The Mechanism Of Celastrus Orbiculatus Extract Synergistic MiR-144 Inhibit Gastric Cancer

In China,gastric cancer(GC)is a malignant tumor with a high mortality rate.Clinically,most patients with gastric cancer do not have obvious symptoms in the early stages of disease,so they often diagnosed with metastatic lesions.Although medical technology is constantly developing,the examination and treatment of gastric cancer is still limited by many factors.Gastric cancer cells are not sensitive to radiotherapy and chemotherapy,and gastric cancer is prone to metastasis and recurrence.Studying the molecular mechanisms of gastric cancer is helpful to establish new diagnostic markers and to discover new therapeutic targets.MicroRNA(miRNA)is a class of non-coding single-stranded RNA molecules of approximately 22(21-25)nucleotides that play an important role in regulating gene function.MiRNA dysfunction is associated with the occurrence and biological behavior of a variety of tumors,including proliferation,metastasis,angiogenesis,and immune escape.MiR-144 has a tumor suppressor effect in a variety of tumor cells,regulating tumor epithelial-mesenchymal transition.But there are few reports on the relationship between miR-144 and gastric cancer.The mammalian target of rapamycin(mTOR)is a member of the phosphoinositide-3-kinase(PI3K)-related kinase(PIKKs)family and has a C-terminal protein kinase similar to the lipid kinase PI3K domain.mTOR is closely related to the process of tumor occurrence and development.mTOR is highly expressed in a variety of solid tumors and promotes tumor cell proliferation,survival,invasion and metastasis by activating ribosomal kinases.There was a negative correlation between miR-144 and mTOR expression in human gastric cancer tissues.However,the interaction between mTOR and miR-144 in gastric cancer is unclear.Our previous study found that the Celastrus Orbiculatus ethylacetate extract(COE)showed obvious antitumor activity,but the molecular mechanisms have not been fully revealed.To explore whether the COE synergy miR-144 inhibit gastric cancer via targeting mTOR,in this paper,the expression levels of miR-144 in gastric cancer tissues were detected firstly.And then,human gastric cancer MGC803 cells with high expression of miR-144 were constructed through transient transfection technology.Finally,the effects of COE on the proliferation and the migration ability of MGC803/miR144+gastric cancer cells were studied.This study is divided into three parts:Chapter I MiR-144 expression in human gastric cancerObjective:To detect the expression level of miR-144 in human gastric cancer tissues.Methods:Twenty patients were from the Affiliated Hospital of Yangzhou University,diagnosed as gastric malignant tumors by pathological examination.The expressions of miR-144(including miR-144-3p and miR-144-5p)in gastric cancer tissues were detected by qRT-PCR,the corresponding adjacent normal tissues were used as negative controls.Results:The 2-??CT values of miR-144-3p and miR-144-5p in gastric cancer tissues accounted for 24.0%and 20.3%of adjacent tissues,respectively.Compared with the negative control,the expression of miR-144 in gastric cancer tissue decreased significantly(P<0.05).Conclusion:The expression level of miR-144 in gastric cancer tissues is significantly reduced.Chapter ? The construction of human gastric cancer MGC803/miR-144+ cells and relationship between miR-144 and mTORObjective:To construct the human gastric cancer MGC803 cells with high expression of miR-144,and to explore the correlation between miR-144 and mTOR signaling pathway.Methods:MGC803 cells of human gastric cancer were cultured to logarithmic phase in vitro.The miR-144 plasmid was transfected into the human gastric cancer MGC803 cells by transient transfection technology,set up the vehicle control and wild cell negative control.48 hours after transfection,the green fluorescence in the cells was viewed under the microscope.Cells were collected and the total RNA was extracted.The expression of miR-144 and mTOR mRNA were determined by using qRT-PCR technology.And the protein expressions of mTOR,p-mTOR,PI3K,AKT,4EBP1,and p-4EBP1 were measured by western blotting in MGC803/miR144+cells,respectively.Results:The green fluorescence proteins were significantly enhanced under the microscope in MGC803/miR144+cells.The results showed that the expression of miR-144 was significantly increase(P<0.05),while the mRNA expression levels of mTOR were significantly reduced(P<0.05)in MGC803/miR144+cells.The protein expression of mTOR,p-mTOR,PI3K,AKT,4EBP1 and p-4EBP1 were all reduced significantly(P<0.05)Conclusion:1)The human gastric cancer MGC803/miR144+cell line was successfully constructed.2)MiR-144 may reduce the expression of mTOR.Chapter ? The effect of Corbiculatus extract on the proliferation and migration in human gastric cancer MGC803/miR-144+cellsObjective:To investigate the effect of COE on the proliferation and the migration in MGC803/miR-144+ cells.Methods:COE(20,40,80,160,320 ?g·mL-1)with different concentrations were added to human gastric cancer MGC803/miR-144+cells.After 24 hours treatment,the effect of COE on the proliferation was measured by MTT,and calculated the half inhibitory concentration(IC50).At the same time,set up the vehicle control group,no treatment as negative group,and positive control group(5-Fu,50 ?g·mL-1).The migration ability of MGC803/miR-144+ cells was analyzed by cell scratch test.The expression of epithelial-mesenchymal transition-related proteins(N-cadherin,Vimentin and E-cadherin)were detected by western blotting.Results:After 24 hours treatment,the proliferation ability of MGC803/miR-144+ cells was inhibited and showed a certain concentration dependence(P<0.05).The half inhibitory concentration(IC50)was about 126.86 ?g·mL-1.Compared with the vehicle control,the migratory ability was significantly reduced in MGC803/miR-144+cells.Compared with the no treatment cells,COE significantly inhibited the migration of MGC803/miR-144+ cells(P<0.05).COE can significantly increase the expression of E-cadherin(P<0.05),and decrease the expression of N-cadherin and Vimentin(P<0.05).Conclusion:COE can inhibit the proliferation and the migration of MGC803/mir-144+cells,and its mechanisms may be related to epithelial-mesenchymal transition(EMT).