Experimental Studies Of The Function Of KLK7 In The Proliferation And Servival Of Pancreatic Cancer Cells

Background The extracellularly secreted protein,Human tissue kallikrein-related peptidases 7(Kallikrein 7,KLK7),a member of the Kallikrein-related peptidases family(KLKs),is abnormally expressed in a variety of malignant tumors.Researchers have shown that high expression of KLK7 gene plays a crucial role in the proliferation,migration,and invasion of pancreatic cancer cells.And our previous study revealed that Compound 42,a suicide substrate inhibitor of KLK7,can significantly block the proliferation and induce ferroptosis in pancreatic cancer cells.In this study,we will focus on the KLK7 gene to further elucidate its vital role in the growth of pancreatic cancer cells.Objective To investigate the effects of KLK7 gene inhibition in various ways on the growth of pancreatic cancer cells.And to clarify the correlation between the KLK7 gene and the ferroptosis of pancreatic cancer cells.and also to uncover the mechanism between the KLK7 and the cell cycle of pancreatic cancer cells.As well as to provide some theoretical support for the future treatment of pancreatic cancer.Methods(1)This study will be followed through by inhibiting the pancreatic cancer cells’ expression of the KLK7 gene by si RNA,sh RNA and CRISPR-CAS9 and the level of KLK7 gene was detected by q RT-PCR.(2)The Real-time cell analysis(RTCA)machine was used to track the proliferation of cells within 72 hours.(3)Western blot was used to detect the protein levels of proliferation cell nuclear antigen(PCNA),glutathione peroxidase 4(GPX4),ribonucleoside-diphosphate reductase subunit M2(RRM2),recombinant thioredoxin reductase 1(TXNRD1),and thioredoxin(TXN)in each group of cells.(4)The flow cytometry was performed to evaluate the effect of apoptosis and necrosis,lipid peroxidation level,and cell cycle.(5)The enzyme marker was used to detect the level of the intracellular iron(6)The comet assay was conducted to observe and ensure cell DNA damage in pancreatic cancer cells after being treated with Compound 42.Results1.Compared with WT cells and negative control cells,the KLK7 m RNA levels of all the KLK7 inhibition groups were greatly decreased.2.The proliferation ability of all KLK7 inhibition groups was considerably suppressed.The RTCA results showed that compared to the control group,the proliferation ability of each KLK7 inhibition group decreased significantly within 72 hours.At the same time,the PCNA protein level in the KLK7 inhibition group decreased after si RNA treatment.3.Inhibited the KLK7 expression of pancreatic cancer cells by si RNA would induce ferroptosis.The flow cytometry results showed that compared to the control groups,there was no significant apoptotic or necrotic observed.Whereas,the test of intracellular Lipid reactive oxygen species(Li-ROS),GPX4 protein levels,and intracellular iron levels all revealed that ferroptosis was induced in the inhibited groups.4.Inhibited the KLK7 expression of pancreatic cancer cells stably does not cause ferroptosis.Compared to the control groups,although the intracellular iron levels in each KLK7 inhibition group showed an upward trend,there were no significant differences in Li-ROS and GPX4 protein levels in the later-generation PANC-1 KD and KO cells.5.Inhibition of KLK7 expression by si RNA did not affect the cell cycle of pancreatic cancer cells.Flow cytometry results showed that there was no difference in cell cycle between the KLK7 inhibition group and the control group after si RNA treatment.6.Compound 42 caused cell cycle arrest in pancreatic cancer cells by inducing DNA damage and reducing the DNA repair capacity of cells.Transcriptomics and proteomics,as well as western blot suggested that there was a significant downregulation of RRM2 protein level in the Compound 42 treatment group compared to the control group.At the same time,Comet assay showed that DNA damage was observed in the Compound 42 treated groups.Further detection by western blot revealed that both TXN and TXNRD1 proteins were significantly upregulated,which was not observed in the si RNA treatment group.Conclusion1.Inhibition of KLK7 expression in pancreatic cancer cells significantly inhibited the proliferation ability of pancreatic cancer cells.2.Inhibition of KLK7 expression by si RNA induced the ferroptosis in pancreatic cancer cells,whereas inhibition of KLK7 expression stably did not lead to ferroptosis of cells.3.Inhibition of KLK7 expression by si RNA did not block the cell cycle of pancreatic cancer cells.4.Compound 42 arrest the cell cycle of pancreatic cancer cells by inducing DNA damage and decreasing the DNA repair ability.