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. 2014 Sep 19:12:234.
doi: 10.1186/s12967-014-0234-x.

miR-10b is overexpressed in hepatocellular carcinoma and promotes cell proliferation, migration and invasion through RhoC, uPAR and MMPs

Cheng-gong Liao, Ling-min Kong, Ping Zhou, Xiu-li Yang, Jian-guo Huang, He-long Zhang, Ning Lu

miR-10b is overexpressed in hepatocellular carcinoma and promotes cell proliferation, migration and invasion through RhoC, uPAR and MMPs

Cheng-gong Liao et al. J Transl Med. .

Abstract

Background: Recently, miR-10b is identified as a miRNA highly expressed in many human cancers, promoting cell migration and invasion. However, the specific function of miR-10b in hepatocellular carcinoma (HCC) is unclear at this point.

Methods: The miR-10b expression levels in 60 paired different TNM Stage HCC tumor tissues compared with adjacent non-tumor (ANT) tissues, normal tissue control (8 benign tumor and 7 normal liver tissues), 3 normal liver and 7 HCC cell lines were measured by real-time quantitative RT-PCR and to evaluate their association with HCC clinicopathologic features. Invasion assay, MTT proliferation assay and wound-healing assay were performed to test the invasion and proliferation of HCC cell after transfection. The effect of miR-10b on HCC in vivo was validated by murine xenograft model.

Results: We found that miR-10b expression was increased in human HCC tissues and cell lines compared with normal control, respectively. The expression of miR-10b was correlated with HCC metastatic ability. Overexpression of miR-10b in MHCC-97L cells increased cell motility and invasiveness, whereas inhibition of miR-10b in MHCC-97H cells reduced cell motility and invasiveness in vitro and in vivo. We also showed that HOXD10 was negatively regulated by miR-10b at the posttranscriptional level, via a specific target site within the 3'UTR by luciferase reporter assay. Furthermore, we found that miR-10b induced HCC cell invasion and migration by modulating the HOXD10 target gene RhoC, uPAR, MMP-2 and MMP-9 expression.

Conclusions: Our results suggested that miR-10b was overexpressed in HCC and promoted HCC cell migration and invasion through the HOXD10/ RhoC/ uPAR/ MMPs pathway which may provide a novel bio-target for HCC therapy.

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Figures

Figure 1
Figure 1
miR-10b is over-expressed in HCC tissues and cell lines. (a) The relative levels of miR-10b in sixty paired of HCC samples were measured by real-time quantitative RT–PCR, and the U6 small nuclear RNA was used as an internal control. Student’s t test was used to analyze the significant differences between the HCC and ANT tissues. (b) The expression of miR-10b in the five tissue groups. Benign tumor tissues and normal liver tissues were counted as control. One-way ANOVA was used to analyze the significant differences among the groups. (c) The expression of miR-10b in metastatic HCC (HHCC and LHCC) tissues and non-metastatic HCC (NHCC) tissues. The expression of miR-10b was calculated as the normalized ratio of the expression level of miR-10b in HCC tissues to the expression level of miR-10b in ANT tissues. Student’s t test was used to analyze the significant differences. (d) The relative levels of miR-10b in the seven HCC and three normal liver cell lines. One-way ANOVA was used to analyze the significant differences.
Figure 2
Figure 2
miR-10b promotes HCC cell migration and invasion. (a) In MHCC-97L cells transfected with pcDNA3.1-miR-10b and control vector and in MHCC-97H cells transfected with anti-miR-10b, miR-10b expression was measured by real-time quantitative RT-PCR. (b) Proliferation of MHCC-97H and MHCC-97L cells transfected with miR-10b inhibitor or miR-22 overexpression vector was measured in the indicated time periods using the MTT assay. (c) Wound healing assay of MHCC-97H and MHCC-97L cells transfected with miR-10b inhibitor or miR-22 overexpression vector. (d) Migration and invasion potential in both cell lines treated as above were measured by Transwell assays. The results were the means of three independent experiments ± S.D. * P < 0.05, Student’s t test was used to analyze the significant differences.
Figure 3
Figure 3
miR-10b increases HCC growth in vivo . (a) Representative figures of tumors in negative control and miR-10b overexpression groups. (b) Determination of tumor volumes at different time points. (c) After the final measure, the mice were sacrificed, and the tumors were excised. Tumor volume was measured and calculated using the formula length × width2/2. Student’s t test was used to analyze the significant differences.
Figure 4
Figure 4
The HOXD10 3′UTR is a target of miR-10b. (a) Upper panel, predicted duplex formation between human HOXD10 3′UTR and miR-10b. Lower panel, diagram of the luciferase reporter plasmids: plasmid with the full length wild-type HOXD10 3′UTR (pmirGLO-HOXD10-3′UTR) insert and plasmid with a mutant HOXD10 3′UTR (pmirGLO-HOXD10-3′UTR-mut) which carried a substitution of four nucleotides within the miR-10b binding site. (b) Luciferase activity assay demonstrates a direct targeting of the 3′UTR of HOXD10 by miR-10b. MHCC-97L and HepG2 cells were transfected with pcDNA3.1-miR-10b and pmirGLO-HOXD10-3′UTR or pmirGLO-HOXD10-3′UTR-mut. pRL-TK Renilla was used for normalization of transfection efficiency.
Figure 5
Figure 5
miR-10b modulates invasion factors RhoC, uPAR and MMPs expression via the target HOXD10 gene. (a) MHCC-97L cells were transfected with pcDNA3.1-miR-10b and control vector. MHCC-97H cells were transfected with anti-miR-10b and nonsense sequence. Then, HOXD10 protein levels were analyzed by Western blot. (b) In parallel, HOXD10 mRNA in the two cell lines treated as above was measured by real time RT-PCR. GAPDH served as internal control. (c) The protein expression levels of RhoC, uPAR, MMP-2 and MMP-9 were measured in the two cell lines treated as above was measured by Western blot, respectively.
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