| ObjectiveTo establish the molecular mechanism of miR-491involved in regulating cancer stem cells-like properties, and influence of invasion and recurrence potential in HCC, which will provide new clues to HCC’s diagnosis, targeted therapy and prognosis prediction.Methods1. MiR-491and CSCs-like properties are associated with malignant behavior of cell lines and tissue samples of HCC.(1) Screening of miRNA expression profiles in well/poorly differentiated HCC tissue:Human miRNA OneArray(?) v4miRNA microarray analysis between15well-differentiated HCC tissues and17poorly-differentiated HCC tissues.(2) Measurement of miR-491and CSCs-like markers expression in HCC cell lines and clinic samples:qRT-PCR was used to evaluate miR-491and CSCs-like markers expression level in HCC cell lines and samples.2. MiR-491attenuates the CSCs-like properties in HCC cells.Assessment of CSCs-like markers expression and capacity for formation of spheroids in transfected HCC cell lines:qRT-PCR assessed CD44, CD90, and EpCAM expression. Spheroid formation experiment estimated capacity for formation of spheroids in HCC cell lines transfected with miR-491-mimic/inhibitor.3. NF-κB is involved in the maintenance of CSCs-like properties of HCC cells and is associated with malignant behavior in HCC tissue samples.(1) Measurement of NF-κB and snail expression in HCC cell lines and clinic samples:qRT-PCR was used to evaluate NF－κB and snail expression level in HCC cell lines and samples.(2) Assessment of snail and EMT-related proteins in transfected HCC cell lines: cell morphology was observed with a microscope. Western blot was used to assess snail、E-cadherin, and Vimentin expression in MHCC97H cells transfected with p65siRNA.(3) Assessment of CSCs-like markers expression and capacity for formation of spheroids in transfected HCC cell lines:qRT-PCR was used to evaluate CD44, CD90, and EpCAM expression level, while spheroid formation experiment was employed to estimate capacity for formation of spheroids in MHCC97H and MHCC97L cells transfected with p65siRNA.4. MiR-491blocks the activation of NF-κB/snail signal pathway by GIT-1-mediated ERKs inactivation.(1) Assessment of NF-κB and snail in transfected HCC cell lines:Western blot was used to assess p-p65and snail expression in MHCC97H cells transfected with miR-491-mimic and HepG2cells transfected with miR-491-inhibitor.(2) Luciferase reporter assays were conducted to assess the relative transcriptional activity in MHCC97H cells co-transfected with pGL3-GIT-13’UTR (wt)-Luc construct and miR-491-mimic.(3) Western blot was used to assess p-ERKs and p-p65expression in MHCC97H cells transfected with GIT-siRNA or con-siRNA.(4) HepG2cells were transfected with GIT-siRNA or con-siRNA_for12h after transfected with miR-491-inhibitor or anti-con for24h before. Then Western blot was used to assess p-p65expression.5. MiR-491and CSCs-like properties are associated with recurrence of HCC.(1) Estimating the metastasis and prognosis of HCC patients:According to previous indexes and clinical diagnosis, we estimated the recurrence and prognosis of HCC patients.(2) we re-divided the HCC tissue samples (the initial tumour tissue samples taken after surgery) into non-recurrence and recurrence groups, and re-determined the expressions of miR-491and CSCs-like markers with qRT-PCR.Results1. MiR-491and CSCs-like properties are associated with malignant behavior of cell lines and tissue samples of HCC.(1) Different miRNA expression between well and poorly differentiated HCC samples:Human miRNA One Array(?) v4miRNA analysis showed41miRNA obviously changed (increase3times/decrease to1/3), miR-491was one of the most significantly changed miRNAs (0.15).(2) The expression of miR-491and CSCs-like markers in HCC cell lines and samples:qRT-PCR indicated miR-491expression was negatively-correlate with HCC cell lines’ invasion potential and differentiation, while CD44, CD90, and EpCAM expression were positively-correlate with HCC cell lines’ invasion potential and differentiation.2. MiR-491attenuates the CSCs-like properties in HCC cells.Compared with HepG2cells, there was relative lower background expression of miR-491in MHCC97H cells. Over-expression of miR-491decreased the expressions of CD44, CD90, EpCAM, and attenuated the capacity for formation of spheroids. Further, in HepG2cells, knockdown of miR-491improved the expressions of CD44, CD90, EpCAM, and elevated the capacity for formation of spheroids. These results suggest that miR-491attenuates the CSCs-like properties in HCC cells. 3. MiR-491blocks the activation of NF-κB/snail signal pathway by GIT-1-mediated ERKs inactivation.(1) Over-expression of miR-491decreased the levels of p-p65and snail in MHCC97H cells, while knockdown of miR-491improved the expressions of p-p65and snail in HepG2cells.(2) Southwestern assay suggested that miR-491attenuated the DNA-binding activity of NF-κB.(3) In MHCC97H, co-transfected with pGL3-GIT-13’UTR (wt)-Luc construct and miR-491-mimic caused a significant decrease in the luciferase activity. It suggested that miR-491directly targeted GIT-1in HCC cell lines.(4) Knockdown of GIT-1decreased the phosphorylation of p-ERKs and p65.(5) In HepG2cells, knockdown of miR-491improved the activation of ERKs and NF-κB, however, inhibition of ERKs blocked such phenomenon. Collectively, these results suggest that miR-491blocks the activation of NF-KB/snail signal pathway by GIT-1-mediated ERKs inactivation.4. NF-κB is involved in the maintenance of CSCs-like properties of HCC cells and is associated with malignant behavior in HCC tissue samples.(1) Activation of NF-κB (as determined by the phosphorylation of NF-KB/p65) in poorly differentiated HCCs was higher than that in well differentiated HCC tissues. In HCC cell lines, we also found that, with more migration behavior of HCC cells, there was more increased phosphorylation of NF-KB/p65.(2) Knockdown of NF-κB improved the expression of E-cadherin but decreased the expression of Vimentin, and changed the cell morphology to an epithelial-like properties(3) Knockdown of NF-κB decreased the expressions of CD44, CD90and EpCAM, and attenuated the capacity for formation of spheroids. Collectively, these data suggest that the NF-κB may be involved in the maintenance of CSCs-like properties in HCC cell lines and tissue samples. 5. MiR-491and CSCs-like properties are associated with recurrence of HCC.HCC’s postoperative clinical follow-up:The recurrence rate of poorly differentiated HCC patients was significantly higher than well-differentiated ones, but the mortality had no significant difference. Recurrence patients had a lower miR-491expression level, but higher expression levels of CSCs-like properties. These results suggest that miR-491is inversely correlated with recurrence in HCC patients, while CSCs-like properties are positively correlated with it.Conclusions1. MiR-491and CSCs-like properties are associated with malignant behavior of cell lines and tissue samples of HCC.2. MiR-491attenuates the CSCs-like properties in HCC cells.3. MiR-491blocks the activation of NF-κB/snail signal pathway by GIT-1-mediated ERKs inactivation,4. NF-κB is involved in the maintenance of CSCs-like properties of HCC cells and is associated with malignant behavior in HCC tissue samples.5. MiR-491and CSCs-like properties are associated with recurrence of HCC.In sum, in HCC cells and tissue samples, miR-491decreases activation of the NF-κB/snail signal pathway by GIT-1mediated ERKs inactivation. Such process blocks the EMT process. Such progression attenuates the CSC-like properties in HCC cells. |
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