Expression And Its Biological Role Of KLF9 In Colorectal Cancer

Colorectal carcinoma(CRC)is one of the most common malignancies which threaten the health of human.It is the third common cancer and the second leading cause of cancer-related death in each year in western developed countries.Numerous genes,associated with the occurrence and development of CRC,have been found since the proposal of Vogelstein model of colorectal carcinogenesis.The incidence and mortality of CRC increase gradually with the changes of lifestyle and diet structure in China in recent two decades.It is of great value to explore the molecular changes related to CRC and investigate their roles in CRC for the prevention, diagnosis and treatment of this disease.Accumulation of genetic changes in the adenoma-carcinoma sequence is the basis for development of CRC.Alterations of three different classes of genes, tumor-suppressor genes(eg,APC,DCC,SMAD4/DPC4,TP53),oncogenes(eg, K-ras,c-myc)and mismatch repair genes(MMRs),are involved in colorectal carcinogenesis.Some of these genes,such as DCC,SMAD4/DPC4 and TP53,belong to transcription factors.The relationship between transcription factors and CRC imply that they play important roles in the occurrence and development of CRC. KLF9(Krüppel-like factor 9,KLF9)zinc finger transcription factor,screened from electronic gene expression libraries developed by our laboratory,is a differentially expressed gene in CRC.KLF9,which is a number of SP/KLF(specificity protein/Krüppel-like factor,SP/KLF)family transcription factors,is implicated in the control of cell proliferation,differentiation and apoptosis.Dysregulation of intestinal crypt cell proliferation and villus cell migration has been observed in mice lacking Klf9.There is growing evidence that some SP/KLF proteins play a critical role in the growth and metastasis of many tumor types,such as breast cancer,colorectal cancer, prostate cancer,esophageal cancer and hepatocellular carcinoma,by regulating growth-related signal transduction pathways,differentiation,apoptosis and angiogenesis.The relationship between KLF9 and CRC has not been explored.This, accompanied by the role of KLF9 in crypt cell proliferation and villus cell migration and well-characterized functions for some KLF proteins in occurrence and development of cancer,prompted us to characterize the expression and in vitro function of KLF9 in CRC.Fresh colorectal cancer tissues and the histologically normal tissues in the distant resection margin to the tumor were collected from 70 patients who underwent the curative colorectal cancer resection at the Second Affiliated Hospital,School of Medicine,Zhejiang University from August 2005 to June 2006.The levels of KLF9 mRNA were detected by quantitative real-time reverse transcriptase PCR(Q-PCR), and the expression of KLF9 protein detected by Western blot.Tissue microarray (TMA),containing normal,adenoma,and adenocarcinoma colorectal tissues,was constructed,and KLF9 protein expression was detected by immunohistochemistry. Of the 50 cancerous tissues examined,86%(43/50)expressed lower levels of KLF9 mRNA than individually-matched normal mucosa(P＜0.0001).Expression of KLF9 protein was down-regulated in CRC by Western blot(P＜0.01)and immunohistochemistry(P＜0.001).Statistically,no correlation was found between KLF9 protein expression and tumor differentiation grade,infiltrating depth,location, and lymph node metastasis(P＞0.05).Expression of KLF9 in HT29,SW480, SW620,LoVo,RKO,and Caco2 colorectal cancer cell lines was also measured by RT-PCR and Western blot.mRNA of KLF9 could be detected among the six CRC cell lines.SW480 expressed the highest levels of KLF9 mRNA,and the rest showed weak expression.Except Caco2,the other five cell lines expressed KLF9 protein. The highest KLF9 protein was observed in RKO and the other four exhibited low levels of expression.KLF9 full-length coding sequence(CDS)in HT29,SW480, SW620 and RKO cell lines was sequenced.CDS of RKO had a mutation of 518T＞C (F173S)and no mutations were detected in others.We analyzed and compared the structure of RKO and normal KLF9 protein(NP₀01197)with the help of some online services and softwares,such as NCBI,ExPASy,PBIL,SWISS-MODEL, ESyPred3D,and Rosmal.A few differences of the primary,secondary and tertiary structures exist.These differences may cause functional alteration of RKO KLF9 protein.Based on down-regulation of KLF9 in CRC,we attempt to explore the function of KLF9 in CRC cell lines,pcDNA3.1-KLF9 eukaryotic expression vector was constructed and applied in transfection by using PolyFect transfection reagent.The expression of KLF9 mRNA and protein in the transfectants was detected by RT-PCR and western blot.No morphological alterations could be observed in KLF9 transfectants using light microscopy when cells were transfected transiently. Apoptosis and cell cycle assay using flow cytometry showed that KLF9 transfection did not induce apoptosis and alteration of cell cycle.The expression of cell cycle-related genes,Cyclin D1,p21(Cip1),CDK2 and PCNA,was detected in KLF9 transfectants using real-time PCR.The results showed that there are no differences in the levels of mRNA expression of these genes.In the stable transfection,cells were selected with G418 for getting the stable cell lines.Unfortunately,cells transfected with pcDNA3.1-KLF9 began to die during the later period of stable transfection,so we could not obtain the transfectants which keep expressing KLF9.From this study,we concluded that:1.Expression of KLF9 is down-regulated,and reduced expression might be involved in the occurrence and development of colorectal carcinoma.2.Post-transcriptional modifications may be related to KLF9 expression in CRC cell lines.3.Continuous expression of KLF9 protein may be lethal for some CRC cell lines,or high levels of KLF9 may lead to increased sensitivity to drug used in stable transfection and cause cell death.