| ObjectivesAs a crucial mechanism for maintaining cellular homeostasis,autophagy plays an essential role in the occurrence and development of gastric cancer and other malignant tumors and is regulated by multiple pathways,such as the PI3K/AKT/MTOR pathway.Our previous studies have found that semaphorin 5A gene expression could promote gastric cancer development and progression through ERK/MMP9 and PI3K/Akt/u PA signaling pathways,and it was hypothesized that semaphorin 5A could affect autophagy by activating the PI3K/Akt signaling pathway.Therefore,this study focused on gastric cancer tissues as well as gastric cancer cell lines to clarify the correlation between semaphorin 5A gene expression and autophagy inhibition,providing new strategies for further investigating the mechanism of gastric cancer occurrence and development.Method1.Establish the correlation between semaphorin 5A gene expression and autophagy inhibition at the tissue level.(1)Gastric cancer tissue and its paracancerous tissue samples were collected,with the former as the experimental group and the latter as the control group.(2)The expression of semaphorin 5A gene in tissues was detected by immunohistochemistry and q PCR.(3)The expression of autophagy-related proteins was detected by immunohistochemistry while conducting an analysis of the relationship between autophagy-related proteins and intracellular semaphorin 5A gene expression in clinical specimens.(4)Intracellular autophagosomes were observed under an electron microscope,along with an analysis of the relationship between the number of autophagosomes and intracellular semaphorin 5A gene expression in clinical specimens.2.Establish the correlation between semaphorin 5A gene expression and autophagy inhibition at the cellular level(1)Semaphorin 5A overexpression,low expression AGS,and 7901 cell lines were constructed.(2)The conversion of autophagy marker protein I/II and the expression of autophagy-related proteins BECLIN 1 and P62 in the cell lines were detected by Western blot.(3)The correlation between semaphorin 5A gene expression and autophagy inhibition was analyzed.Results:1.Semaphorin 5A expression was higher in gastric cancer tissues than in paracancerous tissues,and semaphorin 5A expression was associated with clinical stage,lymph node metastasis,and differentiation.2.BECLIN 1 and LC3 in gastric cancer tissues saw a lower positive expression rate than those in paracancerous tissues,and the expression of P62 in gastric cancer tissues was higher than that in paracancerous tissues.A correlation existed between the expression of these three autophagy-related proteins and the clinical stage of gastric cancer.3.Semaphorin 5A was negatively and positively correlated with the expression levels of BECLIN 1 and LC3 and such levels of P62 in gastric cancer tissues,respectively,while BECLIN 1 was positively correlated with the expression levels of LC3 in gastric cancer tissues.4.Semaphorin 5A positive gastric cancer tissues saw fewer autophagosomes than semaphorin 5A negative gastric cancer tissues.5.The protein expression levels of LC3 Ⅱ and BECLIN 1 were increased,and the protein expression level of P62 was decreased in gastric cancer cells with Semaphorin 5A knockdown expression.Conclusions:1.In gastric cancer tissues,the expression of semaphorin 5A was negatively correlated with the expression of BECLIN 1 and LC3,and positively correlated with the expression of P62;semaphorin 5A expression increased and autophagosome number decreased.2.Semaphorin 5A knockdown decreased the proportion of LC3I/II and the expression of P62 and up-regulated the expression of BECLIN1 in gastric cancer cells.3.Expression of semaphorin 5A inhibits autophagy in gastric cancer. |
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