Studies On The Expression Of MMSET In Endometrial Cancer And The Effects Of Silencing MMSET Gene Expression On The Biological Behavior Of Endometrial Cancer Cells

Background and objective: Endometrial carcinoma is one of the three most common cancers in female reproductive system. The incidence rate has increased worldwide in rescent years, and its prevalence increasing with the age of patients decreasing. Despite endometrial cancer is frequently diagnosed at an early stage and responds well to surgical treatment, advanced and recurrent endometrial cancer always with a poor prognosis remains a serious health problem for women worldwide. Therefore, many studies focused on genes which involved in the carcinogenesis of endometrial cancer. However, the molecular mechanisms underlying carcinogenesis and progression of endometrial cancer have not been comprehensively reviewed till now. Therefore, efforts to identify more molecular markers that could be used to detect endometrial cancer more earlier are of great clinical importance. MMSET(multiple myeloma SET), a novel oncoprotein, has been implicated to be overexpressed in several carcinomas and associated with invasion and migration of some carcinomas, such as neuroblastoma, hepatocellular carcinoma, gallbladder carcinoma, lung cancaer, colon cancer and prostate cancer. It has important roles during the development and progression of certain types of carcinomas, and also the expression of MMSET is associated with the poor prognosis of certain carcinomas. However MMSET expression and its role in the development of endometrial cancer remain unknown. The objectives of our study are as followings:(1) to determine the level of expression of MMSET in endometrial cancer and to analyze its relationship withclinicopathologic features, including the prognosis of patients with endometrial cancer;(2) Western blot were used to detect the level of MMSET protein in three different endometrial cancer cell lines, and to chose the endometrial cancer cell lines with high expression of MMSET. And we constructed specific sh RNA recombinant fluorescence expression plasmid against MMSET gene. And then to harvest endometrial cancer cell lines with stable expression recombinant sh RNA plasmid, which lay a foundation for the further investigation about the relationships between MMSET and the development and progression of endometrial cancer;(3) to inhibit the expression of MMSET in endometrial cancer cells by RNA interference, and to investigate the effects of MMSET silencing on the growth, migration and invasion of endometrial cancer cells.Material and methods:(1) Immunohistochemistry was used to detect the expression of MMSET protein in 62 normal endometrium specimens and 161 endometrial cancer specimens;(2) The expression of MMSET protein in the endometrial cancer cells HEC-1A, HEC-1B, and ECC-1 were detected by westernblot to choose the the endometrial cancer cells with high expression of MMSET. In accordance with the principle of sh RNA design, we designed the sh RNA sequences and cloned it into the p IRES2-EGFP plasmid, then the recombinant plasmid was transfected into the endometrial cancer cells. And we detected MMSET protein expression using westernblot to examine the transfection result;(3) The MTT assay and colony formation assay was performed to evaluate the effect of the MMSET silencing on the cell proliferation of endometrial cancer cells; Cell monolayer scratch assay was performed to evaluate the effect of the MMSET silencing on the migration of endometrial cancer cells; Matrigel invasion assay was performed to evaluate the effect of the MMSET silencing on the invasion of endometrial cancer cells.Results:(1) MMSET expression was significantly elevated in endometrial cancer(P<0.001) compared with the normal controls.(2) The overexpression of MMSET was significantly associated with the tumor histologic grade, International Federation of Gynecology and Obstetrics stage, lymph node metastasis, depth of myometrial invasion, vascular/lymphatic invasion, recurrence and poor prognosis(P<0.05).(3) Western blot showed the relative density of MMSET protein in the endometrial cancer cells HEC-1A, HEC-1B, and ECC-1 was 0.972±0.028 、1.265±0.043 and 0.904±0.023, and in the endometrial cancer cells HEC-1B, the level of MMSET expression was the highest. So we chose the endometrial cancer cells HEC-1B to construct MMSET sh RNA stable cell line.(4) The recombinant expression vector of sh RNA targeted MMSET was suecessfully constructed and transfected into the endometrial cancer HEC-1B cells. Western blot showed that the recombinant plasmids have MMSET gene silencing effect(t=28.975,P<0.001).(5) MTT method result showed that the growth rate of MMSET sh RNA group was significantly lower than the control group(P<0.05) scine the third day.(6) The rate of colony formation of the MMSET sh RNA group was(39.45±4.37)%, however the control group was(84.23±8.62)%. The proliferation of the endometrial cancer HEC-1B cells was suppressed obviously(t=10.361, P<0.001).(7) Cell monolayer scratch assay results showed that the MMSET sh RNA group migrated slowly and closed th wound by(15.72±3.12) % afer 24 h. In constrast, control group cells closed the wound by(23.23±1.75)% after 24 h. The migration of endometrial cancer cells in MMSET sh RNA group was significantly lower than that in the control grup(t=4.694, P<0.01).(8) The numbers of endometrial cancer cell of the MMSET sh RNA group on the lower polycarbonate membrane of transwell chamber covered matirgel were(16.3±2.8), however the control group were(39.4±4.2). The invasive cells number in theMMSET sh RNA group was significantly lower than in control group(t=10.231,P<0.001).Conclusion:(1) MMSET expression was significantly elevated in endometrial cancer, and the overexpression of MMSET was significantly associated with the tumor histologic grade, International Federation of Gynecology and Obstetrics stage, lymph node metastasis, depth of myometrial invasion, vascular/lymphatic invasion, recurrence and poor prognosis(P<0.05). Overexpression of MMSET may be associated with tumor progression in endometrial carcinoma and thus may serve as a new molecular marker to predict the prognosis of endometrial carcinoma patients.(2) We constructed specific sh RNA recombinant fluorescence expression plasmid against MMSET gene successfully, and transfected it into the endometrial cancer cells. And we harvested endometrial cancer cell lines with stable expression recombinant sh RNA plasmid, which lay a foundation for the further investigation about the effect of MMSET silencing on the cell proliferation, migration, and invasion of endometrial cancer cells.(3) The experiments in vitro showed that the MMSET silencing could inhibited the ability of cell proliferation, migration, and invasion in endometrial cancer HEC-l B cells. It indicated that MMSET played an important role in the invasion and migration of endometrial carcinoma and may become a new potential target in the therapy of endometrial cancer.