| 包利利,赵达,俞岩,周俏苗.长链非编码RNA，PTENP1通过miR-3611/PTEN基因途径调控宫颈癌进程的分子机制研究[J].中国医药导报,2024,21(3):19-27 |
二维码（扫一下试试看！） |
| 长链非编码RNA，PTENP1通过miR-3611/PTEN基因途径调控宫颈癌进程的分子机制研究 |
| Study of molecular mechanism of regulation in cervical cancer progression by long non-coding RNA PTENP1 via miR-3611/PTEN gene pathway |
| 收稿日期：2023-04-26 |
| DOI：10.20047/j.issn1673-7210.2024.03.04 |
| 关键词: 长链非编码RNA 微RNA 竞争性结合机制 PTEN基因 宫颈癌 |
| Key Words: |
| 基金项目:海南省卫生健康行业科研项目（22A200105） |
|
| 摘要点击次数: 550 |
| 全文下载次数: 437 |
| 摘要:目的 探讨长链非编码RNA PTENP1（以下简称“PTENP1”）在宫颈癌细胞增殖、迁移和侵袭的分子机制。方法 选取2019年1月至2022年12月海南省妇女儿童医学中心诊断的54例的癌组织与癌旁组织，另选取宫颈癌细胞系（HeLa、SiHa、C33A、Caski）和正常宫颈上皮细胞系（H8），采用实时荧光定量PCR、蛋白质印迹法检测PTENP1、miR-3611、PTEN基因、PTEN蛋白水平。选取合适的宫颈癌细胞系，比较PTENP1在细胞质和细胞核中的表达情况，并进一步将其分为空白组（无处理）、pcDNA3.1组（转染pcDNA3.1）、pcDNA3.1-PTENP1组（转染pcDNA3.1-PTENP1）、NC组（阴性对照，转染模拟物或抑制剂NC）、miR-3611抑制剂组（转染miR-3611 抑制剂）、pcDNA3.1-PTENP1+NC组（转染pcDNA3.1-PTENP1和NC）和pcDNA3.1-PTENP1+miR-3611 mimics组（转染pcDNA3.1-PTENP1和miR-3611模拟物）。比较空白组、pcDNA3.1组、pcDNA3.1-PTENP1组PTENP1、miR-3611、PTEN基因、PTEN蛋白及上皮间质转化的相关指标，采用细胞活力检测及流式细胞仪检测细胞增殖和凋亡情况；比较空白组、NC组、miR-3611抑制剂组miR-3611、PTEN基因、PTENP1水平；比较pcDNA3.1-PTENP1+NC组和pcDNA3.1-PTENP1+miR-3611 mimics组的细胞增殖情况及上皮间质转化的相关指标。采用双萤光素酶报告基因及RNA下拉实验验证miR-3611与PTENP1、PTEN基因的靶向关系。 结果 癌组织PTENP1、PTEN基因、PTEN蛋白水平低于癌旁组织，miR-3611水平高于癌旁组织（P＜0.05）。HeLa、SiHa、C33A、Caski细胞PTENP1、PTEN基因、PTEN蛋白水平低于H8细胞，miR-3611水平高于H8细胞（P＜0.05），后续实验选择HeLa、Caski细胞进行，PTENP1在HeLa、Caski细胞质中高表达。pcDNA3.1-PTENP1组PTENP1、凋亡率、上皮钙黏素、PTEN基因高于空白组，细胞增殖活力（培养48、72、96 h）及ZEB1、Snail、波性蛋白、miR-3611低于空白组（P＜0.05）。miR-3611抑制剂组miR-3611低于空白组，PTEN基因、PTENP1高于空白组（P＜0.05）。pcDNA3.1-PTENP1+miR-3611 mimics组细胞增殖活力（培养48、72、96 h）、上皮钙黏素低于pcDNA3.1-PTENP1+NC组，ZEB1、Snail、波性蛋白高于pcDNA3.1-PTENP1+NC组（P＜0.05）。野生型miR-3611转染生物素标记的HeLa、Caski细胞的PTENP1水平高于转染生物素标记的空白细胞和突变型miR-3611转染生物素标记的细胞，分别转染PTENP1或PTEN野生型和miR-3611模拟物的293T细胞的相对萤光活性低于仅转染PTENP1或PTEN野生型的293T细胞（P＜0.05）。结论 PTENP1通过竞争性结合miR-3611调控PTEN表达影响宫颈癌细胞增殖、迁移和侵袭。 |
| Abstract:Objective To investigate the molecular mechanism of long non-coding RNA PTENP1 (hereinafter referred to as “PTENP1”) in the proliferation, migration, and invasion of cervical cancer cells. Methods Cancer tissues and adjacent tissues of 54 patients with cervical cancer diagnosed in Hainan Women and Children’s Medical Center from January 2019 to December 2022 were selected, and cervical cancer cell lines (HeLa, SiHa, C33A, Caski) and normal cervical epithelial cell line (H8) were selected. The levels of PTENP1, miR-3611, PTEN gene, and PTEN protein were detected by real-time fluorescent quantitative PCR or Western blot. Appropriate cervical cancer cell lines were selected to compare the expression of PTENP1 in cytoplasm and nucleus, and further divided into blank group (no treatment), pcDNA3.1 group (transfected with pcDNA3.1), pcDNA3.1-PTENP1 group (transfected with pcDNA3.1-PTENP1), NC group (negative control, transfected with eilther mimic or inhibitor, negative control), miR-3611 inhibitor group (transfected with miR-3611 inhibitor), pcDNA3.1- PTENP1+NC group (transfected with pcDNA3.1-PTENP1 and NC), and pcDNA3.1-PTENP1+miR-3611 mimics group (transfected with pcDNA3.1-PTENP1 and miR-3611 mimics). The levels of PTENP1, miR-3611, PTEN gene, PTEN protein, and epithelial mesenchymal transformation related indexes in blank group, pcDNA3.1 group, and pcDNA3.1- PTENP1 group were compared. Cell viability and flow cytometry were used to detect cell proliferation and apoptosis. The levels of miR-3611, PTEN gene, and PTENP1 in blank group, NC group, and miR-3611 inhibitor group were compared. The cell proliferation and epithelial mesenchymal transformation related indexes were compared between pcDNA3.1- PTENP1+NC group and pcDNA3.1-PTENP1+miR-3611 mimics group. Double luciferase reporter gene and RNA pull-down experiments were used to verify the targeting relationship between miR-3611 and PTENP1 and PTEN gene. Results The levels of PTENP1, PTEN gene, and PTEN protein in cancer tissues were lower than those in adjacent tissues, and the levels of miR-3611 were higher than those in adjacent tissues (P＜0.05). The levels of PTENP1, PTEN gene, and PTEN protein in HeLa, SiHa, C33A, and Caski cells were lower than those in H8 cells, and the levels of miR-3611 was higher than those in H8 cells (P＜0.05). HeLa and Caski cells were selected for subsequent experiments. PTENP1 was highly expressed in HeLa and Caski cytoplasm. The levels of PTENP1, apoptosis rate, E-cadherin, and PTEN gene in pcDNA3.1-PTENP1 group were higher than those in blank group, while cell proliferation activity (cultured 48, 72, 96 h), and levels of ZEB1, Snail, vimentin, miR-3611 were lower than those in blank group (P＜0.05). The levels of miR-3611 in miR-3611 inhibitor group was lower than that in blank group, and PTEN gene and PTENP1 were higher than those in blank group (P＜0.05). Cell proliferation activity (cultured 48, 72, 96 h) and the levels of E-cadherin in pcDNA3.1- PTENP1+miR-3611 mimics group were lower than those in pcDNA3.1-PTENP1+NC group, and the levels of ZEB1, Snail, and vimentin were higher than those in pcDNA3.1-PTENP1+NC group (P＜0.05). The PTENP1 levels of HeLa and Caski cells transfected with wild type miR-3611 biotin marker were higher than those of blank cells transfected with biotin marker and cells transfected with mutant type miR-3611 biotin marker, the relative fluorescence activity of 293T cells transfected with PTENP1 or PTEN wild type and miR-3611 mimics was lower than that of 293T cells transfected with PTENP1 or PTEN wild type, respectively (P＜0.05). Conclusion PTENP1 regulates the expression of PTEN by competitively binding to miR-3611 and affects the proliferation, migration and invasion of cervical cancer cells. |
| 查看全文 HTML 查看/发表评论 下载PDF阅读器 |
|
|
|
