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. 2016 Oct 18;16(1):806.
doi: 10.1186/s12885-016-2801-4.

miR-10b exerts oncogenic activity in human hepatocellular carcinoma cells by targeting expression of CUB and sushi multiple domains 1 (CSMD1)

Qiao Zhu  1 , Li Gong  1 , Jun Wang  1 , Qian Tu  1 , Li Yao  1 , Jia-Rui Zhang  1 , Xiu-Juan Han  1 , Shao-Jun Zhu  1 , Shu-Mei Wang  1 , Yan-Hong Li  2   3   4 , Wei Zhang  5   6
Affiliations

miR-10b exerts oncogenic activity in human hepatocellular carcinoma cells by targeting expression of CUB and sushi multiple domains 1 (CSMD1)

Qiao Zhu et al. BMC Cancer. .

Abstract

Background: Hepatocellular carcinoma (HCC) is a lethal disease, while the precise underlying molecular mechanisms of HCC pathogenesis remain to be defined. MicroRNA (miRNA), a class of non-coding small RNAs, can post-transcriptionally regulate gene expression. Altered miRNA expression has been reported in HCCs. This study assessed expression and the oncogenic activity of miRNA-10b (miR-10b) in HCC.

Methods: Forty-five paired human HCC and adjacent non-tumor tissues were collected for qRT-PCR and immunohistochemistry analysis of miR-10b and CUB and Sushi multiple domains 1 (CSMD1), respectively. We analyzed the clinicopathological data from these patients to further determine if there was an association between miR-10b and CSMD1. HCC cell lines were used to assess the effects of miR-10b mimics or inhibitors on cell viability, migration, invasion, cell cycle distribution, and colony formation. Luciferase assay was used to assess miR-10b binding to the 3'-untranslated region (3'-UTR) of CSMD1.

Results: miR-10b was highly expressed in HCC tissues compared to normal tissues. In vitro, overexpression of miR-10b enhanced HCC cell viability, migration, and invasion; whereas, downregulation of miR-10b expression suppressed these properties in HCC cells. Injection of miR-10b mimics into tumor cell xenografts also promoted xenograft growth in nude mice. Bioinformatics and luciferase reporter assay demonstrated that CSMD1 was the target gene of miR-10b. Immunocytochemical, immunohistochemical, and qRT-PCR data indicated that miR-10b decreased CSMD1 expression in HCC cells.

Conclusions: We showed that miR-10b is overexpressed in HCC tissues and miR-10b mimics promoted HCC cell viability and invasion via targeting CSMD1 expression. Our findings suggest that miR-10b acts as an oncogene by targeting the tumor suppressor gene, CSMD1, in HCC.

Keywords: CSMD1; Hepatocellular carcinoma; Oncogene; miR-10b.

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Figures

Fig. 1
Fig. 1
Overexpression of miR-10b in HCC tissues and cells. a Relative levels of miR-10b expression in HCC tissues (n = 45) and normal liver tissue (n = 45) were measured using qRT-PCR. miR-10b levels were higher in HCC samples compared to adjacent nontumor tissues (−1.4590 ± 0.69542 vs. -1.7312 ± 0.62758, p < 0.01). b The relative levels of miR-10b expression in normal human hepatocytes and HepG2 cells were measured using qRT-PCR. miR-10b expression was nearly 3-fold higher in HepG2 compared to HL-7702 cells
Fig. 2
Fig. 2
Detection of transient transfection efficiency. We transfected hsa-miR-10b mimics (10b-m), mimics negative control (mnc), hsa-miR-10b inhibitors (10b-i), or inhibitors negative control (inc) into HepG2 cells. Relative levels of miR-10b were measured using qRT-PCR. After transfection of 10b-m, the expression of mir-10b was significantly increased, whereas 10b-i elicited the opposite result
Fig. 3
Fig. 3
Effects of miR-10b on HepG2 cell viability, colony formation, and apoptosis. HepG2 cells were transfected with hsa-miR-10b mimics (10b-m), mimics negative control (mnc), hsa-miR-10b inhibitors (10b-i), inhibitors negative control (inc). a MTT assay. miR-10b mimics increased cell viability after 24–72 h of transfection. In contrast, miR-10b inhibition reduced cell viability. b Colony formation and soft agar colony formation assay. miR-10b inhibitors reduced the rate of colony formation by 17.5 and 4.25 %, respectively (p < 0.01). c Flow cytometry cell cycle assay. 19.3 % of miR-10b mimic-transfected cells were in the S phase of the cell cycle, compared to only 8.02 % of negative control cells (p < 0.01). d Flow cytometry for apoptosis assessment. miR-10b transfected cells exhibited lower rates of cell death (0.48 % of early apoptotic cells and 0.27 % of late apoptotic cells) compared to their negative control transfected counterparts (1.24 % of early apoptotic cells, 1.24 and 0.91 % of late apoptotic cells; p < 0.01)
Fig. 4
Fig. 4
Effects of miR-10b on HCC cell migration and invasion. HepG2 cells were transfected with hsa-miR-10b mimics (10b-m), mimics negative control (mnc), hsa-miR-10b inhibitors (10b-i), or inhibitors negative control (inc). a Transwell migration assay. miR-10b mimics led to a 2-fold increase in cell migration, whereas knockdown of miR-10b reduced migration by 40 %. b Matrigel invasion assay. miR-10b mimics led to over 50 % increase in cell invasion capacity, whereas knockdown of miR-10b reduced invasion by 40 %. c Wound healing assay. miR-10b mimics induced wound healing at 48 h, while the mnc group did not demonstrated wound healing at 72 h. miR-10b inhibitors and inc did not induce wound healing at 72 h, but the inc group demonstrated better wound healing compared to the inhibitors
Fig. 5
Fig. 5
miR-10b binds to CSMD1 3’-UTR and represses CSMD1 expression in HepG2 cells. a Luciferase activity assay. HepG2 cells were co-transfected with pMIR/CSMD1 3’-UTR or mutated pMIR mu-CADM1 3’-UTR plus miR-10b mimics or negative control (NC). miR-10b overexpression markedly suppressed luciferase expression in HepG2 cells transfected with pMIR-CSMD1-3’-UTR-WT but not pMIR-CSMD1-3’-UTR-mut1. b qRT-PCR. Expression of CSMD1 mRNA after transfection with hsa-miR-10b mimics (10b-m) or hsa-miR-10b inhibitors (10b-i) was measured by qRT-PCR in HepG2. miR-10b overexpression decreased CSMD1 expression; miR-10b knockdown increased CSMD1 expression in cultured cells c Immunocytochemistry. CSMD1 expression was low in HepG2 compared to normal liver HL-7702cells. d Immunocytochemistry. CSMD1 expression was higher in HepG2 cells transfected with the miR-10b inhibitors, whereas CSMD1 expression was reduced in HepG2 cells transfected with miR-10b mimics
Fig. 6
Fig. 6
Effects of miR-10b on regulation of tumor cell xenograft growth. miR-10b agomir or agomir negative control (NC) were injected into tumor lesions. a miR-10b injection promoted xenograft growth in nude mice. b The relative levels of miR-10b expression in xenografts and nude mouse liver tissues were measured by qRT-PCR. The expression level of miR-10b was significantly increased in tumor tissues compared to normal liver tissues. c CSMD1 protein expression in xenografts and nude mouse livers was detected by immunohistochemistry. CSMD1 protein expression was decreased in xenografts compared to mouse livers
Fig. 7
Fig. 7
Effects of CSMD1 knockdown on tumor cell viability, invasion, and migration. HepG2 cells were transfected with CSMD1 siRNA or NC a CSMD1 protein levels were decreased in HepG2 cells transfected with CSMD1 siRNA. b MTT assay. Knockdown of CSMD1 using siRNA promoted proliferation in HepG2 cells. c Transwell migration assay. Knockdown of CSMD1 promoted migration in HepG2 cells. d Matrigel invasion assay. Knockdown of CSMD1 expression enhanced invasion of HepG2 cells
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