Studies On The Effects Of NGX6 Gene In Wnt/Beta-catenin Signaling Pathway

Colon cancer is one of the most frequent human gastrointestinal carcinomas.It is one of the leading causes of cancer-related deaths in western countries.The incidence rate of this cancer has the tendency of step-up recent years in China.An understanding of the molecular pathogenesis of this cancer should eventually translate into better diagnosis and treatment and improved prognosis.The typical model of pathogenesis of colon cancer was first established by Vogelstein B,that is 'APC→K-ras→DCC→p53' mutation.With the accumulation of experimental indicator on colon cancer,this model can't elucidate the development of colon cancer completely.So,it has become a critical subject to reveal the tumorigenesis of colon cancer by identifying novel tumor suppressor genes or susceptible genes.Molecular genetics laboratory of Cancer Research Institute of Central South University has isolated a novel candidate tumor suppressor gene on the region of chromosome 9p21-22 by position-candidate cloning strategy,designed human NGX6,Gene Bank accession number: AF188239,the full length of cDNA is 2.1Kb.The loss of heterozygosity of 9p21-22 is a common event in many different tumors.The analysis of bioinformation presume that it encodes a transmember protein including 338 amino acid and processing two transmember regions(234-256 269-291)and a EGF-like domain(185-221),three N-glucosylation sites (92-95,100-103,172-175)and a tyrosine kinase phosphorylation site (257-268).These information suggest NGX6 may be a transmember protein,and play vital roles in cell proliferation,adhesion,and migration by these critical domains.In our early studies,we demonstrated that the mRNA expression level of NGX6 in colon cancer tissues,especially in those with distant metastasis is significantly lower than that of in paracancerous colon tissues.Then its repression roles in the proliferation and metastasis of colon cancer were confirmed by cell transfection,MTT,Soft agar colony formation,and nude mice transplantable tumor experiments.Further studies demonstrated NGX6 can inhibit extracellular matrix adhersion and invasion in colon cancer cells by cell adhersion and invasion experiments,and it also can inhibit liver metastasis of spleen situ focus in nude mice transplantation tumor experiment.FCM analysis indicated that NGX6 could repress the G1→S phase progression by down-regulating the expression of cyclinE,cyclinD1 and up-regulating the expression of p27.All of these suggested us NGX6 may participate into the prevention of tumor proliferation and metastasis in colon cancer.To elucidate its potential role in the gene network,We studied the effects of NGX6 on the expression profile of colon cancer cell line HT-29 by cDNA microarray,and some important clues were found.NGX6 could down-regulateNDRG1,MMP1,ILK,ROCK2,COL11A1 and up-regulate DKK1,AKAP12,CSE1L,Geminin.It's extremely exciting that some of them belonges to Wnt signaling pathway,such as DKK1,ILK,MMP1, and COL11A1.These moleculars can result in the inhibition of Wnt signaling pathway and lead to the inhibition of proliferation and metastasis of colon cancer.As we all know,Wnt signaling pathway has been identified as one of the key signaling pathways in colorectal cancer, while over-expression of NGX6 could change their expression.This result provide us a strongly image that NGX6 may participate the inactivation regulation of Wnt signaling pathway.To confirm the relation between NGX6 and Wnt signaling pathway, we studied the effects of NGX6 on the downstream target genes of Wnt signal pathway,and combined with the repression technique to study the feedback of Wnt signal pathway on NGX6,and then studied the effects of NGX6 on the transcriptional activation of Beta-catenin/TCF/LEF.The final step in Wnt signaling is activation of its downstream target genes.Most of them are moleculars related with cell proliferation, apoptosis,and metastasis.So we chose c-myc,cyclinD1,and c-jun for the target of our study.We detected the expression of above moleculars were obviously down-regulated by NGX6 transient transfection in colon cancer cell line SW620 by western-blot.This result suggested us that NGX6 could play negative role in the regulation of Wnt signaling downstream genes.NGX6 protein is located at cytoplasm and cell membrane according to the presume of bioinformation.In cytoplasm,it is located at the membrane of reticulum and mitochondrion.So we presumed that NGX6 may play its role by the interaction with upstream moleculars of Wnt signaling pathway,which may be direct interaction of protein and protein or rely on the mesomeric molecular.In order to identify whether there is potential feedback regulation of Wnt signaling on NGX6 or not,We constructed eukaryotic expression vector pcDNA3.1(+)-LEF1^DN(without N-terminal beta-catenin binding domain).LEF1 is one of the member of transcriptional factor TCF/LEF family. In order to transduce Wnt signal to the target gene,the full length LEF1 is necessary.In this paper,We detected that the activation of TCF-4 luciferase report gene was down-regulated by TCF-4 luciferase report system,and the expression of downstream target genes were down-regulated by western-blot after transient transfection of pcDNA3.1(+)LEF1^DN.These results indicated that Wnt signaling was inhibited by pcDNA 3.1(+)-LEF1^DN.Then we detected the expression of NGX6 in SW620 by RT-PCR and found its expression was up-regulated after pcDNA3.1(+)-LEF1^DNtransient tranfection.This result suggested the potential feedback regulation between NGX6 and Wnt signal pathway, but its detailed mechanism was not clear.The vital molecular of Wnt signaling pathway is beta-catenin.When Wnt signal is activated,abundant of beta-catenin translocate from cytoplasm to nucleus,and make complex with TCF/LEF and then active its transcription.In order to study the relation between NGX6 and beta-catenin/TCF/LEF,We constructed eukaryotic expression vector pcDNA3.1(+)-beta-catenin(WT),and contransfect with NGX6 in SW620 and COS-7.We found that the activation of TCF-4 luciferase report gene in contransfection group were less than that of in NGX6 alone transfection group(p＜0.05),the same results were found in the condition of no extrogenous beta-catenin.At the same time,We detected the expression of beta-catenin and TCF-4 in nucleus by western-blot,and found both of them were decreased after NGX6 transfection.All of these provided us a information that NGX6 may inhibit Wnt signaling pathway by its negative regulation on beta-catenin.But what is the detailed mechanism of the negative regulation of NGX6 on beta-catenin? According to the typical pathway of beta-catenin's nuclear translocation,We presumed the following potential mechanisms:(1)NGX6 target on one or more components of the destruction complex and increase the degradation of beta-catenin,so can decrease its translocation to nucleus,especially NGX6 may target on GSK-3βor its inhibitor DVL-1.(2)NGX6 may participate the nuclear -cytoplasmic shuttling of APC,so the formation of beta-catenin/TCF complex is repressed and the export of nucleus of beta-catenin is increased and then destructed or combined with E-cadherin to increase the cell-cell adhesion.But the further molecular mechanism is not clarified.To further elucidate the regulation mechanism of NGX6 on Wnt signaling pathway,We will study the effects of NGX6 on the activation of GSK-3βand its phosphorylation level.Then We will find the interaction proteins of NGX6 by co-immunoprecipation and mass -spectrometry technology and analyse their function and their critical interaction domain.These studies would provide more pathogenesis theory for colorectal cancer research.