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Table S1. Primers for qRT-PCR analysis

Table S2. Predicted targets of miR-216a/217

Figure S1. Schematic diagram illustrating the overall strategy of this study. The study was initiated by the identification of miRNAs associated with early recurrent HCC disease by comparing the miRNA expressions between early-recurrent and non-recurrent human HCC tissue samples by miRNA microarrays (Fig. 1A). Among the various differentially expressed miRNAs identified between early-recurrent and non-recurrent HCC samples, the expression of miR-216a/217 was chosen to be further examined in detail and validated by real-time quantitative RT-PCR (Fig 1B and 1C). It was also observed that elevated miR-216a/217 expression was significantly associated with poor disease-free survival by Kaplan-Meier survival analysis (Fig. 1D and 1E). To dissect the molecular roles of miR-216a/217 in early-recurrent HCC disease, we studied the expression of miR-216a/217 in a panel of liver cancer cell lines (Fig. 2A and 2B). It was demonstrated that the up-regulation of miR-216a/217 was associated with cell EMT phenotype (Fig. 2C and 2D) and the migratory ability of these cells (Fig. 2E). Establishing that the over-expression of miR-216/217 was associated with cell EMT phenotype and migratory ability of HCC cell lines enabled us to further study the biological activity of miR-216a/217 in vitro and in vivo. We additionally demonstrated that the stable overexpression of miR-216a/217 increased the stem-like cell population (Fig 3A-3D) and enhanced the metastatic ability of HCC cells with epithelial cell morphology (Fig. 3E and 3F). Subsequent bioinformatics analysis, luciferase report assay and western blotting studies identified PTEN and SMAD7 as two functional targets of miR-216a/217 (Fig. 4A and 4B). This observation was corroborated with the down-regulation of both PTEN and SMAD7 in samples from HCC patients with early recurrent disease (Fig. 4C and 4D) and was significantly associated with disease-free survival of HCC patients (Fig. 4E and 4F). Furthermore, ectopic expression of PTEN or SMAD7 could partially rescue miR-216a/217-mediated EMT (Fig. 5A and 5B), cell migratory ability (Fig. 5C) and stem-like properties of HCC cells with epithelial cell morphology (Fig. 5D and 5E). Acquired resistance to a small molecular inhibitor, such as sorafenib, is a major clinical challenge and since miRNAs have been suggested to be involved in regulating epithelial-mesenchymal transition and cancer stem cells and can serve as potential molecular targets for cancer therapeutics and overcoming drug resistance (Ref: Xia, H and Hui, KM. MicroRNAs involved in regulating epithelial–mesenchymal transition and cancer stem cells as molecular targets for cancer therapeutics. Cancer Gene Therapy 2012; 19(11):723-30), we have therefore further investigated the potential molecular roles of miR-216a/217 in the development of resistance to sorafenib in HCC cancer cells. Our results demonstrated that the over-expression of miR-216a/217 acted as a positive feedback regulator for the TGF-β pathway and the canonical pathway involved in the activation of the PI3K/Akt signaling in HCC cells (Fig. 6A). In addition, the activation of TGF-β and PI3K/Akt signaling pathways in HCC cells resulted in the acquired resistance to sorafenib (Fig. 6B). In comparison, blocking the activation of TGF-β pathway overcame miR-216a/217-induced sorafenib resistance (Fig. 6C and 6D) and decreased the metastatic ability of HCC cells in a mouse model (Fig. 6E and 6F). We concluded that over-expression of miR-216a/217 activated the PI3K/Akt and TGF-β pathways by targeting PTEN and SMAD7, contributing to hepatocarcinogenesis and tumor recurrence (Fig. 7).

Figure S2. (A and B) Expression of epithelial marker E-cadherin and mesenchymal marker Vimentin in a panel of liver cancer cell lines. Epithelial HCC cells such as HepG2 and PLC/PRF/5 gave high expression of E-cadherin and low expression of vimentin while HCC cells with mesenchymal phenotype such as SNU-449 and HLE demonstrated low expression of E-cadherin and high expression of vimentin (P<0.05). (C and D) RT-qPCR was employed to validate the significant increase of miR-216a and miR-217 in stably-transfected HepG2-miR-216a/217 and PLC/PRF/5-miR-216a/217 cells. (E) HepG2-miR-216a/217 and PLC/PRF/5-miR-216a/217 stably-transfected cells demonstrated significant morphological change from an epithelial cobblestone phenotype to an elongated fibroblastic phenotype, indicative of EMT.

Figure S3. Silencing of miR-216a and 217 in the HLE, mesenchymal phenotype, HCC cells. Antagomir-miR-216a/217 was transfected into HLE cells. (A) Expression of miR-216a and miR-217 was examined by qRT-PCR and significant silencing of miR-216a/217 was demonstrated. (B) A dramatic morphological change from mesenchymal to epithelial transition was observed. (C) Up-regulation of E-cadherin, an epithelial biomarker and the reduced expression of vimentin, a mesenchymal biomarker, was detected with the simultaneous increased in the expression of SMAD7 and PTEN was also observed.Figure S4. Effects of miR-216a/217 on the proliferation and apoptosis of liver cancer cells. (A) Significant increase in cell proliferation was observed in PLC/PRF/5 at 72 h following the transfection of the p-miR-216a/217-overexpressing vector while transfection of the antagomir-miR-216a/217 into HLE cells significantly decreased cell proliferation. (B) The number of apoptotic cells (Annexin V+ cells) was not significantly affected in PLC/PRF/5 and HLE cells by modulating the expression of the miR-216a/217 cluster. The Annexin V+ cells decreased from 2.44% to 1.32% by overexpression of miR-216a/217 in PLC/PRF/5 cells and increased from 0.99% to 2.92% following the knockdown of miR-216a/217 in HLE cells (P>0.05).

Figure S5. Expression of SMAD7 (A), PTEN (B), JAK2 (C) and CADM1 (TSLC1) (D) was shown by dot plot analysis, by searching a HCC Gene Expression database established in our laboratory using Affymetrix Human Genome U133 plus 2.0 Arrays (Affymetrix, Santa Clara, CA, USA) comprising of HCC tumor and adjacent histologically normal liver tissue (1).

Figure S6. Potential targeting region of miR-216a/217 predicted for PTEN-3'UTR (A and B) and SMAD7-3'UTR (C and D) using RNAhybrid 2.2. (A-D) The predicted target sequences and mutations generated for the 3'-UTR of PTEN and SMAD7 mRNA are shown. (E) Images to show the morphological changes observed for PLC/PRF/5-miR-216a/217 cells following transfection with wild-type plasmids containing SAMD7 (i and ii) or PTEN (iii and iv) compared to control vectors, the morphological changes were indicative of mesenchymal-epithelial transition (MET).

Figure S7. (A and B) TGF-β1 treatment induced the up-regulation of miR-216a/217 in HepG2 cells. (C and D) Addition of LY2109761, a selective TGF-β receptor type I/II dual inhibitor, inhibited TGF-β1-induced miR-216a/217 expression in HCC cells. (E) Low concentration of LY2109761 (< 1 μM) have insignificant effect on the viability of the PLC/PRF/5 cells.

Figure S8. (A) Kaplan-Meier survival analysis between HCC patients with early-recurrent and non-recurrent disease. Significant difference in disease-free survival (P<0.0001) was observed between HCC patients with early-recurrent and non-recurrent disease. (B) Immunohistochemical studies of the expression of P-Akt in matched normal, early-recurrent and non-recurrent HCC liver tissue samples (20X). Of note, more than 50% of the early-recurrent HCC tissues studied by IHC exhibited elevated P-Akt staining in 25-75% of the tumor tissues examined and revealed that a significant difference in the staining of P-Akt between the early recurrent and non-recurrent HCC tissues (S8B).

Figure S9. Expression of CADM1 (TSLC1) (A) and SMAD7 (B) is down-regulated in liver cancer compared with normal liver tissues (indicated by red arrows) by searching the IST Online system (http://www.medisapiens.com/ist-online-overview/).

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