MYH9 binds to lncRNA gene PTCSC2 and regulates FOXE1 in the 9q22 thyroid cancer risk locus

Abstract

A locus on chromosome 9q22 harbors a SNP (rs965513) firmly associated with risk of papillary thyroid carcinoma (PTC). The locus also comprises the forkhead box E1 (FOXE1) gene, which is implicated in thyroid development, and a long noncoding RNA (lncRNA) gene, papillary thyroid cancer susceptibility candidate 2 (PTCSC2). How these might interact is not known. Here we report that PTCSC2 binds myosin-9 (MYH9). In a bidirectional promoter shared by FOXE1 and PTCSC2, MYH9 inhibits the promoter activity in both directions. This inhibition can be reversed by PTCSC2, which acts as a suppressor. RNA knockdown of FOXE1 in primary thyroid cells profoundly interferes with the p53 pathway. We propose that the interaction between the lncRNA, its binding protein MYH9, and the coding gene FOXE1 underlies the predisposition to PTC triggered by rs965513.

Keywords: MYH9; bidirectional promoter; lncRNA; thyroid cancer; transcriptional regulation.

Fig. S1.

A diagrammatic view of the 9q22 locus. The diagram shows the lead SNP rs965513 in GWAS, the two flanking coding genes (XPA and FOXE1), and the lncRNA PTCSC2 isoforms C and D. Red arrows indicate transcriptional orientations. Blue filled boxes represent exons.

Fig. 1.

Identification of MYH9 as a PTCSC2 binding protein. (A) RNA pull-down experiment with nontumorous thyroid tissue extract. Biotin pull-down assays followed by SDS/PAGE separation were used to isolate the binding protein of PTCSC2 isoform C. Antisense RNA of PTCSC2 isoform C was used as the negative control. The arrows indicate the binding protein bands ∼226 KD and 42 KD in size, respectively. (B) Information for the identification of MYH9 by MS. The full length of MYH9 protein is 1,960 amino acids, as shown in the lower diagram. Blue boxes indicate the peptides identified by MS analysis. (C) qRT-PCR detection of the indicated RNAs retrieved by MYH9 antibody (RIP assay) in the BCPAP cell line with stable PTCSC2 isoform C overexpression. IgG was used as a negative control. The relative fold change was normalized to the IgG control. **P < 0.01. Student’s t test.

Fig. S2.

Identification of MYH9 as a PTCSC2 isoform D binding protein. (A) RNA pull-down experiment with nontumorous thyroid tissue extract. Biotin pull-down assays followed by SDS/PAGE separation were used to isolate the protein binding PTCSC2 isoform D. Antisense RNA of PTCSC2 isoform D was used as the negative control. The arrows indicate the binding protein bands ∼226 KD and 42 KD in size, respectively. (B) Information for the identification of MYH9 by MS.

Fig. S3.

The detailed information of MS analysis for ACTB protein identified in the RNA pull-down assay. Information for the identification of beta-actin by MS.

Fig. 2.

Validation of the MYH9 binding site in the FOXE1 promoter region by ChIP assay. (A) Schematic view of a 900-bp transcription factor enrichment region in the FOXE1 promoter region. Genomic coordinate, chr9:100,615,431–100,616,330 (GRC37/hg19). DNase I hypersensitive sites, CpG islands, and ENCODE data were obtained from the UCSC Genome Browser. Red bars indicate the tested regions in ChIP assays. (B) ChIP assay using KTC1 cell line. The results represent four independent experiments, each in duplicates. Results are shown as means ± SD. *P < 0.05. Student’s t test. (C) ChIP assay using nontumorous thyroid tissue sample. The relative abundance was normalized to the input control.

Fig. 3.

Effect of MYH9 on FOXE1 promoter with bidirectional activity. (A) Schematic view of the vectors used in luciferase assays. Red boxes indicate the UTR regions of FOXE1; the yellow box indicates the FOXE1 coding sequences; blue boxes indicate the exons of PTCSC2; the thick gray line indicates the intron of PTCSC2; the thin black line indicates the intergenic genomic region. All of the position numbers are labeled relative to the FOXE1 TSS as +1. Arrows represent the fragment direction in the constructs according to the position in the genome. Dual reporter luciferase assays in BCPAP cells cotransfected with constructs containing a short promoter fragment (B) or long promoter fragment (C) having either forward or inverted orientation, with or without MYH9 overexpression vector. Values are presented as fold of the no promoter control treated with empty expression vector. Results are shown as means ± SD of four independent experiments, each in four replicates. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t test.

Fig. 4.

Effect of PTCSC2 on FOXE1 bidirectional promoter. Dual reporter luciferase assay using the long promoter constructs with forward (A) or inverted (B) orientations cotransfected with PTCSC2, MYH9, or empty expression vectors. The values at the bottom of each column represent the portion of the total expression vectors. Results are shown as means ± SD of four independent experiments, each in four replicates. *P < 0.05. Student’s t test. (C) qPCR detection of endogenous FOXE1 in the BCPAP cell line and (D) the TPC1 cell line transiently transfected with PTCSC2, MYH9, or empty expression vectors as indicated (n = 3). All values were normalized with the values of the corresponding empty vector-transfected groups. Results are shown as means ± SD. *P < 0.05; NS, not significant. Student’s t test.

Fig. S4.

Expression of MYH9 and FOXE1 in thyroid cancer cell lines and nontumorous thyroid tissue. The protein levels of MYH9 and FOXE1 were detected by Western blotting using ACTB as the loading control. Expression of PTCSC2 spliced isoforms and GAPDH as detected by RT-PCR was described previously in ref. .

Fig. S5.

Transcriptional activity of FOXE1 promoter with different alleles of rs1867277. Dual reporter luciferase assay using long promoter constructs with either G or A allele of rs1867277 and forward (Left) or inverted (Right) orientations cotransfected with MYH9, PTCSC2, or empty expression vectors. All values were normalized with the values of the corresponding groups using promoter plasmids containing the G allele. Results were shown as means ± SD of four independent experiments, each in four replicates. *P < 0.05; ***P < 0.001. Student’s t test.

Fig. 5.

Gene expression profile of FOXE1 knockdown in thyroid primary cells indicating its involvement in the p53 pathway. (A) Gene expression differences of the top 25 genes caused by FOXE1 knockdown in primary thyroid cells on day 5 of culture. The expression is plotted with heat-map color scale using relative expression fold change (FOXE1 knockdown culture vs. control culture) (fold change > 1.5, P < 0.001). Exp. 1, Exp. 2, and Exp. 3 represent three independent primary culture experiments derived from different patient samples. (B) Confirmation by qRT-PCR of two up-regulated genes, IGFBP3 (Left) and THBS1 (Right), in FOXE1 knockdown primary cells. si-Control represents the primary cell sample treated with scrambled siRNA control. Results are shown as means ± SD of three independent experiments, each in three replicates. All values were normalized with the values of the corresponding negative control siRNA-treated groups. *P < 0.05; ***P < 0.001. Student’s t test. (C) Part of the p53 signaling pathway including typical gene members and their functions from Kyoto Encyclopedia of Genes and Genomes (KEGG). THBS1 is labeled in a purple ellipse, and IGFBP3 is labeled in two red ellipses. (D) Cell viability changes of BCPAP cell line at three time points (24, 48, and 72 h) with FOXE1 knockdown. For each time point, experiments were performed in four replicates of three biological replicates. All values were normalized with the values of the corresponding negative control siRNA-treated groups. (E) Example plots of cell apoptosis assays in BCPAP cell line treated with FOXE1 siRNA or negative control siRNA. Cells stained with only annexin V were evaluated as being in early apoptosis stage (lower right quadrant); cells stained with both annexin V and PI (propidium iodide) were evaluated as being in late apoptosis stage (upper right quadrant).

Fig. S6.

The top 10 key biological functional groups predicted by dysregulated genes in FOXE1 knockdown thyroid primary cells by IPA analysis.

Fig. S7.

qRT-PCR of FOXE1, THBS1, and IGFBP3 in FOXE1 knockdown cell lines. Expression levels of BCPAP cell line (Left) or TPC1 cell line (Right) were detected after treatment with FOXE1 siRNA or negative control siRNA for 24 h. Results are shown as means ± SD of three independent experiments, each in three replicates. All values were normalized with the values of the corresponding negative control siRNA-treated groups. *P < 0.05; **P < 0.01; ***P < 0.001. Student’s t test.