| Objective Twist1 and micro RNAs(mi RNAs) have been reported to act as a master regulator of tumour progression, involving in tumour invasion, metastasis, angiogenesis, epithelial-mesenchymal transition(EMT), and so on. However, the relationship between Twist-1 and mi RNAs and the function of mi RNAs in human hepatocellular carcinoma(HCC) remain to be elucidated. We aimed to reveal the Twist-1-related mi RNA expression profile and to determine whether Twist1 functions in tumor invasion, metastasis, and vasculogenic mimicry(VM) by regulating mi RNA expression in HCC. This may provide novel prognostic and predictive factors for HCC disease and the design of novel mi RNA-based therapeutic strategies against HCC.Methods 1. Microarray and Ch IP-seq technology were performed to analyze a panel of mi RNAs with significant differential expression when Twist1 was overexpressed in Hep G2 cells. The results from microarray studies were subsequently validated by quantitative Real-Time Polymerase Chain Reaction(q RT-PCR) analysis and chromatin immunoprecipitation(Ch IP) – PCR.2. q RT-PCR was used to determine the expression of mi RNAs in clinical HCC samples. Associations between mi R-26b-5p expression and clinicopathologic characteristics in HCC patients were analysed.3. q RT-PCR was used to detect the expression of Twist1. The relationship between the expression of mi R-26b-5p and Twist1 was further investigated by q RT-PCR.4. The expression of mi RNAs in a panel of liver cancer cell lines was analyzed by q RT-PCR. The selected HCC cell lines with low or high expression of mi RNAs that screened out by microarray analysis were stably transfected with P-mi RNA and P-mi RNA-inhibitor plasmids, respectively. The expression of mi RNAs in these cells was confirmed by q RT-PCR. Then, the expression of EMT/VM-related transcription factors was detected in the selected HCC cell lines by q RT-PCR,Western blot and immunofluorescence assay(IF).5. Scrape assays, Migration/Invasion Assays, 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT), Cell Adhesion, Colony Formation and Three-dimensional culture assays were performed to analyse the role of the selected mi RNAs in HCC.6. Potential target genes of mi RNAs were first predicted using databases, including Target Scan, Pic Tar, and mi Randa. These targets were subjected to further analysis by a Dual Luciferase Reporter Assay to identify the directly binding between mi RNAs and targets. In HCCcell lines with low expression of mi RNAs, when mi RNAs were upregulated or mi RNAs and targets were co-transfected, q RT-PCR,Western Blot and functional experiments were performed to explore the possible biological significance of mi RNAs in interaction between mi RNAs and targets.7. q RT-PCR was used to detect the expression of the selected target and its related transcription factors. The relationship between the expression of the above transcription factors was further investigated by q RT-PCR.8. HCC cell lines with medium Twist1 and the target gene expression were selected. In these cell lines with the upregulation of mi RNAs or co-transfection of mi RNAs and targets, q RT-PCR,Western Blot and functional experiments were used to reconfirm that mi RNAs and targets affected EMT/VM, cell migration, invasion, adhesion, proliferation, and colony formation.9. Subcutaneous xenograft model by injecting stable transfected cells of mi RNA or mi RNA-inhibitor into nude mice was made to investigate the effects of mi RNAs on the tumor growth and metastatic potential of HCC cells. Expression levels of EMT/VM-associated markers(E-cadherin, VE-cadherin, vimentin, BMP4, Smad1, β-catenin, Snail, and Twist1) and the existance of VM in engrafted mouse HCC samples were detected by immunohistochemical staining(IHC) and endomucin/periodic acid-Schiff(PAS) double staining to analyse the infulence of mi RNAs on EMT/VM in HCC.10. The expression of transcription factors on Upstream and Downstream of mi RNAs were detected by Western Blot to explore the pathway by which mi RNAs and the target genes exert their functions in HCC.Results 1. Microarray analysis revealed a panel of mi RNAs with significant differential expression followed by the upregulation of Twist1. Among these mi RNAs, the expression of mi R-26b-5p was significantly(P=0.00733) downregulated in the Hep G2-Twist1 cell line. The results from microarray studies were subsequently validated by q RT-PCR analysis. In addition, Ch IP-seq analysis indicated that Twist1 could directly bind to the promoter region of mi R-26b-5p and the results were validated by Ch IP-q RT-PCR.2. In HCC tissues, the expression of mi R-26b-5p was significantly downregulated. Kaplan-Meier plots demonstrated that lower mi R-26b-5p expression was significantly associated with early metastasis and recurrence of HCC(Log Rank, P=0.01).3. In HCC tissues, the expression of Twist1 was up-regulated. Pearson correlation analysis showed that mi R-26b-5p expression was inversely correlated with Twist1 expression in the clinical samples(P=0.004, r=-0.572).4. In HCC cell lines(H7402, Bel7402, SMMC7721, Hep G2, PLC, HCCLM3, MHCC97 L, Huh7) and normal liver cell line(L02), the expression of mi R-26b-5p was significantly downregulated in all of the HCC cell lines studied. Thus, four HCC cell lines were selected and transfected as recipient cells: viz.Bel7402 and SMMC7721 with P-mi R-26b-5p or P-mi R-control, and Hep G2 and PLC with P-mi R-26b-5p-inhibitor or P-mi R-inhibitor-control. Stable cell lines over-expressing and down-regulating mi R-26b-5p were established and were tentatively designated Bel7402-mi R-26b-5p or SMMC-mi R-26b-5p, Hep G2-mi R-26b-5p-inhibitor or PLC-mi R-26b-5p-inhibitor. The expression of mi R-26b-5p in these cells was confirmed by q RT-PCR. Bel7402-mi R-26b-5p and SMMC-mi R-26b-5p exhibited high expression of E-cadherin and low expression of vimentin, while Hep G2-mi R-26b-5p-inhibitor and PLC-mi R-26b-5p-inhibitor demonstrated low expression of E-cadherin and high expression of vimentin.5. Bel7402 and SMMC7721 cells that were transfected with mi R-26b-5p displayed a much lower ability to promote migration, invasion, adhesion, proliferation, colony formation abilities and tube formation abilities compared with those that were transfected with empty vector or with non-transfected cells(P<0.05). Compared with those of the controls, Hep G2-mi R-26b-5p-inhibitor and PLC-mi R-26b-5p-inhibitor showed a higher ability to enhance migration, invasion, adhesion, proliferation, colony formation abilities and tube formation abilities(P<0.05).6. Potential target genes of mi R-26b-5p were first predicted using databases, including Target Scan, Pic Tar, and mi Randa. Among them, SMAD1 was chosen for further experimental validation. Dual-luciferase reporter analysis showed that mi R-26b-5p may suppress gene expression through its binding sequence at 3’-UTR of SMAD1. Bel7402-mi R-26b-5p cells with re-expression of SMAD1 demonstrated lower expression of E-cadherin and higher expression of vimentin, VE-cadherin at the m RNA and protein expression levels. Meanwhile, the restoration of SMAD1 inhibited the mi R-26b-5p-suppressed migration, invasion, adhesion, proliferation, colony formation abilities and tube formation abilities(P<0.05).7. In HCC tissues, the expression of SMAD1 was up-regulated. Pearson correlation analysis showed that mi R-26b-5p expression was inversely correlated with SMAD1 expression in the clinical samples(P=0.035, r=-0.442). BMP4 was significantly up-regulated in the HCC tissues, and the level of mi R-26b-5p was inversely correlated with BMP4 expression( P=0.010, r=-0.524), SMAD1 expression was positively correlated with BMP4 expression in the clinical samples(P=0.029, r=0.455).8. HepG2 and MHCC97 L cell lines with medium Twist1 and SMAD1 expression compared with other HCC cell lines were selected. In Hep G2 and MHCC97 L cells, the expression of E-cadherin at the m RNA and protein levels was up-regulated whereas vimentin was down-regulated, following the upregulation of mi R-26b-5p. the expression of E-cadherin was highest in mi R-26b-5p-upregulated group, was lowest in SMAD1-upregulated group, and in mi R-26b-5p/SMAD1-co-upregulated group, fell in between. As for the vimentin expression, opposing results were observed in the above groups.Moreover, in these two transfected HCC cells, cell migration, invasion, adhesion, proliferation, colony formation abilities and tube formation abilities were enhanced by the overexpression of SMAD1 and impaired by the upregulation of mi R-26b-5p. The results of functional assays fell in between when both mi R-26b-5p and SMAD1 were upregulated.9. Compared with those of the controls, the tumours derived from SMMC-mi R-26b-5p cells displayed lower levels of tumorigenicity, showed the decreased expression of EMT and VM associated proteins(vimentin, VE-cadherin, β-catenin, Snail), Smad1, BMP4, and the increased expression of E-cadherin. However, the tumours derived from Hep G2-mi R-26b-5p-inhibitor cells displayed higher levels of tumorigenicity, showed the increased expression of EMT and VM associated proteins(vimentin, VE-cadherin, β-catenin, Snail), Smad1, BMP4, and the decreased expression of E-cadherin. The changes in the Twist1 expression had no statistical significance in both SMMC-mi R-26b-5p transplanted tumours and HepG2-mi R-26b-5p-inhibitor transplanted tumours.10. In Hep G2 and MHCC97 L cell lines, due to increased mi R-26b-5p or the overexpression of SMAD1, or the co-overexpression of mi R-26b-5p and SMAD1, the levels of Eph A2, Wnt5α and SIP1 were not significantly changed in different groups; the expression of Smad1, BMP4, β-catenin, Snail, and Slug, was highest in SMAD1-overexpressed group, was lowest in mi R-26b-5p-overexpressed group, and fell in between when both mi R-26b-5p and SMAD1 were upregulated.Conclusions 1. Twist1 can downregulate the expression of mi R-26b-5p by directly binding the promoter region of mi R-26b-5p.2. Expression of mi R-26b-5p is inversely correlated with Twist1 expression in HCC tissues and its downregulation is associated with patient poor prognosis.3. mi R-26b-5p is associated with the EMT phenotype and impairs EMT /VM.4. mi R-26b-5p suppresses migration/invasion, adhesion, proliferation, colony formation, tube formation abilities of HCC cells in vitro and impairs tumorigenicity and tumour angiogenesis in vivo.5. SMAD1 is identified as a downstream target of mi R-26b-5p, and re-expression of SMAD1 partially abrogated mi R-26b-5p-mediated EMT/VM suppressive effects and metastasis suppression.6. MiR-26b-5p inactivates the BMP4/Smad1 pathway and exerts its inhibitory role in EMT and VM by targeting SMAD1. |
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