miR-16是一种参与真核细胞多种生物学过程的重要分子，然而对其如何行使其生理功能过程缺乏全面认识。本项研究中，来自天津医科大学和南京医科大学的研究者鉴定了新的miR-16靶基因并研究其生物学功能。研究中采用三种miRNA靶基因预测方法，即TargetScan，PicTar和miRanda预测了miR-16靶基因，并结合表达谱芯片筛选和定量PCR验证对候选靶基因及miR-16表达水平进行检测，进而用Western blot对候选靶基因蛋白表达做了深入检测。接下来，研究者通过构建了荧光素酶报告系统和转染实验证实了对靶基因的直接调控作用。最后，通过细胞生存的MTT实验和检测细胞凋亡及细胞周期的流式细胞术检测研究了miR-16及其靶基因的影响。当在一些肿瘤细胞细胞系中过表达miR-16后，mRNA全基因组表达谱芯片鉴定到27个可能被miR-16的基因，通过生物信息学分析过滤得到18个miR-16候选靶基因；通过实验验证确认其中3个，即MAP7，PRDM4和CDS2，是miR-16的直接靶基因。在肿瘤细胞实验中发现miR-16通过靶向MAP7机制在细胞增殖中发挥关键作用，但该机制对于细胞凋亡和细胞周期调控没有影响。该研究已经发表在2013年Molecular Cancer杂志上。本研究中人基因表达谱芯片检测工作由博奥生物集团有限公司完成。
The identification of novel targets of miR-16 and characterization of their biological functions in cancer cells
Background: In eukaryotes, miR-16 is an important microRNA (miRNA) that is involved in numerous biological processes. However, it is not fully understood how miR-16 executes its physiological functions. In the present study, we aimed to identify novel miR-16 targets and study their biological functions.
Methods: Candidate target genes of miR-16 were screened by microarray analysis of mRNA levels in several cancer cell lines with enhanced miR-16. Three bioinformatics algorithms, including TargetScan, PicTar, and miRanda, were used in combination to calculate the miR-16 targets. The expression levels of miR-16 and target mRNA were examined by relative quantification RT-PCR, and the expression levels of target protein were detected by Western blot. Luciferase reporter plasmids were constructed to confirm direct targeting. The effect of miR-16 and target gene on cell viability was evaluated using MTT assays. The effects of miR-16 and target gene on apoptosis and cell cycle distribution were evaluated by flow cytometry analysis.
Results: By overexpressing miR-16 in several cancer cell lines and measuring global mRNA levels using microarray analysis, we identified 27 genes that may be regulated by miR-16. After the bioinformatics filtering process, 18 genes were selected as candidate miR-16 targets. Furthermore, we experimentally validated three of these candidates, MAP7 (microtubule-associated protein 7), PRDM4 (PR domain containing 4) and CDS2 (CDP-diacylglycerol synthase 2), as direct targets of miR-16. Finally, we demonstrated that miR-16 targeting MAP7 played a critical role in regulating proliferation but not apoptosis and cell cycle progression in cancer cells.
Conclusion: In summary, the present study identifies several novel miR-16 targets and illustrates a novel function of miR-16 targeting MAP7 in modulating proliferation in cancer cells.