MOESM1 of Long non-coding RNA MIAT promotes growth and metastasis of colorectal cancer cells through regulation of miR-132/Derlin-1 pathway

Additional file 1: Figure S1. Down-regulation of MIAT inhibited SW480 cell proliferation, migration and invasion. SW480 cells were transfected with si-control, si-MIAT-1 or si-MIAT-2 for different time, (A) MIAT expression and cell viability was measured. SW480 cells were transfected with si-control, si-MIAT-1 or si-MIAT-2 for 72 h, (B) cell apoptosis, (C) cell migration and cell invasion was determined. **P < 0.01, compared to si-control

MOESM2 of Long non-coding RNA MIAT promotes growth and metastasis of colorectal cancer cells through regulation of miR-132/Derlin-1 pathway

Additional file 2: Figure S2.. Down-regulation of MIAT inhibited SW480 cell proliferation, migration and invasion by miR-132/Derlin-1 axis. SW480 cells were transfected with si-MIAT-2, miR-132 inhibitor and Derlin-1 shRNA (shRNA-Derlin-1) for 72 h, (A) cell viability, cell apoptosis, (B) cell migration and cell invasion was determined. **P < 0.01, compared to si-control. ##P < 0.01, compared to si-MIAT-2 + NC. & P < 0.01, compared to si-MIAT-2 + miR-132 inhibitor + shRNA

The mRNA and protein expression of E-cadherin, p-p38, HSP70 and α-SMA in tumors and the correlation between tumor-free survival and expression of HSP70, α-SMA and p-p38.

<p>(A) The mRNA expressions of E-cadherin, α-SMA and HSP70 and p38 (normalized to GADPH expression). (B) Photomicrographs of well-differentiated hepatic cancer (top panel) and poorly differentiated hepatic cancer. p-p38, HSP70 and α-SMA were detected in the cytoplasm; E-cadherin was detected in the plasmalemma. Original magnification, 200´. (C) The protein expressions of α-SMA, HSP70, p38 and p-p38 (normalized to GAPDH expression). Kaplan-Meier tumor-free survival curves for hepatic cancer patients showing that the median tumor-free survival time of patients correlated with HSP70 (D), α-SMA (E) and p-p38 (F) expression.</p

Expression of E-cadherin (red) and α-SMA (green) in Huh-7 cells observed by immunofluorescence.

<p>α-SMA was detected in the cytoplasm; E-cadherin was found in the plasmalemma and the cytoplasm. HSP70/HSP70-PCs treatment reduced the expressions of E-cadherin and promoted the expression of α-SMA.</p

HSP70/HSP70-PCs affect the protein levels of E-cadherin , α-SMA (A), total-p38, phosphor-p38 (C) and mRNA (B) expression of E-cadherin and α-SMA.

<p>24h after induction, cells were harvested for western blotting and real-time RT-PCR. Data are presented as means ± SD from three independent experiments (normalized to GADPH expression).</p

Characterization of the transcription factor binding elements by mutagenesis and luciferase assay.

<p>HEK 293 cells were transfected with the <i>FTO</i> promoter (PGL3-100), three mutant variants (mutated at the <i>Foxa2</i> site, USF site or both of them), pHD vector, pHD-Foxa2, negative control siRNA (NTC siRNA) or Foxa2 siRNA, and the promoter-less PGL3-basic construct. Relative luciferase activity (RLU), expressed as the fold induction relative to PGL3-basic vector, was measured. Results are presented as mean RLU±SE of three independent experiments (*<i>P</i><0.05).</p

Functional analysis of the human <i>FTO</i> promoter.

<p>Different sizes of 5′deletion fragments of the human <i>FTO</i> promoter were cloned into pGL3-Basic luciferase reporter plasmids. The reporter construct was transfected into HEK 293 and Hela cell lines and the reporter activity was measured. Relative firefly luciferase activities were averages of three independent transfections normalized to renilla control activities. Data are presented a mean±S.D. (*<i>P</i><0.05).</p

ChIP assays to test the binding of Foxa2 to the promoter of <i>FTO in vivo</i>.

<p>Formaldehyde cross-linked chromatin prepared from HEK 293 cells was immunoprecipitated with a Foxa2 antibody. PCR was performed with special primers to amplify a 156 bp DNA fragment containing Foxa2 binding sites. Sonicated decrosslinked DNA was used as positive input control for PCR. ChIP analysis reveals that Foxa2 could bind the sequence about 100 bp away from the transcription start site of <i>FTO</i>.</p

The primer sequences for the preparation of promoter-reporter plasmids.

<p>Restriction endonuclease sequences are shown in bold.</p

Putative binding sites in the <i>FTO</i> gene promoter region.

<p>The underlined sequences are putative binding sites for transcription factors in the <i>FTO</i> promoter region, based upon database searches using the TFsearch and Alibaba programs. The transcription start site (TSS) is indicated in the figure.</p