Expression Of HDAC1 In Ovarian Cancer And Effect Of HDAC1-siRNA On Ovarian Cancer Cells Hypoxia And Its Mechanism

Ovarian cancer is the most lethal gynecologic malignancy. In most patients, metastasis occurs by the time of diagnosis. To date, operations are still the main clinical methods, after that platinum and paclitaxel demonstrate the greatest efficacy. However, the duration of response to chemotherapy remains brief. Long-term survival in patients with advanced ovarian cancer is still no more than 20%. Therefore, we urgently need to find new treatments.Under the microenvironment of the solid tumor, hypoxia is the most common phenomena because of the vast energy and oxygen consuming. HIF-1αis the most important factor for the behavior changes under hypoxic condition. HIF-1αexpression can cause tumor cell proliferation and internal hypoxia, resulting in the activation of oncogenes and tumor suppressor gene inactivation, inhibition of HIF-la can significantly inhibit tumor growth.Histone deacetylase inhibitors (HDACIs) have been actively explored as a new generation of chemotherapeutics for cancers, with high efficiency, low toxicity, and can inhibit tumor cell proliferation, induce cell cycle arrest and promote apoptosis of tumor cells. Recent findings indicate that HDACIs repress the function of hypoxia-inducible factors.This study investigate the expression of histone deacetylase 1 (HDAC1) in human epithelial ovarian cancer tissue by immunohistochemical SP method, and approach its clinical significances. HDACl-siRNA lentiviral plasmid vector is successfully built and identified. COC1 cells were treated with deferoxamine mesylate (DFOM) at a concentration of 2.5μmol/L for 48 hours to establish cell hypoxia model. HDAC1-siRNA lentiviral plasmid vector transfect the hypoxia ovarian cancer cells. With MTT, flow cytometry, realtime-PCR, immunofluorescence and Western blot methods, to detect the repression HIF-la function and repression mechanism of HDAC1-siRNA lentiviral plasmid vector on the hypoxia ovarian cancer cells.The experiments as follows: (1) Expression of Histone Deacetylase 1 in Human Epithelial Ovarian CancerThe expression of HDAC1 protein in 45 cases of ovarian cancer tissues,15 cases of borderline ovarian tumor tissues,23 cases of benign ovarian tumor tissues and 9 cases of normal ovarian tissues were detected by immunohistochemical SP method.45 cases of epithelial ovarian cancer patients with clinical data was collected and analysis the clinical and pathological data.Results:There was over-expression of HDAC1 in epithelial ovarian cancer tissue. High expression of HDAC1 is early events during ovarian carcinogenesis. HDAC1 may be the new target markers for early diagnosis and treatment of epithelial ovarian cancer. Patients with HDAC1 positive tumors showed lower postoperative survival rates. HDAC1 is associated with a poor prognosis for ovarian carcinoma and indicates that HDAC1 is an independent index for prognosis.(2) Establishment and identification of HDAC1 siRNA plasmidAccording to mRNA sequence of HDAC1 from NCBI data base and the design philosophy of siRNA, three target interfering sequences were designed. BLAST Search shows no homology with other human gene sequence. This sequence is repeated in the DNA oligo as a reverse compliment. The two sequences are separated by a 9 nt random sequence that will serve as the "loop" in the short hairpin RNA (shRNA). The DNA oligos are annealed such that they get cloned into the pRNAT-U6.2/Lenti backbone using the BamHⅠand XhoⅠsites. The sequences were transformed into E.coli. to amplify, and identify positive clones, plasmid extraction and sequencing.Results:We successfully constructed three plasmids of HDAC1 siRNA interfering lentivirus, and sequencing is exactly right. This lay the foundation for the further experiments.(3) HDAC1 siRNA effect on hypoxia of ovarian cancer cells and the mechanisms.When ovarian cancer cells COC1 is in the logarithmic phase, added Deferoxamine Mesylate with the concentration of 2.5μmol/L for 48h and established the model of cell hypoxia. It is divided into three groups:the first is COC1, the second is COC1+DFOM+ pRNAT-U6.2/Lenti, the third is COC1+DFOM+HDAC1 siRNA. With MTT, flow cytometry, realtime-PCR, immunofluorescence and Western Blot technique, to study the effect of HDAC1 siRNA on HIF-1αand its mechanisms.Results:Deferoxamine Mesylate with the Concentration of 2.5μmol/L treated COC1 cells for 48h can induce the expression of HIF-1α, improve the hypoxia, and cell cycle arrest, apoptosis increased. Transfection HDAC1 siRNA plasmids into COC1 cells can induce the radio of Bcl-2/Bax decreasing and cell apoptosis. This study indicated that inhibition of HDAC1 can induce the regression of HIF-1αand the mechanism may be associated with the induction of apoptosis.Conclusions:(1) There is overexpression of HDAC1 in epithelial ovarian cancer tissue. Patients with HDAC1 positive tumors showed lower postoperative survival rates. HDAC1 is associated with a poor prognosis for ovarian carcinoma.(2) HDAC1-siRNA lentiviral interference plasmids were successfully constructed for further study HDAC1 gene silencing and anti-ovarian cancer mechanism.(3) Deferoxamine Mesylate treated COC1 cells can successfully established a cell model of hypoxia and induced the expression of HIF-1αunder normoxia, so it provides a convenient hypoxia model for we study the function of HIF-1α.(4) The expression of HIF-1αwas increased in hypoxia ovarian cancer cell, and hypoxia can induce the over-expression of HDAC1, and suggest that HDAC1 may be involved in cell adaptation to hypoxia.(5) Transfection HDAC1 siRNA plasmids into COC1 cells can reduce the expression of HIF-la. It decreased anti-apoptotic gene Bcl-2 and upregulation of pro-apoptotic gene Bax, induce the radio of Bcl-2/Bax decreased, which may promote ovarian cancer cells apoptosis.