Effect And Regulatory Mechanism Of NKAP In Colon Cancer

Colon cancer is the world's third most common malignant tumor with the second highest mortality rate,and it is also a common preventable cancer.Approximately 40%-50%of colon cancer patients still recur locally after surgery and die because of distant metastasis.In the past few years,the morbidity and mortality of colon cancer in the target population have declined,and the reason may be related to the increase in cancer screening.Unfortunately,the incidence and mortality of colon cancer have been rising among young people under 50.The early symptoms of colon cancer lack specificity,and most patients have already had lymph node metastasis or distant metastasis at the time of diagnosis.With the development of bioinformatics,gene expression profile of colon cancer has been fully characterized and many of the genetic abnormalities contributing to colon cancer,such as potential oncogenes and tumor suppressor genes,are well known.These efforts have greatly facilitated the early diagnosis and accurate treatment of colon cancer.Therefore,studying the mechanism of colon cancer cell metastasis can provide new targets for discovering the inhibition of cancer metastasis.The NKAP gene is located on human chromosome Xq24 and contains 10 exons.The protein encoded by it contains 415 amino acids and is involved in the activation of the transcription factor NF-?B.In recent years,with the deepening of research,the multiple roles of NKAP in organisms have gradually been revealed.Studies have found that as a transcription factor,NKAP plays a very important role in transcriptional inhibition.In addition,NKAP is essential for the development and maturation of T cells and the process of acquiring functional capabilities.NKAP is very important for the maintenance and survival of hematopoietic stem cells,and the lack of NKAP will lead to reduced proliferation and increased apoptosis of hematopoietic stem cells,thereby destroying stable hematopoietic function.The lack of NKAP will also affect the stability of neurogenesis,indicating that NKAP is also crucial to neurogenesis.NKAP is a key regulator of mitosis,and its imbalance may promote tumorigenesis by causing chromosomal instability.Studies have shown that NKAP can interact with RNA-binding protein,RNA helicase and splicing factors,and play an important role in RNA splicing and processing.In recent years,research on NKAP in cancer is rare.At the beginning of this study,there was no relevant information about NKAP and cancer.However,articles on breast cancer published during the course of this study indicated that NKAP is highly expressed in breast cancer tissues and tumor cell lines.Down-regulation of NKAP can inhibit the proliferation,migration and invasion of breast cancer cells,and promote the apoptosis of cancer cells.The up-regulation of NKAP will promote the proliferation,migration and invasion of breast cancer cells,and inhibit the apoptosis of cancer cells.In addition,NKAP can also promote the occurrence and development of cancer by activating signal pathways such as AKT/mTOR.Therefore,NKAP may affect various biological behaviors of tumors as an oncogene,and play an indispensable role in the occurrence and development of tumors.So far,the role of NKAP in colon cancer is unclear.Therefore,this study used immunohistochemistry,real-time fluorescent quantitative PCR,Western blot,CCK8 detection,clone formation experiment,Transwell experiment,Wound healing experiment,flow cytometry,nude mouse tumorigenicity assay and other experimental techniques to clarify the expression and clinical significance of NKAP in colon cancer.The clinical significance is to explore the influence of NKAP expression changes on the biological behavior of colon cancer and its mechanism research,which provides a theoretical basis for finding biomarkers for early diagnosis of colon cancer and gene targeted therapy.Part ?:The expression and clinical significance of NKAP in colon cancerPurpose:Colon cancer is a common cancer at present.The research on the mechanism of colon cancer occurrence,development and metastasis is of great significance for the prevention and treatment of colon cancer.As a highly conserved nuclear protein,NKAP plays a role in transcriptional inhibition,T cell maturation and functional ability,the maintenance and survival of human hematopoietic stem cells,and RNA splicing.In recent years,NKAP has been studied more and more in cancer.So far,NKAP has been studied in breast cancer,Ewing's sarcoma,glioma,liver cancer,gastric cancer and other cancers.However,the expression of NKAP in colon cancer is still unknown.Therefore,in order to study the expression and clinical significance of NKAP in colon cancer,this study firstly analyzed the expression of NKAP in colon cancer through the method of biological information.Analyze the expression difference in the adjacent tissues and the survival curve of colon cancer patients with high/low NKAP expression.Then collected the paraffin sections of colon cancer tissues and corresponding para-cancerous tissues of 75 patients diagnosed with colon adenocarcinoma,detected the expression of NKAP in colon cancer tissues,and performed the basic clinical data collected with the expression of NKAP Through the analysis of correlation,the role of NKAP in the occurrence and development of colon cancer was discussed,providing a theoretical basis for finding biomarkers for early diagnosis of colon cancer and gene targeted therapy.Materials and Methods:1.Specimen collection2.Collection of basic clinical data3.Tissue protein extraction and Western blot analysis4.Immunohistochemistry5.Statistical analysisResult:1.NKAP correlation analysis based on TCGA databaseGEPIA website analysis found that NKAP was up-regulated in colon cancer tissues compared with adjacent tissues.The prognosis of NKAP low expression group was better than that of NKAP high expression group.2.Localization and expression of NKAP in colon cancerProtein detection results showed that the expression of NKAP protein in colon cancer tissue was significantly higher than that in adjacent tissues of colon cancer.The results of immunohistochemistry further showed that the positive signal of NKAP was located in the nucleus and cytoplasm of tumor cells,and the expression of NKAP in colon cancer tissues was significantly higher than that of NKAP in adjacent tissues.3.Correlation analysis between NKAP expression and clinicopathological features and prognosis of patients with colon cancerStatistical analysis of the relationship between the expression of NKAP in colon cancer tissues of 75 patients and the clinical pathological characteristics found that the high expression of NKAP was not correlated with the age(P=0.2807),gender(P=0.1013),and lymph node metastasis(P=0.8819)of colon cancer patients,but positively correlated with tumor size(P=0.0296),T stage(P=0.0339)and clinical pathological stage(P=0.0288).The overall survival rate of 75 colon cancer patients was evaluated based on the expression of NKAP(P=0.0305).The prognosis of NKAP low expression group was better than that of NKAP high expression group.Conclusion:1.The results of GEPIA website,Western blot and immunohistochemistry showed that the expression of NKAP in colon cancer was significantly higher than that in adjacent tissues.2.The high expression of NKAP in colon cancer tissue is positively correlated with the tumor size,T stage and clinical pathological stage of colon cancer.The low expression group of NKAP has a better prognosis.Part ?:Study on the changes of NKAP expression on the biological behavior of colon cancer and its mechanismPurpose:Research on NKAP and tumors shows that NKAP is related to tumor proliferation,invasion,metastasis,apoptosis and other biological behaviors,and plays a very important role in tumor occurrence and development.NKAP is highly expressed in breast cancer cell lines cultured in vitro.In addition,NKAP can also promote the occurrence and development of cancer by activating AKT/mTOR,NF-?B,Notch and other signaling pathways.In order to explore the effect of NKAP expression changes on the biological behavior of colon cancer and the mechanism of its effect,this study knocked down the expression of NKAP by transfection of NKAP shRNA,and transfected the overexpression plasmid pcDNA3.1-NKAP to induce high expression of NKAP.The effects of NKAP expression differences on colon cancer proliferation,invasion,migration,apoptosis,autophagy,EMT,etc.,and the relationship between NKAP and AKT/mTOR and NF-?B signaling pathways in colon cancer were further studied,and the effect of NKAP on tumor growth in vivo and the expression of Ki67 protein were studied by nude mouse tumorigenicity assay.It provides an important basis for exploring the exact regulatory mechanism of NKAP in colon cancer,and also provides a new perspective for finding the potential application of NKAP inhibitors in the treatment of colon cancer.Materials and Methods:1.Cell source2.Cell culture3.RNA extraction,reverse transcription and qPCR to detect the expression of NKAP gene4.Plasmid transformation and extraction5.Cell transfection experiment6.RNA extraction,reverse transcription and qPCR to detect the expression of NKAP gene7.Total cell protein extraction and Western blot analysis to detect the expression of NKAP8.CCK8 test to detect cell proliferation9.Clone formation test to detect cell proliferation10.Transwell test to detect cell invasion11.Wound healing assay to detect cell migration12.Total cell protein extraction and Western blot analysis to detect the expression of EMT-related proteins13.Flow cytometry to detect apoptosis14.Total cell protein extraction and Western blot analysis to detect the expression of apoptosis-related proteins15.Total cell protein extraction and Western blot analysis to detect the expression of autophagy-related proteins16.Total cell protein extraction and Western blot analysis to detect the expression of AKT/mTOR and NF-?B signaling pathway related proteins17.Nude mouse tumorigenicity assay to detect the effect of NKAP on tumor growth18.Total tumor tissue protein extraction and Western blot analysis to detect the expression of NKAP19.HE staining to detect the effect of NKAP on tumor cells20.Paraffin section and immunohistochemistry to detect the expression and localization of NKAP21.Total tumor tissue protein extraction and Western blot analysis to detect the expression of Ki6722.Statistical analysisResult:1.qPCR detected the expression of NKAP in colon cancer cells.It was found that the expression of NKAP in HT-29 and HCT116 cells was higher than that of other cells,while the expression of NKAP in HCT-15 cells was lower than that of other cells.Subsequently,HT-29 and HCT116 cells were selected for NKAP knockdown experiments,and HCT-15 cells were selected for NKAP overexpression experiments.2.After NKAP was transfected with NKAP shRNA and the overexpression plasmid pcDNA3.1 NKAP,the expression of NKAP was detected at the mRNA level and the protein level respectively.The results showed that the knockdown and overexpression of NKAP in colon cancer cells were successful.3.The results of CCK8 and clone formation experiments found that overexpression of NKAP can promote the proliferation of colon cancer cells,while knockdown can inhibit the proliferation of colon cancer cells.4.Transwell test and Wound healing assay results found that the number of HCT116 and HT-29 cells passing through the matrigel and basement membrane decreased significantly after NKAP knockdown,and the migration area was also significantly reduced,while the number of HCT-15 cells passing through the matrigel and basement membrane after NKAP overexpression was significantly increased,and the migration area was also significantly increased.The result indicated that NKAP can significantly enhance the invasion and migration ability of colon cancer cells.5.NKAP can regulate the EMT process of colon cancer cells in vitro.Western blot detected the expression of adhesion factor ?-catenin,epithelial marker protein E-cad,mesenchymal markers Vimentin and Snail,and N-cad.It was found that ?-catenin,Vimentin,Snail and N-cad were significantly lower after NKAP knockdown,and E-cad was increased,while ?-catenin,Vimentin,Snail and N-cad were significantly higher after NKAP overexpression,and E-cad was reduced.6.Flow cytometry detected the effect of NKAP on the apoptosis of colon cancer cells.It was found that the apoptosis rate of HCT116 and HT-29 cells increased significantly after NKAP knockdown,and the apoptosis rate of HCT-15 cells decreased significantly after NKAP overexpression.Results showed that NKAP can inhibit the apoptosis of colon cancer cells.Western blot detection of the expression of Bcl-2,Bax and Active caspase-3 related to apoptosis showed that the expression of Bcl-2 decreased significantly after NKAP knockdown,while the expression of Bax and Active caspase-3 increased significantly.After overexpression of NKAP,the expressions of Bcl-2,Bax and Active caspase-3 were exactly opposite to those of the knockdown group.The results of Western blot further indicated that NKAP can inhibit the apoptosis of colon cancer cells.7.The expression detection of autophagy-related P62,Beclin and LC3 protein found that the expression of Beclin and LC3II increased significantly after NKAP knockdown,while the expression of P62 and LC3I decreased significantly.After NKAP overexpression,the expression of P62,Beclin and LC3 was exactly the opposite of the knockdown group,indicating that NKAP can inhibit the autophagy of colon cancer cells.8.The expression detection of key proteins in the AKT/mTOR and NF-?B pathways found that the total amount of AKT and mTOR remained unchanged after NKAP knockdown,while the expression of p-AKT and p-mTOR was down-regulated,and the downstream genes Cyclin D1 and the expression of P70 was also significantly reduced,indicating that the AKT/mTOR pathway was inhibited.The protein detection situation after NKAP overexpression was opposite to the knockdown group,indicating that the AKT/mTOR pathway was overactivated.In cells,the expression of P65 decreased after NKAP knockdown,and the expression of P65 increased after NKAP overexpression,indicating that the NF-?B signaling pathway was inhibited when NKAP was knocked down and promoted when NKAP was overexpressed.9.The results from nude mouse tumorigenicity assay showed that NKAP knockdown could significantly suppress the subcutaneous tumorigenesis ability and Ki67 expression of colon cancer cells in nude mice,while NKAP overexpression could promote the subcutaneous tumorigenesis ability and Ki67 expression of colon cancer cells in nude mice.Conclusion:1.NKAP overexpression can promote the proliferation,invasion,migration and epithelial-mesenchymal transition of colon cancer cells,and inhibit the apoptosis and autophagy of colon cancer cells.2.NKAP knockdown can inhibit the proliferation,invasion,migration and epithelial-mesenchymal transition of colon cancer cells,and promote the apoptosis and autophagy of colon cancer cells.3.NKAP can activate the AKT/mTOR and NF-?B signaling pathways in colon cancer cells.4.NKAP knockdown can inhibit tumor growth and Ki67 expression in mice.5.NKAP overexpression can promote tumor growth and Ki67 expression in mice.