KIAA1522 Promotes The Development And Distant Metastasis Of Colon Cancer Cells And Affects The Prognosis Of Patients By Upregulating Notch Signaling Pathway

BackgroundColorectal cancer(CRC)is one of the most common cancers in the world,ranking third in the incidence of new cancers and second in cancer-related mortality.In China,with the development of economy,people’s lifestyle and eating habits have changed,resulting in an increasing incidence and mortality of colorectal cancer.With the development of molecular biology and pharmacy,many advanced adjuvants therapy techniques have achieved good results in the treatment of colorectal cancer,but surgery is still recognized as the most effective radical cure for CRC.Whether because of the disease itself or treatment methods,like surgery,radiotherapy or chemotherapy,CRC not only brings great pain to patients but also increases the economic burden to patients and society.However,because of its high recovery rate and high metastasis rate,the mortality rate of colorectal cancer is still very high,and metastatic disease is still the most common cause of death.Liver is the most common metastatic site of colorectal cancer.About 20%-25%of patients with colorectal cancer may have synchronous liver metastasis,and 50%will develop liver metastasis of colorectal cancer during its course.In the past 20 years,with the introduction of new adjuvant radiotherapy and chemotherapy technology in perioperative period and the continuous progress of comprehensive treatment programs for metastatic colorectal cancer,the prognosis of metastatic colorectal cancer has significantly improved.The 2-year relative survival rate of colon cancer with distant metastasis has increased from 21%in the mid-1990s to 37%at present.About 10%to 20%of patients with metastatic colorectal cancer can receive chemotherapy and surgery,and can be cured in some cases.For those who receive surgery directly,many will recur within 3 years after surgery.As a heterogeneous disease,the occurrence and metastasis mechanism of colorectal cancer involves a variety of genetic changes and multiple pathways,showing different clinical and pathological characteristics,leading to different prognosis.Previously recognized risk factors for CRC recurrence and metastasis,such as the number and size of liver metastases,can’t reflect the biological heterogeneity of liver metastases and cannot adequately predict prognosis.In the current era of genomic medicine and targeted therapy,biomarkers have become important predictors to guide patient selection for systemic therapy and surgery.In recent years,with the widespread application of next-generation sequencing technology,new genes and tumor markers have been discovered.Among them,KIAA1522 was discovered by sequencing human tissues in 2000.Among the few studies reported so far,KIAA1522 has been confirmed to be highly expressed in esophageal squamous cell carcinoma(ESCC),non-small cell lung cancer(NSCLC)and breast cancer cell lines,and related to tumor differentiation,invasion and metastasis.At present,studies on the regulatory mechanism of KIAA1522 are not completely unified,and the upstream miRNA regulatory mechanism or downstream regulatory signaling pathway in a variety of cancers show inconsistencies.At present,the functional mechanism between KIAA1522 and colorectal cancer is still unclear.The biological role and molecular mechanism of KIAA1522 in mediating tumor progression and distant metastasis have not been studied.Based on the clinical data and follow-up data of patients who were followed up for more than 10 years and obtained samples from both primary and liver metastases of colorectal cancer,we concluded that the expression of KIAA1522 gene in primary and metastases of colorectal cancer was associated with clinicopathological factors and poor prognosis.The expression of KIAA1522 gene in colon cancer cell lines is closely related to Notch signal transduction pathway.The Notch gene has been named widely in studies of Drosophila melanogaster.Notch homologues have been found in a variety of metazoans,so the Notch pathway is considered to be an ancient and highly conserved signaling pathway involved in a variety of biological processes,such as organ formation,tissue function and tissue repair.Therefore,abnormal Notch signaling may lead to pathological consequences.Significantly upregulated Notch ligands(DLL1,DLL3,DL4,JAG1,and JAG2)and aberrant activation of Notch receptor(Notchl)have been identified in human colorectal cancer tissues.This aberrant Notch activation is associated with poor prognosis and metastasis of colorectal cancer.As a transmembrane protein,KIAA1522 gene protein may mediate EMT and other forms of colon cancer cells through regulating Notch pathway,thus promoting tumor development and distant metastasis.Therefore,based on the aforementioned analysis of KIAA1522 gene and clinical physical and chemical factors and poor prognosis of patients with colorectal cancer liver metastases,we validated the role of KIAA1522 gene in the proliferation,apoptosis,migration and invasion of colon cancer cells through in vivo and in vitro experiments with next-generation sequencing screening analysis as the fulcrum.The relationship between the expression of KIAA1522 and Notch signaling pathway was further investigated by immunohistochemistry and western blot,and the possible mechanism of KIAA1522 gene mediating the occurrence and development of colorectal cancer and distant metastasis by regulating Notch pathway was further elucidated.Part Ⅰ Expression and clinical significance of KIAA1522 in colorectal cancer and liver metastasesObjectives:Colorectal cancer is a common gastrointestinal malignancy with poor prognosis and a high distant metastasis rate in the world.As a newly discovered and studied gene,the expression of KIAA1522 in colorectal cancer,especially in patients with colon cancer liver metastasis,and its clinical significance are still unclear.Therefore,in this study,by analyzing the expression of KIAA1522 gene in colorectal cancer and liver metastases,we further explored the relationship between KIAA1522 gene expression and clinicopathological factors in patients with colorectal cancer liver metastases and its impact on the prognosis of the patients.Methods:We first used GEPIA to analyze KIAA1522 mRNA expression in colorectal adenocarcinoma(COAD)and rectal adenocarcinoma(READ)tumor samples.Relationship between expression of KIAA1522 and long-term survival and disease-free survival.The correlation between the expression levels of KIAA1522 and MKI67,a typical proliferation marker,in tumor samples of colorectal adenocarcinoma and rectal adenocarcinoma was analyzed.From Jan.2005 to Jan.2016,131 patients with colorectal cancer and liver metastasis who underwent surgery in Jinan and Qingdao Hospitals of Shandong University Qilu Hospital and had postoperative pathological results were collected,and 57 patients were further selected to be included in this study cohort.The expression of KIAA1522 gene was detected by immunohistochemistry,and the expression of KIAA1522 in primary colorectal cancer and liver metastases was statistically analyzed by univariate analysis with clinical and physical factors.Kaplan-meier method was used to draw the survival curve of patients,and the COX proportional hazards model was used to compare the differences in survival of patients,analyze the independent risk factors affecting the prognosis of patients,and explore the clinical significance of KIAA1522 in patients with colorectal cancer liver metastasis.Results:GEPIA analysis revealed that KIAA1522 mRNA expression was significantly up-regulated in colorectal adenocarcinoma(COAD)and rectal adenocarcinoma(READ)tumor samples compared with non-tumor samples.Survival analysis showed that high expression of KIAA1522 indicated poor disease-free survival in CO AD patients.The expression level of KIAA1522 was positively correlated with that of MKI67,a typical proliferation marker,in tumor samples of colorectal adenocarcinoma and rectal adenocarcinoma.By analyzing and comparing the expression of KIAA1522,we found that the expression of KIAA1522 in primary colorectal cancer was significantly positively correlated with its expression in corresponding liver metastases.The expression of KIAA1522 in primary colorectal cancer is associated with T stage and venous invasion of colorectal cancer,indicating that high expression of KIAA1522 in primary colorectal cancer predicts worse tumor stage and more local vascular invasion.The expression of KIAA1522 in liver metastases is correlated with the volume of individual liver metastases,suggesting that high expression of KIAA1522 in liver metastases predicts more rapid metastasis proliferation.In the survival analysis,we found that KIAA1522 expression in primary colorectal cancer was statistically significantly correlated with overall survival,but not with disease-free survival.Multivariate regression analysis showed that the high expression of KIAA1522 in primary colorectal cancer and venous invasion were confirmed to be independent prognostic factors for long-term survival of patients with colorectal cancer after surgery.Conclusions:1.There is a correlation between the expression of KIAA1522 protein in primary colorectal cancer and liver metastasis.2.The expression of KIAA1522 protein in primary colorectal cancer is related to tumor stage and local venous invasion,while the expression of KIAA1522 gene protein in liver metastasis is related to the volume of metastatic tumor.3.The expression of KIAA1522 protein in primary foci is an independent risk factor for colorectal cancer patients with liver metastasis,and its expression level is negatively correlated with the long-term survival of patients.Part Ⅱ KIAA1522 promotes the proliferation,invasion and distant metastasis of colon cancer cells by upregulating Notch signaling pathwayObjectives:Based on the previous studies and analysis in Part Ⅰ,we found that KIAA1522,as an independent prognostic marker for colorectal cancer patients with liver metastasis,was involved in the development and distant metastasis of colorectal cancer,but its regulatory mechanism has not been clearly defined.In this part,we explore the correlation between KIAA1522 gene and Notch signaling pathway,and explore the regulatory mechanism of KIAA1522 gene on Notch signaling pathway and the effect of this regulatory mechanism on the development and distant metastasis of colon cancer cells.Methods:To further confirm the above hypothesis,we first screened the expression of KIAA1522 in different colon cancer cell lines and verified the effectiveness of transfected siRNA and overexpression plasmid.At the level of in vitro experiments,we verified the cancer-promoting effects of KIAA1522 gene on proliferation,apoptosis,migration and invasion of colon cancer cells by CCK-8 assay,colony formation assay,flow cytometry,Transwell assay and scratch assay.Effect of KIAA1522 gene on tumor formation and distant metastasis of colon cancer cells was confirmed through the tail vein injection of metastasis model and subcutaneous tumor bearing model in nude mice.We further analyzed the changes of downstream signaling pathway after deferentially expressing of gene KIAA1522 in colon cancer cell lines through the second-generation sequencing technology,and determined that Notch signaling pathway was involved.Western blotting and immunohistochemical analysis were performed at cellular and animal level to explore the correlation between the expression of KIAA1522 and the expression of Notch signaling pathway.Thus,the possible mechanism of KIAA1522 gene promoting the development and distant metastasis of colorectal cancer through up-regulating Notch signaling pathway was further elucidated.Results:KIAA1522 gene was expressed in SW480,LOVO and HCT116 cells.Compared with SW480 cells,KIAA1522 expression was higher in HCT116 and LOVO cells.After KIAA1522 knockdown,the apoptosis of HCT116 cells was increased compared with the control group,while the apoptosis of SW480 cells was decreased after KIAA1522 overexpression.After KIAA1522 silencing,the level of the apoptosis-related protein Bax increased in the HCT116 cell line,while the level of the anti-apoptotic protein Bcl-2 decreased;the opposite result was observed in the SW480 cell line overexpressing KIAA1522.The results of CCK-8 assay and colony formation assay showed that compared with the control group,KIAA1522 gene silencing resulted in the inhibition of HCT116 cell growth and the reduction of colony formation rate,while KIAA1522 gene overexpression showed the opposite result.In vivo,KIAA1522 silencing inhibited the proliferation of colon cancer HCT116 cells,which further indicated that KIAA1522 gene may play an important role in promoting the progression of colorectal cancer.The results of Transwell assay showed that compared with the control group,the transmembrane migration and invasion ability of HCT116 cells were decreased after silencing KIAA1522 gene expression,while the transmembrane migration and invasion ability of SW480 cells were enhanced after upregulating KIAA1522 gene expression.The results of scratch test showed that down-regulation of KIAA1522 gene expression could reduce the migration ability of HCT116 cells compared with the control group,while up-regulation of KIAA1522 gene expression could increase the migration ability of SW480 cells compared with the control group.In vivo experiments showed that the mice injected with KIAA1522 gene silenced stable transfected cells had less lung metastasis than the mice injected with control cells,and the number of lung metastatic nodules between the two groups was statistically significant.Sequencing and GSEA analysis showed that genes related to Notch signaling pathway were significantly enriched in KIAA1522 gene overexpression group.Subsequent GEPIA database analysis found that KIAA1522 gene expression was positively correlated with the expression of key genes in Notch signaling pathway in colorectal adenocarcinoma and rectal adenocarcinoma samples.Further experiments showed that the key protein levels of Notch pathway decreased in KIAA1522 downregulated cells,while increased in KIAA1522 upregulated cells.These results indicated that KIAA1522 gene promoted the occurrence,development and distant metastasis of colorectal cancer by up-regulating Notch signal transduction pathway.Conclusions:1.KIAA1522 plays an important role in the proliferation,apoptosis,invasion and migration of colon cancer cell lines.2.The KIAA1522 promoted tumor-bearing proliferation and distant metastasis of colon cancer cell lines in nude mice.3.KIAA1522 may promote the development of colon cancer cells and distant metastasis by upregulating the expression of Notch signaling pathway.Therefore,KIAA1522 is promising as a potential therapeutic target for colon cancer liver metastasis.