Effects Of SiRNA Targeted Midkine On Biological Behavior Of Breast Carcinoma

Objectives:Midkine (MK) was found as the product of a retinoic acid-responsive gene discovered by screening for induced gene during the differentiation of embryo carcinoma cells. The distribution of MK is changed with the development of embryo. The expression of MK is stronger in many tissues from the early stage to middle stage of embryo development. The expression of MK decreases gradually after that periods and is localized. MK has diverse biological activites. MK is not only likely to participate in neural development but also modulates the proliferation, differentiation, and migration of carcinoma cells by acted on mitosis,induced the transformation,angiogenesis, and antiapoptosis. MK shows highly increased expression in a number of malignant tumors including pre-cancerous lesions but decreased or no expression in benignant tumors and normal tissues. That shows MK maybe involve in carcinogenesis and development of cancer. People pay much attention for MK gene. Studies indicated that matrix metalloproteinase(MMP)and its corresponding tissue inhibitors had taken an important role in the process of tumor invasion and metastasis. Among all of the MMP discovered recently,MMP-2 and MMP-26 are two of MMPs which has the closest relationship with tumor invasion. Many studies shown that many kinds of malignant tumor has high expression of MMP-2 and MMP-26,and the expression level of MMP- 2 and MMP-26 have close relationship with the degree of tumor invasion and poor prognosis. There is not reported about the interaction between MK and MMPs in the invasion and metastasis of carcinoma. RNA interfere (RNAi) is a process in which double-stranded RNA triggers the degradation of a homologous mRNA. The Double-stranded RNA in cell is diced up a class of 21-23 nucleotide-long RNA( siRNA) by the enzyme Dicer. The siRNA degrades its homologous mRNA to interfere with the gene expression by mixing with and activating multi-component nucleases, which is post-transcriptional gene silencing (PTGS). The main purpose of this study is to suppress MK expression using RNA interference, and explore its effects on the growth and biological behavior of breast cancer cells and learn its function in carcinogenesis and development of carcinoma.Methods and Results1. Construction and identification of MK gene specific siRNA expression Plasmid.According to midkine mRNA sequence in genebank, the target sequence was designed and synthesized, then cloned into the eukaryotic expression PGCsi-U6 vector in definite direction. The constructed recombinant was analyzed and identified by restricted endonuclease digestion and DNA sequencing. We also proved siRNA-MK reduced the expression of MK in level of mRNA and protein by RT-PCR and Western blot.2. Effects of siRNA-MK on the biological behavior of breast carcinoma(1) Cell viability was assessed by CCK-8 Kit that demonstrated the proliferation of cells transfected was decreased than control groups.The results of cell cycle analysis by flow cytometry showed that the siRNA-MK transfected cells were less the number of cells in S phase than those in control groups(2) Adhesion of cells was detected by cell-matrix adhesion assay. The results show the adhesive ability of cells transfected was in decreased.(3) Migration of cells was examined via wound–healing experiment that showed the migrated capability of cells was significantly lower in siRNA-MK group than those in control.(4) Tumor cell migration and invasion was determined by Boyden chamber. Compared with control group, The migration and invasion ability of the cells transfected was inhibited.3. Effects of siRNA-MK on apoptosis(1) The cell apoptosis was evaluated by flow cytometry that showed the rates of apoptotic cells was significantly higher in the group of cells transfected than the group of non-transfected cells. (2)The mitochondrial membrane potential was evaluated by Rodamine 123 staining and flow cytometry that demonstrated the loss of mitochondrial membrane potential of cells transfected.(3)Caspase-3 activity was measured by caspase-3 Colorimetric Assay Kit. The results showed the caspase-3 activity obviously was higher in group of cells transfected than those in control group(4) The expression of bcl-2 mRNA was determined by semi-quatitative RT-PCR that demonstrated the bcl-2 mRNA expression of cells was decreased significantly in siRNA-MK group than those in control.(5)The morphology was examined by DAPI staining. Nuclear fragmentation, irregular rim of nuclear and condensation were observed in MCF-7 cell treated with siRNA-MK.4. Effects of siRNA-MK on matrix metalloproteinase(1) The expression of mmp26 was determined by RT-PCR and Western blot that demonstrated the mmp26 expression of cells transfected was decreased in level of mRNA and protein. The expression of integrin-β1 protein was reduced in siRNA-MK group(2)The gelatin zymography analysis showed mmp-2 activity was reduced in the cell transfected group.5. Expression of MK and MMP-26 in various human breast tissues and its clinical significanceBy immunohistochemistry staining showed that there were higher expression of MK and MMP-26 in breast carcinoma than in benign tumor or normal tissue. There is a positive correlation between the expression of MK or MMP-26 in breast cancer and the lymph node metastasis. There is a positive correlation between expression of MK and expression of MMP-26 in breast carcinoma.Conclusions:(1) Construction of MK gene specific siRNA expression plasmid was successful and it could efficiency depress the expression of MK in mRNA and protein level(2) siRNA-MK inhibits the proliferation of tumor cells, decreased adhesion ability of cell-matrix, and migration and invasion of tumor cell that may affect carcinogenesis and development of cancer.(3) siRNA-MK could induce apoptosis via down-regulation of Bcl-2, mitochondrial depolarization, and increased caspase-3 activity(4) Effects of siRNA-MK reduced migration and invasion of tumor cell maybe through down-regulated matrix metalloproteinases(mmp26和mmp-2)and integrin-β1 protein expression.(5) There was a positive correlation between the expression of MK and MMP-26 in breast cancer tissue and the between expression of MK/ MMP-26 and lymph node metastasis.