Mechanisms Of Hydroxy-3-oxoolean-12-en-29-oic Acid Synergistic MiR-451 Inhibit The Metastasis In Gastric Cancer

Gastric Cancer(GC)is a malignant tumor that occurs in the epithelial tissue of the stomach.It has the characteristics of high morbidity,high metastasis,and high mortality.Traditional anti-tumor and targeted therapies have slightly improved prognosis,but have no significant improvement in patients with metastases and advanced stages.By monitoring tumor-related miRNAs,target proteins and genome-related metastatic genes,it can be used as a new breakthrough point in the treatment of gastric cancer.As a plant of the genus Celastrus orbiculatus thumb.,it has anti-tumor,anti-inflammatory,anti-virus,anti-oxidation,bactericidal and other activities.On the basis of total terpenes of C.orbiculatus,the monomer components of various terpenes were successfully isolated in our laboratory.By improving the extraction process,the drug sensitivity test of the newly separated monomer component was carried out,and it was found that the biological activity of 28-Hydroxy-3-oxoolean-12-en-29-oic Acid was strong,so the component was selected for further study.MiRNA is a recognized new tumor marker.Studies have shown that after high expression of miR-451,gastric cancer cell cycle stagnates in G0/G1 phase.After inhibiting the expression of miR-451,it promotes the cell division of gastric cancer.Therefore,it is speculated that miR-451 plays a role of tumor suppressor gene in gastric cancer.Our research team found that the expression of Mammalian Target of Rapamycin(mTOR)increased significantly in gastric cancer.This study first analyzed the relationship between the expression of miR-451 and mTOR in gastric cancer tissues and the clinicopathological characteristics of gastric cancer.Then,the human gastric cancer AGS cell line with miR-451 overexpress(AGS/miR-451+)was constructed.Finally,study the mechanisms of Hydroxy-3-oxoolean-12-en-29-oic Acid synergistic miR-451 inhibit the metastasis in the human gastric cancer AGS/miR-451+cells.This study provides a scientific theoretical basis for the clinical treatment of gastric cancer.This study is divided into three parts.Part Ⅰ Expression of miR-451 and mTOR in gastric carcinoma and its relationship with clinicopathological featuresObjective:To detect the expression of miR-451 and mTOR in gastric cancer tissues and their relationship with clinicopathological characteristics.Methods:Collect 20 pairs of gastric cancer surgical samples from the Affiliated Hospital of Yangzhou University,and record their clinicopathological characteristics.The expression of miR-451(miR-451 a and miR-451b)were detected by using qRT-PCR.The positive expression of mTOR was detected through immunohistochemistry.The relationship between miR-451 and mTOR and clinicopathological characteristics was analyzed.Results:The expression levels of miR-451a and miR-451b in the samples were 10.4%and 43.8%,respectively.The positive expression rate of mTOR was 90%.The expression of miR-451 and mTOR was correlated with clinicopathological characteristics.The results showed that the expression of miR-451 in tissue samples was significantly reduced,and mTOR was positively expressed in gastric cancer tissue.The expression of miR-451 and mTOR were both related to tumor differentiation,TNM staging and lymph node metastasis.Conclusion:The expression of miR-451 is reduced in human gastric cancer tissues,and the expression of mTOR is increased.The expressions of miR-451 and mTOR are both related to tumor differentiation,TNM stage and lymph node metastasis,but not to the patient’s age,gender and tumor diameter.Part Ⅱ Construction the human gastric cancer AGS cells with miR-451 overexpressingObjective:To construct a human gastric cancer AGS cell line with miR-451 stably overexpressing.Method:Using the modified lentiviral small RNA expression vector pTRIPZ(Open Biosystems),the recombinant plasmid pTRIPZ-miR-451 and two auxiliary packaging plasmids were constructed by the method of molecular cloning,and 293 T cells were co-transfected by the Lipofectamine 2000 method.Collect and concentrate the cell supernatant rich in lentiviral particles,and measure and label the virus titer.After the virus infects the AGS cells of the logarithmic stage human gastric cancer,puromycin selects the positive cells.After DOX induction,the changes in cell morphology and fluorescence expression were observed under an inverted fluorescence microscope.Results:After DOX induction,human gastric cancer AGS/miR-451+with red fluorescence.Compared with the control cells,there is no significant difference in cell morphology,the red fluorescent signal is significantly enhanced,and the infection efficiency is about 90%.When the DOX concentration is 100 ng/mL,the expression intensity of miR-451 is the highest.Conclusion:Successfully constructed the human gastric cancer AGS/miR-451+cells.Part Ⅲ Mechanisms of 28-Hydroxy-3-oxoolean-12-en-29-oic Acid inhibit the metastasis in human AGS/miR-451+cellsObjective:To explore the molecular mechanisms of 28-Hydroxy-3-oxoolean-12-en-29-oic Acid inhibits the metastasis in human AGS/miR-451+cells.Methods:The proliferation of different concentrations of 28-Hydroxy-3-oxoolean-12-en-29-oic Acid was detected by MTT in human AGS/miR-451+cells.Scratch test was used to detect the effect of drugs on cell migration ability.The effects of apoptosis and mTOR related proteins were analyzed by Western blot method.Results:28-Hydroxy-3-oxoolean-12-en-29-oic Acid significantly inhibited the proliferation of AGS/miR451+cells(P<0.05),which was time-and concentration-dependent.The scratch test results showed that the drug significantly inhibit the migration of AGS/miR451+cells.Western blot showed that the expression of mTOR,PI3K,AKT,pAKT,4EBP1 and other proteins was significantly reduced(P<0.05),and was concentration-dependent.With the increase of drug concentration,the expression of apoptosis pathway related proteins increased.The expression of Bax,Caspase-1,Caspase-3,and Cyt-c increases,and the ratio of Bcl-2/Bax decreases,which is concentration-dependent.Conclusion:28-Hydroxy-3-oxoolean-12-en-29-oic Acid significantly inhibit the proliferation,invasion and metastasis of AGS/miR451+ cells,and its molecular mechanism is related to the PI3K/Akt/mTOR signal pathway.