Expression Of THY1 Gene In Ovarian Cancer And Its Effect On Growth Of Ovarian Cancer Cells

Background and Objective Epithelial ovarian cancer is the first leading cause of gynecologic malignancy-related deaths. Despite all therapeutic efforts, it is still the tumor with the poorest prognosis and most fatal outcome compared with other gynecologic malignancies. Even though extensive studies have shown numerous genetic alterations in ovarian cancer cells, no specific tumor suppressor gene has been identified to date.A number of independent studies have demonstrated a high frequency of nonrandom loss of heterozygosity(LOH) of chromosome 11q in human ovarian carcinoma cells. The LOH data suggests that chromosome 11q may carry a tumor suppressor gene or genes for ovarian carcinoma.THY1 (CD90, cluster of differentiation 90) which is in human first found by Hamann A in 1980 is a surface glycoprotein of 25-28 kDa, which is expressed on the cytoplasmic membrane of different cell types. THY1 which is located 11q22.3 triggers a variety of cellular functions including proliferation, lymphokine release, differentiation, and apoptosis. Despite extensive investigation, the exact function and physiologic role of THY1 in the cell remains unknown. Introduction of a normal chromosome 11 into SKOV3 ovarian cancer cells resulted in complete tumor suppression and THY1 could be designated as a novel tumor suppressor gene for ovarian cancer. However, direct evidence that THY1 is a candidate TSG in ovarian cancer is still lacking. THY1 is a good candidate tumour suppressor gene in nasopharyngeal carcinoma, which is significantly associated with lymph node metastases.The aim of this study was to investigate the role of THY1 in epithelial ovarian cancer development, so as to provide some theoretic basis for gene therapy that target at THY1 which includes: 1. To detect the expession of THY1 protein in ovarian serous cystadenocarcinoma tissues; 2. To construct THY1 eukaryotic expression plasmid and transfect into ovarian serous cystadenocarcinoma cell line SKOV3; 3. To detect the effects of THY1 transfection on the growth of SK0V3 in vitro and in vivo.Material and Methods1. Immunohistochemistry was performed to detect the expession of THY1 gene in formalin-fixed, paraffin-embedded specimens of normal ovaries(n=25), ovarian serous cystadenoma(n=25), and ovarian serous cystadenocarcinoma (n=53). Correlations of expression of THY1 with clinicpathological parameters of epithelial ovarian cancer were statistically analyzed.2. The gene fragment coding for THY1 was obtained from human normal ovarian tissue using RT-PCR, and inserted into the eukaryotic expression plasmid pcDNA3.1(+) to construct the recombinant plasmid pcDNA3.1(+)-THY1, which was transformed into E. coli JM109, followed by selection of the positive clones containing the target inserts. The eukaryotic expression plasmid was analysed by PCR, restriction endonucleases digestion and DNA sequencing. The recombinant plasmid pcDNA3.1(+)-THY1 was transfected into SKOV3 cells by liposome protocol. The experimental cells were classified into three groups: SKOV3-THY1, SKOV-3-Null and SKOV3. The expression of THY1 mRNA and its protein were examined by RT-PCR and Western blotting methods.3. In vitro the cell growth and apoptosis of three groups were evaluated by MTT assay and flow cytometry. For in vivo tumorigenicity studies, 1 x 107 cells/site (1 site/animal) from each of the respective cell lines were injected subcutaneously into SCID mice. A total of five mice were inoculated with each cell population on day zero and the mice were observed daily to determine the latency period before detectable tumor growth. Once tumors were palpable, the tumor size was measured weekly for a period of 28 days to assess tumor growth rate. After completion of the study the mice were euthanized, the tumors were removed, and a section of each tumor was placed in formalin for histopathologic examination. Sections of the tumors were detected to assess THY1 expression in these tumor cells. T test and X~2 test were used for statistical analysises.Results Established an experimental model to study the relationship betweenepithelial ovarian cancer and THY1 gene, we found:1. The positive expression rates of THY1 protein in normal ovaries, ovarian serous cystadenoma and ovarian serous cystadenocarcinoma were 60.0% (15/25), 72.0% (18/25) and 34.0% (18/53) respectively. The value of IOD of THY1 protein expression were 288449.1±60087.25,271655.5±66588.74 and 252087.5+45559.42 respectively. The expression of THY1 protein down-regulated in ovarian serous cystadenocarcinoma tissues compared to normal ovarian tissues and ovarian serous cystadenoma tissues( P < 0.05). THY1 expression correlated with surgical-pathological staging, histological differention and lymph node involvement negatively( P<0.05).2. The gene fragment of exogenous THY1 was correctly inserted into the eukaryotic expression plasmid pcDNA3.1(+). An eukaryotic expression vector of pcDNA3.1(+)-THY1 was constructed successfully and verified by PCR, restriction endonucleases digestion and DNA sequencing. The recombinant expression plasmid pcDNA3.1(+)-THY1 has been transfected into SKOV3 cells and obtained stable expression verified by RT-PCR and Western blotting methods.3. The apoptotic rate of SKOV3-THY1(31.8%) was higher than SKOV3-Null(10.5%) and SKOV3 (9.8%). There was significant difference between SKOV3-THY1 and SKOV3-Null or SKOV3(P< 0.05), but there was no significant difference between SKOV3-Null and SKOV3( P>0.05). The cell inhibitory rate of SKOV3-THY1(56.6% at the fifth day) was higher than the SKOV3-Null(12.5%), there was significant difference between them(P<0.05). The latency period for tumor formation for both of the cell lines is 5 days. The tumors grew quite rapidly in the null transfectants compared with the THY1-transfected cells over the next 4 weeks. At the time of euthanasia, the tumors from SKOV3-Null measured 340.74 mm~3 on average, but those from SKOV3-THY1 were significantly smaller (almost half the size), measuring only 152.88 mm3 on average. The tumor inhibitory rate of SKOV3-THY1 was 50.00%. THY1 protein expression was positive in tumor cells of SKOV3-THY1 group.Conclusion The levels of THY1 expression reduced in ovarian serous cystadenocarcinoma tissues, and this may be related with the occurrence and development of ovarian serous cystadenocarcinoma. THY1 transfection can inhibit the growth of SKOV3 cells in vitro and in vivo. THY1 gene may play an important role in generation and development of ovarian cancers.