| Objectives:Micro RNAs(miRNAs)are associated with the development of gastric cancer,and play a role in promoting or inhibiting cancer by regulating target genes.Helicobacter pylori(Hp)is the main pathogenic factor leading to the occurrence of gastric cancer.Hp infection will inhibit autophagy and play a protective role in the development of gastric cancer cells.Our research team found that miR-1224-5p and c AMP responsive element binding protein 1(CREB1)were differentially expressed in cag A~+Hp gastric cancer tissues and adjacent tissues,but the specific carcinogenic mechanism is still unclear.The purpose of this study is to verify whether CREB1 is the target gene of miR-1224-5p,how miR-1224-5p adjusts the expression of CREB1in gastric carcinoma cells,investigate whether miR-1224-5p and CREB1 in gastric cancer cells regulate the development of gastric cancer by autophagy through PI3K/Akt/m TOR pathway.Methods:1.We collected 17 samples of gastric cancer tissue and paracancerous tissue removed from the First Affiliated Hospital of Kunming Medical University from May2021 to May 2022,and normal gastric epithelial cells GES-1,gastric cancer cell lines AGS and SGC7901,by q RT-PCR to detect the expression of CREB1.2.The CREB1-WT report plasmid and CREB1-MUT report plasmid were constructed and co-transfected with miR-1224-5p and miR-NC respectively,the luciferase activity of each group was detected by fluorescence enzyme marker.The overexpression and low expression of miR-1224-5p cell lines were constructed by cell transfection,q RT-PCR verified the transfection efficiency,and the expression level of CREB1 in each group was detected by q RT-PCR and Western Blot.3.Using cell transfection technology,different cell lines were constructed and divided into h-CREB1 group,miR-1224-5p group,h-CREB1+miR-1224-5p group and NC group.The effects of miR-1224-5p and CREB1 on the proliferation,invasion and migration of gastric carcinoma cells AGS and SGC7901 were detected by CCK-8cell proliferation test,clone formation test,scratch test,invasion test and transwell migration.4.Si-CREB1 was transfected by cells,and the transfection efficiency was verified by q RT-PCR and Western Blot.Western Blot was used to detect the expression level of autophagy-associated proteins Beclin1,LC3B,p62,ATG5 in each group.After co-transfection of knockdown CREB1 with over-expression and under-expression miR-1224-5p,the expression level of autophagy-associated proteins Beclin1,LC3B,p62 and ATG5 in each group was detected.5.Western blot method was used to detect the expression of PI3K,Akt,p-Akt,m TOR and p-m TOR after knocking down CREB1.The expression of PI3K,Akt,p-Akt,m TOR and p-m TOR were detected after co-transfection of knockdown CREB1with over-expression and under-expression miR-1224-5p respectively.6.Akt inhibitor was used to treat the over-expression and low-expression miR-1224-5p cells,we used Western blot method to detect the expression of autophagy-associated proteins Beclin1,LC3B,p62,ATG5 in each group.Results:1.In clinical tissue samples,the expression of CREB1 in cag A~+Hp gastric cancer tissue is higher than that in adjacent tissues,and the expression of CREB1 in AGS and SGC7901 cells is significantly higher than that in GES-1 cells.2.The results of double luciferase report experiment showed that the luciferase activity of CREB1-WT report plasmid in the miR-1224-5p group was obviously lower than that in the miR-NC group,and after overexpression of miR-1224-5p in the cell line,in terms of the m RNA and protein levels the expression of CREB1 was decreased,and after low expression of miR-1224-5p,the expression of CREB1 at the m RNA and protein levels increased,indicating that CREB1 is the target gene of miR-1224-5p,and is negatively regulated by miR-1224-5p.3.Compared with NC group,the proliferation,invasion and migration ability of gastric cancer cells in h-CREB1 group was enhanced,and the proliferation,invasion and migration ability of cells in miR-1224-5p group was decreased,while the proliferation,invasion and migration ability of cells in h-CREB1+miR-1224-5p group was weaker than that in h-CREB1 group but stronger than that in miR-1224-5p group.The above results indicate that miR-1224-5p can inhibit the proliferation,invasion and migration of gastric cancer cells,CREB1 can promote the proliferation,invasion and migration of gastric cancer cells,and the combined effect can reverse the prometion of CREB1 on the proliferation,invasion and migration of gastric cancer cells.4.Compared with NC group,the expression of Beclin1,LC3 II/I,ATG5 protein increased and the expression of p62 protein decreased after knocking down CREB1.Compared with si-CREB1 group,the expression of Beclin1,LC3 II/I,ATG5 protein increased and the expression of p62 protein decreased after co-transfection of knockdown CREB1 and overexpression miR-1224-5p.However,after co-transfection of knockdown CREB1 and miR-1224-5p inhibitor,the expression of Beclin1,LC3 II/I,ATG5 protein decreased,and the expression of p62 protein increased.These results suggest that miR-1224-5p inhibits autophagy by regulating the expression of CREB1gene.5.Compared with NC group,the total protein expression levels of PI3K,Akt and m TOR did not change significantly after knocking down CREB1,while the protein expression levels of p-Akt and p-m TOR decreased.Compared with si-CREB1 group,the expression level of PI3K,Akt and m TOR total protein did not change significantly after co-transfection of knockdown CREB1 and miR-1224-5p,while the expression level of Akt and m TOR phosphorylated protein decreased.However,after co-transfection of knockdown CREB1 and miR-1224-5p inhibitor,the total protein expression levels of PI3K,Akt and m TOR did not change significantly,while the phosphorylated protein expression levels of Akt and m TOR increased.After Akt inhibitor MK-2206 was used to treat the overexpression and low expression of miR-1224-5p,the expression of autophagy-associated proteins Beclin1,LC3 II/I,ATG5 increased,and the expression of p62 decreased.These results suggest that miR-1224-5p regulates CREB1 activating PI3K/Akt/m TOR pathway to inhibit autophagy and promote the occurrence and development of gastric cancer.Conclusions:1.CREB1 is the target gene of miR-1224-5p and is negatively regulated by miR-1224-5p.2.CREB1 promotes the proliferation,invasion and migration of gastric cancer cells.3.miR-1224-5p regulates CREB1 to activate PI3K/Akt/m TOR pathway to inhibit autophagy and promote the occurrence and development of gastric cancer. |
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