Department of Clinical Laboratory, Tianjin Hospital of ITCWM Nankai Hospital, Tianjin, China.
Department of Clinical Laboratory, Tianjin First Center Hospital, Tianjin, China.
Curr Cancer Drug Targets. 2022;22(2):153-168. doi: 10.2174/1568009622666220111090715.
Dysregulation of miR-130b expression is associated with the development of different cancers. However, the description of the biological roles of miR-130b in the growth and survival of cervical cancer cells is limited.
The miR-130b levels in cervical cancer cells during different stages of growth were determined using reverse transcription-quantitative PCR. The methylation level of DNA sequences upstream of the miR-130b gene was measured using an SYBR Green-based quantitative methylation- specific PCR. Reverse transcription-quantitative PCR, Western blotting, and fluorescence report assays were used to identify the miR-130b-targeted gene. Cell counting kit-8 and comet assays were used to determine cell viability and DNA damage levels in cells, respectively. EdU Apopllo488 in vitro Flow Cytometry kit, propidium iodide staining, anti-γ-H2AX antibody staining, and Annexin-V apoptosis kit were subsequently used to determine DNA synthesis rates, cell cycle distribution, count of DNA double-strand breaks, and levels of apoptotic cells.
miR-130b levels increased at exponential phases of the growth of cervical cancer cells but reduced at stationary phases. The methylation of a prominent CpG island near the transcript start site suppressed the miR-130b gene expression. MiR-130b increased cell viability, promoted both DNA synthesis and G1 to S phase transition of the cells at exponential phases, but reduced cell viability accompanied by accumulations of DNA breaks and augmentations in apoptosis rates of the cells in stationary phases by targeting cyclin-dependent kinase inhibitor 1A mRNA.
miR-130b promoted the growth of cervical cancer cells during the exponential phase, whereas it impaired the survival of cells during stationary phases.
miR-130b 表达失调与多种癌症的发生发展有关。然而，miR-130b 在宫颈癌细胞生长和存活中的生物学作用描述有限。
采用反转录定量 PCR 检测不同生长阶段宫颈癌细胞中 miR-130b 的水平，采用 SYBR Green 定量甲基化特异性 PCR 检测 miR-130b 基因上游 DNA 序列的甲基化水平，采用反转录定量 PCR、Western blot 和荧光报告实验鉴定 miR-130b 的靶基因。细胞计数试剂盒-8 和彗星实验分别用于检测细胞活力和 DNA 损伤水平，EdU Apopllo488 体外流式细胞术试剂盒、碘化丙啶染色、抗 γ-H2AX 抗体染色和 Annexin-V 凋亡试剂盒分别用于检测 DNA 合成率、细胞周期分布、DNA 双链断裂计数和凋亡细胞水平。
宫颈癌细胞生长的指数期 miR-130b 水平升高，而静止期 miR-130b 水平降低。靠近转录起始位点的一个显著 CpG 岛的甲基化抑制了 miR-130b 基因的表达。miR-130b 在指数期增加细胞活力，促进细胞 DNA 合成和 G1 期到 S 期的转变，但在静止期降低细胞活力，同时伴有 DNA 断裂的积累和凋亡率的增加，其作用靶点是细胞周期蛋白依赖性激酶抑制剂 1A mRNA。
miR-130b 在指数期促进宫颈癌细胞的生长，而在静止期则损害细胞的存活。
