Emerging evidence has indicated that deregulation of long non\coding RNAs (lncRNAs)

Emerging evidence has indicated that deregulation of long non\coding RNAs (lncRNAs) can contribute to the progression and metastasis of human cancer, including hepatocellular carcinoma (HCC). suppress the metastasis of HCC cells by interacting with FUS, which indicates potential of for the prognosis and treatment of HCC. that was downregulated in HCC tumor tissues, compared to the adjacent noncancerous tissues. Low expression of was correlated with poor prognosis of HCC patients. could suppress the migration of HCC cells and lung metastasis bound to FUS and inhibition of this binding between and FUS could contribute to promoting migration in HCC cells. Materials and Methods Ethics statement The present study was approved by the research ethics committee of Zhongshan hospital, and the experiments were undertaken with the understanding and written consent of each subject. Patients and cell lines Frozen samples of HCC tissues and paired adjacent noncancerous liver tissues were randomly selected from patients undergoing hepatectomy at Zhongshan hospital (Shanghai, China) between 2004 and 2005. Ethical approval was obtained from the research ethics committee of Zhongshan hospital, and informed consent was obtained from each patient. All patients were followed up until October 2010. Overall survival (OS) was defined as the interval between the dates of surgery or the last follow\up. Cell culture The human HCC cell lines HepG2, Hep3B were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). HCC cell line SMMC7721 was purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Huh7 was purchased from RIKEN BRC cell Bank, Tsukuba, Japan. All the cell lines were maintained in Dulbecco’s Modified Eagle’s Medium BTZ038 (DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics (100?U/mL penicillin, 100?mg/mL streptomycin), in a 5% CO2 BTZ038 atmosphere at 37C. Quantitative real\time PCR Real\time PCR analyses were performed according to the manufacturer’s instructions (Takara Biotechnology, Dalian, China). The primers used are listed in Table?S1. The expression levels were normalized to \actin, and the relative expression levels were calculated using the 2?Ct method. Western blotting Total cell lysates were prepared in 6 SDS loading buffer. Proteins were separated by sodium dodecyl sulfate\polyacrylamide gel electrophoresis (SDS\PAGE) and transferred onto nitrocellulose membranes. Membranes were incubated with the specific primary antibodies and then with HRP\conjugated secondary antibodies. Protein bands were visualized using chemiluminescence detection. The following antibodies were used: FUS (1:500; Abcam, Cambridge, MA, USA), \actin (1:10?000; Sigma, St. Louis, MO, USA). Race 5\RACE and 3\RACE were performed using SMART RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. The gene\specific primers used for RACE analysis were presented in Table?S2. overexpression and RNA interference For overexpression, was cloned into pWPT vector. HepG2 and Hep3B cells were infected with virus containing the Rabbit polyclonal to Synaptotagmin.SYT2 May have a regulatory role in the membrane interactions during trafficking of synaptic vesicles at the active zone of the synapse. plasmid hybridization Cells were fixed in 4% formaldehyde then incubated in 5% acetic acid for 15?min followed by washes with phosphate\buffered saline (PBS). The fixed cells were further treated with pepsin (1% in 10?mM HCl) and subsequent dehydration through 70%, 90%, and 100% ethanol. The air\dried cells were subjected to incubation with 40?nM FISH probe in hybridization buffer (100?mg/mL dextran sulfate, 10% formamide in 2 saline sodium citrate [SSC]) at 80C for 2?min. The hybridization was performed at 55C for 2?h and the slide was washed with 2 SSC at 65C followed by dehydration through 70%, 90%, and 100% ethanol. The air\dried slide was mounted with Prolong Gold Antifade Reagent with DAPI for detection. RNA FISH probes were designed and synthesized by Shanjing Biological Science and Technology Co., Ltd (Shanghai, China). Probe sequences are listed in Table?S3. Transwell assays For the transwell migration assay, cells were trypsinized and resuspended in BTZ038 serum\free DMEM. 5??104?cells (300?L) were planted on the top chamber of each insert (BD Biosciences, Franklin Lakes, NJ, USA) with 8\m\diameter pores on its membrane. To conduct migration assay of HCC cells, 800?L DMEM supplemented with 10% FBS was injected into the lower chambers. After incubation at 37C, cells remaining in the top chamber of the inserts were carefully removed. After fixation and staining in a dye solution containing 0.1% crystal violet, cells adhering to the lower side of the inserts were counted and imaged through an IX71 inverted microscope (Olympus, Tokyo, Japan). Cell proliferation assays Cell proliferation was assayed by Cell Counting Kit\8 (Dojindo Laboratories, Kumamoto, Japan). HCC cells were plated in BTZ038 96\well plates (1C2??103 cells per well), and the numbers of cells per well were measured at the indicated time points. metastasis assays The tail vein metastasis.

Comments are closed.