Objective: To investigate the influence of circ_BACH2 on the malignant biological behavior of papillary thyroid cancer and its molecular mechanism. Methods: 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. Results: 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 (r=-0.634), and the expression level of circ_BACH2 was positively correlated with that of GIT1 (r=0.635). The expression level of circ_BACH2 was negatively correlated with that of miR-370-3p (r=-0.394, P＜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 G0/G1 cells in papillary thyroid cancer cells TPC-1 and SW579 was increased, the proportion of S-phase cells was decreased and the proportion of G2/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 (P＜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) mm3 vs (857±114) mm3] after 35 days of culture; tumor weight was decreased [(0.62±0.13) mg vs (1.06±0.15) mg, P＜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. Conclusion: 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.
目的： 探讨circ_BACH2对甲状腺乳头状癌恶性生物学行为的影响及分子机制。 方法： 收集天津市第四中心医院2017—2019年51例甲状腺乳头状癌患者的癌组织和癌旁正常组织。实时荧光定量PCR法检测癌组织、癌旁正常组织和细胞中circ_BACH2、miR-370-3p和GIT1 mRNA的表达水平，流式细胞术检测细胞凋亡情况和细胞周期变化，平板克隆形成实验检测细胞克隆形成数，细胞计数试剂盒8检测细胞增殖能力，Transwell检测细胞迁移和侵袭能力，Western blot法检测蛋白表达，双荧光素酶报告实验检测circ_BACH2、miR-370-3p和GIT1之间的靶向关系，裸鼠成瘤实验检测抑制circ_BACH2的表达对小鼠体内肿瘤的影响。 结果： 甲状腺乳头状癌组织中circ_BACH2、GIT1 mRNA和蛋白表达水平高于癌旁正常组织，miR-370-3p表达水平低于癌旁正常组织，甲状腺癌细胞TPC-1、SW579中circ_BACH2、GIT1 mRNA和蛋白表达水高于人甲状腺正常细胞Nthy-ori3-1，miR-370-3p表达水平低于Nthy-ori3-1细胞（均P＜0.05）。GIT1 mRNA表达水平与miR-370-3p表达水平呈负相关（r=-0.634），circ_BACH2表达水平与GIT1 mRNA表达水平呈正相关（r=0.635），circ_BACH2表达水平与miR-370-3p表达水平呈负相关（r=-0.394，均P＜0.05）。circ_BACH2与miR-370-3p具有结合位点，GIT1的3'UTR与miR-370-3p具有结合位点。si-circ_BACH2组TPC-1、SW579细胞G0/G1期细胞比例高于si-NC组，S期细胞比例、细胞吸光度值低于si-NC组，细胞克隆形成数［分别为（43±5）个和（54±8）个］低于si-NC组［分别为（100±6）个和（100±9）个］，细胞凋亡率［分别为（19.60±2.40）%和（18.10±2.10）%］高于si-NC组［分别为（4.30±0.20）%和（5.10±0.23）%］，细胞迁移数［分别为（61±7）个和（58±6）个］、细胞侵袭数［分别为（45±6）个和（47±4）个］均低于si-NC组［分别为（134±15）个、（112±11）个、（113±11）个和（92±9）个，均P＜0.001］。si-circ_BACH2组细胞中Snail、Twist1表达水平低于si-NC组，E-cadherin表达水平高于si-NC组（均P＜0.001）。抑制miR-370-3p的表达逆转敲减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逆转过表达miR-370-3p对甲状腺癌细胞增殖、迁移、侵袭和凋亡的影响。注射稳定转染sh-circ_BACH2的TPC-1细胞的裸鼠在培养35 d 后肿瘤体积缩小［（535±91）mm3和（857±114）mm3］，肿瘤重量减少［（0.62±0.13）mg和（1.06±0.15）mg，均P＜0.05］。裸鼠肿瘤组织中circ_BACH2、GIT1 mRNA表达水平降低，miR-370-3p mRNA表达水平升高（均P＜0.05）。 结论： 抑制circ_BACH2可能通过靶向调控miR-370-3p/GIT1从而抑制体外甲状腺乳头状癌细胞的增殖、迁移、侵袭，促进细胞凋亡，并抑制体内肿瘤的生长。.