Upregulation of polycistronic microRNA-143 and microRNA-145 in colonocytes suppresses colitis and inflammation-associated colon cancer

Because ADAM17 promotes colonic tumorigenesis, we investigated potential miRNAs regulating ADAM17; and examined effects of diet and tumorigenesis on these miRNAs. We also examined pre-miRNA processing and tumour suppressor roles of several of these miRNAs in experimental colon cancer. Using TargetScan, miR-145, miR-148a, and miR-152 were predicted to regulate ADAM17. miR-143 was also investigated as miR-143 and miR-145 are co-transcribed and associated with decreased tumour growth. HCT116 colon cancer cells (CCC) were co-transfected with predicted ADAM17-regulating miRNAs and luciferase reporters controlled by ADAM17-3’UTR. Separately, pre-miR-143 processing by colonic cells was measured. miRNAs were quantified by RT-PCR. Tumours were induced with AOM/DSS in WT and transgenic mice (Tg) expressing pre-miR-143/miR-145 under villin promoter. HCT116 transfection with miR-145, −148a or −152, but not scrambled miRNA inhibited ADAM17 expression and luciferase activity. The latter was suppressed by mutations in ADAM17-3’UTR. Lysates from colonocytes, but not CCC, processed pre-miR-143 and mixing experiments suggested CCC lacked a competency factor. Colonic miR-143, miR-145, miR-148a, and miR-152 were downregulated in tumours and more moderately by feeding mice a Western diet. Tg mice were resistant to DSS colitis and had significantly lower cancer incidence and tumour multiplicity. Tg expression blocked up-regulation of putative targets of miR-143 and miR-145, including ADAM17, K-Ras, XPO5, and SET. miR-145, miR-148a, and miR-152 directly suppress colonocyte ADAM17 and are down-regulated in colon cancer. This is the first direct demonstration of tumour suppressor roles for miR-143 and miR-145 in an <i>in vivo</i> model of colonic tumorigenesis.

鉴于解整合素-金属蛋白酶17（ADAM17）可促进结肠肿瘤发生，本研究旨在筛选调控ADAM17表达的潜在微小RNA（microRNAs，miRNAs），并探究饮食与肿瘤发生对这些miRNAs的影响。同时，我们还在实验性结肠癌模型中分析了部分miRNAs的前体加工过程及其肿瘤抑制功能。利用靶标扫描（TargetScan）数据库预测，miR-145、miR-148a及miR-152可调控ADAM17的表达。由于miR-143与miR-145共转录且与肿瘤生长抑制相关，本研究同时纳入miR-143进行探究。将预测可调控ADAM17的miRNAs与受ADAM17 3'非翻译区（3'UTR）调控的荧光素酶报告基因共转染HCT116结肠癌细胞（CCC）。此外，我们检测了结肠细胞对pre-miR-143的加工过程。采用逆转录聚合酶链式反应（RT-PCR）对miRNAs进行定量分析。在野生型（WT）及携带绒毛蛋白启动子调控的pre-miR-143/miR-145转基因小鼠（Tg）中，通过氧化偶氮甲烷/葡聚糖硫酸钠（AOM/DSS）诱导肿瘤发生。结果显示，转染miR-145、miR-148a或miR-152（而非乱序miRNA对照）可抑制HCT116细胞中ADAM17的表达及荧光素酶活性，且该抑制效应可被ADAM17 3'UTR的位点突变所阻断。结肠上皮细胞裂解液可加工pre-miR-143，但HCT116结肠癌细胞裂解液无此活性；混合实验结果提示，HCT116细胞缺乏某种加工必需因子。肿瘤组织中miR-143、miR-145、miR-148a及miR-152的表达水平均下调，而小鼠喂食西式饮食后，这些miRNAs的表达仅出现中等程度下调。转基因小鼠对DSS结肠炎具有抵抗性，其肿瘤发生率及肿瘤多发率均显著降低。转基因表达可阻断miR-143与miR-145推定靶标的上调，包括ADAM17、K-Ras、输出蛋白5（XPO5）及SET蛋白。本研究证实，miR-145、miR-148a及miR-152可直接抑制结肠上皮细胞中ADAM17的表达，且在结肠癌中表达下调。这是首次在结肠肿瘤发生的体内（in vivo）模型中直接证实miR-143与miR-145发挥肿瘤抑制功能。