Table2_MiR-372-3p Functions as a Tumor Suppressor in Colon Cancer by Targeting MAP3K2.DOCX

MicroRNAs (miRNAs) as small non-coding RNA transcripts bind their complementary sequences in the 3′-untranslated region (3′-UTR) of target messenger RNAs (mRNAs) to regulate their expression. It is known that miR-372 belongs to the miR-371–373 gene cluster and has been found to be abnormally expressed in a variety of cancers, but its precise mechanism in cancer remains to be discovered. In this study, miR-372-3p expression was assessed in 153 frozen tissue samples, including primary diagnosed colon cancer and matched normal and adjacent tissues, using real time quantitative polymerase chain reaction (qPCR). An analysis of qPCR data revealed a significant reduction in miR-372-3p expression (by >2-fold) in colon cancer tissues in 51.5% (34/66) of patients. Consistent with this, mimicking the increased miR-372-3p levels in SW480 colon cancer cells significantly suppressed cell growth and proliferation. Although no direct correlation was found between the low level of miR-372-3p and certain tumor-related factors, such as p53, HRE-2, PMS2, MLH1, MSH2, MSH6, HDAC4, p21, and Wee1, in colon cancer tissues, an inverse relationship between miR-372-3p and Ki67 (a marker of proliferation) or miR-372-3p and MAP3K2(MEKK2), which plays a critical role in the MAPK signaling pathways, was confirmed using tissue samples. The target relationship between miR-372-3p and MAP3K2 was verified using luciferase assays in SW480 colon cancer cells. As expected, miR-372-3p mimics significantly suppressed the luciferase activity of pMIR-luc/MAP3K2 3′-UTR in cells, suggesting that miR-372-3p modulates the expression of MAP3K2 by directly targeting its 3′-UTR. Overall, the results obtained herein suggest that miR-372-3p may function as a tumor-suppressor miRNA in colon cancer by targeting MAP3K2.

微小RNA（MicroRNAs, miRNAs）是一类小型非编码RNA转录本，可通过结合靶信使RNA（messenger RNAs, mRNAs）3'非翻译区（3′-untranslated region, 3′-UTR）的互补序列调控其表达。已知miR-372属于miR-371–373基因簇，且在多种癌症中存在异常表达，但其在癌症中的精确作用机制仍有待阐明。本研究采用实时定量聚合酶链反应（real time quantitative polymerase chain reaction, qPCR），对153份冰冻组织样本中的miR-372-3p表达水平进行检测，样本包括初诊结肠癌组织及配对的正常组织与癌旁组织。对qPCR数据的分析显示，51.5%（34/66）的患者结肠癌组织中miR-372-3p表达显著下调（下调幅度超过2倍）。与此一致的是，在SW480结肠癌细胞中过表达miR-372-3p模拟物可显著抑制细胞生长与增殖。尽管在结肠癌组织中未发现miR-372-3p低表达与部分肿瘤相关因子（如p53、HRE-2、PMS2、MLH1、MSH2、MSH6、HDAC4、p21及Wee1）存在直接关联，但通过组织样本证实，miR-372-3p与增殖标志物Ki67，以及在MAPK信号通路中发挥关键作用的MAP3K2（MEKK2）均呈负相关关系。本研究采用荧光素酶报告基因实验（luciferase assays）在SW480结肠癌细胞中验证了miR-372-3p与MAP3K2的靶向结合关系。正如预期，miR-372-3p模拟物可显著抑制细胞中pMIR-luc/MAP3K2 3'-UTR的荧光素酶活性，提示miR-372-3p可通过直接靶向MAP3K2的3'-UTR调控其表达。综上，本研究结果表明，miR-372-3p可能通过靶向MAP3K2在结肠癌中发挥抑癌微小RNA的功能。