| The incidence of prostate cancer has been high in Europe and America,it is still rankedsecond in the incidence of male cancer. The etiology of prostate cancer is complex and the latency is long, patients are often no obvious clinical symptoms. In recent years the incidence of prostate cancer is also increasing, about one-third of the patients had symptoms of bone metastases.In the past 10 years, there is a new way in the treatment of prostate cancer. Focus only on cancer treatment in the past change to the treatment of tumors at the same time as focus on regulation in microenvironment in patients. Based on the new understanding between in vivo microenvironment of tumor cells and the interaction, the treatment of prostate cancer has changed. Recently, monocyte chemoattractant protein-1 (MCP-1) has been shown to play an important role in the progression and metastasis of prostate cancer. Foreign scholars have recently found MCP-1 not only has raised the ability of monocytes, but also promote the proliferation and metastasis of prostate cancer. Some scholars have pointed out that the MCP-1 in prostate cancer metastasis positive regulatory role played. Monocyte chemoattractant protein-1 is cons-idered to have the most closely related to the occurrence of bone metastasis. Osteoblasts,endothelial cells, stromal cells and prostate cancer cells can produce MCP-1.MCP-1 can also regulate tumor-associatiated macrophages and promote osteoclastmaturation.In addition,MCP-1 and its receptor(CCR2) complex can induce prostate cancer cellsproliferation, migration and invasion. MCP-1 can activate the survivin gene through PI3K/AKT mechanisms to reduce prostate cancer cell apoptosis. Double-stranded RNA blocks gene expression known as RNA interference effect,also knowm as RNAi.Double-stranded RNA will be digested to many siRNA.Messenger RNA of homologous sequence can occur with these siRNA sequences,and then the mRNA lose their funcion,so translation of mRNA can not produce proteins.RNAi as a powerful tumor blocking tool for the gene has been more and more attention. Objective:In this experiment, a highly metastatic human prostate cancer PC-3M cells, and based on RNAi technology to build pFIV-H1/U6-MCP-1 siRNA vector (pFIV-si-MCP-1 vector). Our aim was to investigate the mechanisms underlying the functional role of MCP-1 in prostate cancer progression and metastasis., while application pFIV-si-MCP-1 vector for RNAi inhibition, attempt to provide a new treatment for prostate cancer clues. Methods:The control groups, si-scramble group, MCP-1 group and the si-MCP-1 group give to the appropriate disposal respectively.Using MTT assay to observe the inhibitory rate of tumor cells in changes.Using scratch experiments to observed cell migration changes. Using Transwell cell invasion assay to observe tumor cell invasion changes. Using ELISA to detect tumor cells MCP-1 protein content. Using TUNEL and Annexin V-FITC flow cytometry to cell observe apoptosis rate changes. Using RT-PCR and Western blot to detect VEGF, Caspase-3, MMP-9 and MCP-1 gene transcription and expression.Results:With the control group and the si-scramble group, the results show that MCP-1 was observed with tumor cell proliferation in the MTT test, cell proliferation rate of MCP-1 group was 30.4±0.12%.The pFIV-si-MCP-1 vector can inhibit tumor cell proliferation, the cell inhibition rate of si-MCP-1 group was 56.9±0.10%. Observed in cell scratch experiment MCP-1 can promote tumor cell migration, and pFIV-si-MCP-1 vector could inhibit tumor cell migration. In the transwell cell invasion assay MCP-1 can enhance the invasive ability of tumor cells, MCP-1 group significantly increased the number of plastic through the Matrigel (P<0.01), while the pFIV-si-MCP-1 vector can inhibit tumor cell invasion, Tumor cells transfected pFIV-si-MCP-1 vector significantly reduced the number of invasive cells (P<0.01). In TUNEL and Annexin V-FITC test in each group were observed under confocal laser microscopy and flow cytometry have shown that MCP-1 can reduce the tumor cell apoptosis, and pFIV-si-MCP-1 can promote tumor cell to apoptosis, the apoptosis rate in each group were 5.4±1.12%(control group),7.0±3.24% (si-scramble group),1.0±0.59%(MCP-1 group) and 40.8±3.02%(si-MCP-1 group). In the ELISA assay, the concentrations of MCP-1 were,63.5±11.60 pg/μg (control group),64.9±11.07 pg/μg (si-scramble group),88.1±8.45 pg/μg (MCP-1 group) And 44.5±11.29 pg/μg (si-MCP-1 group), MCP-1 showed an effect with its own promotion, pFIV-si-MCP-1 MCP-1 vector can reduced MCP-1. RT-PCR and Western blot results suggest that MCP-1 can downregulates Caspase-3 gene transcription and translation, while promoting MMP-9, VEGF and MCP-1 gene transcription and translation, pFIV-si-MCP-1 vector can be The Caspase-3 gene transcription and translation upregulates, and downregulates MMP-9, VEGF and MCP-1 gene transcription and translation. Conclusion:1. Experiments show that MCP-1 can promote the proliferation of PC-3M cells and reduced apoptosis, and increase the invasive ability of PC-3M cells.2. The reason of these effects was MCP-1 increased with VEGF, MMP-9 expression and decreased the expression of Caspase-3.3. pFIV-si-MCP-1 vector can reduce PC-3M cell invasion and promote apoptosis. 4. The reason of these effects was VEGF, MMP-9 expression decreased and the expression of Caspase-3 upregulated after MCP-1 expression was inhibited.
|
PDF Full Text Request