Post-transcriptional Regulation Of HAb18G/CD147 Expression By MicroRNA And Its Molecular Mechanism

Hepatocellular carcinoma (HCC) is one of the most common malignancies that seriously threaten human health. Although the risk factors for HCC are well characterized, the molecular pathogenesis of HCC is poorly understood. The deep understanding of the key moleculars involved in cancer development and progression will facilitate the establishment of effective targeted therapeutic strategies for HCC. HAb18G/CD147 is a novel target for cancer treatment identified in our lab, which is a member of CD147 family and belongs to the immunoglobulin superfamily. This molecule is highly expressed in most of the cancer tissues, while expressed negatively or at a very low level in normal tissues. Preliminary study showed that HAb18G/CD147 in HCC cells could not only induce fibroblasts to secret matrix metalloproteinases (MMPs), which could degrade matrixes surrounding cancer cells and promote the invasion and metastasis of HCC cells, but also induce VEGF expression in endothelial cells to promote the angiogenesis. The previous studies indicate that HAb18G/CD147 may play an important role in occurrence and development of cancer.MicroRNAs (miRNA), a new class of small noncoding RNAs, are 20~25 nucleotide long, which come from hairpin precursors and play important role in regulating gene expressions. The miRNAs are capable of binding to complementary sequences in 3'untranslated regions (3'UTR) of several target mRNAs to induce their degradation or translational repression at post-transcriptional level, and miRNAs have been shown to play important roles in cellular proliferation, and participate in the regulation of a wide range of biological functions such as, cell differentiation, apoptosis process and cell cycle regulation. Recent evidences indicate that miRNAs are involved in tumorigenesis and cell canceration, suggesting that they may represent a novel class of oncogenes or tumor suppressor genes. Our study also tries to examine the post-transcriptional regulation mechanism of HAb18G/CD147 in HCC cell. The study is composed of three parts.Part 1. Identification of miRNA molecules regulating HAb18G/CD147 expression at post-translational levelWe firstly isolated and compared the miRNA expression profile from normal liver cell line and HCC tumor cell line by miRNA array. The miRNAs that are down-expressed differentially in HCC cell line, AS-Scrluded miR-338-3p,miR-193a-3p and miR-146a. The analysis of HAb18G/CD147 predicted targets miRNA was determined using the algorithms TargetScan, the results indicate that miR-146a may bind to the 3'UTR of HAb18G/CD147, the putative binding site of miR-146a in 80~86nt of HAb18G/CD147 3'UTR region. miR-146a is highly expressed in QZG, compared with in SMMC7721 detected by Stem-loopRT-PCR and real-timePCR, and has the inverse correlation with HAb18G/CD147 mRNA at the expression level. The results indicate that miR-146a possibily negatively regulates the HAb18G/CD147 expression. Part 2. microRNA146a negatively regulates HAb18G/CD147 expression at post-translational levelThe results of western blot showed that transfect with miR-146a into HCC cells (FHCC98 and SMMC7721) could significantly inhibit the expression of HAb18G/CD147. On the contrary, transfect with AS-miR-146a into normal liver cell line-QZG, to block the endogenous miR-146a could upregulate the expression of HAb18G/CD147 in protein level. Based on the results obtained, miR-146a was assumed to play a role in the negative regulation of HAb18G/CD147 expression. Semi-quantitative RT-PCR and realtimePCR results showed that the level of HAb18G/CD147 mRNA have not significant differences after transfected with miR-146a or AS-miR-146a in HCC cells or in QZG cell. Those results indicated that miR-146a negatively regulate HAb18G/CD147 expression which was not involved in the level of HAb18G/CD147 mRNA, the mechanism of miR-146a negatively regulate HAb18G/CD147 expression may be a posttranscriptional regulation. Gelatinase zymography and In-vitro invasion assay showed that transfect with miR-146a to downregulate the expression of HAb18G/CD147 could both significantly inhibit the secretion of MMPs and the invasion of the cells (p < 0.05) in the co-culture of HCC cells and fibroblasts, however, the secretion of MMPs and the cellular invasive potential was obviously enhanced (p < 0.05) after knocking-down miR-146a. Above all, miR-146a negatively regulates HAb18G/CD147 expression and was involved in the regulation of MMPs secretion and cell migration.Part 3. Identification of miR-146a target site in the 3'UTR of HAb18G/CD147 mRNA by Luciferase activity assayTo test whether the predicted miR-146a target site in the 3'UTR of HAb18G/CD147 mRNA was responsible for its regulation,We constructed the luciferase reporter vectors, pGL3-18G wt and pGL3-18G mut, which AS-Scrlude putative 3'UTR of HAb18G/CD147 and mutantive 3'UTR of HAb18G/CD147, respectively. Co-transfected those luciferase vectors, respectively, together with Renilla luciferase report vector and miR-146a into HEK-293. A Renilla luciferase vector (pRL-TK) was used as a reference control. Twenty-four hours after transfection, firefly and Renilla luciferase activity were measured using the Dual-Luciferase Reporter Assay (Promega). Luciferase activity of cells transfected with miR-146a and pGL3-18G wt was decreased about 2-fold, a statistically significant difference, when compared with vector alone (P = 0.0008, t test), however, Luciferase activity of cells had not a significant difference in cotransfected with miR-146a and pGL3-18G mut, when compared with vector alone. Above all, luciferase data provided strong indications that HAb18G/CD147 is a target of miR-146a.In summary, our investigation revealed the mechanisms in which miR-146a regulates HAb18G/CD147 expression at post-transcriptional level, and demonstated that miR-146a could inhibit cell migration in vitro. These results further indicate that miR-146a might have a therapeutic potential to suppress cancer metastasis.