Effections And Mechanism Of Isomerase Pin1 On Invasion And Metastasis Of Hepatocellular Carcinoma Cells Before And After Regorafenib Resistance

Objective: Liver cancer is the sixth-largest cancer in the world,but it has become the second leading cause of cancer-related deaths.In the first-line targeted drug Sorafenib,resistance,metastasis and recurrence of hepatocellular carcinoma have emerged.However,whether the newly approved second-line treatment drug Regorafenib is resistant and the relevant mechanisms after drug resistance are unclear.Our previous studies showed that the prolyl cis-trans-isomerase Pin1 plays an important role in the occurrence and development of liver cancer.Therefore,this article focuses on the effect of Pin1 on invasion and metastasis before and after the treatment of Regorafenib resistance in hepatocellular carcinoma cells,and discusses the relevant mechanisms from the perspective of EMT to discover potential strategies for the remission of Regorafenib resistance in liver cancer.Methods:(1)Western Blot and immunofluorescence assays were used to detect the relationship between Pin1 and EMT related protein expression in liver cancer specimens.(2)Five-plasmid system packaging knockdown of Pin1 or over-expression of lentivirus,infection of MHCC-97 H,Huh7,THLE3 cells,screening established stable knockdown or overexpression of Pin1 hepatoma cell lines.(3)Using SMMC-7721 liver cancer cells as the research object,using Regorafenib drug low concentration(0.5μM)and gradient intermittent induction method to screen out the Regorafenib resistant strain SMMC-7721-R-R;calculate the corresponding resistance Drug index(RI = IC50 for drug resistant cells/IC50 for parental cells).(4)Phase contrast microscopy and photographs were taken to collect the changes of cell morphology before and after drug resistance and Pin1 knockdown.(5)To observe the effect of Pin1 knockdown or overexpression on the biological behavior of MHCC-97 H,Huh7,THLE3,and SMMC-7721-RR cell lines: growth curve and plate cloning experiments to detect Pin1 knockdown or overexpression on cell proliferation effect;Transwell chamber assay and scratch test to detect the effect of Pin1 knockdown or overexpression on cell migration and invasion ability;(6)Western Blot and immunofluorescence assay to detect the influence of Pin1 knockdown on EMT-related protein,stem-related protein and Gli1 pathway protein.(7)To establish a tail vein injection model of lung metastasis in nude mice,HE staining and immunofluorescence were used to detect the effect of Pin1 knockdown on hepatoma cell metastasis.(8)In the Stem cell culture conditions.,The effects of Pin1 knockdown and Pin1 inhibitor ATRA on hepatoma cells of the stem viability were detected.Results:(1)In clinical specimens of liver cancer,Pin1 expression was correlated with the expression level of EMT-related proteins.(2)A hepatoma cell line stably knocked down Pin1 was successfully constructed,and the efficiency of Pin1 knockdown was high.(3)SMMC-7721 Regorafenib resistant cell line SMMC-7721-R-R was successfully constructed.(4)After Pin1 knockdown,there was a significant phenotypic change in the cells,which was reflected in the restoration of cell polarity.The cells changed from irregular shapes to regular,paved stone cells.(5)After Pin1 knockdown,cell proliferation and colony formation ability were significantly inhibited;Transwell chamber and scratch experiments showed that Pin1 knockdown reduced cell migration and invasion.(6)Western Blot and immunofluorescence showed that Pin1 expression was significantly down regulated after Pin1 knockdown.Epithelial marker E-cadherin expression was significantly up-regulated,Vimentin expression was significantly down-regulated,Snail was significantly down-regulated,and Gli1 was down-regulated.Bmi1 was significantly downregulated in the resistant strain of Regorafenib.(7)Pin1 knockdown effectively reduced the ability of cells to metastasis in nude mice.(8)The cell-forming ability of the SMMC-7721-R-R was significantly enhanced and the ability of stem was increased.After Pin1 knockdown and Pin1 inhibitor ATRA was applied,the ability is obviously suppressed.Conclusion: Pin1 knockdown can effectively inhibit the ability of migration,invasion and metastasis in vivo of hepatocarcinoma cells and Regorafenib-resistant cells,and provides effective guidance for clinical use of Regorafenib and inhibition of in vivo metastasis of Regorafenib-resistant liver cancer.The clinical guidance plays a important role for the treatment of HCC.