Transcriptional Regulation Of CD147 By Snail Family Involved In Epithelial-mesenchymal Transition

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide with an annual deaths rate of about 662 000 cases, among which 80% are in developing countries. HCC is characterized by rapid progression, highly malignant and poor prognosis. The 5-year overall survival rate of individuals with HCC is less than 10%.CD147, a highly glycosylated cell surface transmembrane protein, belongs to immunoglobulin superfamily. CD147 have been reported closely related with tumor invasion, metastasis, tumor cell growth and survival of tumor cells. CD147 induces tumor cell invasion by stimulating production of matrix metalloproteinases by fibroblasts. Overexpression of CD147 in tumor cells was significantly associated with poor prognosis of liver cancer and other tumors.Epithelial-mesenchymal transition (EMT) is an important phenomenon in the process of cancer. During EMT, stationary polarized epithelial cells are converted to motile mesenchymal-like cells, accompanied with activation of invasive phenotype and neoplastic cell behaviours, gain of mesenchymal cell markers (fibronectin, vimentin, -smooth muscle actin, and N-cadherin), and loss of epithelial markers (E-cadherin, and - andγ-catenin). A prominently morphologic alteration of hepatocytes induced by TGF-βis EMT which is involved in hepatic fibrogenesis and carcinogenesis. TGF-βtriggers EMT via the Smads-dependent signaling cooperating with other Smads-independent oncogenic pathways, such as Ras, p38 MAPK, PI3K/Akt to maintain the mesenchymal phenotype of invasive cells. Despite all these evidences, the molecular mechanism of EMT in liver carcinogenesis remains unclear.Among transcriptional and post transcriptional mechanisms, the transcription factors play an important role in gene regulation. Snail superfamily, the master transcription factor involved in the EMT, is divided into the Snail and Scratch families, with three members of the Snail family having been described in vertebrates to date: Snail1, Snail2 (Slug) and Snail3. Members of the Snail family are zinc-finger transcription factors that share a common organization: a highly conserved C-terminal region. The central region of the Snail proteins has a serine–proline-rich region that is highly divergent between Snail members. Snail2 proteins contain the so-called slug domain in this region, the function of which remains elusive. The zinc fingers serve as the sequence-specific DNA-binding domains that recognize consensus E2-box type elements CAGGTG.Our previous experiments demonstrated that CD147 is involved in TGF-β1-stimulated EMT. The purpose of this study was to identify the transcription factors that regulate CD147 expression.The first aim of our study was to identify the binding sites of Snail family members in the promoter of CD147. The second was to know whether the expression of transcription factor family members can influence the expression of CD147. The third purpose was to analyze the correlation of expression of cancer-associated molecule CD147 and Slug by immunohistochemical. TGF-β1With stimulation of TGF-β1, both Snail and Slug mRNA levels were enhanced in QZG cells with corresponding elevation of CD147 mRNA. By si-Snail1 transfection, Snail1 mRNA level was downregulated either in the absence or presence of TGF-β1, but we did not observe decreased CD147 expression. However, si-Slug transfection caused CD147 down-expression efficiently. In addition, expression of Slug protein was inhibited by si-Snail1 indicating Slug as a downstream factor of Snail signaling. By western blot analysis,the pattern of CD147 expression affected by Slug was consistent with that of real-time PCR. Part 2. Slug directly regulates CD147 expressionTo determine whether Snail1or Slug directly regulates CD147 expression at a transcriptional level, we co-transfected the CD147 P(-1761?+37) Plasmid with pEGFP-Snail1 or pcDNA3.1–Slug into HEK-293 cells. CD147P(-1761?+37) is a luciferase reporter plasmid that contained a 1 798 bp genomic DNA fragment spanning the 5'upstream region of CD147.. Co-transfection of pcDNA3.1-slug and CD147 promoter region (-1761?+37) led to a dose-dependent increase in luciferase activity. However, the activity of the CD147 promoter was not activated by Snail1 in HEK-293 cells. The result indicates that Slug has an inducible effect on the CD147 promoter activity, and regulates the transcriptional expression of CD147 directly. Deletion of nucleotides from -644 to +37 on the 5-end led to moderated reduction of transcription activities; however, deletion in the region from -338 to +37 completely abolished the activity of the reporter gene. Thus, the most critical region for the basal transcriptional activity of the CD147 promoter is located within this 306 bp region between positions -338 to -644. To further define the role of Slug in transcriptional regulation CD147 promoter activity, we generated a construct of CD147 P(-644? +37 Slug mt) that contained E-box2 mutations in the Slug binding site by site directed mutagenesis. The consensus sequence CAGGTG Part 1. Upexpression of Snail family members and CD147 stimulated with was changed to AAGGTA in the respective mutant E-boxes used in the competition experiments. The promoter activity was completely abolished upon the mutation of Slug binding site. On the other hand, the CD147 with E-box2 site mutation responded normally to Slug activation, suggesting that E-box2, but not E-box1, is required for the activation of the CD147 promoter by Slug. ChIP analysis demonstrated that Slug activates CD147 transcription through direct interaction with the E2-box located in the proximal promoter of the CD147 gene.Part 3. Expression of cancer-associated molecule CD147 and Slug in HCC tissues by immunohistochemicalWe examined the expression level of CD147 in human liver tissues adjacent to hemangioma, HBV and HCV hepatitis, and cirrhosis tissues. Expression of CD147 was shown to be negative in all examined hemangioma (25 cases) and hepatitis (12 cases) specimen. Thirty-seven five of 49 HCC cases including well, moderately and poorly differentiated samples showed overexpression of CD147. The P value of hemangioma is less than 0.001. In HCC tissues, Slug expression is positived with CD147 (rs=0.3064, P=0.0323).