Altered Ezrin Expression In Mice Lymphatic Metastatic Hepatocellular Carcinoma And Subcellular Fractions Upon Annexin7 Modulation In-Vitro And In-vivo

Background: Tumor metastasis defines as a potential ability of tumor cell to move from the original tumor site which called primary tumor to other location which called metastatic tumor or secondary tumor.Tumor metastasis is a very complicated procedure that's many factors were required for cell metastasis to be done.Adhesion,invasion,and migration are the main steps included in metastasis procedure,and many pathways including different genes and proteins in different steps of metastasis phenomenon.Lymphatic metastasis is a more complicated process which includes many factors that enhance cancer cells to migrate through the lymphatic system.All lymphatic metastatic tumors are malignant and always combine with high mortality and worst output.Hepatocellular carcinoma(HCC)ranks as the second malignant tumor presenting as a single case leading to death universally,that's about 746,000 patients dying annually from HCC disease worldwide.HCC epidemiology and geographical endemic areas are East Asia,North and West Africa,and some in Europe and America.The big challenge in HCC is the nature of the disease which defines as a highly vascularized tumor,and phenotypically heterogeneous polyclonal disease which makes most conventional chemotherapy is not functional.Early recognition of HCC patient is one of a critical required for gaining a good prognosis and increasing the survival rate of the patient,in addition,to giving patients a good life status.Ezrin and Annexin seven(A7)have been expected to be involved in several roles in cancers metastasis process.However,the role of Ezrin and the effect of A7 on Ezrin expression in lymphatic metastatic hepatocellular carcinoma(LNM-HCC)have not been extensively explored yet.In continuation to our lab efforts on finding a novel marker that can help in the early diagnosis or as a target treatment for hepatocellular carcinoma lymphatic metastasis disease,this study applied to evaluate the expression levels of Ezrin as diagnostic and/or prognostic biomarker of LNM-HCC in both primary and secondary tumors.Subcellular fraction is the one of most modified and highly effective biological technique which helps on targeting cell's genes and proteins in their functional localizations,that's could help on studying their actual roles and the interaction between the genes and proteins,also subcellular fraction technique could help in finding the low-copy number proteins.Subcellular fractionation also could improve the differential proteins identification and their roles in many cellular activities like;nucleic acid processes,oxidoreductase,cytoskeleton proteins,signal transduction pathways,cell structure and motility nucleoside,focal adhesion,etc.This study reports the expression of Ezrin in high lymphatic metastasis(Hca-F >70%)and low lymphatic metastasis(Hca-P <30%),HCC cell lines,and the influence of A7 on Ezrin expression.Objectives: 1.To demonstrate Ezrin expression in different lymphatic Metastasis Hepatocellular Carcinoma(LNM-HCC)in-vitro and in-vivo.2.To study the effect of Annexin 7 on Ezrin in different levels of and cell cellular fractions of LNM-HCC in-vitro and in-vivo.3.To fractionate the cells into their subcellular compartments.4.To fortify and comparing Ezrin expression in different subcellular fractions of LNMHCC cell lines.4.5.To evaluate Ezrin expression as diagnostic and/or prognostic bio-marker of LNMHCC in-vitro and in-vivo.Methods: Mouse syngenic,high lymphatic metastasis hepatocellular carcinoma(Hca-F < 70% metastasis)and low lymphatic metastasis hepatocellular carcinoma(Hca-P > 30% metastasis)cell lines,Chinese inbred 615 mice;the two models were used in this study.Therefore,both Hca-F and Hca-P cell lines were revived from liquid nitrogen tank of Pathology Department of Dalian Medical University,the refreshed by washing three times with sterile cold phosphate buffer saline(PBS)and cultured with sterile 10% fetal bovine serum(FBS)in sterile 1460 cell culture medium for 24 hours in 5% CO2 at 37?C in humidified atmosphere.Annexin 7 gene was up-regulated in Hca-F cells(A7-c DNA-Hca-F)and down-regulated in Hca-P cells(A7-sh RNA-Hca-P)were designed into p Silencer vector to silence Annexin7 gene,the transfections were selected and Geneticin 418(G 418)drug 400 ?g/ml(In cells culture media)was used for three weeks to select stably transfected cells;q RT-PCR and western blot techniques were applied to confirm stability expressions of Annexin A7 gene and protein.Furthermore,Annexin 7 modulated and parent cells were fractionated to their subcellular compartments to study the proteins in their functional localizations.RealTime PCR,Western blot,Immunocytochemistry and Immunofluorescence techniques were done to detect the expressions of Ezrin and Annexin 7 genes and proteins in parent and transfected cell lines.In addition,migration and invasion assays were applied to show the role of our interesting proteins on the metastatic phenomenon.From each cell lines,2 x 106 cells/ mouse were inoculated in the left footpad of equal groups of Chinese inbred 615-mice,the mice were harvested after five weeks of inoculation.The primary and secondary tumors were collected and inserted in Hematoxylin and Eosin staining to determining the histological changes,Ezrin expression was assessed by Immunohistochemistry,q RT-PCR and western blot in primary and secondary tumors,in addition to,the serum Ezrin expression was evaluated for different groups using Enzyme-linked Immunosorbent Assay(ELISA).Results: In-vitro model: The result had been shown that Annexin 7 gene was stably transfected,by up-regulated in Hca-F(high lymphatic metastasis)and down-regulated in Hca-P(low lymphatic metastasis)cell lines,as it appears in q RT-PCR and western blot results.Mice ascites implanted cells were palpable within 10 days post-inoculation for both Annexin 7 modulated and parent cell lines.Subcellular fractionation was successfully done using mammalian proteome extraction kit on different lymphatic metastasis cell lines,yielding five subcellular particles;cytoplasm1,cell membrane2,nucleus membrane and nucleus soluble3,nucleoli and histone bands4 and cytoskeleton5 from each cell lines.Ezrin expression in lymphatic metastatic hepatocellular carcinoma LNM-HCC cell lines was investigated;we found that Ezrin expression was associated with the metastasis and aggressiveness of the tumor.The critical role both Ezrin and Annexin 7 in LNM-HCC showed significant differences in proteins expression,however,the attendance of Ezrin and Annexin7 proteins in unusual subcellular locations with unequal expressions,correlated to lymphatic metastasis;could explain Ezrin and Annexin7 roles in different cellular bioactivities as well as cell metastasis.Furthermore,the increase of Ezrin expression was observed in association with Annexin7 down-regulated cells,whereas,the decrease of Ezrin expression was correlated with Annexin 7 up-regulated cells.These findings could confirm the tumor suppression role of Annexin 7 gene.Also,the results impacted a positive correlation between Ezrin expression and LNM-HCC metastasis and negative relationship between the expression of Ezrin and Annexin 7 in LNM-HCC(P <0.05).Western blot analysis of different lymphatic metastasis hepatocellular carcinoma cell lines revealed that Ezrin expression was higher in Hca-F(high lymphatic metastasis)than Hca-P(low lymphatic metastasis)(p=0.0046).Cell fractionation analysis reveals that Ezrin expression was highly present in the cytoplasm,nucleus,and cytoskeleton of Hca-F cells.However,Ezrin expression was highly observed in the cell membrane,nucleus,and cytoskeleton of Hca-P cells.Annexin7-up-regulated in Hca-F suppressed Ezrin expression(p= 0.0248)but leading to increasing the migration and invasion.In addition,Ezrin was mainly located in the cytoplasm and nucleus fractions.Downregulation of Annexin7 in Hca-P cells enhanced Ezrin expression(p<0.0001),Ezrin expression was mainly concentrated in cytoplasm and nucleus fractions,and suppressed tumor cell migration and invasion.In-vitro model : The tumor starts growth two weeks post-implantation of lymphatic metastasis hepatocellular carcinoma cell lines in mice left feet-pads,after five weeks post-implantation,the mice were harvested,primary and secondary tumors of each group were collected separately.We determined that the size of primary tumors and the number of metastatic lymph nodes in Hca-F was greater than in Hca-P mice.Furthermore,in Annexin 7 modulated groups;the size of primary tumors and the number of metastatic lymph nodes was less than Hca-F,regarding in the Hca-PAnnexin7-down-regulated group,the primary tumor was bigger than Hca-P,whereas,there is no difference in the numbers of metastatic lymph nodes.Hematoxylin and Eosin stain was applied to evaluating tissue histological abnormalities.The results have been shown;there was no histological abnormality on the feet and the lymph nodes of the normal control mice.But,the high tissue necrosis was observed in association with primary and secondary tumors of Hca-F mice comparing to Hca-P mice.About Annexin7 regulated groups,tissue abnormalities on A7-up-regulated-Hca-F was less than Hca-F mice in both primary and secondary tumors.In the case of A7-downregulated-Hca-P,the histological damage in primary tumor was massive than Hca-P,while,there are no differences in lymphatic metastatic tissues.In-vivo experiments of Ezrin expression gave credit to our in-vitro findings,thats Ezrin expression was positively correlated with LNM-HCC and negatively with Annexin7.Western blot results registered significant differences in Ezrin expression between the collection primary and collection secondary tumors among the different groups(p=0.0001),as well as,Annexin7 expression(p=0.021).Immunohistochemistry results showed;high Ezrin expression in Hca-P A7 downregulated primary tumors comparing to Hca-P primary tumors,whereas,Low Ezrin expression was observed in Hca-F-A7-up-regulated secondary tumors comparing with Hca-F secondary tumors(P= 0.004).Serum ELISA test illustrated the differential expression of Ezrin among the study groups(P= 0.0005).Also,serum Ezrin expression was higher in Hca-F than Hca-P(p=0.0090),While,serum Ezrin expression was suppressed in Hca-F-A7-up-regulated group comparing to Hca-F group(P=0.0010),it was enhanced in the case of Hca-P-A7-down-regulated group(P=0.0510).Conclusion l The subcellular localization of Ezrin and Annexin7 proteins were varied according to lymphatic metastasis levels,that's,Ezrin expression and localization could help on determining the level of lymphatic metastasis hepatocellular carcinoma which Ezrin always associated with the lymphatic metastasis and aggressiveness of the disease.l Ezrin may play a role in lymphatic metastasis hepatocellular carcinoma and might be inversely associated with Annexin 7 expression.l Ezrin expression was a negative association with Annexin7 expression in lymphatic metastasis hepatocellular carcinoma.l Ezrin expression in the subcellular compartments of LNM-HCC is directly associated with the grade of lymphatic metastasis in both in-vitro and in-vivo models.Ezrin level could be fast,easy and sensitive assessed in tissue and serum samples.Ezrin level could be lighting a clear prediction and help to take a good decision about the kind of treatment and choosing a suitable treatment protocol.l Ezrin level may serve as a differential diagnostic and/or prognostic biomarker for high and low LNM-HCC and may be beneficial in early recognition of HCC disease.l In high metastatic cells,Ezrin comparable was highly concentrated in the cytoskeleton comparable.However,in Hca-P cells,it was mostly located was mostly in the membranes and nucleus.Whereas,high nuclear localization coupled with low cytoskeletal Ezrin expression in low metastasis.The features of flexibility,concentration,and cross-linkage of cytoskeleton-dependent and filament sizeare necessary for cellular appearance and cellular interactions.That's,cytoskeletal Ezrin might play a role in the regulation of dynamic reformation of actin cytoskeleton cross-linkageand subsequent cellular activities as cells migration and invasion.l The linkage between high total Ezrin,high cytoskeletal Ezrin and high LNM-HCC as compared with low total Ezrin,low cytoskeletal Ezrin and low LNM-HCC suggest as the major role for Ezrin in lymphatic metastasis mechanisms.l A7 is inversely associated with cytoskeletal Ezrin expression,cell migration,and invasion.This could be a contributing factor to lymphatic metastasis of HCC.l A7 could suppress the expression of Ezrin to suggest that it might have tumor suppressor activity and could be targeted for direct or indirect therapeutic activities.