| Part I Effect of hydromorphone hydrochloride on biological behavior of gastric cancer cellsObjective: To observe the effect of hydromorphone hydrochloride on the biological behavior of gastric cancer cells,and and to explore the effect of hydromorphone on PI3K/Akt/mTOR signaling pathway proteins.Methods: Human gastric cancer cell lines(HGC-27,MGC-803,SGC-7901,AGS)and normal gastric epithelial cells(GSE-1)were cultured in vitro with 50μM,100 μM,200 μM,400 μM,800 μM hydromorphone After incubating for 24 h,48 h,72 h.cell viability was detected by CCK-8 method,cell line and drug concentration were screened for follow-up experiments.The effect of hydromorphone on the proliferation of gastric cancer cells was detected by cell clone formation assay and Brd U cell proliferation assay.The effect of hydromorphone on the invasion and migration ability of gastric cancer cells was detected by cell scratch test and transwell assay.The effect of hydromorphone on apoptosis and cycle distribution of gastric cancer cells was detected by flow cytometry.The effects of hydromorphone on the protein expressions of p-PI3 K,p-Akt and p-mTOR in gastric cancer cells were detected by western blot.Results: Hydromorphone hydrochloride can inhibit the activity of gastric cancer cells in vitro in a concentration-dependent and time-dependent manner,but has little effect on normal human gastric mucosal epithelial cells GSE-1.Hydromorphone can significantly reduce the ability of gastric cancer cells to formcolonies on the plate(P<0.05),hydromorphone can significantly inhibit gastric cancer cell DNA synthesis and cell proliferation(P<0.05).hydromorphone can significantly inhibit scratch healing and inhibit cells The number of invasion and migration inhibits the invasion and migration ability of gastric cancer cells(P<0.05).Hydromorphone significantly increases the rate of apoptosis and promotes tumor cell apoptosis(P<0.05).G0/G1 cells after hydromorphone treatment The proportion increased,the proportion of S-phase cells decreased,and the proliferation rate PI% was significantly reduced(P<0.05).hydromorphone can significantly inhibit the expression of p-PI3 K,p-Akt and p-mTOR(P<0.05).Conclusion: Hydromorphone hydrochloride can inhibit the proliferation,invasion and migration of gastric cancer by promoting apoptosis and arresting the cell cycle.These effects may be related to the inhibition of PI3K/Akt/mTOR signaling pathway.Part II Effects of miR-140-5p on the inhibition of proliferation,invasion and migration of gastric cancer cells by hydromorphoneObjective: To explore the effect of miR-140-5p on hydromorphone in inhibiting the proliferation,invasion and migration of gastric cancer cellsMethods: The differential miRNAs of hydromorphone inhibiting the progression of gastric cancer were screened and verified by RT-q PCR through the literature and GEO data set.After miR-140-5p was silenced and expressed by miRNA inhibitor and lentivirus,the effect of hydromorphone on gastric cancer cell viability was detected by CCK-8 method.The effect of hydromorphone on gastric cancer cell proliferation was detected by plate cell clone formation test and Brd U cell proliferation test.The effect of hydromorphone on the invasion and migration of gastric cancer cells was detected by the cell scratch test and Transwell cell invasion and migration test.The effect of hydromorphone on the apoptosis of gastric cancer cells was detected by flow cytometry.The effects of hydromorphone on the protein expressions of p-PI3 K,p-Akt and p-mTOR in gastric cancer cells were detected by Western blot.Results: It was found that miR-140-5p was involved in the process of hydromorphone inhibiting the progression of gastric cancer.After miRNA inhibitor and lentivirus silenced the expression of miR-140-5p,hydromorphone inhibited the activity of gastric cancer cells significantly decreased(P<0.05).Hydromorphine inhibits the ability of gastric cancer cell plate clone formation to be significantly reduced(P<0.05),hydromorphone inhibits DNA synthesis of gastric cancer cells and weakens cell proliferation(P<0.05).hydromorphone inhibits scratch healing and inhibits the invasion and migration of gastric cancer cells Weakened(P<0.05).The ability of hydromorphone to promote cell apoptosis was weakened(P<0.05).the ability of hydromorphone to inhibit the expression of p-PI3 K,p-Akt and p-mTOR proteins was weakened.Conclusion: Hydromorphone inhibits the proliferation,invasion and migration of gastric cancer cells by up-regulating the expression level of miR-140-5p,induces cell apoptosis,and acts by regulating the PI3K/Akt/mTOR signaling pathway.Part III Hydromorphone hydrochloride inhibits the growth and proliferation of human gastric cancer a subcutaneous xenograft tumors in nude miceObjective: To explore the effect of hydromorphone hydrochloride on the growth and proliferation of human gastric cancer subcutaneous xenograft tumors in nude mice.Methods: The subcutaneous tumor model of gastric cancer in nude mice was established to observe the effects of NC group(1.5 mg/kg normal saline),H1 group(2.1 mg/kg hydromorphone),H2 group(4.2 mg/kg hydromorphone)and H3 group(8.4 mg/kg hydromorphone)on the growth and proliferation of subcutaneous tumor in nude mice.Tumor growth curve was plotted,tumor weight was measured,the effect of hydromorphone on subcutaneous tumor proliferation was detected by HE staining and IHC,the expression of miR-140-5p in subcutaneous tumor tissue was detected by RT-q PCR,and the effect of hydromorphone on PI3K/Akt/mTOR pathway protein in transplanted tumor was detected by western blot.The subcutaneous tumor model of gastric cancer in nude mice was established by silencing miR-140-5p to observe the effect of hydromorphone on tumor growth and proliferation after silencing miR-140-5p.Results: Compared with NC group,H2 group(4.2 mg/kg)and H3 group(8.4 mg/kg)could significantly inhibit tumor volume growth.After 28 days,the subcutaneous tumor mass was taken and the wet weight was measured.Compared with the NC group,the weight of subcutaneous tumor in the H2 group and the H3 group was significantly reduced(P<0.05),while there was no significant difference in the weight change of the H1 group(P>0.05).Compared with NC group,the expression of Ki-67 in subcutaneous tumors in H2 group and H3 group was significantly decreased,which inhibited the growth of subcutaneous tumors.Compared with NC group,the level of miR-140-5p in subcutaneous tumors in H2 group and H3 group was significantly increased(P<0.05),while the level of miR-140-5p in subcutaneous tumors in H1 group was not significantly changed(P>0.05).Compared with NC group,the protein expressions of p-PI3 K,p-Akt and p-mTOR in subcutaneous tumors in H2 group and H3 group were significantly decreased(P<0.05).After miR-140-5p silencing,the inhibitory effect of hydromorphone on the growth and proliferation of subcutaneous tumor of gastric cancer was weakened,and the inhibitory effect of hydromorphone on the phosphorylation of PI3K/Akt/mTOR pathway protein was weakened.Conclusion: Hydromorphone inhibits the growth and proliferation of human gastric cancer a subcutaneous xenograft tumors in nude mice by up-regulating miR-140-5p and inhibiting PI3K/Akt/mTOR pathway activation. |
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