Effect of miR-130b on repair of TNF-α-induced genomic DNA double strand breaks in cervical cancer cells
YANG Lei
LIU Tao
DUAN Jihui
MU Hong
Abstract：Objective To observe the impact of miR-130 b on the repair of DNA double strand breaks in cervical cancer cells and to discuss the mechanism underlying them. Methods Siha cells were classified into 6 groups. The cells transfected with pc DNA 3. 1-CDKN1A ( cyclin-dependent kinase inhibitor 1A) , pc DNA 3. 1, miR-130 b mimics and the negative control of miR-130 b were taken as the CDKN1 A, pc DNA 3. 1, miR-130 b and NC groups in turn. The ones cotransfected with miR-130 b and pc DNA 3. 1-CDKN1 A and those with miR-130 b and pc DNA 3. 1 were taken as the CDKN1 A + miR-130 b and pc DNA 3. 1 + miR-130 b groups. Cells were stimulated with TNF-α after the transfection. DNA oliver tail moments ( OTM) were determined in comet assays and the phosphorylated H2 AX histone protein variant ( γ-H2AX) levels were measured by flow cytometry. The CDKN1 A mRNA and its protein levels were measured by using semiquantitative real-time PCR and Western blotting. pc DNA 3. 1/EGFP, pc DNA 3. 1/EGFP-CDKN1A-wtUTR ( wild type) and pc DNA 3. 1/EGFP-CDKN1A-mutUTR ( mutant type) were transfected into Siha cells with miR-130 b mimics or NC, and the expression levels of reporter genes were determined. We used TNF-α and DNA damage enhancer AZD2461 to stimulate the cells in each group, and then used flow cytometry to determine the level of apoptosis. Results Compared with the pc DNA 3. 1 group, both the oliver tail moment and the γ-H2 AX protein level reduced in the cells of pc DNA 3. 1-CDKN1 A group ( both P < 0. 05) ; compared with the NC group, the oliver tail moment and the γ-H2 AX protein level increased whereas the CDKN1 A mRNA and protein levels decreased in the cells of miR-130 b group ( all P < 0. 05) . Compared with the pc DNA 3. 1 + miR-130 b group, the oliver tail moment, γ-H2 AX protein level and apoptosis rate decreased ( all P < 0. 05) ; compared with NC cotransfection, the cotransfection with miR-130 b mimics reduced the fluorescence levels within cells, but did not change the fluorescent relative activity of the mutant cells. Conclusion The miR-130 b hampers the repair of DNA double strand breaks in cervical cancer cells by inhibiting CDKN1 A gene expression through directly targeting CDKN1 A mRNA to increase the apoptosis rates.
Keywords：cervical carcinomamicroR-130bDNA repaircyclin-dependent kinase inhibitor 1Aphosphorylated H2AX histone protein variantapoptosis
Publication Date：2019-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：4( 28-31 )