Cheng B, Ding F, Huang C-Y, Xiao H, Fei F-Y, Li J
Department of Clinical Laboratory, Shanghai Pudong New Area People's Hospital, Shanghai, China.
Eur Rev Med Pharmacol Sci. 2019 Jan;23(1):137-145. doi: 10.26355/eurrev_201901_16757.
The aim of this study was to investigate the role of miR-16-5p in hepatocellular carcinoma (HCC), and to explore the possible underlying mechanism.
100 pairs of cancerous and para-cancerous tissues surgically removed in our hospital were collected. Real Time quantitative-Polymerase Chain Reaction (qRT-PCR) was used to detect the expression level of miR-16-5p in tissues. Bioinformatics and Dual-Luciferase reporter gene assay were used to screen and verify the potential target genes of miR-16-5p, respectively. Human HCC SMMC-7721 cells were used for functional experiments. Cell proliferation was detected by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay. Cell invasion and migration were evaluated by transwell and scratch wound-healing assay, respectively. The protein expression levels of epithelial-mesenchymal transition (EMT) associated markers were measured by Western blot (WB) assay.
QRT-PCR showed that miR-16-5p expression in HCC tissues was significantly lower than that of adjacent normal liver tissues. At the cellular level, miR-16-5p was lowly expressed in HCC cells (SMMC-7721). Bioinformatics websites (including Targetscan, PicTar, miRanda) predicted that insulin-like growth factor 1 receptor (IGF1R) was a potential target gene of miR-16-5p. Meanwhile, IGF1R was selected for further investigation due to its metastatic function. The results showed that no significant difference was found in the mRNA expression level of IGF1R in HCC tissues. However, the protein level of IGF1R was significantly up-regulated, which was negatively correlated with miR-16-5p. Combined with Dual-Luciferase reporter gene assay, it was confirmed that miR-16-5p could regulate the expression of IGF1R in a targeted manner. Furthermore, down-regulation of IGF1R significantly reduced the inhibitory effect of miR-16-5p on the proliferation and metastasis of SMMC-7721 cells.
We showed that miR-16-5p suppressed invasion and migration of HCC cells, mechanically by directly targeting and inhibiting IGF1R protein expression. The newly identified miR-16-5p/IGF1R axis might provide new insights into the pathogenesis of HCC and novel potential therapeutic targets for the treatment of HCC.
本研究旨在探讨miR-16-5p在肝细胞癌（HCC）中的作用，并探索其可能的潜在机制。
收集我院手术切除的100对癌组织和癌旁组织。采用实时定量聚合酶链反应（qRT-PCR）检测组织中miR-16-5p的表达水平。分别运用生物信息学和双荧光素酶报告基因检测筛选并验证miR-16-5p的潜在靶基因。采用人肝癌SMMC-7721细胞进行功能实验。通过MTT（3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐）法检测细胞增殖。分别通过Transwell实验和划痕伤口愈合实验评估细胞侵袭和迁移能力。采用蛋白质免疫印迹（WB）法检测上皮-间质转化（EMT）相关标志物的蛋白表达水平。
qRT-PCR显示，HCC组织中miR-16-5p的表达明显低于相邻正常肝组织。在细胞水平上，HCC细胞（SMMC-7721）中miR-16-5p低表达。生物信息学网站（包括Targetscan、PicTar、miRanda）预测胰岛素样生长因子1受体（IGF1R）是miR-16-5p的潜在靶基因。同时，由于IGF1R的转移功能，选择其进行进一步研究。结果显示，HCC组织中IGF1R的mRNA表达水平无显著差异。然而，IGF1R的蛋白水平显著上调，且与miR-16-5p呈负相关。结合双荧光素酶报告基因检测，证实miR-16-5p可靶向调控IGF1R的表达。此外，下调IGF1R可显著降低miR-16-5p对SMMC-7721细胞增殖和转移的抑制作用。
我们发现miR-16-5p通过直接靶向并抑制IGF1R蛋白表达，抑制HCC细胞的侵袭和迁移。新发现的miR-16-5p/IGF1R轴可能为HCC的发病机制提供新的见解，并为HCC的治疗提供新的潜在治疗靶点。
