Fig. 2

MeCP2 contributes to SNORD33 mediated cisplatin resistance in mTNBC. aRPL13A, DICER enzyme, or SNORD32a were knocked down in MDA-MB-231 cells. bRPL13A, DICER enzyme, or SNORD32a knockdown do not change the cell viability in response to platinum. These results indicated that platinum resistance arising from loss of SNORD33 may be not caused by alterations of host gene, forming functional miRNA or rRNA modification. c The silver-stained bands showed the bands of the sense and antisense strands. The different bands were concentrated in the range of 50–80 kDa and 30–40 kDa. Mass spectrometry show that MeCP2 is a candidate protein for SNORD33 interaction. d MeCP2 immunoprecipitates were enriched by SNORD33. RNA pull-down (upper panel) and RIP (RNA immunoprecipitation) assays (lower panel) were performed in MDA-MB-231 cells. Anti-IgG was used as a negative control. n = 3; *** represents P < 0.001; two-tailed t test. e SNORD33 knockdown in MDA-MB-231 cells doesn’t alter MeCP2 mRNA and protein levels. f mRNA levels of MeCP2 target genes are decreased in MDA-MB-231 cells knocking down of SNORD33. The mRNA levels of GADD45α, MYOD1, FOXF1, CDKL5, CLDN6 in MDA-MB-231 cells were determined by qRT-PCR. n = 3; * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001; two-tailed t test. g SNORD33 knockdown promotes MeCP2 binding to downstream target gene promoter. SNORD33 was knocked down in MDA-MB-231 cells and chromatin immunoprecipitation (ChIP) assay was performed. qRT-PCR quantification of the immunoprecipitated DNA were measured. Normal rabbit IgG were used as a negative control. Values represented enrichment relative to input DNA. Data are presented as mean ± SD; ** represents P < 0.01, *** represents P<0.001; one-way ANOVA. h, i SNORD33 knockdown does not change the binding of co-repressor mSIN3A and HDAC1 to MeCP2. SNORD33 was knocked down in MDA-MB-231 cells and co-immunoprecipitation was performed by MeCP2 antibody. MeCP2, mSIN3A and HDAC1 proteins were detected with western blot (h). The binding affinity between MeCP2/mSIN3A and MeCP2/HDAC1 was quantified (i). n = 3; n.s. represents not significant; two-tailed t test. j Down-regulation of MeCP2 rescues SNORD33 knockdown induced cell death. The proliferation of cisplatin treated MDA-MB-231, MDA-MB-468, SUM149PT cells was determined by CCK8 assay. n = 3; *** represents P < 0.001; two-tailed t test. k, l Down-regulation of MeCP2 rescues SNORD33 knockdown decreased cell apoptosis (k) and induced alteration of apoptotic markers (l). Apoptosis of cisplatin treated cells was determined by flow cytometric analysis. Western blot was used to detect the indicated apoptotic markers in cisplatin treated cells. 10 μM cisplatin for MDA-MB-231, 8 μM cisplatin for MDA-MB-468 and 3 μM cisplatin for SUM149PT cells