| Hepatocellular carcinoma (HCC) is the sixth common malignant solid tumors and the third cause of cancer-related death in the world. Especially in China, it ranks second in various tumors'lethality. Frequent recurrence and metastasis is the determent factor causing failure in HCC therapy. At present, exairesis is still the effective therapy for HCC, but its outcome is very limited. In general, HCC's recurrence rate reaches as high as 61.5% within 5 years after radical excision, even if the small liver cancer also reaches 43.5%. The recurrence and metastasis of HCC is a multigenetic, multifactoral and complicate process. In this process, an initial step of tumor cell migration is the detachment of epithelial cells from the extracellular matrix, which normally trigger an apoptotic processes termed anoikis. Now, anoikis is thought to play a safeguard role in metastasis and determined its frequence. However, metastatic tumor cell is commonly resistant to anoikis and able to survive in the absence of normal matrix adhesion.OPN is a secreting glucoprotein and plays critical role in cell chemotaxis, adhesion and migration. Previous researches showed that OPN is close relative with HCC metastasis, but its regulative mechanism remains underdetermined. Therefore, it is very important for elucidating anoikis'role in HCC metastasis to explore the OPN and the pathways trigged by it. This is a hopeful route to successful HCC therapy and will give opportunities for target therapy for multiple malignant tumors.Part oneRelative analysis of OPN and its related signal molecules with HCC metastatic potential. Object: to analyze the expression of OPN, intergrinaV, CD44v6, P-FAK, FAK, P-Src, Src, P-ERK, P-AKT,further to explore the relationship between them and HCC metastatic potential. Methods:the expression of OPN, intergrinaV, CD44v6, P-FAK, FAK, P-Src, Src, P-ERKand P-AKT were assayed through the using of TMA analysis. The correlation of them with HCC's clinical attributes was drawn out from statistical data of TMA staining. Results:1. In HCC tissue, there were no significance among OPN's expression with patients'sex, age, tumors's size and number, level of AFP, HBV transfection, tumor's differentiation, degree of cirrhosis, while it had close relation with tumor's TNM grade, tumor capsule and portal vein tumor thrombi (P<0.05).2. The expression of CD44v6 had no significant relationship with patients' sex, age, tumors's size and number, level of AFP, HBV transfection, tumor's differentiation, degree of cirrhosis and tumor capsule, while it had close relation with tumor's TNM grade and portal vein tumor thrombi (P<0.05)。3. The expression of P-Src had no significant relationship with patients'sex, age, tumors's size and number, level of AFP, HBV transfection, tumor's TNM grade,portal vein tumor thrombi, degree of cirrhosis and tumor capsule,, while it had close relation with tumor's differentiation, (P<0.05)。4. The expression of Src and p-ERK had no significant relationship with patients'sex, age, tumors's size and number, level of AFP, HBV transfection, tumor's TNM grade, portal vein tumor thrombi, degree of cirrhosis and tumor capsule, while they had close relation with tumor's differentiation (P<0.05)。5. The expression of P-Akt had no significant relationship with patients'sex, age, tumors's size and number, level of AFP, HBV transfection, tumor's differentiation, portal vein tumor thrombi and degree of cirrhosis, while it had close relation with tumor's TNM grade and tumor capsule (P<0.05)。6. The expression of Integrin aV had no significant relationship with patients'sex, age, tumors's size and number, level of AFP, HBV transfection, tumor's TNM grade, tumor capsule, tumor's differentiation,portal vein tumor thrombi and degree of cirrhosis。Conclusion:OPN expression is the indicator of HCC recurrence/metastasis after surgery. Besides, it was showed thataV, CD44v6, pSrc, pFAK, pAKT played crucial role in HCC metastasis. Part twoRelative analysis of OPN and its related signal moleculesObject: to study the relationship of OPN与IntegrinαV,CD44v6, P-FAK, P-Src,P-ER,andP-AKT and to study the possible pathways of OPN in HCC metastasis. Methods:based on the TMA analysis from part one, the relationship of OPN and pERK, pSrc and pAKT were explored and their role in HCC metastasis were also discussed in this study. Results:OPN was significantly associated with Integrinav,CD44V6 and P-ERK.Though P-FAK,P-Src,P-AKT were not significantly associated with OPN, but they were significantly associated with OPN's receptors Integrinav and CD44V6.Conclusion:OPN activated the pathways of PI3K/AKT and Raf/MEK/ERK through its receptors Integrinav and CD44V6 in HCC metastais.Part threeThe Significance of Osteopontin (OPN) in Anoikis Resistance of Hepatocellular Carcinoma.Objective:Osteopontin (OPN) has been thought as involving in cancer metastasis including hepatocellular carcinoma (HCC). However, the underlying mechanisms are not fully understood. Cancer cells usually are resistant to anoikis (apoptosis induced after detachment from extracellular matrix) which is related with metastasis potential. Here, a high metastasis potential HCC cell line (MHCC-97H) was studied for sensitivity to anoikis after OPN was inhibited with small interfere RNA (RNAi) so as to clarify mechanisms of OPN promoting HCC metastasis. Methods:MHCC-97H cells were cultured in suspension condition in the Poly-Hemo coated dishes and the sensitivity to anoikis was observed after three specific RNAis (RNAi1,RNAi2,RNAi3) of OPN were transfected via Lipofectamine. The anoikis was measured with Flow cytometry (FCM) labeled with Annexin V/PI. The downstream signals of OPN were checked for phosphor-FAK (tyr397), phosphor-Src (tyr416) and phosphor-ERK (thr202/tyr204) with western blotting.Results:MHCC cells were resistant to anoikis when cultured in suspension and the sensitivity to anoikis was significantly restored after OPN was inhibited with RNAi2 and RNAi3 (p<0.05) followed with OPN protein expression inhibition. Phosphor-FAK, phosphor-Src and phosphor-ERK were significantly reduced after knockdown of OPN with RNAis.Conclusion:The present results indicate that resistant to anoikis is one of the mechanisms for OPN promoting HCC metastasis through FAK/Src-ERK pathway. |
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