Effect And Mechanisms Of Celastrus Orbiculatus Extract Inhibit EMT Of Gastric Cancer Through MiR-144/451 Targeting MTOR

Gastric cancer(GC)is one of the most common gastrointestinal malignancies.Compared with paracancerous tissue,the expression of miR-144/451 in gastric cancer tissue was significantly decreased.Using bioinformatics,mammalian target of rapamycin(mTOR)may be a molecular target of miR-144/451.The Celastrus orbiculatus extract(COE)is a patent of our study team.It can inhibit the early metastasis of gastric cancer cells by inhibiting the process of epithelial-mesenchymal transition(EMT),but the specific mechanism has not been elucidated.This study mainly studies whether the extract of Celastrus orbiculatus extract inhibits the EMT of gastric cancer by miR-144/451 targeting mTOR,which is of great significance to the research and development of new anti-tumor Chinese medicine.The research is divided into three parts:Part I:Relationship among miR-144/451,mTOR and EMTObjective:to study the relationship among miR-144/451,mTOR and EMT in miR-144/451 knockout(KO)mice.Methods:The gastric tissue of 3 miR-144/451KO mice was taken,gastric tissue of the same strain wild C57BL/6 mice as control.The expressions of mTOR and EMT related proteins were detected by Western blot and immunohistochemistry.The total RNA was extracted,the mTOR mRNA expression level was quantitatively detected by qRT-PCR.Results:Compared with the wild C57BL/6 mice,the protein expression of E-cadherin in miR-144/451KO mice decreased significantly.The expression of Vimentin、N-cadherin、mTOR、p-mTOR、PI3K、p-PI3K、Akt and p-Akt protein was significantly increased(P<0.05).The immunohistochemical results showed that after miR-144/451 was knock out,the expression of E-cadherin wasdown-regulated,and the expression of Vimentin、N-cadherin and mTOR related proteins was up-regulated(P<0.05).Conclusion:miR-144/451 may be closely related to the expression level of mTOR and EMT-related proteins.Part II:Effects of miR-144/451 deletion on spontaneous gastric cancer in miceObjective:To study the relationship and molecular mechanism between miR-144/451 and gastric carcinogenesis in miR-144/451KO mice.Methods:Method:BaP was prepared with corn oil for 5 mg/mL,gavage 10μL/g body weight doses.Forty mice,randomly divided into four groups,10 in each group.The wild type and miR-144/451KO mice were set up corn oil control group and BaP group,respectively.Gavage twice a week for 6 weeks.After the last gavage,Week 2,week 8,week 16,week 24 and week 30,one mouse was randomly executed in each group.Take the whole stomach,observe histological changes under microscope.Western blot and immunohistochemistry were used to detect the expression of mTOR related proteins in gastric cancer tissues.The mTOR mRNA expression was detected by qRT-PCR.Results:Under microscope,the gastric mucosa,submucosa,muscular layer and serous layer of wild type C57BL/6 mice were clear.After BaP gavage group had obvious inflammatory cell infiltration,other no obvious changes.The gastric tissue of miR-144/451 KO mice was close to that of normal C57BL/6 mice,but the gastric differentiation was found under microscope in the miR-144/451 KO group of mice perfused with BaP,which could be regarded as carcinoma in situ.The results of Western blot and qRT-PCR showed that the mTOR expression was significantly increased after BaP stimulation,and the mTOR expression level was the highest in miR-144/451 KO mice(P<0.05).Part III:Effects of Celastrus orbiculatus extract in human gastric cancer cells with miR-144/451 highly expressedObjective:To construct 4 human gastric cancer cell models(AGS/miR-144+,AGS/miR-451+,HGC-27/miR-144+,HGC-27/miR-451+),and to explore its molecular mechanism underlying the effect of Celastrus orbiculatus extract on the proliferation and migration ability in human gastric cancer cells.Methods:MTT method was used to detect the effects of COE(20,40,80,160,320 μg/mL)on the proliferation in 4 human gastric cancer cells(AGS/miR-144+,AGS/miR-451+,HGC-27/miR-144+,HGC-27/miR-451+).Logarithmic growth cell,after 200μg/mL COE treatment for 24 h,detect the expression level of mTOR using qRT-PCR method.Western blot analysis the effects of COE on mTOR signal pathways and EMT related proteins.Transwell migration experiments investigated the effects of COE on migration ability.Results:COE significantly inhibited the proliferation of 4 human gastric cancer cells(AGS/miR-144+,AGS/miR-451+,HGC-27/miR-144+,HGC-27/miR-451+)in a time and concentration-dependent manner.The migration ability was significantly inhibited after COE treatment,the expression of E-cadherin was significantly increased,and the expression of Vimentin,N-cadherin,mTOR,p-mTOR,PI3K,p-PI3K,Akt,p-Akt,4EBP1 and p-4EBP1 proteins was significantly decreased(P<0.05).Conclusion:COE synergistic miR-144/451 targeting mTOR,inhibit the EMT through PI3K/Akt/mTOR signal pathway in gastric cancer cells.