Functional studies revealed that miR-140-5p targets transforming learn more growth
factor β receptor 1 (TGFBR1) and fibroblast growth factor 9 (FGF9), by which it suppresses HCC cell proliferation and metastasis. FGF9, fibroblast growth factor 9; HCC, hepatocellular carcinoma; NHCC, nodular hepatocellular carcinoma; qRT-PCR, real-time quantitative PCR; SHCC, small hepatocellular carcinoma; SLHCC, solitary large hepatocellular carcinoma; TGFR1, transforming growth factor β receptor 1. Matched fresh HCC specimens and ANLTs were obtained from 120 patients during hepatic resection at the Department of Surgery, Xiangya Hospital of Central South University (CSU) from January 2004 to October 2007. Details are described in the Supporting Materials. Prior informed consent was obtained and the study protocol was approved by the Ethics Committee of Xiangya Hospital of
CSU. miRCURY LNA microRNA chips (v. 8.0, Exiqon, Vedbaek, Denmark) were used to profile the differences for miRNA expression among SLHCC, SHCC, and NHCC. The array contained a total of 840 specific probes in triplicate. It was performed according to the protocol of miRCURY LNA microRNA Array Power Labeling kit (Exiqon).18 The image analysis was conducted in Genepix Pro 6.0 (Axon Instruments) as described19 RAD001 order (details in the Supporting Materials and Methods). HCCLM3 and MHCC97-L cell lines were used for this study. Details are described in the Supporting Materials and Methods. qRT-PCR was performed using the TaqMan MicroRNA reverse transcription kit and TaqMan Universal PCR Master Mix (Ambion, Austin, TX). Details are described in the Supporting Materials and Methods. Details are described in the Supporting Materials and Methods. Follow-up data were obtained after hepatic resection for all 120 patients. Details are described in the Supporting Materials. The DNA fragment for miR-140-5p was amplified from genomic DNA and inserted into Age I / EcoR I site of a lentiviral expression vector pGCSIL-GFP (GeneChem, Shanghai, China). The TGFBR1 and FGF9 expression vectors
PtdIns(3,4)P2 were constructed by inserting their ORF sequence into the pGCL vector (GeneChem, Shanghai, China). siRNAs were purchased from GenePharma (Shanghai, China) and the sequences of these siRNAs are provided in Supporting Table 1. Transfection was performed according to the manufacturer’s protocol. Viruses were harvested 72 hours after transfection and viral titers were 1 × 109 TU/mL; 1 × 105 cells were infected with 2 × 106 lentivirus in the presence of 6 μg/mL polybrene (Sigma, St. Louis, MO). In the present study, the infection efficiency of lentivirus was over 90% (Supporting Fig 1). No significant cell death was observed after virus infection. Bulk transfectants were used for subsequent assays.