The Roles Of MiRNAs In The Anti-tumor Effects Of Triptolide In Hepatocellular Carcinoma Cells

Background&Aims: At present Hepatocellular carcinoma (HCC) is the third leading causeof cancer death worldwide with annual death exceeding600000. HCC is associated with avery poor prognosis because of its aggressive growth, metastasis, and resistance to the mostof current therapeutic approaches. Therefore, there is an urgent need to develop effectivetherapeutic strategies for the large number of HCC patients. MicroRNAs (miRNAs) areendogenous～23nt RNAs that negatively regulate gene expression by pairing to the mRNAsof protein-coding genes to direct their posttranscriptional repression. As key negativeregulators in gene expression, miRNAs play an important role in many cellular processes,such as differentiation, proliferation, and apoptosis. Importantly, a large body of evidence hasshown that miRNAs regulate molecular pathways in cancer by targeting various oncogenesand tumor suppressors, thus forming central nodal points in cancer development andprogression. Not surprisingly, miRNAs have also been discovered to be aberrantly expressedin HCC and some of them are functionally implicated in hepatocarcinogenesis andprogression. Combinations of genomic analyses and functional studies have identified somemiRNAs, such as miR-17-92, miR-21, and miR-221, function as oncogenes in initiation andmaintenance of HCC. In contrast, some miRNAs, including let-7, miR-122and miR-26, havebeen identified as tumor suppressors. Therefore, targeting oncogenic miRNAs and restoringtumor-suppressive miRNAs would be a reasonable therapeutic strategy for HCC patients.Triptolide is a structurally unique diterpene triepoxide isolated from Tripterygium wilfordiiHook F, a Chinese medicinal plant used for treating a wide range of diseases for centuries. Triptolide has been shown to possess potent anti-inflammatory, immunosuppressive andanticancer activity. The antitumor activity appears quite broad in that triptolide is capable ofkilling cancer cells originated from different tissues, including blood, colon, breast, brain,ovary, kidney and prostate, with IC50values in the low nanomolar range. The mechanismsresponsible for antitumor activity of triptolide have been extensively investigated in the pastfew decades. Triptolide is demonstrated to cause transcriptional inhibition, initially believedto target specific transcription factors but recently revealed to cause global transcriptioninhibition via targeting RNA polymerase I and II. More recently, Titov et al. reported thattriptolide covalently bound to a human90kD protein ERCC3(also known as XPB), andinhibited its DNA-dependent ATPase activity, which led to the inhibition of RNA polymeraseII–mediated transcription. It seems that the transcription inhibition accounts for most of theaforementioned biological activities of triptolide. However, besides downregulating theexpression of a lot of genes, triptolide has also been revealed to increase the mRNA or proteinlevels of several molecules, including p53, RIZ1and HIF-1a, and such an increase has beenshown to contribute to the anticancer activity of triptolide.To explain this discrepancy, we hypothesized that triptolide inhibits the transcription of someoncogenic miRNAs, which in turn increase the protein level of their target genes. We usedmiRNA microarrays and observed that the miRNAs expression profiles are significantlyaltered by the treatment of triptolide. Among the modulated miRNAs, up to94%aredown-regulated. Two oncogenic miRNA cluster, mir-17-92and mir-106b-25, aredownregulated by the treatment of triptolide simultaneously. On the contrary, the putative target genes of these miRNA clusters are upregulated by triptolide. We further proved thatboth of the two miRNA clusters are directly transactivated by c-Myc, and triptolidedownregulates the expression of these miRNA clusters in a c-Myc dependent manner.Importantly, the modulation of c-Myc/miRNA clusters/target genes cascade contributes totriptolide-induced cell death.METHODS: Cells were treated with triptolide, and the anti-HCC activity of triptolide wasevaluated using flow cytometry, western blot, and xenograft studies. microRNA microarray andquantitative reverse-transcription polymerase chain reaction was used to identify differentialmicroRNAs induced by triptolide. Chromatin immunoprecipitation assay was employed tostudy the interaction between c-Myc and genomic regions of miR106b-25. microRNAsoverexpression and knockdown experiments was performed to determine the role of thesemicroRNAs in triptolide-induced apoptosis.RESULTS: Triptolide induces cell proliferation inhibition and marked apoptosis in multipleHCC cell lines with different p53status. Several signaling molecules belonging to differentpathways are altered by the treatment of triptolide. Xenograft tumor volume was significantlydecreased in triptolide-treated group when compared with vehicle control group. Two miRNAclusters, miR-17-92and miR-106b-25, were significantly repressed by triptolide, resulting inthe upregulation of their common target genes, including BIM, PTEN, and p21. In HCCsamples, high levels of these miRNA clusters correlated with shorter recurrence free survival.Triptolide inhibits the expression of theses miRNAs in a c-Myc-dependent manner, whichenhances triptolide-induced cell death. We further show that triptolide downregulates the expression of c-Myc through targeting ERCC3, a newly indentified triptolide-binding protein.CONCLUDSIONS: The triptolide-induced modulation of c-Myc/miRNA clusters/targetgenes axis enhances its potent antitumor activity, thereby making triptolide an attractivechemotherapeutic agent against HCC.