MicroRNA-630 suppresses tumor metastasis through the TGF-β- miR-630-Slug signaling pathway and correlates inversely with poor prognosis in hepatocellular carcinoma

The epithelial-mesenchymal transition (EMT) is the key process that drives tumor metastasis. Accumulating evidence suggests that the deregulation of some microRNAs (miRNAs), is implicated in this process. Here, we highlight the function and molecular mechanism of miR-630 and its potential clinical application in hepatocellular carcinoma (HCC). First, we identified the clinical relevance of miR-630 expression in a screen of 97 HCC patient tissues. Patients with low miR-630 expression had higher recurrence rates and shorter overall survival than those with high miR-630 expression. Functional studies demonstrated the overexpression of miR-630 in HCC cells attenuated the EMT phenotype in vitro. Conversely, inhibition of miR-630 promoted EMT in HCC cells. Mechanistically, our data revealed that miR-630 suppressed EMT by targeting Slug. Knockdown of Slug expression reversed miR-630 inhibitor-mediated EMT progression. Furthermore, we found that the TGF-β-Erk/SP1 and JNK/c-Jun signaling pathways repressed miR-630 transcription through occupying transcription factor binding sites. Ectopic expression of miR-630 restored the TGF-β-activated EMT process. Taken together, these findings demonstrate, in HCC cells, miR-630 exerts its tumor-suppressor functions through the TGF-β-miR-630-Slug axis and provides a potential prognostic predictor for HCC patients.


Patients and specimens
Ninety-seven human liver tumor tissues were obtained between 2010 and 2012 from patients who underwent liver resection surgery at the Hepatic Surgery Center of Tongji Hospital affiliated with the Huazhong University of Science and Technology. The pathology of all of the tissues specimens was verified and all of the specimens were stored at -80°C until used for analysis. Overall survival (OS) was defined as the interval between the date of resection and the date of patient death or last follow-up. For surviving patients, the data were censored at the last follow-up. Time to recurrence (TTR) was defined as the interval between the date of surgery and the date of diagnosis of any type of relapse (intrahepatic recurrence and extra-hepatic metastasis).The study protocol was approved by the ethics committee of the Huazhong University of Science and Technology and Tongji Hospital and a written informed consent were obtained from all participants involved in this study.

Cell proliferation assay
Two thousand cells per well were plated in 96well plates and incubated at 37°C. Cell proliferation was assessed at various time points after transfection using the Cell Counting Kit-8 (Beyotime Institute of Biotechnology) according to the manufacturer's instruction. Each assay was repeated three times.

Cell migration and invasion assays
For Transwell cell migration and invasion assays, tumor cells (2x10 4 ) in 0.2 ml of serum-free medium were seeded onto the 8μm insert (upper chamber) of a Transwell plate (Corning Costar, NY, USA). The lower chamber was filled with 0.5 ml of medium containing 10% FBS. For the migration assay, cells were incubated for 24 h postseeding, after which, the cells remaining in the upper part of the Transwell were removed with a cotton swab. Cells that had migrated to the lower chamber were then stained with 0.5% Crystal Violet dye, and the number of cells per high field (×100) was counted using a Nikon microscope. For cell invasion assays, Transwell chamber inserts were pre-coated with Matrigel (BD Biosciences, NJ, USA). After a 48 h incubation period post-seeding, the number of cells invading the Matrigel-coated insert was counted.

Wound healing assay
Cells (4 × 10 5 /well) were seeded in 12-well plates, cultured overnight. After forty-eight hours, a 5-mm-wide scratch was made across the cells using a sterile plastic tip, and the cells were washed twice with culture medium. Fresh serum-free medium was then added, and the imaging was performed after 0 h and 24 h using an NIKON Digital ECLIPSEC microscope and a Retiga-4000DC camera. The images were analyzed using Cell Profiler.

Cell counting assay
Cells were digested by trypsin. The number of cells was counted by Cellometer Mini (Nexcelom Bioscience, Massachusetts USA). Each assay was repeated three times.

Luciferase reporter assay
We used the Dual-Luciferase Reporter Assay System (Promega, USA) to verify the precise target of miRNAs and the specific response element that participated in the down-regulation of miR-630 promoter activity. Briefly, 1x10 5 Bel7402 cells were seeded in 24-well plates. After 24 h, the luciferase reporter plasmid was co-transfected with small RNA molecules or TGF-β using the riboFect TM CP Transfection Kit (333T) (Invitrogen). Firefly and Renilla luciferase activities were measured 24 h posttransfection using the DualGlo Luciferase Assay System (Promega, USA). Firefly luciferase was normalized to Renilla luciferase activity. All experiments were performed three times.

Animal studies
Six-week-old male BALB/c athymic nude mice were housed under specific pathogen free (SPF) conditions and cared for according to the institutional guidelines for animal care. All animal studies conformed to the National Institutes of Health guidelines (NIH publication 86-23 revised 1985) and were approved by the Committee on the Ethics of Animal Experiments of the Tongji Medical College, HUST. The Bel7402-shmiR-630 and NC-transfected

SUPPLEMENTARY MATERIALS AND METHODS
www.impactjournals.com/oncotarget/ Oncotarget, Supplementary Materials 2016 clone cells were harvested and resuspended to 2 × 10 6 cells/ml in serum-free DMEM. For the in vivo tumorigenicity assay, we injected 2 × 10 6 cells in a total volume of 100μl subcutaneously into the right flanks of athymic nude mice (n=10). All mice were monitored once every three days and were sacrificed four weeks later. Tumor weight was monitored. For in vivo tumor metastasis assays, subcutaneous tumor tissue was minced into small pieces of equal volume (1 mm3) and transplanted into the livers of nude mice (10 mice per group). All mice were monitored once every three days, then sacrificed eight weeks later. Their liver and lung tissues were dissected, fixed and prepared for histological examination.