Study Of Regulation Mechanism Of Invasion And Metastasis Via ELMO1Expression In Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignancies in the world. Because of the high incidence, high recurrence and high mortality, HCC has already become an important risk for the people’s health all over the world. Metastasis and recurrence in HCC patients who have been implemented operation have been the key issues that prevent the improvement of prognosis.Therefore, studing molecular mechanism and exploring further inhibition of invasion and metastasis are of great importance in the improvement of HCC postoperative survival.ELMO1(engulfment and cell motility protein1) is a class of newly discovered transmembrane protein. Recent studies have shown that a complex ELMO1and Dock180play a key role in activating Racl, which promote cell phagocytosis, suggesting that ELMO1may involve in invasion and metastasis of cancer. Nck(adaptor protein)is an important binding protein of WASP family. Nck can cause tyrosine phosphorylation of ELMO1, which has a powerful ability in the regulation of cell motility, suggesting that ELMO1possibly involve in the process of invasion and metastasis of HCC through interaction with Nck, but its mechanism is still unclear.In our study, the expressions of ELMO1were firstly detected in HCC tissues, para-cancer tissues and normal liver tissue samples. Further, to investigate the role and molecular mechanism of ELMO1in regulating of invasion and metastasis of HCC, comprehensive utilization of a series of molecular biology and cell biology methods were implemented after inhibiting the expression level of ELMO1in hepatocellular carcinoma cell lines HCCLM3by RNA interference technology. Chapter I The expression of ELMO1in hepatocellular carcinoma, para-cancer and normal liverObjective To explore the expression difference of ELMO1in hepatocellular carcinoma tissues, para-cancer tissues and normal liver tissues.Methods(1) Immunohistochemical staing was employed to detect distribution and positive expression rate of ELMO1protein in57cases of hepatocellular carcinoma tissues, para-cancer tissues and15cases of normal liver tissues (NLs) were regarded as control.(2) Real-time quantitative PCR (qRT-PCR) and western blotting were respectively employed to detect expression of ELMO1mRNA and protein in57cases of fresh hepatocellular carcinoma tissues, corresponding para-cancer and15cases of NLs were regarded as control.Results(1) Immunohistochemical analysis showed that the distribution of ELMO1protein is mainly located in cytoplasm, and the positive expression rate and scores mean of ELMO1in hepatocellular carcinoma tissues were both significantly higher than those in para-cancer tissues and NLs (P<0.05). While, there is no significant difference of ELMO1positive levels between para-cancer tissues and normal liver tissues. (2) qRT-PCR and Western blotting analysis showed that expression levels of ELMO1mRNA and protein in fresh hepatocellular carcinoma tissues were significantly higher than those in the corresponding para-cancer non tumor tissues and NLs (P＜0.05). But there is no significant difference of ELMO1levels between para-cancer tissues and NLs.ConlusionELMO1is highly expressed in hepatocellular carcinoma tissues. But it is lowly expressed in para-cancer tissues and NLs. And there is no significant difference in expression levels of ELMO1between para-cancer tissues and NLs. Chapter Ⅱ Target to ELMO1expression effect on the biological behavior of hepatocellular carcinoma cells by small interfering RNAiObjective To investigate the expression of ELMO1effect on the biological behavior of hepatocellular carcinoma cells.Methods(1) A retroviral siRNA expression vectors pSUPERELMO1RNAi+and placeb plasmid pSUPERELMO1RNAi-were constructed, then transfected into PT67cells to acquire recombinant retroviral particles, further transfected into HCCLM3cell lines.(2) Western blotting assay was employed to detect the expression of ELMO1protein in L02normal liver cell lines and HCCLM3ELMO1RNAi+, HCCLM3ELMO1RNAi-cell lines.(3) Wound-healing assay, transwell invasion assay, adhesion test were employed to detect migration, invasion and adhesion in HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cell lines.(4) immunofluorescence assay was employed to detect cell morphology and pseudopodia in HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cell lines.(5) MTT, colony formation and flow cytometry assay were employed to detect proliferation and apoptosis in HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cell lines.Results(1) We got satisfactory transfection efficiency and successfully acquired HCCLM3ELMO1RNA1+with downregulated expression of ELMO1and HCCLM3ELMO1RNAi-as invalid control.(2) Western blotting analysis showed that the expression of ELMO1proteins in HCCLM3ELMO1RNAi-was markedly higher than those in HCCLM3ELMO1RNAi+and L02cell lines (P<0.05), and suggested inhibition efficiency of ELMO1in HCCLM3ELMO1RNAi+was apparently higher than that in HCCLM3ELMO1RNAi-(3) The adhesion test confirmed HCCLM3ELMO1RNAi+cells had significantly higher homogeneity adhesion and lower heterogeneity adhesion ability than HCCLM3ELMO1RNAi-cells. Wound-healing assay showed that the closure of HCCLM3ELMO1RNAi+was significantly slower than that of HCCLM3ELMO1RNAi-. Transwell invasion assay showed the number of HCCLM3ELMO1RNAi+cells that passed through matrigel was fewer as compared with HCCLM3ELMO1RNAi-cells (P＜0.05).(4) Cell immunofluorescence assay showed that there was significant difference in cell morphology and pseudopodia between HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cells (P<0.05).(5) There was no significant difference in cell proliferation and apoptosis between HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cells through MTT, colony formation experiments and flow cytometry assay (P>0.05).ConlusionThe down-regulated expression of ELMO1may significantly inhibit invasion and metastasis of hepatocellular carcinoma cell lines in vitro, but may not affect the proliferation of hepatocellular carcinoma cell lines. Chapter Ⅲ The molecular mechanism of ELMO1control invasion and metastasis in hepatocellular carcinomaObjective To study the phosphorylation sites between ELMO1and Nck and investigate the molecular mechanisms which control invasion and metastasis of HCC cells.Methods(1) Western blotting assay was employed to detect the expression of Racl, Fibronectin and Nck protein in HCCLM3ELMP1RNAi+and HCCLM3ELMO1RNAi+cells lines.(2) One wild-type plasmid (pcDNA-HA-ELMOl) and five mutation plasmid (pcDNA-HA-ELMO1-Y18F、Y216F、Y395F、Y511F、 Y720F) were constructed and transfected into HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cell lines.(3) Co-immunoprecipitation technique and western blotting were used to detect tyrosine phosphorylation site between ELMO1and Nck.Results(1) Western blotting analysis showed that the expression of Rac1and Nck protein in HCCLM3ELMO1RNAi+cells were both significantly lower than those in HCCLM3ELMO1RNAi-cells. But the expression of Fibronectin protein in HCCLM3ELMO1RNAi+cells was significantly higher than that in HCCLM3ELMO1RNAi-cells (P<0.05). (2)With mutant and wild type plasmids co-transfected into HCCLM3ELMO1RNAi+and HCCLM3ELMO1RNAi-cells, Tyrosine sites Y720F of ELMO1were confirmed to be interaction site between ELMO1and Nck by co-immunoprecipitation and Western blotting.Conclusion(1) Down-regulated expression of ELMO1may cause Rac1and Nck protein levels to be down-regulated, while induce Fibronectin protein levels to be up-regulated, which play an important role in cell adhesion and invasion of hepatocellular carcinoma cell.(2) Tyrosine sites Y720F of ELMO1may regulate the tyrosine phosphorylation between ELMO1and Nck.(3) ELMO1-Nck signaling pathway play an important role in regulating motility of hepatocellular carcinoma cell, suggesting this signaling pathway is expected to be a target of potential drug which prevent recurrence and metastasis of HCC.