The Research Of The Function Of BMP7-Ids Signaling Pathway In The Proinvasive Activity And Proliferative Ability Of Colon Cancer

INTRODUCTIONColorectal cancer(CRC) is becoming one of the most prevalent cancers in the world. The investigation of the mechanism of the genesis and development of CRC at the molecular level has become the hot spot nowadays. The function of the BMP7-Ids signaling pathway has been paid more attentions recently. However, many problems have not been clarified yet.In this research we focused on the function of the BMP7cytokine. Firstly we detected the expression of the mRNA and protein of BMP7、ALK2(which is the type Ⅰ receptor of BMP7) and their downstream cytokines Id2and Id3in the colon cancer tissues to investigate the their clinical significance and their relationships with each other. Secondly we observed the changes of the proinvasive activity and proliferative ability of the colon cancer cell in the presence of increasing concentration BMP7. We also investigated the changes of the expression of the cyclin-dependent kinase(CDK) and cyclin-dependent kinase inhibitors(CDKI). Lastly we investigate the function of Id2and Id3in the increase of the proinvasive activity and proliferative ability of the cancer cell promoted by BMP7through the silence of Id2and Id3genes.PART Ⅰ The Expressions of the Molecules Related With the BMP7-Ids Signaling Pathway and Their Clinical SignificanceObjectiveTo investigate the expression of the mRNA and protein of BMP7、ALK2(which is the type Ⅰ receptor of BMP7) and their downstream cytokines Id2and Id3in the colon cancer tissues to investigate their clinical significance and their relationships with each other. To detect the expression of the protein of BMP7in the colon cancer cell lines.Methods We collected60cases of colon cancer samples and their matched normal tissues. These patients’ paraffin sections were also collected from the pathology department. Real-Time PCR and IHC were used to detected the expression of the mRNA and protein of BMP7、ALK2、Id2and Id3. Their clinical significances were analyzed. ELISA was used to detected the expression of BMP7protein in the colon cancer cell lines of SW480、HCT116and HT29.ResultsThe expression of the mRNA and protein of BMP7was higher in the colon cancer tissues than in the normal tissues significantly. The expression of the mRNA of BMP7was related with the depth of the invasiveness of tumor、the metastasis of lymph and liver and the stage of Duke. The expression of the protein of BMP7was related with the expression of mRNA of BMP7. The expression of the mRNA and protein of ALK.2was higher in the colon cancer tissues than in the normal tissues significantly. The expression of the protein of ALK2was related with the expression of the mRNA of BMP7. The expression of the mRNA and protein of Id2was higher in the colon cancer tissues than in the normal tissues significantly. The expression of the mRNA of Id2was related with the depth of the invasiveness of tumor and the metastasis of liver. The expression of the protein of Id2was related with the expression of mRNA of Id2. The expression of the mRNA and protein of Id3in the colon cancer tissues has no differences with that in the normal tissues. The expression of the mRNA of Id2was related with the expression of the mRNA of BMP7. The HT29cell line hardly expresses any BMP7cytokine. The SW480cell line expresses only a small amount of BMP7cytokine.ConclusionThe expression of mRNA of BMP7was high in the colon cancer tissues and was related with the depth of the invasiveness of tumor、the metastasis of lymph and liver and the stage of Duke. The expression of mRNA of Id2was high in the colon cancer tissues and was related with the depth of the invasiveness of tumor and liver metastasis. The expression of the mRNA of these two cytokines had a close relationship. The expression of BMP7、ALK2and Id2was high in the colon cancer tissues and related closely with the expression of their mRNA separately. The expression of mRNA and protein of Id3was not elevated in the colon cancer tissues. PART Ⅱ The Effects of BMP7Cytokine on the Proinvasive Activity and Proliferative Ability of Colon Cancer CellObjectiveTo explore the effects of BMP7on the proinvasive activity and proliferative ability of colon cancer cell HT29. To observe the impact of BMP7on the expression of Id2and Id3which are the downstream cytokines of BMP7and the expression of cyclin-dependent kinase(CDK) and cyclin-dependent kinase inhibitors(CDKI).MethodsWe explored the changes of the proinvasive activity and proliferative ability of colon cancer cell and the expression of cyclin-dependent kinase(CDK) and cyclin-dependent kinase inhibitors(CDKI) and Id2and Id3in the presence of increasing concentration of BMP7by using cellular invasion assay、MTT、flow cytometry assay and Western Blot.ResultsBMP7can promote the proinvasive activity and proliferative ability of colon cancer cell through concentration gradient dependent manner. BMP7can induce the expression of the protein of cyclin Dl and CDK4and repress the expression of the protein of P21. Besides, BMP7can induce the expression of its downstream Id2and Id3cytokines, especially for Id2.ConclusionBMP7can increase the proinvasive activity and proliferative ability of colon cancer cell. This effect may be related with the induction of cyclin Dl and CDK4and repression of P21. BMP7can also induce the expression of its downstream Id2and Id3cytokines, especially for Id2. PART Ⅲ The Functions of Ids Cytokines in the BMP7-Ids Signaling Pathway ObjectiveTo explore the functions of Id2and Id3in the process of invasiveness and proliferation of colon cancer cells promoted by BMP7.MethodsWe used the technique of siRNAto silence the Id2and Id3genes of colon cancer cell line HT29, then Western Blot was used to verified the effects of transfection. We compared the transfected cell line with the positive and negative control cells by using cellular invasion assay、MTT、flow cytometry assay and Western Blot to observe the changes of proinvasive activity and proliferative ability of the cells and the changes of the expression of the proteins of cyclin-dependent kinase(CDK) and cyclin-dependent kinase inhibitors(CDKI).ResultsAfter transfection, we verified through Western Blot that the expression of the proteins of Id2and Id3was weakened significantly. After the silence of Id2, the proinvasive activity and proliferative ability of the colon cancer cells were reduced, the expression of the protein of cyclin Dl was reduced and the expression of the protein of P21was elevated compared with the positive control cells. After the silence of Id3, the similar scenario mentioned above was also observed. However, the extent of the changes was much smaller. After the silence of Id2and Id3simultaneously, we never observed additive or synergistic effects.ConclusionId2plays an important role in the process of invasiveness and proliferation of colon cancer cells promoted by BMP7. However, Id3plays a less important role compared with Id2. These two cytokines have no additive or synergistic effects with each other.