Additional file 3: Figure S1. of Down-regulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson’s disease-like motor dysfunction

Detection of a selection marker cassette knock-in by PCR. Genotyping by PCR was performed by using the primers listed in Table S3. For the detection of alleles of wildtype or indels, 5’PARK2-PCR-Fw and 3’PARK2-PCR-Rv, PARK2-Exon2-PCR-Fw and PARK2-Exon2-PCR-Rv were used. A large 5082-bp and 3464-bp fragment derived from knock-in allele is also detectable in this primer set. For the detection of alleles of knock-in, 5’PARK2-PCR-Fw and PGKp-Rv for detection of the 5′ knock-in fragment, and PuroR-Fw and 3’PARK2-PCR-Rv for detection of the 3’knock-in fragment were used. (JPEG 429 kb

Additional file 2: Table S2. of Down-regulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson’s disease-like motor dysfunction

List of primers used for qRT-PCR analysis. (JPEG 308 kb

Additional file 1: Table S1. of Down-regulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson’s disease-like motor dysfunction

List of antibodies used for immunocytochemical analysis. (JPEG 244 kb

Additional file 4: Table S3. of Down-regulation of ghrelin receptors on dopaminergic neurons in the substantia nigra contributes to Parkinson’s disease-like motor dysfunction

List of primers used for PCR analysis. (JPEG 291 kb

Chronic treatment of non-small-cell lung cancer cells with gefitinib leads to an epigenetic loss of epithelial properties associated with reductions in microRNA-155 and -200c

<div><p>Background</p><p>The EGFR tyrosine kinase inhibitor gefitinib is used in therapy for non-small-cell lung cancer (NSCLC). However, its application is limited by resistance-accelerated disease progression, which is accompanied by the epithelial-to-mesenchymal transition (EMT). In the present study, we performed multiple expression analyses of microRNAs (miRNAs) and quantified the expression of several related EMT players in gefitinib-resistant NSCLC cells.</p><p>Methods and results</p><p>To establish gefitinib-resistant NSCLC cells, gefitinib-sensitive HCC827 cells, which exhibit an in-frame deletion [E746-A750] in EGFR exon 19, were exposed to gefitinib for at least 1.5 months. Next, to profile “gefitinib-resistant HCC827 (HCC827GR)” cells, which have a secondary T790M mutation in EGFR exon 20, a miRNA array analysis was performed in HCC827 and HCC827GR cells. The greatest differences were seen in the levels of miR-155 and miR-200c, which essentially disappeared in HCC827GR cells. In addition to these reductions, the levels of smad2 and zeb1, which are both key players in EMT and targets for miR-155 and miR-200c, respectively, were dramatically increased in HCC827GR cells. In HCC827GR cells, the expression of epithelial-cadherin (E-cadherin) was greatly reduced with repressive histone modifications, whereas vimentin, which is expressed in mesenchymal cells, was dramatically increased with active histone modifications. In another gefitinib-resistant NSCLC cell line (H1975 cells), similar to the findings in HCC827GR cells, both miR-155 and miR-200c were absent, and the EMT was induced along with epigenetic modifications. Interestingly, the inhibition of both miR-155 and miR-200c in HCC827 cells without gefitinib induced significant increases in smad2 and zeb1 along with a dramatic decrease in E-cadherin and a slight increase in vimentin. Furthermore, although the inhibition of these miRNAs in HCC827 cells decreased gefitinib sensitivity, this dual-inhibition in HCC827 cells without gefitinib did not produce a secondary T790M mutation in EGFR exon 20.</p><p>Conclusion and implications</p><p>These results suggest that chronic treatment of NSCLC cells with gefitinib changes the expression of miRNAs, including dramatic reductions in miR-155 and miR-200c along with an EGFR mutation. Furthermore, this depletion of miR-155 and miR-200c may be associated with the EMT along with histone modifications, and may contribute to the decrease in the sensitivity to gefitinib independent of a secondary EGFR mutation.</p></div

Effects of miR-155 and miR-200c inhibitors on HCC827 cells.

<p>(A, B) The HCC827 cells were co-transfected with a specific miR-155 inhibitor and a specific miR-200c inhibitor, or the negative control. Transfection of each inhibitor almost completely abolished the expression of miR-155 (A) and miR-200c (B), respectively (**p<0.01, ***p<0.001 vs. HCC827 or ###p<0.001 vs. HCC827 transfected with a negative control (HCC827-NC)). (C-F) In HCC827 cells transfected with the inhibitors (HCC827-miR155/200c IH), mRNA levels of smad2 (C), zeb1 (D), E-cadherin (E) and vimentin (F) were quantified by qRT-PCR in comparison to the results in the negative control (###p<0.001 vs. HCC827-NC). Each column represents the mean with S.E.M of 6 samples. (G-J) In HCC827 cells transfected with the inhibitors (HCC827-miR155/200c IH), the protein levels of smad2 (G), zeb1 (H), E-cadherin (I) and vimentin (J) were quantified by western blot in comparison to the results in the negative control (#p<0.01, ###p<0.001, vs. HCC827-NC).</p

Changes in predicted targeting gene of miR-200c in HCC827GR cells.

<p>(A) miR-200c targeting predictions were quantified in three target prediction databases. (B) Result of the analysis of 433 predicted targets for miR-200c. (C) Assessment of luciferase reporter activity in HEK293 cells co-transfected with pre-miR-200c mimics and reporter vector of zeb1 3’UTR or mutation zeb1 3’UTR. The data represent the relative expression level of renilla luciferase expression standardized to firefly luciferase (***p<0.001 vs. psiCHECK2 zeb1 3’UTR miR-NC). (D) The mRNA expression level of zeb1 was quantified by qRT-PCR in HCC827GR cells compared to HCC827 cells (***p<0.001 vs. HCC827 cells). (E) Change in protein levels of zeb1 in HCC827GR cells compared to HCC827 cells. Results are shown as the ratio of the density of zeb1 to that of fibrillarin. Each column represents the mean ± S.E.M. of 3 independent experiments (***p<0.001 vs. HCC827 cells).</p

Changes in histone modifications at the E-cadherin or vimentin promoter in HCC827GR cells.

<p>(A-D) ChIP-qPCR analysis of (A) AcH3, (B) H3K4me3, (C) H3K9me3 and (D) H3K27me3 at E-cadherin loci was performed in HCC827GR cells compared to that in HCC827 cells. (E-H) ChIP-qPCR analysis of (E) AcH3, (F) H3K4me3, (G) H3K9me3 and (H) H3K27me3 at vimentin loci was performed in HCC827GR cells compared to that in HCC827 cells. The value for ChIP/Input was normalized by that for the internal standard in each control. Each column represents the mean with S.E.M of 3 samples (**p < 0.01, ***p < 0.001 vs. HCC827 cells).</p

Molecular profiling of another geftinib-resistant H1975 cells compared to HCC827GR cells.

<p>(A,B) qRT-PCR for miR-155, miR-200c and RNU44, an internal standard, in HCC827 cells and H1975 cells (***p<0.001 vs. HCC827 cells). (C,F) The mRNA expression levels of E-cadherin (C) and vimentin (F) were quantified by qRT-PCR in H1975 cells compared to HCC827 cells (***p<0.001 vs. HCC827). Each column represents the mean with S.E.M of 6 samples. (D,E) ChIP-qPCR analysis of AcH3 (D) and H3K27me3 (E) at E-cadherin loci was performed in H1975 cells and the results were compared to those in HCC827 cells. (G,H) ChIP-qPCR analysis of AcH3 (G) and H3K27me3 (H) at vimentin loci was performed in H1975 cells and the results were compared to those in HCC827 cells. The value for ChIP/Input was normalized by that for the internal standard in each control. Each column represents the mean with S.E.M of 3 samples (**p < 0.01, ***p < 0.001 vs. HCC827 cells).</p

miRNA expression analysis using microarrays.

<p>(A) Pearson’s correlation scatter plot of miRNA levels between HCC827 and HCC827GR cells. (B) List of down-regulated miRNAs in HCC827GR cells compared to HCC827 cells (fold change of p<0.05). (C,D) qRT-PCR for miR-155, miR-200c and RNU44, an internal standard, in HCC827 cells and HCC827GR cells (***p<0.001 vs. HCC827 cells).</p