Journal: Aging (Albany NY)

Article Title: Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression

doi: 10.18632/aging.103642

Figure Lengend Snippet: DLEU1 sponges miR-421 in PTC cells. ( A ) The diagram shows the predicted miR-421 binding sites in the 3’UTR of DLEU1 and the mutations in the miR-421 binding sites. ( B ) Dual luciferase reporter assay shows the relative luciferase activity of TPC-1 cells co-transfected with miR-421 mimic or miR-NC plus luciferase reporter plasmid with the wild-type DLEU1 (WT-DLEU1) or mutant DLEU1 (MUT-DLEU1). The miR-421 binding sites are mutated in the mutant DLEU1. ( C ) QRT-PCR results show DLEU1 levels in the RNA pull down extracts using biotinylated wild-type or mutant miR-421. ( D ) QRT-PCR analysis shows miR-421 levels in control and DLEU1-silenced TPC-1 cells. ( E ) QRT-PCR analysis shows DLEU1 levels in control and miR-421 mimic-transfected TPC-1 cells. ( F ) QRT-PCR analysis shows miR-421 expression in 54 paired PTC and adjacent normal thyroid tissues.( G ) QRT-PCR analysis shows the expression of miR-421 in four PTC cell lines (BHP5-16, 8505C, TPC-1, and SW1736) and the human thyroid follicular epithelial cell line, Nthy-ori3-1. ( H ) Spearman’s correlation analysis shows that DLEU1 expression is inversely related to miR-421 expression in PTC tissues (n=54). Note: The data are represented as the means ± SD of at least three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: The negative control mimics (miR-NC) and Hsa-miR-421 mimics (miR-421) were purchased from RiboBio (Guangzhou, China).

Techniques: Binding Assay, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Expressing

Journal: Aging (Albany NY)

Article Title: Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression

doi: 10.18632/aging.103642

Figure Lengend Snippet: ROCK1 is a direct target of miR-421 in PTC cells. ( A ) The predicted miR-421 binding sites in the 3’UTR of ROCK1 and the mutated sequence are shown. ( B ) Dual luciferase reporter assay shows the relative luciferase activity in TPC-1 cells co-transfected with miR-421 mimic or miR-NC and luciferase reporter plasmid with wild-type ROCK1-3’-UTR (WT-ROCK1) or mutant ROCK1-3’-UTR (MUT-ROCK1). ( C ) QRT-PCR analysis shows the ROCK1 mRNA levels in miR-NC- and miR-421 mimic-transfected TPC-1 cells. ( D ) Western blot analysis shows ROCK1 protein levels in miR-NC- and miR-421 mimic-transfected TPC-1 cells. ( E ) QRT-PCR analysis shows ROCK1 mRNA expression in 54 paired PTC and adjacent normal thyroid tissues. ( F ) Spearman correlation analysis shows that ROCK1 expression is inversely related to miR-421 expression in PTC tissues (n=54). Note: The data are represented as the means ± SD of at least three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: The negative control mimics (miR-NC) and Hsa-miR-421 mimics (miR-421) were purchased from RiboBio (Guangzhou, China).

Techniques: Binding Assay, Sequencing, Luciferase, Reporter Assay, Activity Assay, Transfection, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Western Blot, Expressing

Journal: Aging (Albany NY)

Article Title: Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression

doi: 10.18632/aging.103642

Figure Lengend Snippet: DLEU1 regulates PTC cell growth and progression through the miR-421/ROCK1 axis. ( A ) Western blot analysis shows ROCK1 protein levels in sh-NC-, sh-DLEU1- and sh-DLEU1 plus miR-421 inhibitor-transfected TPC-1 cells. ( B ) Spearman correlation analysis shows that ROCK1 mRNA expression is inversely related to DLEU1 expression in PTC tissues (n=54). ( C ) CCK-8 assay analysis shows proliferation rates of TPC-1 cells transfected with sh-NC, sh-DLEU1, sh-DLEU1 plus miR-421 inhibitor, and sh-DLEU1 plus ROCK1 overexpression plasmid. ( D ) Flow cytometry analysis shows percentage apoptosis (% Annexin-V + cells) in TPC-1 cells transfected with sh-NC, sh-DLEU1, sh-DLEU1 plus miR-421 inhibitor, and sh-DLEU1 plus ROCK1 overexpression plasmid. ( E ) Wound healing assay results show the numbers of migrating cells in the TPC-1 cells transfected with sh-NC, sh-DLEU1, sh-DLEU1 plus miR-421 inhibitor, and sh-DLEU1 plus ROCK1 overexpression plasmid. ( F ) Transwell invasion assay results show the numbers of invading cells in the TPC-1 cells transfected with sh-NC, sh-DLEU1, sh-DLEU1 plus miR-421 inhibitor, and sh-DLEU1 plus ROCK1 overexpression plasmid. Note: The data is represented as the means ± SD of at least three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: The negative control mimics (miR-NC) and Hsa-miR-421 mimics (miR-421) were purchased from RiboBio (Guangzhou, China).

Techniques: Western Blot, Transfection, Expressing, CCK-8 Assay, Over Expression, Plasmid Preparation, Flow Cytometry, Wound Healing Assay, Transwell Invasion Assay

Journal: Aging (Albany NY)

Article Title: Long non-coding RNA DLEUI promotes papillary thyroid carcinoma progression by sponging miR-421 and increasing ROCK1 expression

doi: 10.18632/aging.103642

Figure Lengend Snippet: DLEU1 knockdown reduces in vivo growth of xenograft tumors in nude mice model. ( A ) The curve shows the rate of growth of xenograft tumors generated from subcutaneously injected control and DLEU1 knockdown TPC-1 cells in the nude mice (n=5 each). Tumor growth was measured every 7 days for 28 days. ( B ) The representative images show the xenograft tumors in nude mice that are subcutaneously injected with control and DLEU1 knockdown TPC-1 cells for 28 days. ( C ) The histogram plot shows the weight of xenograft tumors derived from nude mice subcutaneously injected with control and DLEU1 knockdown TPC-1 cells. ( D ) Representative images show IHC staining with the anti-Ki67 antibody of xenograft tumor tissue sections derived from nude mice subcutaneously injected with control and DLEU1 knockdown TPC-1 cells. ( E – F ) QRT-PCR analysis shows the levels of DLEU1 and miR-421 in the xenograft tumor tissues derived from nude mice subcutaneously injected with control and DLEU1 knockdown TPC-1 cells. ( G ) QRT-PCR analysis shows ROCK1 mRNA levels in the xenograft tumor tissues derived from nude mice subcutaneously injected with control and DLEU1 knockdown TPC-1 cells. ( H ) Western blot analysis shows ROCK1 protein levels in the xenograft tumor tissues derived from nude mice subcutaneously injected with control and DLEU1 knockdown TPC-1 cells. Note: The data are represented as the means ± SD of at least three independent experiments. * P < 0.05 and ** P < 0.01.

Article Snippet: The negative control mimics (miR-NC) and Hsa-miR-421 mimics (miR-421) were purchased from RiboBio (Guangzhou, China).

Techniques: In Vivo, Generated, Injection, Derivative Assay, Immunohistochemistry, Quantitative RT-PCR, Western Blot