摘要
目的探讨miR-146a对人甲状腺乳头状癌细胞系(TPC-1)细胞侵袭转移的调控作用及内在机制。方法采用流式细胞术、细胞活性检测试剂(CCK-8)检测TPC-1细胞活性及凋亡情况;Transwell实验和划痕实验检测TPC-1细胞侵袭和迁移能力;实时荧光定量PCR检测免疫球蛋白超家族成员1(Immunoglobulin super family member1,IGSF1)、miR-146a的mRNA表达水平;免疫组化法检测IGSF1的表达水平;Western-Blot检测β-连环蛋白(β-catenin)与骨桥蛋白(Osteopontin,OPN)表达水平;双荧光素酶基因检测IGSF1和miR-146a交互作用。结果细胞增殖活性结果显示,与Control组(100.00±0.00)%比较,miR-146a mimics组TPC-1细胞增殖活性(84.77±3.71)%显著降低(P<0.05);而miR-146a inhibitor组TPC-1细胞增殖活性(119.20±5.81)%显著升高(P<0.05)。流式细胞术检测结果显示,与Control组(6.37±0.27)%比较,miR-146a mimics组TPC-1细胞凋亡率(20.33±1.26)%显著增加,而miR-146a inhibitor组TPC-1细胞凋亡率(3.35±0.93)%显著降低(F=217.1,P<0.05)。Transwell实验和细胞划痕实验结果显示,与Control组比较,miR-146a mimics组在24 h和48 h的相对迁移距离、侵袭细胞数显著降低(P<0.05),而miR-146a inhibitor组在24 h和48 h的相对迁移距离、侵袭细胞数显著增加(P<0.05)。与Control组比较,miR-146a mimics组miR-146a表达水平显著增加,IGSF1表达水平显著降低(P<0.05);而miR-146a inhibitor组miR-146a表达水平显著降低,IGSF1表达水平显著增加(P<0.05)。与Control组(0.92±0.09)比较,miR-146a mimics组β-catenin(0.54±0.08)和OPN(0.30±0.09)表达水平均显著降低(P<0.05),而miR-146a inhibitor组β-catenin(1.26±0.07)和OPN(1.07±0.13)表达水平显著增加(P<0.05)。双荧光素酶报告基因检测结果显示,miR-146a mimics组野生型(0.64±0.11)与miR-146a mimics NC组野生型(1.16±0.07)比较,差异有统计学意义(P<0.05)。结论miR-146a可交互作用IGSF1,调控OPN及β-catenin的表达水平,影响甲状腺乳头状癌的发生发展进程。
Objective To investigate the regulation and mechanism of miR-146a on invasion and metastasis of TPC-1 cells.Methods CCK-8 and flow cytometry were used to detect cell activity;Transwell was used to detect cell invasion and apoptosis ability;scratch assay was used to detect cell healing ability;real-time fluorescence quantitative PCR was used to detect miR-146a and IGSF1 mRNA expression,and immunohistochemistry was used to detect IGSF1 expression.The protein expression of OPN andβ-catenin was detected by Western Blot,and the binding of miR-21 and IGSF1 was analyzed by dual luciferase reporter gene assay.Results The results of cell proliferation activity showed that the proliferation activity of TPC-1 cells(84.77±3.71)%was significantly reduced compared with of control group(100.00±0.00)%(P<0.05).However,the cell proliferation activity of TPC-1 cells(119.20±5.81)%was significantly increased in miR-146a inhibitor group(P<0.05).The flow cytometry results showed that the apoptosis rate of TPC-1 cells(20.33±1.26)%were increased significantly in the miR-146a mimics group compared with the control group(6.37±0.27)%,and the TPC-1 cell apoptosis rate(3.35±0.93)%was significantly decreased in the miR-146a inhibitor group(F=217.1,P<0.05).The results of Transwell experiments and cell scratch experiment showed that the relative migration distance and the number of invading cells were decreased significantly at 24 h and 48 h(P<0.05),while the relative migration distance and the number of invading cells in the miR-146a inhibitor group were increased significantly at 24 h and 48 h(P<0.05).Compared with the control group,the miR-146a expression level was significantly increased;and the IGSF1 expression level was significantly decreased in the miR-146a mimics group(P<0.05);and miR-146a expression level and IGSF1 expression level were significantly increased in miR-146a inhibitor group(P<0.05).Compared with control group(0.92±0.09),β-catenin(0.54±0.08)and OPN(0.30±0.09)expression levels were all significantly reduced in the miR-146a mimics group(P<0.05),andβ-catenin(1.26±0.07)and OPN(1.07±0.13)were significantly increased in miR-146a inhibitor group(P<0.05).Dual-luciferase reporter gene assay showed that the results were statistically significant difference between wild type of miR-146a mimics group(0.64±0.11)and wild type of miR-146a mimics NC group(1.16±0.07)comparisons(P<0.05).Conclusion miR-146a can interact with IGSF1 and regulate OPN andβ-catenin.The expression level of catenin affects the progression of papillary thyroid carcinoma.
作者
龚烨
程超
王慧琴
GONG Ye;CHENG Chao;WANG Huiqin(Department of Breast and Thyroid,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China;Medical Research and Transformation Center,People's Hospital of Xinjiang Uygur Autonomous Region,Urumqi 830001,China)
出处
《新疆医科大学学报》
CAS
2024年第1期6-13,共8页
Journal of Xinjiang Medical University
基金
新疆维吾尔自治区自然科学基金项目(2022D01C140)。
