The Function And Targeting Therapy Of TIMELESS In Cervical Cancer

Objective: To investigate the expression of TIMELESSin cervical cancer, and to study the effect of TIMELESS on cell proliferation,apoptosis,senescence,clonogenesis and cisplatin responsein cervical cancer.Methods:TIMELESS m RNA expression was analyzed in three independent,open-access gene expression profiles of cervical cancer,TIMELESS protein expression was detected by immunohistochemistryin Cervical Intraepithelial Neoplasia(CIN, including CIN I, CIN II,CIN III) and cervical cancer tissue specimens; the expression of TIMELESS was further validated in cervical tissue microarraysby immunohistochemistry and in cervical cancer cell lines(He La, Si Ha, Caski and C33A) by Western Blot. After TIMELESS knocking-down by targeting si RNA(si RNA#1, si RNA#2, si RNA#3), TIMELESS expression was detectedby Real-time PCR and Western Blot. Cell proliferation and the chemosensitivityto cisplatin of these cell groupswere assessed by CCK8 assay.Cell apoptosis was analyzed by flow cytometry in three cell groups.The colony formation assay was conducted to test the clonogenic ability of each group.β-Galactosidase Staining assay was performed to assess cell senescence in the three cell groups.Theexpression of γ-H2 AX, Rad51 and cleaved caspase-3, which wasrespectively involved in DNA damage repair and apoptosis, were detected by Western Blot in each group.Results:1.The probe against TIMELESS(203046_s_at) was elevated in cervical cancer compared to normal cervix, indicating the increase of TIMELESSm RNA(P<0.05);2. The expression of TIMELESS protein was also elevated in the progression of CIN to cervical cancer(P<0.05);3. TIMELESS was overexpressed in 52.5%(21/40) cases of cervical cancer tissues;4. TIMELESS protein expression was determined in He La, Si Ha, Caski and C33 A cervical cancer cell lines. Si Ha showed the most robust expression of TIMELESS in these cell lines; 5. All three si RNAs targeting TIMELESS knocked down TIMELESS expression effectively, the respective inhibitory efficacy were(0.263±0.018),(0.157±0.013) and(0.286±0.021) at m RNA level, and were(0.162±0.052),(0.134±0.057) and(0.173±0.062)(P<0.01)at protein level in Si Ha cell line, and si RNA#1 and 2 showed more efficiency than si RNA#3;6.The cell proliferation of cells in both si RNAgroupswas dramatically inhibited;7.Flow cytometry assay showed that TIMELESS knockingdown induced cell apoptosis; 8. Colony formation assay showed decreased colonies of Si Ha cells(111±3.786),(129.667±3.590) in both si RNA groups compared with the group of Ctrl si RNA(209.333±3.383);9. In Senescence β-Galactosidase Staining assay, cell senescence rate wasincreased in both si RNA groups(P<0.0001) with or without cisplatin treatment. 10 si RNA#1 and si RNA#2 both sensitized Si Ha cells to cisplatin, the half maximal inhibitory concentration(IC50, μg/ml)was 5.052 in si RNA#1 and 2.565 in si RNA#2, compared to 7.907 in Ctrl si RNA; 11. Cleaved caspase-3 was also increased in both groups, indicating the activated apoptosis pathway;12. γ-H2 AX was increased and Rad51 was inhibited when the TIMELESS was knocked down, indicating the impaired DNA repair.Conclusions: TIMELESS is overexpressed in cervical cancer tissue and cell lines.Targeting TIMELESS may inhibit cervical cancer cell proliferation clonogenesis ability, induce cell apoptosis and senescence, and sensitize cervical cancer to cisplatin treatment.