Table_8_MiR-29b-3p Inhibits Migration and Invasion of Papillary Thyroid Carcinoma by Downregulating COL1A1 and COL5A1.xls

IntroductionMicroRNAs (miRNAs) are small noncoding RNA molecules that regulate genetic expression and are also vital for tumor initiation and development. MiR-29b-3p was found to be involved in regulating various biological processes of tumors, including tumor cell proliferation, metastasis, and apoptosis inhibition; however, the biofunction and molecule-level mechanisms of miR-29b-3p inpapillary thyroid carcinoma (PTC) remain unclear.
MethodsThe expression of miR-29b-3p in PTC samples was tested via qRT-PCR. Cellular proliferation was analyzed by CCK-8 and EdU assays, and cellular migratory and invasive abilities were assessed utilizing wound-healing and Transwell assays. In addition, protein expressions of COL1A1, COL5A1, E-cadherin, N-cadherin, Snail, and Vimentin were identified via Western blot (WB) assay. Bioinformatics, qRT-PCR, WB, and dual luciferase reporter assays were completed to identify whether miR-29b-3p targeted COL1A1 and COL5A1. In addition, our team explored the treatment effects of miR-29b-3p on a murine heterograft model.
ResultsOur findings revealed that miR-29b-3p proved much more regulated downward in PTC tissue specimens than in adjacent non-cancerous tissues. Meanwhile, decreased expression of miR-29b-3p was strongly related to the TNM stage of PTC patients (p<0.001), while overexpression of miR-29b-3p in PTC cells suppressed cellular migration, invasion, proliferation, and EMT. Conversely, silencing miR-29b-3p yielded the opposite effect. COL1A1 and COL5A1 were affirmed as the target of miR-29b-3p. Additionally, the COL1A1 and COL5A1 were highly expressed in PTC tumor samples than in contrast to neighboring healthy samples. Functional assays revealed that overexpression of COL1A1 or COL5A1 reversed the suppressive role of miR-29b-3p in migration, invasion, and EMT of PTC cells. Finally, miR-29b-3p agomir treatment dramatically inhibited Xenograft tumor growth in the animal model.
ConclusionsThese findings document that miR-29b-3p inhibited PTC cells invasion and metastasis by targeting COL1A1 and COL5A1; this study also sparks new ideas for risk assessment and miRNA replacement therapy in PTC.

引言：微小RNA（miRNAs）是一类小型非编码RNA分子，可调控基因表达，且在肿瘤发生与发展过程中发挥关键作用。已有研究证实miR-29b-3p参与调控肿瘤的多种生物学过程，包括肿瘤细胞增殖、转移及抗凋亡；然而，miR-29b-3p在甲状腺乳头状癌（PTC）中的生物学功能及分子层面的调控机制仍未明确。
方法：采用实时荧光定量聚合酶链反应（qRT-PCR）检测甲状腺乳头状癌组织样本中miR-29b-3p的表达水平。通过细胞计数试剂盒-8（CCK-8）和5-乙炔基-2'-脱氧尿苷（EdU）实验分析细胞增殖能力，采用划痕愈合实验和Transwell实验评估细胞的迁移与侵袭能力。此外，采用蛋白质印迹（Western blot, WB）实验检测COL1A1、COL5A1、E-钙粘蛋白（E-cadherin）、N-钙粘蛋白（N-cadherin）、Snail及波形蛋白（Vimentin）的蛋白表达水平。通过生物信息学分析、qRT-PCR、WB及双荧光素酶报告基因实验，验证miR-29b-3p是否靶向调控COL1A1与COL5A1。此外，本团队还在小鼠异种移植瘤模型中探究了miR-29b-3p的治疗效果。
结果：本研究结果显示，相较于癌旁正常组织，miR-29b-3p在甲状腺乳头状癌组织标本中显著低表达。与此同时，miR-29b-3p的低表达与甲状腺乳头状癌患者的TNM分期密切相关（p<0.001）；在甲状腺乳头状癌细胞中过表达miR-29b-3p可抑制细胞的迁移、侵袭、增殖以及上皮间质转化（EMT）。反之，沉默miR-29b-3p则会产生相反的生物学效应。实验证实COL1A1与COL5A1是miR-29b-3p的靶基因。此外，COL1A1与COL5A1在甲状腺乳头状癌肿瘤样本中的表达水平显著高于邻近正常组织样本。功能实验结果显示，过表达COL1A1或COL5A1可逆转miR-29b-3p对甲状腺乳头状癌细胞迁移、侵袭及上皮间质转化的抑制作用。最后，miR-29b-3p激动剂（agomir）处理可显著抑制动物模型中的异种移植瘤生长。
结论：本研究结果表明，miR-29b-3p通过靶向调控COL1A1与COL5A1，抑制甲状腺乳头状癌细胞的侵袭与转移；本研究同时为甲状腺乳头状癌的风险评估及miRNA替代治疗提供了新的思路。