Change The Expression Of The Smad4to Study The Function Of The BMP7-Smad4-Id2Signaling Pathway In The Proinvasive Activity And Proliferative Ability Of Colorectal Cancer

PART1. Smad4protein expression in colorectal cancer and its clinical significanceObjective To investigate the expression of the Smad4protein in colorectal cancer tissues and the relationship between the expression of Smad4and the clinical pathological data of the patient. Methods Detected60cases of colorectal carcinoma tissue and27cases of the corresponding adjacent normal tissue expression of Smad4by the Immunohistochemistry S-P method, collected the corresponding clinicopathological data of patients. Results Compared with normal tissues, the immunohistochemical biopsy score of Smad4in colorectal cancer primary tumors, was a significant difference (Mann-Whitney test, u=294.50, P<0.001). Smad4expression was relationship with the tumor invasion, lymph node metastasis, and Dukes staging (P <0.05). Conclusion Low expression of Smad4may be related to the progression of the colorectal cancer. The Smad4can be used as predictors of the prognosis of colorectal cancer, and the gene can be an effective target in gene therapy. PART2. Down-regulation of Smad4affected proliferation and invasion of colorectal carcinoma HCT116cells and BMP7-Smad4-Id2pathwayObjective The aim of this study was to determine whether the suppression of Smad4by short hairpin RNA (shRNA) regulates the proliferation and invasion of colorectal carcinoma HCT116cells and BMP7-Smad4-Id2pathway. Methods The Smad4-shRNA expression vectors were constructed and stably transfected to HCT116cells. The expression of mRNA and protein of Smad4and Id2was detected using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Cellular proliferation inhibitory activity was determined by methyl thiazolyl tetrazolium (MTT) assay. Transwell assay was used to detect the effect of the inhibition of Smad4-shRNA on migration and invasion.Tht flow cytometry was used to assess the change of the cell cycle. Results The Smad4-shRNA vector, which inhibited Smad4expression, was constructed and successfully transfected to HCT116cells. The levels of mRNA and protein expression of Smad4were markedly decreased following transfection of shRNA compared with the control groups (P<0.05). The abilities of proliferation, migration and invasion were increased following transfection of shRNA (P<0.05). The expression of Id2was increased following transfection of shRNA (P<0.05). For the Smad4-down-regulated HCT116cells, treated with or without BMP7(25ng/ml), no difference was found. Conclusion shRNA-mediated silencing of Smad4was able to enhance the abilities of proliferation, migration and invasion in the HCT116cell line. Therefore, Smad4may act as a tumor-suppressor gene in colorectal carcinoma. BMP7affects Id2by Smad4to change the activity of the HCT116cell. PART3. Down-regulation of Smad4affected human colorectal carcinoma HCT116growth and BMP7-Smad4-Id2pathway in vivoObjective The reduction of Smad4by short hairpin RNA (shRNA) resulted growth Promotation of cancer cells. The present study was to evaluate the effect of reduction of Smad4expression by shRNA on growth of human colorectal carcinoma (CRC) in tumor-bearing nude mice in vivo. Methods To establish HCT116cell, HCT116-HK cell,HCT116-Smad4-shRNA cell transplantable model, the nude mice were subcutaneously inoculated with1.0×10(7) cells and kept growing till the tumor xenografts reached5-7mm in diameter. Then the mice were randomly assigned to five groups(three mice in each group:(1) HCT116cell group;(2) HCT116-HK group;(3) HCT116+BMP7group;(4) HCT116-Smad4-shRNA group; and (5) HCT116-Smad4-shRNA+BMP7group.0.2ml/mouse BMP7(100ng/ml) or Saline was injected intratumorally four times once every two day. The weight and volumes of tumor xenografts were recorded. The levels of Smad4and Id2mRNA transcripts and proteins in tumor xenografts were detected by reverse quantitative transcription polymerase chain reaction (QRT-PCR) and immunohistochemical staining respectively. The terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was used to detect the death of cells. Results The xenografts in mice could be seen at5th day from the implantation of HCT116cells and all had reached5-7mm in size. After injection intratumorally, the growth speed of tumor xenografts in HCT116-Smad4-shRNA group was significantly fast compared with those in HCT116and HK group(P<0.05). The results of QRT-PCR showed that mRNA levels of Smad4reduced more in HCT116-Smad4-shRNA group than those in HCT116and HK group. The mRNA levels of Id2increased more in HCT116-Smad4-shRNA group than those in HCT116and HK group. For the tumor which was growing from the Smad4-down-regulated HCT116cells, treated with or without BMP7, no difference was found. Immunohistochemical analyses of tumor xenograft sections also revealed the decreased Smad4expression in HCT116-Smad4-shRNA group and the change of Id2. TUNEL assay also showed lower death of tumor xenograft tissue cells in HCT116-Smad4-shRNA group. Conclusion Smad4-shRNA may inhibit the growth of human colorectal tumor xenografts in vivo. BMP7-Smad4-Id2pathway was working on the colorectal tumor. PART4. Re-expression of Smad4affected proliferation and invasion of colorectal carcinoma sw480cells and BMP7-Smad4-Id2pathway:Objective The bone morphogenetic proteins (BMPs) Smad4and Id2exert their effect on colorectal carcinoma via several uncharacterized mechanisms. In this study, we determine whether the re-expression of Smad4by cDNA regulates the proliferation and invasion of colorectal carcinoma SW480cells, and investigated whether the transcription factor Id2, which has been implicated in colorectal carcinoma proliferation and metastasis, is involved in BMP7-Smad4signaling, or whether it is regulated by BMP7via another mechanism in this cell type. Methods A Smad4-cDNA vector was constructed and stably transfected into SW480cells. Protein levels of Smad4and Id2were examined by Western blotting. Inhibitory effects on cellular proliferation activity were determined by the methyl thiazolyl tetrazolium (MTT) assay, and invasion and migration potential was detected using the in vitro Matrigel-coated invasion and migration assay. Results Levels of Smad4protein were significantly increased in SW480cells transfected with Smad4-cDNA, compared to those transfected with empty vector. Growth curve analysis revealed that live cell numbers were lower in the Smad4-expressing group than in the control group after36h, and that BMP7treatment caused an increase in live cell numbers in Smad4-expressing cells. Transwell chamber analysis revealed that migration/invasion activity was significantly suppressed when Smad4was expressed. Finally, Smad4-expressing cells treated with BMP7expressed a higher level of Id2protein than the controls. Conclusion The results indicate that Smad4expression may inhibit the growth and invasion of SW480cells, and that BMP7affects Id2levels through Smad4. Therefore, BMP7-Smad4-Id2signaling may play a significant role in the development of colorectal carcinoma.