The Role Of NGX6 Gene In SPAK/JNK Signal Pathway

â– BackgroundColorectal cancer (CRC) is a common malignant tumor in digestive tract, the exact mechanisms of which still remain unknown. But at present, it is generally held that the development of CRC is a multistep process with an accumulation of different genetic alterations. NGX6 is a novel tumor suppressor candidate gene isolated by Molecular genetics laboratory of Cancer Research Institute of Central South University with position- candidate cloning strategy(GenBank accession number: AF188239). Previous studies obtained the following findings: NGX6 was saliently down-regulated in colorectal carcinomas; it can not only postpone the process of cell cycle, suppress tumor propagation and implanted neoplastic growth but also suppress the production of tumor vessel and down regulate the expression of VEGF; Meanwhile, the overexpression of NGX6 can suppress the invasion and adhension of the colon carcinoma cell and recuperate the tumor cell spaces; In addition, NGX6 plays a negative role in thebeta- catenin/Wnt Pathway. The previous research of cDNA microarray also showed that NGX6 can down regulate the expression of two genes over three times - MADD(MAP-kinase activating death domain) and Rhoc(Ras homolog gene family, member C). These two genes are related to MAPK (mitogen-activated protein kinases, MAPKs) pathway, especially to the ERK (P42/P44MAPK) pathway and the SAPK/JNK pathway. Based on the previous studies, this project will investigate the molecular mechanism of how NGX6 suppress the propagation, invasion and metastasis of Colorectal cancer. We will mainly explore how P-JNK and P-ERK expresse in colorectal cancer and what significance they have in clinic applications, and further probe into the way how NGX6 effect the SAPK/ JNK and ERK /MAPK signal pathway, and change the expression of nuclear transcription factor.â– The expression of P-JNK and P-ERK in colorectal tissueIn the present study, we examined the expression of the protein level of P-JNK and P-ERK in 336 Colorectal tissue cores using tissue microarray analysis by Immunohistochemical analysis. Further research was done to know the relationship Between the protein expression and clinicopathological parameters in colorectal Tissue Microarray. RESULTS:Immunohistochemistry in colorectal carcinoma and metastatic carcinoma showed higher levels of activated MAPKs compared with the adjacent mucosa and adjacent normal mucosa of primary tumor. Moreover, a close association was observed between P-JNK expression and sex, differentiation grade, Duke's stages and distant metastasis in the lymph node metastasis tissue. The high protein expression of P-ERK was correlated with Duke's stages in preinvasive carcinoma and associated with distant metastasis in the lymph node metastasis tissue in the adjacent mucosa of primary tumor. However, no correlation between the expression of p-JNK and p-ERK was found.â– NGX6 and MAPK signal pathwayThe HT-29, pcDNA3.1(+)/HT-29, pcDNA3.1(+)/NGX6/HT-29 cell lines were taken as the object of this study. Western blot, cell immunofluorescence staining or immunocytochemistry staining analysis were adopted to detect the effect of NGX6 on the expression of JNK,P-JNK,ERK,P-ERK and on the nuclear translocation of them. RESULTS: NGX6 overexpression can suppress the activation and inhibit the nuclear translocation of JNK1/2 in HT-29 cell line. It is also able to assemble the P-JNK1/2 in the cytoplasm while reducing its nuclear translocation. But NGX6 can't suppress the activation of ERK1/2 or inhibit its nuclear translocation.â– The effect of NGX6 to AP-1(c-Jun,c-los) and Ets-1It is found that the protein expression of AP-l(c-Jun,c-los), Ets-1 were all downreglated after having transfected NGX6 in HT-29 by Western blot analysis, suggesting that NGX6 can suppress the expression of transcription factor on the downstream of MAPK signal pathway. After using PD98059 and SP600125 to inhibit the ERK/MAPK and SAPK/JNK signal pathway, we found both the ERK/MAPK and SAPK/JNK pathway can regulate the expression of c-Jun and Ets-1,but the expression of c-fos is mostly regulated by ERK/MAPK signal pathway while the SAPK/JNK signal pathway can only inhibit it partially. These findings indicate that NGX6 may suppress the expression of transcription factor through the effect of SAPK/JNK pathway. We also detected the effect of NGX6 on AP-1, a key molecule in MAPK signal pathway by luciferase reporter gene assays in SW620 of colon carcinoma cell line. The result shows that transient transfection of NGX6 in colon carcinoma cell line SW620 can inhibit the activity of AP-1 trascription factor.â– The effect of NGX6 on CyclinD1With Westem blot analysis, we detected the effect of NGX6 on the protein expression of CyclinD1, a cell cycle modulin, in HT-29 cell line. We also detected the effect of NGX6 on CyclinD1 by luciferase reporter gene assays in SW620 of colon carcinoma cell line. The result confirmed that NGX6 can suppress the expression of cyclinD1 and inhibit the activity of CyclinD1 trascription factor.CONCLUSION: this study confirmed that tow key molecules of SAPK/JNK and ERK/MAPK signal pathway, P-JNK and P-ERK, have a high expression in colorectal cancer. NGX6 can suppress the expression of transcription factor and inhibit the activity of AP-1 trascription factor through the SAPK/JNK signal pathway. NGX6 also can postpone the process of cell cycle by suppressing e-Jun conjuncted CyclinD1. In this way, NGX6 can suppress the propagation, invasion and metastasis of Colorectal cancer.