The Effect Of RNAi Targeting CCR2Gene Expression On Proliferation, Apoptosis, Migration And Invasion Of PC-3M Cells

Background: Prostate cancer (PCa) is one of the most malignancythat likely develops in older males. PCa became the second leading causeof cancer-related deaths in Western countries, and the incidence of PCa isincreasing annually in China. The number of new cases and deaths due toPCa were estimated at241,740and28,170, respectively in United Statesfrom the current year statistics. The major cause of PCa deaths attributedto the complications resulting from cancer metastases to distant organs inthe body. Early-stage or localized PCa can be cured effectively via radicalprostatectomy, and the5-year survival rate for the patients is more than99%. However, the prognosis of the patients with metastatic PCa is notgood, and the5-year survival rate is only approximately31%for thesepatients. To a large extent, lack of effective treatment for advanced PCa isassociated with poor understanding of the molecular mechanismsunderling progression of the disease. Therefore, the indentification ofalternative pathways that regulates PCa growth and metastasis couldresult in the development of therapeutic targets.Chemokines, through binding to their receptors, play primary rolesin cell migration during inflammatory responses in normal development.In addition, chemokines on various aspects of tumor cell biology,including modulation of cancer cell proliferation, apoptosis, angiogenesis,metastasis and immune response to tumor, has begun to emerge.Substantial data have linked CCL2, a chemokine, with tumor progressionand metastasis. Chemokine (C-C) motif ligand2(CCL2), also known as monocyte chemoattractant protein-1(MCP-1), is a member of the CCchemokine superfamily that plays a basic role in recruitment andactivation of monocytes during acute inflammation and angiogenesis.CCL2is produced by tumor cells and multiple different host cells,including stromal cells, leukocytes, and endothelial cells. Since discovery,CCL2has been implicated in the development, invasion and metastasis ofvarious cancers, especially in PCa, and CCL2is being explored as apotential target for the treatment of these cancers. The data in ourprevious studies have reported CCL2might be functionally important inpromoting the growth and invasion of PC-3M, and inhibition of CCL2could modulated PC-3M cells invasion and apoptosis. Specifically, theCCL2receptor, CCR2is expressed in various cancers, including PCa,indicated CCR2is also a novel target for PCa treatment.Objective: In the present work, we examined the effect ofsiRNA-mediated knockdown of CCR2on the growth, apoptosis andinvasion of PC-3M cells.Method: Based on the CCR2mRNA bases searched by NCBI, weadopted three plasmids that carried different short hairpin RNA reversedto the CCR2gene by RNAi technology and selected the most effectiveone by RT-PCR. Using immunofluorescence microscopy, we detected thetransfection efficiency of recombinant plasmid pGCSi-CCR2, while usingRT-PCR and Western blot assays, we detected interference efficiency ofPC-3M cells transfected with the recombinant plasmid pGCSi-CCR2.The proliferation of PC-3M transfected with the recombinant plasmidpGCSi-CCR2was evaluated by MTT assay. The effect of recombinantplasmid pGCSi-CCR2on apoptosis of PC-3M was detected via TUNELassay. We also explored the mechaniam of CCR2inhibition on cellviability by analyzing several cell proliferation and apoptosis related genes expression, c-Myc, cyclin D, Bcl-2, Bax and Caspase-3, as detectedby RT-PCR and Western blot assays. Using wound-healing assay, wedetected the effect of CCR2inhibition on the migration of PC-3M. UsingTranswell analysis, we detect the invasion of PC-3M transfected with therecombinant plasmid pGCSi-CCR2. Finally, we also analyzed the MMP2and MMP9expressions which involved in tumor metastasis usingRT-PCR and Western blot assays.Result: RT-PCR and western blot assay showed that transfectionwith the plasmid pSi-CCR2successfully inhibited the CCR2expression.The cell proliferation rate was significantly slow and the apoptotic ratewas increased in PC-3M cells treated with CCR2-siRNA, indicating byMTT cell viability and TUNEL assay, respectively. As expected, CCR2knockdown also reduced the migration and invasion of PC-3M cells, asillustrated through wound healing assay and transwell assay. The possiblemolecular mechanism was the regulation of several signal pathwaysinvolved in survival, apoptosis, migration and metastasis.Conclusion: The present finding suggest that CCR2expression iscrucial for CCL2induced proliferation and invasion of PC-3M cells, andCCR2could also be a promising molecular target for prevention of PCagrowth and metastasis.