(H, I) The mice were sacrificed, and subcutaneous tumors were excised and weighed at the 28th day after inoculation

(H, I) The mice were sacrificed, and subcutaneous tumors were excised and weighed at the 28th day after inoculation. RNA was detected by qRT/PCR described above. 2.6. Vectors construction The cDNA encoding LINC00662 was PCR\amplified with Thermo Scientific Phusion Flash High\Fidelity PCR Master Mix (Thermo Fisher, Waltham, MA, USA) and subcloned into the Kpn I and EcoR I sites of pcDNA?3.1(+) vector (Invitrogen), termed as pcDNA3.1\LINC00662. The primer sequences are presented in Table S1. pcDNA3.1\LINC00662 with mutations in miR\15a/16/107 binding sites was produced by GenScript (Nanjing, China), termed as pcDNA3.1\LINC\mut. pcDNA3.1\LINC00662 and pcDNA3.1\LINC\mut were double\digested using Kpn I and EcoR I, and the lncRNA coding sequences were inserted into pSPT19 (Roche), termed as pSPT19\LINC00662 and pSPT19\LINC\mut, respectively. In addition, pcDNA3.1\LINC00662 and pcDNA3.1\LINC\mut were double\digested using Nhe I and Not I, and the lncRNA coding sequences were inserted into pSL\MS2\12X (Addgene, Watertown, MA, USA), termed as pSL\MS2\LINC00662 and pSL\MS2\LINC\mut, respectively. The oligonucleotides for shRNAs targeting LINC00662 were produced and inserted into the shRNA expression vector pGPU6/GFP/Neo (GenePharma, Shanghai, China), termed as shRNA\LINC\1 and shRNA\LINC\2. The shRNA sequences are presented in Table S1. The LINC00662 sequences containing miR\15a/16/107 binding sites were PCR\amplified with Thermo Scientific Phusion Flash High\Fidelity PCR Master Mix (Thermo Fisher) and subcloned into the Sac I and Xho I sites of pmirGLO vector (Promega, Madison, WI, USA). pcDNA3.1\LINC00662 and pcDNA3.1\LINC\mut were used as (S)-3,4-Dihydroxybutyric acid template, and therefore, the constructed vectors were named as pmirGLO\LINC00662 and pmirGLO\LINC00662\mut, respectively. 3′ untranslated region (UTR) of WNT3A containing miR\15a/16/107 binding sites was PCR\amplified with Thermo Scientific Phusion Flash High\Fidelity PCR Master Mix (Thermo Fisher) and subcloned into the Sac I and Xho I sites of pmirGLO vector (Promega), termed as pmirGLO\WNT3A. 2.7. Stable cell line construction To obtain LINC00662 stably overexpressed HCC cells, pcDNA3.1, pcDNA3.1\LINC00662, and pcDNA3.1\LINC\mut were transfected into HCCLM3 and MHCC97H cells using Lipofectamine 3000 (Invitrogen) following the provided protocol. To obtain LINC00662 stably silenced HCC cells, shRNA\NC, shRNA\LINC\1, and shRNA\LINC\2 were transfected into Huh7 (S)-3,4-Dihydroxybutyric acid and SK\HEP\1 cells using Lipofectamine 3000 (Invitrogen). Forty\eight hours after transfection, the cells were selected with neomycin (800?gmL?1) for 4?weeks. The overexpression and silencing efficiencies of LINC00662 were detected by qRT/PCR. 2.8. Luciferase reporter assay pmirGLO, LAMA1 antibody pmirGLO\LINC00662, or pmirGLO\LINC00662\mut were cotransfected (S)-3,4-Dihydroxybutyric acid with (S)-3,4-Dihydroxybutyric acid miR\15a mimics, miR\16 mimics, miR\107 mimics, miR\NC (negative control of miRNA mimics), miR\15a inhibitors, miR\16 inhibitors, miR\107 inhibitors, or inh\NC (negative control of miRNA inhibitors) into HCCLM3 cells using Lipofectamine 3000. pmirGLO\WNT3A was cotransfected with pcDNA3.1, pcDNA3.1\LINC00662, or pcDNA3.1\LINC\mut into HCCLM3 cells using Lipofectamine 3000. pmirGLO\WNT3A was cotransfected with shRNA\NC, shRNA\LINC\1, or shRNA\LINC\2 into SK\HEP\1 cells using Lipofectamine 3000. Forty\eight hours later, the luciferase activities were detected by the Dual\Luciferase Reporter Assay System (Promega) following the provided protocol. 2.9. RNA pull\down Wild\type and miR\15a/16/107 binding sites mutated LINC00662 were < 0.05. (S)-3,4-Dihydroxybutyric acid 2.19. Statistical analysis graphpad prism 6.0 software was used to conduct statistical analyses. One\way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test, KruskalCWallis test followed by Dunn's multiple comparisons test, Wilcoxon matched\pairs signed rank test, log\rank test, Pearson correlation analysis, MannCWhitney test, and Pearsons chi\square test were conducted as indicated in the figure and table legends. in a miR\15a/16/107\dependent manner. Open in a separate window Figure 4 LINC00662 promotes hepatic tumor growth via activating Wnt/\catenin signaling. (A) Wild\type or mutated LINC00662 stably overexpressed and control HCCLM3 cells were subcutaneously inoculated into nude mice. Tumor volumes were measured every 7?days. (B, C) The mice were sacrificed, and subcutaneous tumors were excised and weighed at the 28th day after inoculation. (D) Ki67 IHC staining of tumors derived from (C). (E) Cleaved caspase\3 IHC staining of tumors derived from (C). (F) The expression of LINC00662, WNT3A, cyclin D1, and c\Myc in the tumors derived from (C) was measured by qRT/PCR. (G) LINC00662 stably silenced and control SK\HEP\1 cells were subcutaneously inoculated into nude mice. Tumor volumes were measured every 7?days. (H, I) The mice were sacrificed, and subcutaneous tumors were excised and weighed at the 28th day after inoculation. (J) Ki67 IHC staining of tumors derived from (I). (K) Cleaved caspase\3 IHC staining of tumors derived from (I). (L) The expression of LINC00662, WNT3A, cyclin D1, and c\Myc in.