Related Study Of The Effect Of Abnormal Myosin On The Pathogenesis Of Uterine Myoma

Background Uterine myoma is the most common benign tumor of female reproductive organs, the clinical incidence of20%to40%. The main clinical symptoms include genital tracts bleeding, infertility, abortion and anemia, brought great pain to the patient and seriously affect their quality of life. Uterine fibroids are mainly by the formation of uterine smooth muscle cell proliferation, its pathogenesis is complex.The traditional view is that uterine fibroids is due to complex interaction between hormones and local growth factor, lead to abnormal hyperplasia of uterine smooth muscle cells,then produce fibroids, so uterine fibroids belong to hormone-dependent tumor. However, with the deepening of research in recent years,the phenomena of uterine smooth muscle cells of uterin myoma patients exist abnormal contraction show that,myosin dysfunction may have played an important role in the incidence of uterine fibroids. Myosin exist in smooth muscle cells, play an important function in the muscle movements, therefore, the relationship between myosin dysfunction and the pathogenesis of uterine fibroids become the research focus of this project.Smooth muscle cell shrinkage in "muscle silk slip" principle, each contracting unit is composed of thick filament（myosin） and thin filament （actin）.When smooth muscle cell contracted, the muscle thick filaments overlaped, spiralled and twisted,it make smooth muscle cell shorter and bolder.Myosin is the basic component protein of thick filaments,aggregate by about200-250molecular, about1.5microns long. And myosin is also the most abundant structural protein and contractile proteins in all kinds of muscle cells, about25%of its total protein bank. Myosin is a six protein macromolecule polymers, composed of two heavy chain （MHC） and two light chain （MLC）. Each heavy chain including a complete globular heads （N-end） and a long fibrous alpha helix tail （C-end）,the rest of heavy chain with light chain form the head together, most of the two heavy chains spiral wound each other,the remaining two heavy chains interwined with the light chains,they can break down the ATP after they were actived.As a kind of multifunctional protein, myosin plays a important role in the organism.Myosins belong to a superfamily proteins, at least18species were found at present, the structure and function of them is different. In recent years, exploration show that myosin closely related with tumor proliferation, invasion and migration.Myosin may serve as a regulators span cell life activities in different levels.At the same time, tumor cell proliferation, invasion and migration need a lot of extracellular matrix as a support.Hallmarks of the myofibroblast are secretion of extracellular matrix（ECM）, ECM is synthesized and secreted by cells,located on the cell surface or macromolecules between cells, is mainly composed of polysaccharide, protein and proteoglycan. These substances constitute a complex space truss structure, support and connection tissue, regulate cell physiological activities. Adhesions between the cell and the ECM are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix.These combined functions in force transduction,signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. Myosin involved in muscle movement, not only to provide power for muscle contraction, but also myosin is the component of the cytoskeleton,interact with ECM components regulating cell proliferation and invasive process. There are excess expression of extracellular matrix in patients with uterine fibroids, therefore, myosin abnormalities whether affect the accumulation of extracellular matrix, thereby further aggravate the onset of uterine fibroids has also become the important research content of this project.So myosin as the intersection of smooth muscle cell contraction pathway and proliferation pathway, inspires us to think deeply of the pathogenesis of uterine fibroids. At present myosin in the pathogenesis of uterine fibroids, there is no in-depth study, this subject is divided into two parts and discussed the role of myosin in the occurrence and development of uterine fibroids. Part OneObjective By detecting the protein and mRNA expressions of MLC,MHC and MLCK in normal uterine smooth muscle tissue and uterine myoma tissues,to compared the expression differences of above three kinds of molecular,then to investigate there is abnormal contraction in uterine myoma,and to explore the roles of them on the development of uterine fibroid.Method1. Use immunohistochemical method to detect the expressions of MLC,MLCK,p-MLCK and MHC protein in30cases of uterine myoma tissues of the patients with uterine fibroids and30cases normal uterine myoma smooth muscel tissue,and compare the expressions of the three indicators in uterine myoma tissue and normal smooth muscel tissue.2. Apply RT-PCR method to detect the expressions of MLC,MLCK,p-MLCK and MHC mRNA in30cases of uterine myoma tissues and30cases normal uterine myoma smooth muscel tissue.Compare the expressions of MLC,MLCK,p-MLCK and MHC mRNA in uterine myoma tissue and normal smooth muscel tissue.3. Use Western blot method to detect the expressions of MLC,MLCK,p-MLCK and MHC protein in30cases of uterine myoma tissues and30cases normal uterine myoma smooth muscel tissue,and compare the expressions of the three indicators in uterine myoma tissue and normal smooth muscel tissue.Results Immunohistochemical results show that the morphology of uterine myoma cells are similar to normal smooth muscle cells, but uterine myoma cells permutation are more intense. Immunohistochemical slices in tan for a positive result, the deeper dyeing showed expression quantity is higher,immunohistochemical staining positive expression site is located in the cytoplasm. The differences of the three indicators between uterine myoma tissue and normal smooth muscle tissue had statistical significance （P<0,05）.2. The relative abundance of MLC, MLCK and MHC mRNA in the uterine myoma tissue all increased. The differences of all the indexes above between the expression in uterine myoma tissue and normal smooth muscle tissue had statistical significance （P<0,05）.3. Specific protein bands were detected in both uterine myoma and smooth muscle tissue by using Western Blot method,but MLC, MLCK, p-MLCK, MHC relative content was significantly higher in uterine myoma tissue than in normal smooth muscle tissue, differences exist statistical significance （P<0,05）.Conclusions The expression of the MLC, MLCK, p-MLCK, MHC protein and mRNA was upregulated in uterine myoma tissue, and the expression of MHC is the most significant changed.The result suggests that the abnormal expression of every composition of myosin may be associated with the onset of uterine fibroids. Significance Our experiments used immunohistochemistry, RT-PCR and Western Blot method confirmed that there was abnormal expression of MLC,MHC and upstream MLCK in uterine myoma tissue, and it may cause arrangement and contraction dysfunction of smooth muscle cells.Uterine smooth muscle cell dysfunction has certain correlation with the production of uterine fibroids, at the same time, the results for the further study of myosin dysfunction in uterine smooth muscle cells proliferation and promote the secretion of extracellular matrix laid a foundation. Part TwoObjective Observe abnormal expression of MHC on the influence of dysfunction proliferation and the secretion of extracellular of uterine fibroid cell.Method Using RNAi technology, knock down MYH11gene encoding MHC,interfere the expression of MHC, observe loss expression of MHC on the influence of uterine fibroid cell lproliferation.; Through the detection of protein involved in cell proliferation, observe the relationship between MHC and cell proliferation; By detecting the extracellular matrix related proteins, observe the role of MHC in the regulation of extracellular matrix, thereby further understand the expression of extracellular matrix protein change in the role of MHC related cell proliferation.1. Adherence method for uterine myoma cells in vitro, and identified using immunohistochemical method.2. Using the pcDNATM6.2-GW/EmGFPmiR and pLENT6.3/V5-SR60₁expression vector to construct containing MYH11siRNA slow virus vector.3. Containing MYH11siRNA slow virus vector was transfected into human uterine myoma cells. After transfection at72h cells were collected,then fluorescence microscopy was used to measure and observe transfection conditions.According to the green fluorescent protein （GFP） expressing green fluorescent characteristics, observed the GFP expression under the fluorescence microscope.4. RT-PCR determined the MHC mRNA expression after MYH11RNA interfere and observed interferenceing efficiency.5. The proliferation ability of MYH11RNA interfered uterin myoma cell and normal control group cell was examined by MTT method,Analysis MYH11RNAi for the influence of uterine myoma cell proliferation ability.6, Western blot method for detection the change of the expression of proliferating cell nuclear antigen（PCNA）, P53and serine/threonine kinase AKT IN uterine myoma cells before and after MYH11RNA interfere, further observation loss expression of myosin heavy chain impact on uterine myoma cell proliferation.7. RT-PCR method was applied to detect the mRNA expression of Collagen I, IGF, EGF, fibronectin and proteoglycan uterine myoma cells after MYH11RNA interfere and normal control group,and analyze the influence on the mRNA expressions of Collagen I, IGF, fibronectin,EGF and proteoglycan after MYH11RNAi.8. ELISA method was used to detect the expression of Collagen I, IGF, fibronectin, EGF and proteoglycan uterine myoma cells after MYH11RNA interfere and normal control group,and analyze the influence on the expressions of Collagen I, IGF, EGF, fibronectin and proteoglycan after MYH11RNA interfere.Results1. The shape of uterine myoma cell is spindle under a microscope,in vitro, in good condition.2. Green fluorescence（GFP） could be observed in the successftilly transfected cells through a microscope, the efficiency of infection is greater than70%by fluorescence microscope counting.3. RT-PCR method for detection uterine myoma cell after MYH11RNAi72h, the amount of mRNA expression of MHC decreased about72%compared to the normal control group, so we considered we downregulated the expression of MHC successfully.4. MTT assyay showed that, after72h of the transfection, compare with the normal control group, the uterine myoma cells in MYH11RNA interference group decreased significantly （P<0.05）.5. According to the results of Western blot, the expression of PCNA, p-AKT and AKT was low in MYH11interference groups compare with the fibroid cells control group, and the expression of P53is increased in the interferenced fibroid cells group, there are statistically significance between groups（P<0.05）.6.RT-PCR assyay showed that the mRNA expression of Collagen I in MYH11RNA interferenced uterine myoma cells decreased significantly compared with the normal control group （P<0.05）. RT-PCR assyay showed that the mRNA expression of IGF-1in MYH11RNA interferenced uterine myoma cells decreased significantly compared with the normal control group （P<0.05）. RT-PCR assyay showed that the mRNA expression of fibronectin in MYH11RNA interferenced uterine myoma cells decreased significantly compared with the normal control group （P＜0.05）. RT-PCR assyay showed that the mRNA expression of EGF in MYH11RNA interferenced uterine myoma cells increased significantly compared with the normal control group （P<0.05）. RT-PCR assyay showed that there is no significant difference of the mRNA expression of proteoglycan in MYH11RNA interferenced uterine myoma cells and the normal control group（.P>0.05）.7. ELISA was performed to detect the extracellular secretion of collagen I, IGF-1, fibronectin, EGF and proteglycans. The results showed that collagen I secretion was maintained at a stable level after MYH11RNAi. In control cells, collagen I production increased gradually, compared with control group cells, so that over time the secretion of collagen I was significantly decreased after MYH11RNAi （P<0.05）.IGF-1secretion was also reduced after MYH11RNA interference groups compared to control cells. In control group cells, IGF-1remained at a high level at24and48h, after which it decreased gradually, but levels remained higher than those in MHY11RNA interference groups （P<0.05）.In addition, fibronectin secretion was also reduced after MYH11RNA interference groups compared to control cells,fibronectin production increased gradually over time （P<0.05）. EGF production increased gradually over time compared with control group cells （P<0.05）. But proteoglycans did not shown any significant change compared to control cells（P>0.05）.Production increased gradually in either MHY11RNA interference groups cells or control group cells.Conclusion Loss expression of MHC can affect the uterine myoma cells to produce protein involved in cell proliferation,such as PCNA,P53and AKT,and secrete some characteristics of protein, the expression of PCNA,AKT,collagen I,IGF1,fibronectin decreases obviously with the down expression of MHC,but P53and EGF markedly increase, no obvious changes in the content of proteoglycans.Significance Through this experiment, we had knocked down the expression of MHC, indirect damage the structure and activity of myosin, detected uterine myoma cell proliferation ability markedly reduced; In addition, some of the extracellular matrix proteins and cytokines secreted abnormally, directly or indirectly changed the physiological characteristics of smooth muscle cells and microenvironment around the cells.Therefore, we hypothesized that myosin not only played an important role in the contraction of uterine smooth muscle cells, the expression of MHC changes has produced a certain influence on uterus smooth muscle cell proliferation and extracellular matrix secretion, so as to inspire us to do more depth research and discussion on the pathogenesis of uterine fibroids.