contributed to drafting of the manuscript. malignancy. It has also been reported that LINC00312 can inhibit the invasion and metastasis of bladder malignancy cell by down-regulating [14]. LncRNAs and miRNAs have been found to be significantly associated with TC. For example, lncRNA H19 regulates YES1 expression by binding and polymorphism predisposing patients to TC [15,16]. However, the effects of LINC00312 and have not been proven on TC. Therefore, this research was conducted to investigate the involvement of LINC00312 and in TC and demonstrate their effect on the proliferation, invasion, and migration ability of TC cells. Materials and methods Ethical statement The study was approved by the ethical committee of the First Affiliated Hospital of Nanchang University or college. All research tissues were obtained from patients who experienced signed informed consent forms. Study subjects The study included 211 TC tissues and 70 adjacent normal tissues (2 cm away from the tumor site) obtained from 211 TC patients (99 females and 112 females) who were diagnosed with TC. All patients received primary surgical resection at the First Affiliated Hospital of Nanchang University or college between October 2013 and August 2015. All the samples were confirmed via pathological examination, all patients had not received any previous treatment and experienced no severe systemic diseases such as malignant tumors or severe systemic infections. The average age of patients was 46.43 14.27 years (ranging from 20 to 75 years). According to the tumor node metastasis (TNM) staging requirements [17] published by the Union for International Malignancy Control (UICC), there were 190 patients in phase I/II and 21 patients in phase III/IV [17]. Rabbit polyclonal to ACTN4 Sixty-nine patients experienced lymph node metastasis and 142 patients did not show lymph node metastasis. Seventy-two patients had tumor diameter 1.0 cm and 139 patients had tumor diameter 1.0 cm. One hundred and eight patients experienced papillary TC, 54 patients experienced follicular TC, 36 patients experienced squamous TC, and 13 Oncrasin 1 patients experienced anaplastic TC. The samples were preserved at C70C for further use. Cell culture K1 (papillary TC), SW579 (squamous TC), and 8505C (anaplastic TC) cell lines (Chinese Academy of Sciences, Shanghai, China) were used in our study. Cells were cultured in Dulbeccos altered Eagles medium (DMEM) (Promega, Madison, WI, U.S.A.) containing 15% FBS (HyClone, Logan, Utah, U.S.A.) and 1% streptomycin at 37C with 95% relative humidity and 5% CO2. Cells with 80% adherence were utilized for subculturing. Cells were then rinsed twice Oncrasin 1 with PBS and digested with trypsin (Gibco Organization, Grand Island, NY, U.S.A.). The trypsin was removed when the intercellular space was enlarged. Cells were routinely passaged without suspension cells in the above-mentioned culture medium. Luciferase reporter gene assay The potential target gene and fragment sequences made up of reaction sites were analyzed using microRNA.org. The DNA was extracted in rigid accordance with the instructions of the TIANamp Genomic DNA Kit (Tiangen Biotech, Beijing, China). The p120 3-UTR wild-type (WT) sequence named p120-3-UTR-WT was 5-CACTTTTATTTTTTGGTGGTGAAT-3 and the mutant sequence of p120 3-UTR missing the binding site with named p120-3-UTR-Mut was 5-CACTTTTATTTTTTGACAAGTCCT-3. The luciferase reporter gene vector was constructed and TC cells were transfected. Luciferase reporter gene assay kits (Promega, Madison, WI, U.S.A.) were used to detect the luciferase activity of samples. At 48 h after transfection, the culture medium was removed and the samples were washed twice with 0.1 M PBS (8 g NaCl, 0.2 g KCl, 3.58 g Na2HPO4.12H2O, and 0.24 g KH2PO4 mixed and dissolved with double distilled water to 100 ml, pH 7.4). Passive lysis buffer (100 l) was added into each well. Samples were slightly oscillated at room heat for 15 min and then the cell lysis buffer was collected. Two seconds of prereading Oncrasin 1 was conducted before 10 s of reading. The sample volume of Luciferase Assay Reagent II (LARII) and Stop & Glo? Reagent was 100 l. The luminotron or luminous plate (20 l per sample) which had been added with LARII and Stop & Glo? Reagent was placed into the biological luminous detector (type Modulus?, Turner BioSystems, Inc., Sunnyvale, CA, U.S.A.). Cell transfection and grouping The primer sequences of the unfavorable control (NC) plasmid, inhibitors plasmid, and LINC00312 overexpression + mimics plasmid were constructed by Sangon Biotech, Shanghai, China (Table.