Mechanism Research Of LETM1 Regulating Autophagy And Apoptosis Through AMPK Phosphorylation-mediated Beclin-1/Bcl-2complex Dissociation In Hepatocellular Carcinoma

Objective:1.To analyze the level of LETM1 in hepatocellular carcinoma(HCC)tissues and adjacent tissues,and to explore the correlation between LETM1 expression and clinicopathological features and prognosis.2.To observe the effects of LETM1 on proliferation,apoptosis and autophagy in HCC cell lines.3.To explore the underlying molecular mechanism of LETM1 regulating apoptosis and autophagy of HCC cell lines.Method:1.Oncomine database was used to analyze the expression of LETM1 in HCC tissues and normal liver tissues and its prognosis.Then,q RT-PCR and Western blot were applied to detect the expression of LETM1 of clinical samples of HCC tissues and corresponding adjacent tissues.Immunohistochemistry was used to detect the level of LETM1 in matched HCC tissues and adjacent tissues,and the relationship between LETM1 expression level and clinical features as well as overall survival rate of HCC patients was analyzed.2.Lentivirus transfection technology and RNAi technology were used to establish stable LETM1-knockdown Huh7 and QGY-7701 cell lines.CCK8 assay was used to detect cell survival rate to reflect cell proliferation.Flow cytometry with PI staining was used to detect cell cycle.Annexin V-FITC/PI staining and AO/EB double fluorescence staining were used to detect cell apoptosis.Immunofluorescence of LC3 and transmission electron microscope(TEM)were used to assess the level of autophagy.Western blot was applied to test the expression levels of apoptosis-related marker proteins(including p-Bcl-2,Bcl-2,Bax and Caspase-3)and autophagy-related marker proteins(including Beclin-1,p62 and LC3).3.Western-blot was used to detect the expression of apoptotic and autophagy-related proteins after administrating AMPK inhibitor;Co-IP technique was used to detect the interation between Beclin-1 and Bcl-2.Results:1.The results of Oncomine database analysis showed that the expression of LETM1 was increased in liver cancer tissues compared with normal liver tissues,and the overall survival of patients with high LETM1 expression group was worse than those with low LETM1 expression group.RT-q PCR,Western blotting and immunohistochemistry results showed that the m RNA and protein levels of LETM1 in HCC patient tissues were significantly higher than those in adjacent tumor tissues,and highly increased LETM1 was associated with poor prognosis in HCC patients and correlated with tumor size,portal vein emboli,metastasis and TNM stage of HCC.2.Sh RNA lentivirus was used to establish stable Huh7 and QGY-7701LETM1-knockdown cell lines.CCK-8 assay revealed that proliferation was significantly decreased in the sh-LETM1 group compared with the NC group in both Huh7 and QGY-7701 cells.Cell cycle detection results showed that compared with sh-NC group,the proportion of cells in G0/G1 phase in sh-LETM1 group was increased,while the proportion of cells in S phase and G2/M phase were decreased.In addition,Annexin V-FITC/PI staining showed that the apoptosis rate of Huh7 and QGY-7701 cells was significantly increased in the sh-LETM1 groups compared with the sh-NC groups.The immunofluorescence assay demonstrated that the fluorescence intensity of LC3 was significantly higher in the sh-LETM1 group than in the NC group in both Huh7 and QGY-7701 cells.And TEM showed that the number of autophagosomes in sh-LETM1 group was higher than that in sh-NC group.Besides,the results of Western blot revealed that compared with sh-NC group,the level of p-Bcl-2/Bcl-2 ratio,Bax,Caspase-3,Beclin-1 and LC3Ⅱ/Ⅰ were increased in the sh-LETM1 group,while the level of p62 was decreased.3.Western-blot results showed that compared with sh-NC group,the expression of p-AMPK in sh-LETM1 group was upregulated,while total protein level of AMPK had not difference.In addition,compared with sh-NC group,the expression of p-Bcl-2/Bcl-2 ratio,Bax,Caspase-3,Beclin-1,LC3Ⅱ/Ⅰ in sh-LETM1 group were significantly increased and the level of p62 was downregulated.Nevertheless,the expression of p-Bcl-2/Bcl-2 ratio,Bax,Caspase-3,Beclin-1,LC3Ⅱ/Ⅰ in Dorsomorphin+sh-LETM1 group were significantly decreased and the level of p62 was upregulated compared with sh-LETM1 group.The results of co-IP experiment showed that compared with sh-NC group,the abundance of Beclin-1 in precipitates of Bcl-2 in sh-LETM1 group was decreased while the level of Bcl-2 had no obvious change.Furtherly,Beclin-1 was also immunoprecipitated,and Bcl-2 was detected.The results suggested that the expression of Bcl-2 in precipitates of Beclin-1 was downregulated while the level of Beclin-1 was upregulated.Besides,inhibition of AMPK may reverse the effect of LETM1 sh RNA on Beclin-1/Bcl-2 complex dissociation.Conclusion:1.LETM1 expression was highly expressed in HCC tissues than in paracancerous tissues,and the upregulated level of LETM1 was correlated with tumor size,portal vein tumor thrombus and metastasis,and TNM stage.Additionally,patients with high LETM1 expression had a shorter overall survival than those with low LETM1 expression.2.LETM1 knockdown inhibited the proliferation of HCC cell lines,and promoted the apoptosis and autophagy of cells.3.LETM1 knockdown regulated autophagy and apoptosis by phosphorylating AMPK.4.LETM1 knockdown phosphorylated AMPK to regulate autophagy and apoptosis possibly through the dissociation of Beclin-1/Bcl-2 complex and the phosphorylation of Bcl-2.