Effect On Biological Behaviors Of MCF-7Cell After RNA Interference Of Nonmuscle Myosin Heavy Chain ⅡB(NMHC ⅡB)

Malignant tumor is grave threat to human health. In recent years, malignant tumoris proved to be the major death factor in many countries. Breast cancer is one ofclinically common malignant tumors in female. Recurrence and metastasis are primarycourse of death of breast cancer. Capability of invasiveness and metastasis is one ofthe tumors’ biological characteristics, and major topics of tumor research and treatment.Invasiveness and metastasis of tumor cell depends on cell motility。 Therefore，thestudy on factors and proteins involved in cell motility turns into major orientation oftumor research.The nonmuscle myosinⅡ(NMⅡ) is main composition of the cytoskeleton.involved in the process of cell motility, including cell polarity, cell migration and celladhesion. While, tumor metastasis also undergoes that process. So we deduce that theNMⅡ is involved in tumor metastasis. NMⅡ is composed of two heavy chain(NMHC) and two pairs of light chains (NMLC). NMLC can regulate and stabilize cellmovement, and NMHC maybe the direct work point of tumor metastasis. NMHCⅡhas three subtypes, respectively NMHCⅡA, NMHCⅡB and NMHCⅡC. Currentlystudies on NMHCⅡA are a lot, while the functions of NMHCⅡB is not clear. In thisresearch we observe breast cancer cells biological behavior by silence NMHCⅡBgene in MCF-7cells.Objective: We applied RNAi to silence NMHCⅡB gene in MCF-7cells，andobserved the effection of NMHCⅡB on the biological behaviors of breast cancer celllines MCF-7.Methods: MCF-7cells were transfected with plasmids of RNA interference ofNMHC Ⅱ B(plasmid2#, plasmid3#, plasmid5#) and the control vector bylipofectamine2000. We observed transfection sfficiency with fluorescence microscope,and detected the expression of NMHCⅡB gene through immunohistochemicalstaining and Western Blot after the transfection, and then screened out the plasmid which was the optimal effectiveness of inhibition of NMHCⅡB. We valuated theeffect of NMHCⅡB on the cell viability, migration and invasion of MCF-7: the abilityof cell viability was measured by CCK-8kit; cell migration was tested by scratchwound healing assay; the capabilty of cell invading through the matrigel-coated filterwas detected in Boyden chamber. To investigate the mechanism of NMHCⅡB, theexpression of cell skeleton and MMP-9was observed by Immunofluorescent andImmunohistochemical stainingResults:1. Under the fluorescence microscope, the transfection efficiency of each group wasnearly80%.2. The results of immunohistochemical staining and Western Blot confirmed that3plasmid provided could deduce the expression of NMHCⅡB, meanwhile, plasmid5#showed optimum efficiency.3. CCK-8kit tested that, compared with control group and control vector group,,cells in RNAi group showed no visible defference on cell viability.4. Scratch wound healing assay showed that the migration rate of cells in RNAigroup was lower than that in the control and control vector group.5. Boyden chamber results showed: the number of cell passing through the Metrigelin RNAi group is significant less than that in the other two groups;6. Immunofluorescence staining of β-tublin to watch the cytoskeleton arrangementsof cells in3groups showed, cytoskeleton of cells in RNAi group seemed to be blurryand without clear network state.7. Immunohistochemical staining to test MMP-9expression showed that theexpression of MMP-9deceased after silence the NMHCⅡB gene in MCF-7cell.Conclusion:1. NMHCⅡB can not effect the viability of MCF-7cell.2.NMHCⅡB involved cell migration through regulating.cytoskeletal structure.3. NMHCⅡB regulated tumor migration and invasion through ability of synthesisand secretion of MMP-9.