Effects Of CCNO Inhibition On Biological Behaviors Of Cervical Cancer Hela Cells

Background:Cervical cancer is one of the most common gynecological malignant tumors, and the 4th cause of death in female cancer patients, which has a rising morbidity in recent years, especially in developing countries. In addition to HPV infection, the anomaly gene expression related to cell growth differentiation in host cell also plays an important role in the pathogenesis of cervical cancer. In recent years, many preclinical basic studies show that gene therapy is a potential treatment to cervical cancer, including p53 gene therapy which is very important in cell cycle regulation and apoptotic signaling pathway, Bax gene therapy mostly studied in cervical cancer, HPV E2 and p21 gene, antisense-RNA therapy aimed directly at oncogene, FHIT and PTEN gene therapy in cervical cancer. We used RNA-Seq technique and found that the expression of cyclin O which is a cell cycle proten in cervical cancer tissue is significantly higher than adjacent tissues, indicating its encoded gene CCNO may relative to the development of cervical cancer.Objectives:To investigate the effects of downregulation of CCNO expression on the biological behaviors (such as growth, proliferation and apoptosis) of cervical cancer Hela cells.Materials:Pathological slices include cervical cancer tissues and adjacent tissues, coming from patients with cervical cancer in department of Obstetrics and Gynaecology, PUMCH, inaddition to those who had radiotherapy, chemotherapy and didn’t get satisfied paraffin-embedded blocks.Hela cell line is Human cervical adenocarcinoma cell line, coming from Shanghai cell bank。Methods1. Detect the expression of CCNO in cervical cancer by hematoxylin-eosin (HE) and immunofluorescence.2. Design a small interfering RNA targeting CCNO (CCNO-siRNA), and transfect into Hela cells in vitro, downregulating the endogenous expression of CCNO.3. Examine the transfection efficiency of CCNO-siRNA by western blotting, confirming the inhibition of CCNO expression which could be used in next experiments.4. Detect the effect of CCNO on proliferation of cervical cancer Hela cells by CCK-8 assay.5. Analysis cell cycle and cell apoptosis by flow cytometry.Results1. Increased expression of CCNO was detected in cervical cancer tissues than adjacent tissues.2. After transfection, the designed siRNA could efficiently downregulate the endogenous CCNO expression in Hela cells proved with Western blotting.3.72h and 96h after transfection, the proliferation of CCNO-siRNA transfected cells were dramatically inhibited and were observed by CCK-8 assay(P<0.05, P<0.01).4. CCNO deficiency caused a cell accumulation in G0-G1 phase and a cell reduction by 12%(P＜0.05) in S phase cell number than those in the negative control group.5. The percentage of apoptotic Hela cells in CCNO-siRNA transfected cells is higher than those in the negative control group (P＜0.05).Conclusions1. CCNO expression in cervical cancer tissues is higher than adjacent tissues.2. CCNO-siRNA could efficiently downregulate the endogenous CCNO expression in Hela cells.3. CCNO could promote cell proliferation, cell cycle progression and inhibit apoptosis of Hela cells.