Study On The Clinicopathological Significance And Stemness Of LETM1 Expression In Gastric Adenocarcinoma

Background:Gastric cancer(GC)is one of the most common malignant tumors in humans,and its lethality ranks among the top three common tumors.Patients with GC are usually diagnosed as advanced,with a median 5-year survival rate of no more than 20%.At present,the best method for the treatment of GC is surgical resection,and radical treatment and adjuvant chemotherapy or radiotherapy are used.However,the recurrence rate of GC is still high and the prognosis is poor.Therefore,further research on the molecular mechanisms that promote GC progression is the key to unveiling new diagnostic or prognostic indicators,as well as treatment goals to improve clinical outcomes.The leucine zipper EF-hand domain transmembrane protein 1(LETM1)is closely related to the occurrence and development of many human malignancies.Many research reports indicate that in several types of human cancers,the expression of LETM1 is significantly enhanced and has a close relationship with poor clinical prognosis,which also leads to lower survival rate.However,the clinical significance of LETM1 and its stemness relationship in gastric adenocarcinoma(GA)are not fully understood.Objectives:This study aimed to investigate the expression of LETM1 in GA and its clinicopathological significance.Furthermore,we investigate the role of LETM1 in regulating the stemness of GA cells through PI3K/Akt signaling pathway.Methods:In this study,we evaluated the expression of LETM1 in 189 paraffin-embedded GA tissue samples using immunohistochemical techniques,and analyzed the expression of LETM1 and clinicopathological parameters,tumor stemness-related proteins,and tumor microenvironment related factors,cell cycle,and signaling pathway related proteins.Then the expression of relevant stem cell markers,cell cycle-related proteins and signal pathway-related proteins in GA-derived cell lines was examined.Kaplan-Meier survival analysis was used to explain the value of LETM1 in prognosis of patients with GA.Second,western blot and immunofluorescence were used to detect the expression of LETM1 and tumor stemness-related proteins in GA-derived cell lines.Then we treated the AGS cells with CoCl2,simulating the hypoxic environment of the cells,and detected the expression of LETM1 and related stemness proteins by western blot.Then small molecule interfering RNA(esiRNA)transfection technology was used to silence the expression of LETM1 and perform MKN28 spheroid formation experiments to detect the tumorigenic characteristics of stem cells.Single staining flow cytometry and migration invasion experiments were used to detect changes in cell cycle and migration invasion ability.And after inhibiting the expression of PI3K protein,the expression of PI3K/Akt signaling pathway related proteins and LETM1 in MKN28 and MKN74 cells was detected by western blot.Results:1.LETM1 expression in primary GA tissue is significantly higher than normal gastric epithelial tissue and chronic gastritis and intestinal metaplasia tissue.The expression of LETM1 is closely related to tumor differentiation,pathological tumor stage,lymph node metastasis,clinical stage and general tumor type of GA.Kaplan-Meier survival analysis showed that the overall survival rate of GA patients with LETM1 positive expression was significantly lower than those with negative expression.In addition,the positive expression of LETM1 is closely related to the increase of tumor microvessel density.2.LETM1,CD44,and LGR5 and other tumor-related stem markers are highly expressed in GA cell lines MKN28,MKN74.In GA cell lines MKN28 and MKN74,the use of esiRNA to mediate LETM1 silencing reduced the expression levels of tumor stem-related proteins CD44 and LGR5 in the cells.At the same time,silencing LETM1 expression significantly inhibited the spheroid formation ability of MKN28 cells.3.Under the condition of hypoxia induced by CoCl2,the expression levels of LETM1,CD44,LGR5 and hypoxia-inducible factor-1α(HIF-1α)in GA cell line AGS increased significantly with the prolonged hypoxia(6h,12h).4.LETM1 and cell cycle related protein cyclin D1 were positively expressed in MKN28 and MKN74 cells.At the same time,silencing LETM1 expression reduced the proportion of S-phase subpopulations in the MKN28 and MKN74 cell cycle,and reduced the invasion and migration ability of MKN28 and MKN74 cells.5.LETM1 and PI3K/Akt signaling pathway proteins(such as pPI3K-p85 and pAkt-Thr308)are highly expressed in GA cell lines MKN28 and MKN74.In addition,PI3K inhibitors significantly down-regulated the expression levels of LETM1,pPI3K-p85 and pAkt-Thr308 in MKN28 and MKN74 cells.Conclusion:1.LETM1 is involved in the occurrence and development of GA and is a biological indicator to determine the progress and poor prognosis of GA.2.LETM1 regulates the expression of CD44 and LGR5 in GA cells,which is closely related to the formation of tumor stem characteristics.3.LETM1 regulates the invasion and migration ability and cell cycle of GA cells.4.PI3K/Akt signaling pathway regulates LETM1 expression in GA cells.