The Mechanism Of Notch Signaling Pathway Participating In Hepatocellular Carcinoma Cells Migration And Invasion

Hepatocellular carcinoma (HCC) is the one of the most common malignancies.Despite advances in the detection and treatment of HCC, the mortality rate remains highbecause the metastasis. Hence, to explore the mechanism of HCC cell’s migration andinvasion is more important. The main process of HCC cell’s migration and invasion is asfollows：The activity of HCC cell is enhanced; The adhesiveness of HCC cells decrease;HCC cells migrate through the extracellular matrix and vascular wall into vascularcirculation and it also can promote the formation of blood vessels.Many molecules areinvolved in this process such as Fascin, E-cadherin, MMP-2, MMP-9and VEGF. Notch isa classic signaling pathway, its family members are highly conserved in structure. Traditionally, Notch signaling pathway is mainly involved in the individual developmentand tissue regeneration. Along with the study of Notch signaling pathway goes deeper,some evidences indicate that this pathway not only participates in the development processof biological individual, but also participates in different occurrence and development oftumor. Up to now, the mechanism of Notch signaling pathway in HCC is rare reportedwhether domestic or abroad. Especially the mechanism of Notch signaling pathwayparticipating in metastasis and invasion of HCC cell is still unclear.In our experiments, we used inhibitor of Notch signaling pathway to treat differentHCC cell lines. From three parts including the change of cytoskeleton, cell migration andinvasion, we explore the mechanism that Notch signaling pathway participates in theinvasion and metastasis of HCC cell. The results suggest that Notch signaling pathwayplays an important role in invasion and metastasis of HCC cell via regulating differentmolecules. This study adds to the mechanism of Notch signaling pathway in HCC and laysthe foundation for clarifying the mechanism of invasion and metastasis of HCC.Part1. The state of Notch signaling pathway in HCC cells and the effect ofblocking Notch signaling pathway in invasion and migration of HCC cell.Object: To explore the state of Notch signaling pathway in HCC cells and the effectof blocking Notch signaling pathway in invasion and migration of HCC cell. Methods:Using Transwell assay to test the ability of cell invasion and migration; Using PCR todetect the expression of Hes1,Hes5and Hey1; Using Western Blo to detect the expressionof N1ICD; Using cell counting and MTT assay to detect cell proliferation; Using DAPT toblock Notch signaling pathway. Results: We found that the high expression of Hes1, Hes5,Hey1and N1ICD in HCC cell and it was related to the ability of invasion and metastasis;Moreover,both1μmol/L and5μmol/L DAPT could inhibit Notch signaling pathway,thiseffect can effectively inhibit invasion and migration of HCC cell but no influence on cellproliferation. Conclusion: Notch signaling pathway was activated in HCC cell and thisstate enhanced along with the ability of cell invasion and metastasis increased. However,the invasion and migration of HCC cell decreased after blocking Notch signaling pathway. Part2. The influence of blocking Notch signal pathway on HCC cell’s skeleton.Object: To investigate the mechanism of Notch signaling pathway in regulating HCCcell’s skeleton. Methods: Using immunofluorescence and scanning electron microscope todetect the change of cell skeleton and pseudopod; Blocking Notch signaling pathway viaDAPT. Results: Both1μmol/L and5μmol/L DAPT could effectively inhibit the formationof HCC cell skeleton and pseudopod and could suppress the protein expression of Fascin.Conclusion: The structure of HCC cell’s skeleton was changed and the number ofpseudopod decreased after blocking Notch signaling pathway. The potential mechanismmay be that Notch signaling pathway could regulate the expression of cytoskeletal proteinFascin.Part3. The mechanism of blocking Notch signaling to reduce HCC cellmigration.Object: To investigate the mechanism of Notch signaling pathway in HCC cell’smigration. Methods: Using Transwell assay to detecte the ability of cell’s migration；Using PCR to detect the mRNA expression of Snail and E-cadherin；Using Western Blot todetect the protein expression of Snail,E-cadherin and COX-2; Using siRNA to interferethe expression of Snail；Using DAPT to blocking Notch signaling pathway; Using NS-398to inhibit COX-2；The effect of COX-2was simulated by PGE2. Results: Both Snail andE-cadherin were involved in the migration process of HCC cells and Snail can regulate theexpression of E-cadherin. The effect of DAPT could up-regulate the expression ofE-cadherin and down-regulate the expression of Snail and COX-2. PGE2could reverse theregulatory effect of DAPT on Snail/E-cadherin. DAPT and NS-398played the same rolein the migration process of HCC cell. PGE2could reverse the inhibition of DAPT on HCCcell migration. Conclusion: Notch signaling pathway may regulate the migration processof HCC cell through regulating the COX-2/Snail/E-cadherin pathway. Part4. The mechanism of blocking Notch signaling to reduce HCC cell invasion.Object: To investigate the mechanism of Notch signaling pathway in HCC cell’sinvasion. Methods: Using Transwell assay to detect the ability of HCC cell’s invasion;Using Western Blot to detect the protein expression of MMP2, MMP-9, VEGF, ERK1/2and p-ERK1/2; The protein viability of MMP2, MMP-9, VEGF, ERK1/2and itsphosphorylation p-ERK1/2was detect ed by ELISA; Using siRNA to interfere theexpression of MMP2, MMP-9, VEGF; Using DAPT to inhibit Notch signaling pathway；Using PD98059to inhibit ERK1/2pathway. Results: MMP2, MMP-9and VEGFparticipated in the invasion process of HCC cells. The effect of DAPT coulddown-regulate the protein expression and protein viability of MMP2, MMP-9, VEGF andp-ERK1/2. In the invasion process of HCC cell, DAPT and PD98059played the sameroles. Conclusion: Notch signaling pathway may regulate the invasion process of HCCcell through regulating ERK1/2/MMP2, MMP-9, VEGF pathway.In summary, the present results demonstrated that the effect of Notch signalingpathway in HCC cell invasion and metastasis from several aspects. It also suggested thatNotch signaling pathway can participate in the process of HCC cell invasion andmetastasis via multiple pathways and targets. This study lays a theoretical foundation forrevealing the mechanism of invasion and metastasis of HCC and provides new ideas forclinical treatment of HCC.