| Purpose:Invasion,metastasis and drug resistance of liver cancer is the main reasons for poor prognosis and progression.Hepatocellular carcinoma is a typical vascular malignant tumor with angiogenic mimicry.Regorafenib was approved by the FDA in2017 as a second-line treatment for unresectable liver cancer,but the therapeutic efficacy and mechanism of Regorafenib in HCC metastasis are still unclear.The first part of this thesis aims to investigate the effect of Regorafenib on the invasion,metastasis and the formation of VM of HCC cells and to explore the relevant molecular mechanisms.With long-term medication,Regorafenib faces the risk of drug resistance.In the early stage,we have screened Regorafenib-resistant HCC cells,but its drug resistance mechanism still needs in-depth study.The second part of this thesis aims to characterize the invasion,metastasis and VM formation of Regorafenib-resistant HCC cells,to screen potential targets for the reversal of Regorafenib resistance and to explore its related mechanisms.The above studies will provide effective strategies for the treatment of recurrence and metastasis of HCC and the rational use of Regorafenib.Method:1.Part 1:(1)CCK8,Transwell chamber,tube formation and Western blot experiments were used to investigate the effects of Regorafenib on the proliferation,migration and invasion,VM formation,EMT and VM-related protein expression of HCC cells;(2)RNA-seq,Western blot and q RT-PCR were used to screen and verify the potential targets of Regorafenib in the treatment of HCC.CETSA,gene knockdown,and rescue experiments were used to prove that ID1 is the target of Regorafenib;(3)Western blot,immunohistochemistry,CD34-PAS double staining were used to analyze the correlation between ID1,EMT and VM protein expression in HCC samples;(4)Gene sh RNA and overexpression experiments were used to explore the mechanism of ID1 regulating Snail-mediated hepatocarcinoma cell proliferation,migration,invasion and VM formation,and above results were further verified in the model of lung metastasis via the tail vein in nude mice;(5)The effects of Regorafenib on EMT and VM were investigated in HCC PDX models.2.Part 2:(1)MHCC-97H/Rego drug-resistant cells was induced by Regorafenib with low concentration gradients intermittently.CCK8,Transwell chamber,tube formation and Western blot experiments were used to analyze the drug resistance,proliferation and migration,invasion,VM formation,EMT and VM-related protein expression of drug-resistant cells;(2)RNA-seq,Western blot and q RT-PCR were used to screen and verify that CD90 is an important target of Regorafenib-resistant HCC cells;(3)Tumor formed subcutaneously in nude mice and CD34-PAS double staining were used to investigate the tumor growth and VM structure of drug-resistant cells,Western blot and immunohistochemistry were used to investigate the expression of CD90,EMT and VM-related proteins in tumor tissues;(4)Gene sh RNA experiment was used to investigate the influence of CD90 knockdown on the characteristics of drug-resistant cells,and it was further verified in a lung metastasis model of nude mouse;(5)The GEPIA database was used to analyze the correlation between CD90 and Pin1 expression in clinical liver cancer samples,and Western blot was used to detect the effect of CD90knockdown on Pin1 expression in drug-resistant cells;(6)Western blot was used to analyze the correlation between Pin1 and Gli1 expression in liver cancer specimens,and further use of gene sh RNA,overexpression and point mutations,Co-IP,GST-pull down,CHX and other experiments to explore the Pin1/Gli1 signal axis for the influence and mechanism of drug-resistant cell characteristics;(7)CCK8,Transwell chamber,tube formation and Western blot experiments were used to investigate the reversal of Dasatinib on drug-resistant cell proliferation,migration and invasion,VM formation,EMT and VM-related protein expression.Result:1.Part 1:(1)Pretreatment of HCC cells with Regorafenib for 48 h,the cell migration,invasion and VM formation ability was weakened,but the cell proliferation ability was not affected;Regorafenib inhibited the expression of EMT-related proteins Snail,Vimentin and VM-related protein VE-cadherin.(2)Regorafenib significantly down-regulated the expression of ID1 based on RNA-seq;CETSA experimental results showed that the thermal stability of ID1 was enhanced after combining with Regorafenib,ID1 knockdown reduced the inhibitory effect of Regorafenib on the migration of HCC cells,and ID1 rescue could restore the effect of Regorafenib on the ability of cell migration.These results indicates that ID1 is a direct target of Regorafenib.(3)ID1 was positively correlated with Vimentin and VE-cadherin expression in clinical liver cancer specimens,and negatively correlated with E-cadherin expression;In liver cancer tissues with high ID1 expression,there were more CD34~-PAS~+VM positive cells than tissues with low ID1 expression.(4)ID1 and Snail expression were positively correlated;after ID1 knockdown,HCC cell migration,invasion,VM formation and metastasis ability in vivo were weakened.Snail overexpression could enhance the migration and invasion and VM formation ability of sh ID1 cells,and restore its EMT and VM associated protein expression.ID1overexpression enhanced EMT,VM-related biological behavior and protein expression of HCC cells,and Snail knockdown could inhibit its function.(5)In the PDX model of HCC,after 15 mg/kg Regorafenib treatment,the tumor volume becomed smaller and the VM structure decreased.Regorafenib significantly inhibited the expression of ID1,Vimentin,Snail,and VE-cadherin in tumor tissues and promoted the expression of E-cadherin.2.Part 2:(1)MHCC-97H/Rego drug-resistant cells were successfully screened.The drug-resistant cells had the characteristics of enhanced proliferation,migration,invasion and VM formation,and increased expression of EMT and VM-related proteins.(2)CD90 was high expressed in HCC Regorafenib-resistant cells based on RNA-seq,suggesting that CD90 is an important target for reversing Regorafenib resistance of HCC.(3)Regorafenib-resistant cells of HCC had enhanced tumor-forming ability in vivo,CD34~-PAS~+cells in the tissues increased,and the expression of CD90,Snail,Vimentin and VE-cadherin increased.(4)CD90 knockdown reduced the migration,invasion and VM formation ability of Regorafenib-resistant cells of HCC,reduced lung metastasis ability in vivo,and down-regulated the expression of Snail,Vimentin and VE-cadherin.(5)The GEPIA database analysis showed that CD90 was positively correlated with Pin1 expression,and CD90 knockdown could reduce Pin1 expression.(6)Pin1 knockdown reduced the migration and invasion of drug-resistant cells,weakensed the ability of VM formation,and down-regulateed EMT and VM-related proteins.Pin1 overexpression enhanced EMT and VM-related biological behaviors of HCC cells and up-regulated the expression of EMT/VM-related proteins.In liver cancer specimen,Pin1 was positively correlated with Gli1,Pin1 interacted with Gli1 and regulated the stability of Gli1 protein,mediated drug-resistant cell migration and invasion,VM formation and related protein expression.(7)Dasatinib could reduce the migration and invasion of HCC Regorafenib-resistant cells,weaken the formation of VM,down-regulate the CD90/Pin1/Gli1 signal and downstream EMT and VM related proteins,suggesting that it can be used to reverse Regorafenib resistance.Conclusion:Regorafenib inhibits the invasion,metastasis and VM formation of HCC cells by targeting ID1-mediated EMT,providing theoretical support for Regorafenib in the treatment of HCC metastasis.CD90 affects the expression of Pin1 and regulates the invasion,metastasis and VM formation of HCC Regorafenib-resistant cells.Dasatinib can reverse Regorafenib resistance in liver cancer by inhibiting the CD90/Pin1/Gli1 axis,revealing a new molecular mechanism of Regorafenib resistance.This study provides a treatment strategy for the recurrence and metastasis of liver cancer,and has guiding significance for the rational use of Regorafenib and the reversal of drug resistance in the clinic. |
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