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