Effect Of Gene Silence Of NMHC Ⅱ Isoforms On Biological Behaviors In Breast Carcinoma

Breast cancer is one of the malignant tumors harm to women’s health.Tumormetastasis is the main characteristic of malignant tumor.Metastasis and recurrenceare the the biggest obstacle for cure as well as the primary causes of death.Recentstudies show that the capability of invasion and metastasis of tumor is associatedwith the regulation of migration protein.Nonmuscle myosin,however,as a member ofmotor protein family,is a critical monitor in tumor cell activity.thus,The role ofnonmuscle myosin in tumor metastasis has drawn the attention of the researchers.Nonmuscle MyosinⅡ,NMⅡas the main component of cytoskeleton,is amember of motor protein super family,not only supplying power for intracellularmolecular movement,but also participating in mulriple physiological activity.Theinitial stage of tumor metastasis includes tomor cell polarization,the change of celladhesion and cell migration. NMⅡ not only involved in the cell movement, but asan important regulator of cell polarization and cell adhesion.Thus, we deduce thatthe NMⅡis involved in the regulation of tumor metastasis.NMⅡ is composed oftwo230kd nonmuscle myosin heavy chain(NMHC),a couple of20kd regulatorylight chain(RLC)and17kd essential light chain(ELC).In the process of cellmovement, light chain has play a role on regulation,heavy chain which is combinedwith actin directly regulate cell movement.According to the differeence of heavychain,NMHCⅡ has three isoforms,namely NMHCⅡA,NMHCⅡB andNMHCⅡC,which are encoded by MYH9,MYH10and MYH14respectively.In the previous work,we only detected two isoforms,NMHCⅡA andNMHCⅡB,NMHCⅡC was not detected,In addition,the expression of bothNMHCⅡA and NMHCⅡB in breast tumor tissue and breast tumor cell MCF-7were higher than in the normal breast tissue and cells.The expression of lymph glandmetastasis group was obviously increased.Therefore,we consider NMHCⅡis closely related to the malignant degree of tumor and metastasis of breast cancer.Objective:We established stable silence NMHCⅡ different isoforms of cell lines by RNAinterference,Through the experiment in vitro and NOD/SCID mice transplantedtumor model study the effect of silencing different isoforms of MCF-7cell onbiology behaviors such as cell proliferation, cell invasion.Methods:1.We used the technique of RNA interference respectively to silenceNMHCⅡA gene and NMHCⅡB gene of MCF-7cell,and selected out their stablecell lines.The RNAi group as the experimental group and the control group whichwas consisted of empty vector transfection and non-transfection MCF-7cell line,The efficiency of silence was detected via Western Blot and immunocytochemistry.2.Through experiments in vitro studey the effect of gene silence of NMHCⅡisoforms on biological behaviorsof MCF-7cell.(1)Detect the effect of MCF-7cell proliferation on NMHCⅡgene via CCK-8kit.(2)Detect the change of cell cycle after silence of each isoform cell line via flowcytometry.(3)Detect the change of cell migration ability by cell wound scratch assay.(4)Detect the effect of NMHCⅡon cell invasion ability of MCF-7cell viaBoyden.3.By constructed a NOD/SCID mice animal model of spontaneous metastasis invivo and observed the influence of different isoforms of NMHCⅡabout tumorgrowth.In vivo experiments, silencing of NMHC II isoforms have been verified.(1)Through the immunohistochemical staining to observe the expression ofNMHCⅡAand NMHCⅡB.(2)Through the appearance of tumors, histological observation to detect theinfluence of transfer in different isoforms of NOD/SCID mice which were NMHC Ⅱ deficiency.(3)Through the immunohistochemical staining to examine PCNA, Caspas3andMMP2expression which reflect the proliferation,apoptosis and invasion of thetumors.(4)Through the observation of various organs, to detect whether there is distantmetastasis.Results:1. We obtained the stable transfection cell line.The results of Western Blot andthe immunocytochemistry both showed that the expression of NMHCⅡB decreasedobviously,while have no effect on the expression of NMHCⅡA of the RNAiⅡBgroup which were compared with control;the expression of NMHCⅡA as well asNMHCⅡB both were decreased of RNAiⅡAgroup compared with control.2.Experimental results in vitro showed that:(1)The result of CCK-8kit and flowcytometry indicated that the proliferation ability were decreased no matter whichNMHCⅡisoform was absent.(2)The cell wound scratch assay showed that themigration efficiency of group RNAiⅡA andⅡB were slightly higher than thecontrol group.(3)The boyden chamber results showed that the artificial basementmembrane cells of the experimental group were less than the control group.Therewere no difference between the two experimental groups in the above results.3.Animal models of spontaneous metastasis in immunodeficient NOD/SCIDmice in vivo experiment results showed that the NMHCⅡsilenced group in micetumor necrosis were serious, PCNA expression were weaker than the control group,the Caspase3expression of the tumor tissue in mice were better than the controlgroup,the expression of MMP2were weaker than the control group,each group hadno obvious metastasis.Conclusions:1.There is a relationship of regulation between NMHCⅡA and NMHCⅡB,NMHCⅡAplays a role on the expression of NMHCⅡB.2.NMHCⅡA and NMHCⅡB have a regulation on the proliferation and invasion of MCF-7.3.Lack of NMHCⅡMCF-7cell in NOD/SCID mice in the formation of theobvious necrosis of tumor tissue might be associated with the suppression ofproliferation and the promotion of apoptosis.