NKAP Regulates Growth And Metastasis Of Pancreatic Cancer Through MTOR Pathway And Its Sensitivity To Gemcitabine

Research BackgroundPancreatic adenocarcinoma(PAAD)is one of the most common malignant tumors of the digestive system and a major global public health concern because of its poor clinical prognosis,due to lack of effective treatment,metastasis,recurrence and chemotherapy resistance.Genetic and epigenetic abnormalities are commonly found in pancreatic cancer and are associated with abnormal activation of tumor driver genes.Therefore,the search for molecular proteins related to the occurrence and development of pancreatic cancer is of great significance to improve the overall survival rate of pancreatic cancer patients.NKAP(NF-κB activating protein)is a highly conserved nuclear protein with a molecular weight of 47 kd.This molecule was first discovered in yeast two-hybrid screening.As for its functional annotation,NKAP was initially found to be related to the development of T cells and its role in the immune system.With further study,it was found that NKAP is a transcription factor capable of regulating the expression of a large number of genes,including some genes involved in immune regulation,inflammatory response and anti-apoptotic effect.In particular,genes involved in activating the NF-κB pathway are regulated by TNF and IL-1.In recent years,NKAP has opened up a new and very promising research direction in the field of malignant tumor.NKAP has been confirmed to play a role in cancer as an oncogene mediated by AKT/mTOR,Notch and NF-κB signaling pathways.This study aims to clarify the expression of NKAP in pancreatic cancer,explore the effect of NKAP on malignant phenotypes such as proliferative activity,migration and invasion,apoptosis level of pancreatic cancer cells and the mechanism of action on chemotherapy drug sensitivity,and reveal the effect of NKAP on biological function of pancreatic cancer cells.It provides theoretical basis and new drug target for accurate treatment of pancreatic cancer.Research methods:Gene Expression Profiling Interactive Analysis(GEPIA)and DepMep database were used to preliminally analyze the mRNA expression of NKAP in pancreatic cancer tissues and the protein expression level in pancreatic cancer cells.Western blot and real-time quantitative polymerase chain reaction(qRT-PCR)were used to detect differences in the expression of NKAP protein and mRNA in pancreatic cancer cell lines.si NKAP and PEX-3 overexpression plasmids were used to establish knockdown and overexpression models of NKAP in pancreatic cancer cells.A series of in vitro experiments,including CCK-8,clonogenesis,Transwell,matrix gel assay and flow cytometry,were conducted to determine the effects of NKAP on the cell biological functions of pancreatic cancer cells,such as the proliferation ability,migration and invasion ability,apoptosis level and so on.Later,this study explored how NKAP regulates the biological function of pancreatic cancer cells through signaling pathways,predicted the related signaling pathways of PAAD regulation by NKAP through bioinformatics combined with existing literature studies,and identified mTOR as the main signaling pathway for regulation.The effect of NKAP knockdown or overexpression on mTOR phosphorylation and total protein expression was detected by western blot.Then,the mTOR pathway inhibitor Rapamycin(RaPa)was used to perform rescue experiments,and the regulatory effect of NKAP on mTOR signaling pathway was verified in reverse.At the same time,CCK-8,Transwell and western blot were used to detect the changes of pathway inhibitors on the proliferation,migration and invasion ability of pancreatic cancer cells and the expression levels of related molecular proteins.In addition,combined with the current status of clinical diagnosis and treatment,this study also explored the regulatory influence of NKAP on the sensitivity of PAAD chemotherapy drugs.CCK-8 and flow cytometry were used to observe the changes of the effects of chemotherapy drug gemcitabine on the proliferation ability and apoptosis level of pancreatic cancer cells after NKAP knockdown.Research results:1.Bioinformatics results showed that NKAP was highly expressed in pancreatic cancer tissues.Western blot and qRT-PCR results showed that compared with normal pancreatic duct epithelial cells,NKAP was highly expressed in pancreatic cancer cell lines(P<0.05).2.CCK-8,clone formation,Transwell,matrix gel test and flow cytometry showed that knockdown NKAP could inhibit the proliferation,migration and invasion of pancreatic cancer cells,block cell cycle progression and induce apoptosis,while overexpression of NKAP showed opposite detection results(P<0.05),indicating that NKAP could regulate the growth and metastasis of pancreatic cancer cells.3.GEIPA prediction showed a correlation between NKAP and mTOR signaling pathway in pancreatic cancer(P<0.05,R=0.3).western blot results showed that NKAP knockdown could inhibit the phosphorylation level of mTOR and the expression of mTOR downstream target p70S6 K,while overexpression of NKAP could significantly up-regulate the expression of above molecules(P<0.05).Subsequently,the mTOR pathway inhibitor RaPa was used to carry out rescue experiments.Both CCK-8 and Transwell results showed that RaPa could reverse the promoting effect of overexpression of NKAP on the proliferation and migration of pancreatic cancer cells(P<0.05).Protein level experiments also showed that RaPa could reverse the up-regulation effect of overexpressed NKAP on the expression of signaling pathway proteins and cycle-related proteins,suggesting that the effect of NKAP on the growth and metastasis of pancreatic cancer cells is regulated through the mTOR signaling pathway.4.The detection results of CCK8 and flow cytometry showed that knocking down NKAP could increase the inhibition rate of gemcitabine on pancreatic cancer cells and increase the level of apoptosis of cells(P<0.05),indicating that regulating the expression of NKAP could affect the cytotoxicity of gemcitabine on pancreatic cancer cells.Research conclusion:This studie have shown that NKAP is highly expressed in pancreatic cancer cells and regulates the growth and metastasis of pancreatic cancer cells through mTOR signaling pathway;It was found that knocking down NKAP could increase the killing effect of gemcitabine on pancreatic cancer cells.