Study On Application Of Analytical Method For Functional Genes In Malignant Tumor

With the development of molecular biology, cell biology and analytical chemistry, and the advanced study of functional genomics studies in "post-genome era", more and more genes promoting the development of cancer genes have been known. This paper focuses on the function identification and expression detection of tumor genes, and the rules of promoting tumor cell growth.1. Analysis of UHRF1 Expression and Function in Human Ovarian Cancer CellLentivirus-mediated short hairpin RNAs (lv-shRNAs-UHRF1) were designed to trigger the gene silencing RNA interference (RNAi) pathway. Small interfering RNA (si-RNA) was introduced in four human ovarian cancer cell lines (SKOV-3, OVCAR-3, HO-8910 and HO-8910 PM) to downregulate the expression of UHRF1, and study the function of UHRF1 in ovarian cancer cells. UHRF1 expression in infected ovarian cancer cell lines was evaluated by real-time PCR and Western blot analysis. The cell counting Kit-8 (CCK-8) assay was used to measure cell proliferation, and flow cytometry and Hoechst 33342 assay were applied to measure cell cycle arrest and apoptosis. Cell invasion was assessed using transwell chambers. Western blot analysis was used to measure the expression of the cell cyclin protein D2, E and B1. flow cytometric apoptosis assay was used to characterize the cell cycle behaviour of ovarian cancer cells depleted of UHRF1. Our results demonstrated that the loss of UHRF1 promoted ovarian cancer cells apoptosis, while inhibited cell proliferation and invasion. A small percentage of cells remains blocked in G2/M phase and an increase in the mitotic cyclin B1. Based on these results, we presumed that UHRF1 might act as a new oncogene in ovarian cancer.2. Analysis of UHRF1 Expression in Human Ovarian Cancer TissueUHRFl levels in paired ovarian cancer tissues and adjacent normal tissues from ovarian cancer patients were detected using relative quantitatively PCR and western blot. Immunohistochemistry was used to detect the UHRF1 protein in cases of paraffin-embedded ovarian cancer tissues and cased of ovarian innocent tumours. According to the clinical and pathological data of 56 cases of ovarian cancer patients, we analysed the relationship between UHRF1 gene and ovarian cancer. RT-PCR and Western blot results showed that the expression of UHRF1 mRNA and protein in ovarian cancer tissues were higher than corresponding paracancerous (P<0.01; P<0.05). Immunohistochemistry results showed that in ovarian innocent tumour tissues, the positive expression of UHRF1 was 6.67%(1/15); in ovarian carcinoma tissues, the positive expression rate was 42.86%(24/56), which was higher than that of ovarian innocent tumour tissues, the difference was statistically significant (P<0.05). According to the clinical and pathological data of 56 cases of ovarian cancer patients, we found the expression of UHRFlwas related to the classification of ovarian cancer (P<0.05). However, there was no relationship between UHRF1 and ovarian cancer patient’s age, tumor size, tumor stage and lymph node metastasis (P>0.05).3. RNAi-mediated Downregulation of Cyclin Y to Attenuate Human Breast Cancer Cell GrowthCyclin Y (CCNY) is a newly identified PFTK1 interacting protein and has been found to be associated with the proliferation and tumorigenesis of human non-small-cell lung cancer. In this work, we analyzed the expression levels of CCNY in 65 cases of breast cancer (BC) tissues and four BC cells, BT-474, MDA-MB-231, T-47D and MCF-7 cells. Lentivirus-mediated short hairpin RNA (shRNA) was employed to knock down CCNY expression in MCF-7 and MDA-MB-231 cells. The effects of CCNY depletion on cell growth were examined by MTT, colony formation and flow cytometry assays. The results showed that immunohistochemical expression of CCNY in tumor tissues is stronger than normal tissues.CCNY was also expressed in all four BC cell. The knockdown of CCNY resulted in a significant reduction in cell proliferation and colony formation ability. Cell cycle analysis showed that CCNY knockdown arrested MDA-MB-231 cells in the G0/G1 phase. Furthermore, depletion of CCNY could inhibit BC cell growth via the activation of Bad and GSK3β, as well as cleavages of PARP and caspase-3 in a p53-dependent manner. Therefore, we believe that CCNY has biological effect on BC development, and its inhibition via an RNA interference lentiviral system may provide a therapeutic option for BC.