Over-Expression of miR-106b Promotes Cell Migration and Metastasis in Hepatocellular Carcinoma by Activating Epithelial-Mesenchymal Transition Process

<div><p>Hepatocellular carcinoma (HCC) is one the the most fatal cancers worldwide. The poor prognosis of HCC is mainly due to the developement of distance metastasis. To investigate the mechanism of metastasis in HCC, an orthotopic HCC metastasis animal model was established. Two sets of primary liver tumor cell lines and corresponding lung metastasis cell lines were generated. <i>In vitro</i> functional analysis demonstrated that the metastatic cell line had higher invasion and migration ability when compared with the primary liver tumor cell line. These cell lines were subjected to microRNA (miRNAs) microarray analysis to identify differentially expressed miRNAs which were associated with the developement of metastasis <i>in vivo</i>. Fifteen human miRNAs, including miR-106b, were differentially expressed in 2 metastatic cell lines compared with the primary tumor cell lines. The clinical significance of miR-106b in 99 HCC clinical samples was studied. The results demonstrated that miR-106b was over-expressed in HCC tumor tissue compared with adjacent non-tumor tissue (p = 0.0005), and overexpression of miR-106b was signficantly correlated with higher tumor grade (p = 0.018). Further functional studies demonstrated that miR-106b could promote cell migration and stress fiber formation by over-expressing RhoGTPases, RhoA and RhoC. <i>In vivo</i> functional studies also showed that over-expression of miR-106b promoted HCC metastasis. These effects were related to the activation of the epithelial-mesenchymal transition (EMT) process. Our results suggested that miR-106b expression contributed to HCC metastasis by activating the EMT process promoting cell migration <i>in vitro</i> and metastasis <i>in vivo</i>.</p> </div

Orthotopic HCC metastasis animal model.

<p>(A) Primary liver tumor was detected after 1-week orthotopic implantation using luciferase labeled HCC cell line, PLC8024. (B) Metastatic nodules were formed in the lung of the SCID mice at week 10. (C) H&E staining for the lung of the SCID mice show that luciferin signal from the lung nodules are orginated from the tumor cells.</p

Alteration of RhoGTPases and EMT markers expression attributed to miR-106b expression.

<p>(A) High miR-106b expressing cells (PLC-LM, PLC-PT/106b+, PLC-PT/LNA-Scr, PLC-LM/LNA-Scr, Huh7/106b+, and Hep3B/106b+) show over-expression of RhoA and RhoC by western blotting. (B) Expression of EMT markers, E-cadherin, N-cadherin, Vimentin, and TWIST1, were analyzed by western blot. PLC-LM shows higher expression of N-cadherin, Vimentin, and TWIST1, but less E-cadherin, indicating EMT process is activated in the metastatic cell line. miR-106b knock-down cells decrease mesenchymal markers expression and increase epithelial marker expression indicating miR-106b expression can activate EMT process. N-cadherin and TWIST1 expression are too low to be detected in PLC-PT. EMT activation was also observed in miR-106b over-expressing cell lines, Huh7/106b+ and Hep3b/106b+, but not in its corresponding vector control.</p

<i>In vitro</i> functional studies for PLC-PT and PLC-LM cell lines.

<p>(A) Cell migration ability were compared by wound healing assay. Wound closure was observed in the PLC-LM but not in the PLC-PT at 24 and 48 hours. (B) Trans-well invasion assay for PLC-PT and PLC-LM. The number of invading cells in PLC-LM are significantly higher than the PLC-PT (p = 0.01). (C) Phalloidin staining for PLC-PT and PLC-LM. Stress fiber (white arrow) was observed in PLC-LM but absent in PLC-PT.</p

<i>In vitro</i> functional studies for miR-106b in Huh7 and Hep3B cell lines.

<p>Cell migration ability was compared in Huh7 (A) and Hep3B (B) cell lines with or without over-expression of miR-106b. miR-106b over-expressing cells, Huh7/106b+ and Hep3B/106b+, show enhanced cell migration ability when compare to its corresponding vector control, Huh7/V.C. and Hep3B/V.C., at 24 and 48 hours interval. Stress fiber formation was visualized by phalloidin staining (C & D). More stress fiber (white arrow) was observed in miR-106b over-expression cells in Huh7/106b+ (C) and Hep3B/106b+ (D) cell lines compare to the vector control cell lines, Huh7/V.C. and Hep3B/V.C.</p

Candidate miRNAs identified from miRNA microarray analysis.

<p>Candidate miRNAs identified from miRNA microarray analysis.</p

Clinicopathological analysis for miR-21 and miR-106b expression in HCC clinical samples.

1<p><i>Chi-square test</i>.</p