150 Complex Antithrombotic Therapy (CAT) and the Risk of Upper Gastrointestinal Events (UGIE) Among Vulnerable Elders

Abstract

Background. Cholangiocarcinoma (CCA) is an aggressive epithelial cancer of the biliary tree with survival measured in months. The only potentially curative treatment for CCA is surgical resection. Unfortunately, the vast majority of patients are diagnosed at late stages, when surgery is not an option. MicroRNAs (miRs) are short single stranded RNA sequences, recently shown to have major regulatory functions in cancer. TIMP3 is one of 4 members of the metalloproteinases family and its role was associated with cancer invasion and metastasis. We aimed at elucidating the regulatory mechanisms of TIMP3 in CCA. Methods. Fifteen histologically confirmed CCAs and 9 normal liver specimens were collected at surgery at Johns Hopkins Hospital after informed consent was obtained from all patients. The NLs were obtained at the time of surgery for CCA from normal liver. Quantitative RT-PCR (qRTPCR) for messenger RNA (mRNA) and miRs and quantitative Methylation Specific PCR (qMSP) were performed as previously described. Results. We measured the qRT-PCR level of TIMP3 mRNA and found it to be underexpressed in CCA vs. NL specimens, suggesting a tumor-suppressive role for TIMP3 in the biliary tree. In contrast, our previous work revealed that miR-21 is overexpressed in CCA vs. NL, suggesting a possible negative regulation of TIMP3 by miR-21. In silico searches suggested that TIMP3 is indeed a putative target of miR-21. The CCA cell lines CAK1, TFK1 and HuCCT1 were transfected with a miR-21 inhibitor and TIMP3 protein levels were measured. TIMP3 was significantly elevated in all 3 cell lines transfected with the inhibitor, strongly suggesting that miR-21 downregulates TIMP3 protein levels. Interestingly, miR-21 does not directly bind to TIMP3 mRNA, as evinced by luciferase assays, suggesting the presence of an intermediary molecule or a different mechanism of miR-21 regulation of TIMP3. To investigate a potential co-regulation of TIMP3 in CCA by hypermethylation, we measured the promoter methylation in all 24 specimens. TIMP3 promoter was found to be hypermethylated in only 1/15 CCA (6.6%), 0/3 CCA cell lines and 0/9 NL specimens. Conclusions: TIMP3 mRNA is elevated in NL and decreased in CCA specimens implicating TIMP3 as a tumor suppressor gene in the biliary tree. Our data also suggests that TIMP3 promoter hypermethylation does not appear to play a major role in its downregulation in human CCA specimens. In contrast, miR-21 is a major regulator of TIMP3 in CCA. Furthermore, miR-21 may exert at least part of its oncogenic, pro-invasive effects in CCA by inhibiting TIMP3.