Effect Of LEPR (Leptin Receptor) On Biological Behavior Of Hepatocarcinoma Cells And Interaction Between LEPR And ANXA7

Background:Our previous study has screened the differentially expressed genes Annexin A7(ANXA7)and LEPR(leptin receptor)from Hca-F(lymph node metastasis >70%)and Hca-P(lymph node metastasis <30%).It was found that the expression of these two genes may be closely related to tumor lymphatic metastasis.LEPR and ANXA7 play important roles in many cancer,but the molecular association between them in cancer cells has not been studied thus far.Suppression of ANXA7 expression in Hca-F cells induced decreased proliferation,migration and invasion ability,and increased the number of apoptotic cells.In this study,the silencing of LEPR had the same impact on the biological behaviors of Hca-F cells as Annexin A7 down-regulation.Further q RT-PCR,Western blot,Enzyme-linked immunosorbent assay and coimmunoprecipitation assays revealed that the expression level of LEPR was positively correlated with ANXA7 regulation,which mediated the function of LEPR in promoting liver cancer lymphatic metastasis.Furthermore,we also found that LEPR was in complex with ANXA7 by Coimmunoprecipitation(co-IP)and immunofluorescence staining.Together,these findings unveiled a previously unappreciated role of LEPR in the development of tumor cells,assigning LEPR as a promising therapeutic target for liver cancer treatments.Objective1.Using q PCR,Western blot,ELISA and cellular immunofluorescence assay to detect the expression of LEPR in Hca-F/Hca-P hepatoma cells(F/P)and ANXA7 silenced andoverexpressed cells.2.CCK-8,transwell and flow cytometry were used to detect the changes of biological behaviors such as proliferation,migration,invasion and apoptosis of Hca-F hepatoma cells.3.Application of immunoprecipitation,co-localization to verify that ANXA7 and LEPR are interacting proteins.Method1.Culture of mouse liver cancer with different lymphatic metastatic potential cell lines Hca-F and Hca-P.2.Construction of Hca-F/P cell ANXA7 silencing and overexpression transfected cell line and Hca-F cell LEPR silencing transfected cell line3.q PCR,Western blot,and cellular immunofluorescence were used to detect the expression of LEPR in FA7,FNC,PA7 up-regulation and PNCcells at m RNA and protein levels.4.The expression of LEPR in cell supernatant of F/P,FA7 down-regulation,FNC,PA7up-regulation and PNC cells were detected by ELISA5.CCK-8,transwell and flow cytometry experiments were used to detect the effects of LEPR on the biological behaviors of Hca-F hepatoma cells such as proliferation,migration,invasion and apoptosis6.Co-immunoprecipitation and co-localization confirmed that ANXA7 and LEPR were interacting proteins in F cells.Results:1.The expression of LEPR in different lymphatic metastatic potential cells of hepatocellular carcinoma:(1)q PCR,Western blot and cellular immunofluorescence experiments showed that LEPR expression in Hca-F cells with high lymphatic metastasis potential was significantly higher than that in low lymphatic metastasis Potential tumor cell Hca-P cells(2)ELISA results showed that the expression of LEPR in Hca-F cells was significantly higher than that in Hca-P cells in the supernatant of hepatocellular carcinoma with high lymphatic metastatic potential.2.The effect of LEPR gene expression regulation on the biological behavior of hepatocellular carcinoma cells:(1)CCK-8 result that silenced of LEPR reduced the proliferation in Hca-F cells,indicating that LEPR expression can enhance cells Proliferative capacity.(2)Transwell results indicated that the silencing of LEPR expression decreased the migration and invasion of Hca-F cells,indicating that LEPR can promote the migration and invasion of tumor cells(3)Flow cytometry detection of LEPR expression silencing can induce apoptosis of Hca-F cells in liver cancer,indicating that LEPR may promote the growth of tumor cells.3.Mutual regulation between ANXA7 and LEPR gene expression:(1)The regulation of ANXA7 gene on LEPR gene expression.The results of q PCR,Western blot and cellular immunofluorescence showed that the expression level of LEPR in ANXA7 silencing group was significantly lower than that in Hca-F cell group and FNC cell group at m RNA level and protein level.Compared with the Hca-P cell group and the PNC cell group,the expression level of LEPR in the ANXA7 overexpression group was significantly increased at m RNA level and protein level.The results of ELISA showed that the expression of LEPR in supernatant of ANXA7 silencing group was significantly lower than that of Hca-F cells and FNC cells.The expression of LEPR in ANXA7 overexpressing cell supernatant was higher than that in Hca-P cells and PNC cells.(2)The regulation of LEPR gene on ANXA7 gene expression.The results of q PCR and Western blot showed that there was no significant difference in the expression level of ANXA7 between LEPR silencing group,Hca-F cell group and FNC cell group at m RNA level and protein level.4.ANXA7 and LEPR are interacting proteins:(1)The results of co-immunoprecipitation experiments showed that ANXA7 interacts with LEPR in Hca-F cells(2)immunofluorescence colocalization experiments showed that ANXA7 and LEPR protein were colocalized on the cell membrane.Conclusion:Our experiments show that the expression levels Of LEPR in Hca-F cells are higher than those Hca-P cells at m RNA and protein levels as well as cytoimmunofluorescence and ELISA demonstrated that LEPR expression had a sametendency.Moreover,LEPR promotes proliferation,migration,invasion and inhibits apoptosis of hepatocarcinoma cells.Furthermore,With silencing or overexpression of ANXA7,the expression levels of LEPR are uniformly decreased or increased,but LEPR does not influence the expression of ANXA7 in liver cancer cells,suggesting that LEPR is a important downstream target for ANXA7-mediated.Furthermore,our study is the first to demonstrate LEPR interacts with ANXA7.LEPR-ANXA7 interaction may contribute that the LEPR promotes proliferation,migration,invasion and inhibits apoptosis of hepatocarcinoma cells.LEPR-ANXA7 complex may represent a potential target for tumor growth suppression and metastasis prevention,which influences the occurrence and development of tumors.