The Role And Mechanism Of KLK7 In Invasion And Metastasis Of Pancreatic Cancer

Background Globally,pancreatic cancer is the twelfth most common malignant tumor and the seventh most lethal cancer,with a 5-year survival rate of only 10%.At present,the main strategy for the treatment of pancreatic cancer is surgical resection,but only 15～20% of patients are suitable for surgical resection.Because it is difficult to detect pancreatic cancer at early stage,nearly 80% of patients had distant metastasis by the time of diagnosis.Although systematic chemotherapy can delay the local progress and metastasis of pancreatic cancer,it does not prolong the overall survival time of patients.Previous studies have shown that human tissue kallikrein 7(KLK7)can affect the migration and invasion of pancreatic cancer cells.Therefore,studying the mechanism of KLK7 affecting the invasion and metastasis of pancreatic cancer may help to explore effective treatment strategies for pancreatic cancer.Objective To inhibit the activity and expression of KLK7,observe the changes of invasion and metastasis of pancreatic cancer,and initially explore the corresponding molecular mechanism,so as to provide a new therapeutic strategy for the treatment of pancreatic cancer.Methods(1)After transfection of siRNA and shRNA knocks down the expression of KLK7 in pancreatic cancer PANC-1 cells and BxPC-3 cells,cell scratch test and real-time cell analysis(RTCA)test were used to detect the changes of migration and invasion ability.(2)The research group used CRISPR/Cas9 gene editing technology to construct KLK7 knockout PANC-1 cells(KO)in the early stage.Scratch experiments were used to detect changes in the migration ability of KO cells,and RTCA experiments were used to detect changes in the migration and invasion ability of KO cells.(3)After inhibiting the activity of KLK7 in pancreatic cancer PANC-1 cells and BxPC-3 cells with compound 42(C42),a KLK7 targeted inhibitor synthesized by our research group in the earlier stage,we used scratch test to detect the change of Cell migration ability.After treating BxPC-3 cells with different concentrations of C42,RTCA experiments were performed to detect changes in their migration and invasion abilities.(4)After inhibiting KLK7 activity and knocking down KLK7 expression in PANC-1cells and BxPC-3 cells,Western blot was used to detect changes in phenotype related indicators of interstitial cells.(5)Four-week-old BALB/c nude mice were used to construct the liver metastasis model of pancreatic cancer with PANC-1 cells.Randomly divide the constructed nude mouse model into a DMSO control group and a C42 medication group,and observe the mental state and weight changes of the nude mice.After the experiment,draw the survival curve of nude mice and observe the structural changes of important tissues and organs in each group using H&E staining;Observation of the expression of KLK7,ki67,and stromal cell phenotype related indicators in liver metastases by IHC.(6)Four-week-old BALB/c nude mice were used to construct the liver metastasis model of pancreatic cancer(KO)with KO cells,and the control group was the liver metastasis model of pancreatic cancer(WT)with wild type PANC-1 cells.After the experiment,draw the survival curve of nude mice and observe the structural changes of important tissues and organs in each group using H&E staining;IHC observed the expression of KLK7,ki67,and stromal cell phenotype related indicators in liver metastases.Results(1)After transfection of siRNA and shRNA and reduction of KLK7 expression,the migration and invasion ability of pancreatic cancer PANC-1 cells and BxPC-3 cells were significantly reduced.(2)After KLK7 gene knock-out was performed with CRISPR/Cas9 gene editing technology,the migration and invasion ability of KLK7 cells were significantly decreased.(3)C42(50 μM,100 μM)After acting on pancreatic cancer PANC-1 cells and BxPC-3 cells,the scratch test results showed that the migration ability of the two cells was reduced.BxPC-3 cells were treated with C42 at concentrations of 22 μM and 44 μM.RTCA results showed that the migration and invasion ability of BxPC-3 cells were decreased.(4)After treatment with C42(50 μM,100 μM)and siRNA,compared with the control group,the expression of N-cadherin,Vimentin,α-SMA and MMP9 in PANC-1 and BxPC-3 cells was significantly decreased.(5)Compared with the DMSO control group,there was no significant difference in the survival rate and body weight of nude mice treated with C42;There were no significant changes in the heart,spleen,lungs,kidneys of nude mice;The number of liver metastases decreased significantly;Compared to the liver tissue adjacent to the metastatic lesion,the expression of ki67 and KLK7 in the metastatic lesion was significantly increased;The expression of N-cadherin,Vimentin,α-SMA and MMP9 was significantly reduced in liver metastases.(6)Compared with the WT group,there was no significant difference in survival rate and body weight of nude mice in the KO group;There were no significant changes in important organs such as the heart,spleen,lungs,and kidneys of nude mice;The number of liver metastases significantly decreased;Compared to the liver tissue adjacent to the metastatic lesion,the expression of ki67 and KLK7 in the metastatic lesion was significantly increased;The expression of N-cadherin,Vimentin,α-SMA and MMP9 was significantly reduced in liver metastases.Conclusion(1)Inhibiting the activity of KLK7 and knocking down the expression of KLK7 can inhibit the migration and invasion of pancreatic cancer cells at the cell level by reducing the expression of phenotype related proteins in mesenchymal cells.(2)Inhibiting the activity of KLK7 and knocking out the expression of KLK7 can inhibit the liver metastasis of subcutaneous transplanted tumor of pancreatic cancer in nude mice at the animal level by reducing the expression of phenotype related proteins in mesenchymal cells.