Study On The Role And Mechanism Of MiR-199b-5p In The Occurrence Of Gastric Cancer

Background and objective:Gastric cancer(GC)is one of the most common malignant tumors.The mortality of GC patients is still high.The main reason for poor prognosis of GC patients is the low detection rate of early GC.The traditional methods of GC examination mainly include histopathology and imaging,but patients are often in the advanced stage after diagnosis.Therefore,it is urgent to find new detection methods to improve the detection rate of patients with early GC,and the most valuable application of liquid biopsy is early tumor screening.More and more evidences support that micro RNAs(miRNAs)play an important role in tumorigenesis and development.However,the role of miRNAs in human GC remains largely unknown.Therefore,this study aims to screen out potential risk miRNAs associated with gastric cancer through bioinformatics,so as to find potential diagnostic markers in plasma of patients with GC,and preliminarily explore their role and mechanism in the occurrence of GC.Methods: Based on the published GC expression profile data,combined with bioinformatics analysis,the potential risk miRNAs in the progression of GC were screened,and their expression differences in plasma of GC patients and normal people were detected by RT-PCR.The expression of miR-199b-5p in GC cells(SGC7901,MGC803 and AGS)was detected by RT-PCR.Meanwhile,stable transfected miR-199b-5p-mimic and miR-199b-5p-inhibitor cell models were established in SGC7901 and MGC803 Cells to detect the effect of overexpression/ inhibition of miR-199b-5p on the expression of miR-199b-5p in SGC7901 and MGC803 Cells.The biological effects of miR-199b-5p on gastric cancer cells in vitro were investigated by edu cell proliferation assay,colony formation assay,Transwell migration assay,wound healing assay and Western blot.The biological effect of miR-199b-5p on GC cells in vivo were study by establishing subcutaneous xenograft tumor model and lung metastasis tumor model in nude mice.The candidate target genes of miR-199b-5p were predicted by bioinformatics,and the expression levels of HHIP in GC cells and tissues were detected by RT-PCR and immunohistochemistry.The interaction between miR-199b-5p and its potential target gene HHIP was verified by dual luciferase assay.The effect of miR-199b-5p on HHIP expression was detected by RT-PCR and cell immunofluorescence.The stable transfected cell models of miR-NC+lv-NC、miR-199b-5p-mimic+lv-NC、miR-NC+lv-HHIP、miR-199b-5p-mimic+ lv-HHIP were established in SGC7901 and MGC803 Cells,and the expression level of HHIP was detected by RT-PCR.Through a series of recovery experiments,we further proved the mechanism of miR-199b-5p in GC.Results: Through bioinformatics analysis,we screened the risk miR-199b-5p that was significantly up-regulated in GC tissue and associated with poor prognosis of GC patients.RT-PCR results showed that miR-199b-5p was significantly overexpressed in plasma of GC patients.RT-PCR results showed that miR-199b-5p was significantly up-regulated in SGC7901 and MGC803 Cells compared with GES1 cells(p < 0.001),but there was no significant difference in AGS(p > 0.05).Compared with the miR-NC group,the expression of miR-199b-5p in the mimic group was increased in SGC7901 and MGC803 Cells(p < 0.001),but decreased in inhibition group(p < 0.05).Compared with miR-NC group,overexpression of miR-199b-5p significantly promoted the proliferation(p <0.05),migration(p < 0.05)and EMT process of SGC7901 and MGC803 Cells,while knockdown of miR-199b-5p significantly inhibited the proliferation(p <0.05),migration(p < 0.05)and EMT capacity of SGC7901 and MGC803 Cells.The results of subcutaneous transplantation tumor model and lung metastasis tumor model in nude mice showed that compared with miR-NC group,miR-199b-5p-mimic group significantly promoted SGC7901 cells proliferation(p < 0.05)and lung metastasis(p < 0.05).miR-199b-5p-inhibitor group significantly inhibited SGC7901 cells proliferation(p < 0.05)and lung metastasis(p < 0.01).Bioinformatics predicted that the potential target of miR-199b-5p was HHIP;RTPCR results showed that compared with GES1,HHIP m RNA expression levels were significantly lower in SGC7901,MGC803 and AGS(p < 0.01);immunohistochemistry results showed that HHIP was mainly expressed in cytoplasm,and the positive reaction in GC was weak.The result of dual luciferase assay showed that miR-199b-5p could directly bind to HHIP;RT-PCR result showed that compared with miR-NC group,HHIP expression level in miR-199b-5p-mimic group of SGC7901 and MGC803 Cells was down-regulated(p < 0.05),and it was up-regulated in inhibitor group(p < 0.05).Cell immunofluorescence results showed that compared with mir-nc group,HHIP protein expression in miR-199b-5p-mimic group of SGC7901 and MGC803 Cells was inhibited,while HHIP expression in miR-199b-5p-inhibitor group was increased.RT-PCR results showed that co-transfection of lv-HHIP in miR-199b-5p-mimic group of SGC7901 and MGC803 Cells could partially reverse the expression of miR-199b-5p(p < 0.05).The results of cell recovery experiment showed that co-transfection of lv-HHIP in SGC7901 and MGC803 Cells with miR-199b-5p-mimic could partially reverse the effects of miR-199b-5p on proliferation(p < 0.05),migration(p < 0.05)and EMT of GC cells.Conclusion: miR-199b-5p screened by bioinformatics can be used as one of the potential molecular markers to predict the occurrence of GC.miR-199b-5p can promote the proliferation,migration and EMT of GC cells in vitro and in vivo.miR-199b-5p can play the role of oncogene in GC by targeting HHIP.miR-199b-5p may be a potential therapeutic target for GC.