Investigation On The Function And Molecular Mechanism Of MTMR14 In Liver Cancer

We checked that MTMR14 as a diagnostic biomarker for liver cancer based from GEO data previously.MTMR14 encodes a myotubularin(MTM)-related protein.The encoded protein is a phosphoinositide phosphatase that specifically dephosphorylates phosphatidylinositol 3,5-biphosphate(PI(3,5)P2)and phosphatidylinositol 3-phosphate(PI3P).We founded MTMR14 was over-expressed in liver cancer tissue compared with nornal tissue,suggested that MTMR14 could promote the development of tumor,but however,the underlying mechanism remained elusive.In this research,we investigated the expression of MTMR14,the function and molecular mechanism in liver cancer.Part 1: The expression of MTMR14 and clinical significance analysis in liver cancerObjectives: To detect the expression of MTMR14 in liver cancer tissues and normal tissues.The relationship between MTMR14 expression level and clinicopathologic features was analyzed.Methods: We downloaded chip information of liver cancer patients from the GEO database,divided them into vascular metastasis group and non vascular metastasis group,analyzed abnormal gene expression.The abnormal expressed genes were analyzed by gene ontology enrichment analysis,pathway enrichment analysis and co-expression network analysis,and checked by q-PCR and IHC.Results: Expression analysis by quantitative real-time polymerase chain reaction(qRT-PCR)and immunohistochemistry demonstrated that MTMR14 expression is obviously overexpressed in liver cancer(P <0.05),and positively correlated with clinical stage(P <0.05).Conclusion: MTMR14 expression is obviously overexpressed in liver cancer,it may have an oncogenic role in human liver cancer.Part 2: The role of MTMR14 in liver cancer cellsObjectives: To study the effects of MTMR14 on cell proliferation,migration,invasion,colony formation,cell cycle and apoptosis in liver cancer cells,and also tumorigenesis in vivo.Methods: Using small interference RNA silenced MTMR14 expression,after then,We used flow cytometry technique,CCK 8 and transwell respectively to detect the cell cycle,apoptosis,proliferation and invasive ability.We used slow virus to built liver cancer cell which lower expression stability,tested MTMR14 effects on cells migration ability in vivo.Results: A loss-of-function study showed that knockdown MTMR14 promotes cell apoptosis and inhibits cell migration.MTMR14 knockdown also inhibits tumor migration in vivo in liver cancer peritoneal implantation nude mouse model(P <0.05).But however,knockdown MTMR14 influence cycle,proliferation and invasion was not significant(P>0.05).Conclusion: MTMR14 involved in liver cancer cell migration and apoptosis at the same time,the tip in the development of tumor may play an important role.Part 3: MTMR14 regulation autophagy and apoptosis in liver cancer cells through ER stressObjectives: To study the effects of MTMR14 can also influence autophagy,clarify the mechanism of MTMR14 induce apoptosis and the autophagy at the same time.Preliminary study the mechanism of MTMR14 affected the ability of the cell migration.Methods: We used flow cytometric analysis of cell apoptosis,laser scanning confocal microscopy for autophagy flow,transmission electron microscope scanning autophagosome,and West-blot analysis protein about autophagy,apoptosis and migration,detected endoplasmic reticulum stress related indicators.Results: MTMR14 knockdown also induced liver cancer cell autophagy(P<0.05).A molecular mechanistic study by western blot showed that knockdown MTMR14 causes downregulation of N-cadherin and E-cadherin,and promotes the cleavage and activation of caspase12,caspase9 and caspase3,but not includes caspase8.Western-blot also showed knockdown MTMR14 causes ATF6,DAPK1 and Beclin1 upregulation.inhibiting cell autophagy can promote more apoptosis based on knockdown MTMR14 by reduced mitochondrial membrane potential(P<0.05).Loading fluorescent calcium probe showed MTMR14 may trigger intracellular calcium overload(P<0.05).Conclusion: Knockdown MTMR14 can induce autophagy and apoptosis at the same time,it may regulation autophagy and apoptosis in liver cancer cells through ER stress.Part 4: Preliminary discussion MTMR14 interaction with other proteinsObjectives: To study MTMR14 may be combined with protein,proteomics provide direction for further.Methods: Extraction of HuH7 cell total protein,was dealed by precipitation,silver stain,West-blot experiments and LC-MS mass spectrometry.Results: According to the result of mass spectrometry and software analysis,keratin family/cytoskeleton protein got the highest score.Conclusion: It suggested that MTMR14 and keratin family exercise the relevant functions together.