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环状RNA BACH2通过调控miR-370-3p影响甲状腺乳头状癌的恶性生物学行为

[circ_BACH2 affects the malignant biological behavior of papillary thyroid cancer by regulating miR-370-3p].

作者信息

Sun P, Feng Y, Zhou L Z, Pei F, Su B, Qiao X C

机构信息

Departemnt of Otolaryngology, the Fourth Central Hospital of Tianjin, Tianjin 300140, China.

Departemnt of Otolaryngology, Shidong Hospital, Affiliated to Shanghai University of Technology, Shanghai 200438, China.

出版信息

Zhonghua Zhong Liu Za Zhi. 2024 Jul 23;46(7):663-675. doi: 10.3760/cma.j.cn112152-20231024-00245.

DOI:10.3760/cma.j.cn112152-20231024-00245
PMID:39034802
Abstract

To investigate the influence of circ_BACH2 on the malignant biological behavior of papillary thyroid cancer and its molecular mechanism. Cancer tissues and paracancer tissues of 51 patients with papillary thyroid carcinoma from the Fourth Central Hospital of Tianjin between 2017 and 2019 were collected. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_BACH2, miR-370-3p and G protein coupled receptor kinase interacting factor 1 (GIT1) mRNA in tissues and cells; flow cytometry to detect cell apoptosis and cell cycle; plate clone formation experiment to detect the number of cell clones; cell counting kit 8 (CCK-8) to detect cell proliferation; Transwell array to detect cell migration and invasion; western blot to detect protein expressions; dual luciferase report experiment to detect the targeting relationship between circ_BACH2, miR-370-3p and GIT1; the nude mouse tumor formation experiment to detect the effect of circ_BACH2 on tumors in mice. Compared with adjacent tissues, the expressions of circ_BACH2 and GIT1 in papillary thyroid cancer tissues was increased, while the expression of miR-370-3p was decreased. Compared with Nthy-ori3-1 cells, the expressions of circ_BACH2 in papillary thyroid cancer cells TPC-1 and SW579 were increased, the mRNA and protein levels of GIT1 were increased, miR-370-3p expression was decreased. The expression level of GIT1 mRNA was negatively correlated with that of miR-370-3p (=-0.634), and the expression level of circ_BACH2 was positively correlated with that of GIT1 (=0.635). The expression level of circ_BACH2 was negatively correlated with that of miR-370-3p (=-0.394, <0.05). Circ_BACH2 and miR-370-3p has a binding site at the 3' UTR of GIT1. After knocking down circ_BACH2, the proportion of G/G cells in papillary thyroid cancer cells TPC-1 and SW579 was increased, the proportion of S-phase cells was decreased and the proportion of G/M-phase cells did not change significantly. The cell absorbance value was lower than that in si-NC group. The number of cell clone formation was decreased (43±5 vs 100±6, 54±8 vs 100±9); the cell apoptosis rate was increased [(19.60±2.40)% vs (4.30±0.20)%, (18.10±2.10)% vs (5.10±0.23)%]; cell migration number was decreased (61±7 vs 134±15, 58±6 vs 112±11), the invasion number was also decreased (45±6 vs 113±11, 47±4 vs 92±9); the expressions of Snail and Twist1 were decreased, and the expression of E-cadherin was increased (<0.000). Inhibition of miR-370-3p expression reversed the effect of circ_BACH2 knockdown on proliferation, migration, invasion and apoptosis of thyroid papillary cancer cells. Overexpression of GIT1 reversed the effects of overexpression of miR-370-3p on proliferation, migration, invasion and apoptosis of thyroid papillary cancer cells. Mice injected with TPC-1 cells stably transfected with sh-circ_BACH2 showed a reduction in tumor volume [(535±91) mm vs (857±114) mm] after 35 days of culture; tumor weight was decreased [(0.62±0.13) mg vs (1.06±0.15) mg, <0.05]; the expressions of circ_BACH2 and GIT1 were decreased, and the expression of miR-370-3p was increased in nude mouse tumor tissue. Silencing circ_BACH2 may inhibit the proliferation, migration and invasion of papillary thyroid cancer cells in vitro, promote cell apoptosis, and inhibit tumor growth in vivo through targeted regulation of miR-370-3p/GIT1.

摘要

探讨环状RNA BACH2(circ_BACH2)对甲状腺乳头状癌恶性生物学行为的影响及其分子机制。收集2017年至2019年期间天津市第四中心医院51例甲状腺乳头状癌患者的癌组织及癌旁组织。采用逆转录-定量实时聚合酶链反应(RT-qPCR)检测组织和细胞中circ_BACH2、微小RNA-370-3p(miR-370-3p)及G蛋白偶联受体激酶相互作用因子1(GIT1)mRNA的表达;采用流式细胞术检测细胞凋亡和细胞周期;采用平板克隆形成实验检测细胞克隆数;采用细胞计数试剂盒8(CCK-8)检测细胞增殖;采用Transwell小室检测细胞迁移和侵袭;采用蛋白质免疫印迹法检测蛋白表达;采用双荧光素酶报告实验检测circ_BACH2、miR-370-3p与GIT1之间的靶向关系;采用裸鼠成瘤实验检测circ_BACH2对小鼠肿瘤的影响。与癌旁组织相比,甲状腺乳头状癌组织中circ_BACH2和GIT1的表达升高,而miR-370-3p的表达降低。与Nthy-ori3-1细胞相比,甲状腺乳头状癌细胞TPC-1和SW579中circ_BACH2的表达升高,GIT1的mRNA和蛋白水平升高,miR-370-3p表达降低。GIT1 mRNA的表达水平与miR-370-3p呈负相关(r=-0.634),circ_BACH2的表达水平与GIT1呈正相关(r=0.635)。circ_BACH2的表达水平与miR-370-3p呈负相关(r=-0.394,P<0.05)。circ_BACH2与miR-370-3p在GIT1的3'非翻译区(UTR)有结合位点。敲低circ_BACH2后,甲状腺乳头状癌细胞TPC-1和SW579中G/G期细胞比例升高,S期细胞比例降低,G/M期细胞比例无明显变化。细胞吸光度值低于si-NC组。细胞克隆形成数减少(43±5比100±6,54±8比100±9);细胞凋亡率升高[(19.60±2.40)%比(4.30±0.20)%,(18.10±2.10)%比(5.10±0.23)%];细胞迁移数减少(61±7比134±15,58±6比112±11),侵袭数也减少(45±6比113±11,47±4比92±9);Snail和Twist1的表达降低,E-钙黏蛋白的表达升高(P<0.000)。抑制miR-370-3p表达可逆转敲低circ_BACH2对甲状腺乳头状癌细胞增殖、迁移、侵袭和凋亡的影响。过表达GIT1可逆转过表达miR-370-3p对甲状腺乳头状癌细胞增殖、迁移、侵袭和凋亡的影响。注射稳定转染sh-circ_BACH2的TPC-1细胞的小鼠在培养35天后肿瘤体积减小[(535±91)mm³比(857±114)mm³];肿瘤重量减轻[(0.62±0.13)mg比(1.06±0.15)mg,P<0.05];裸鼠肿瘤组织中circ_BACH2和GIT1的表达降低,miR-370-3p的表达升高。沉默circ_BACH2可能通过靶向调控miR-370-3p/GIT1抑制甲状腺乳头状癌细胞的体外增殖、迁移和侵袭,促进细胞凋亡,并抑制体内肿瘤生长。

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