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1 Supplementary Figures and Tables Figure S1. Validation Supplementary Figures and Tables Figure S1. Validation of miR-4423 expression in the human airway via qRT-PCR. The mature 3p and 5p forms of miR-4423 are detected in human bronchial epithelium (n=9). The average Ct values were 26.6 for the 3p form and 30.1 for the 5p form. Expression of RNU44 was measured as a positive control (average Ct value= 24.3). 1 Figure S2. MiR-4423 overexpression. A) Stable and transient overexpression of a plasmid containing the miR-4423 precursor plus 200 bps of flanking region, results in the production of processed 3p and 5p forms. An empty vector was used as a control. Figure S3. Validation of miR-4423 biogenesis. A) Transfection with a siRNA targeting Dicer decreases Dicer mRNA levels in H1299 cells stably overexpressing miR-4423 (P=0.0001)(n=3). B) Levels of processed miR-4423-5p, miR-4423-3p, miR-10b, miR-21 and miR-26 are significantly decreased in Dicer knocked-down cells compared to control (P= 0.0014, P= 0.0013, P=0.04, P=0.01; P=0.019, respectively). Error bars indicate standard error and P values were determined using Student’s t test. 2 Figure S4. RNA-Seq profile of the miR-4423 locus using reads that associated with the AGO complex. Using a small RNA sequencing dataset from Hafner et al (1), one read that associated with TNRC6C and two reads that associated with AGO3 aligned uniquely to the 5p form while one read that associated with TNRC6A aligned uniquely to the 3p form. Grey background indicates the genomic location of the 5p and 3p forms of miR-4423. Red text indicates a mismatch at that position in the read compared to the genome. Figure S5. In situ hybridization of U6 and scramble controls. In situ hybridization in tissue sections from the mainstem bronchus shows strong expression of the positive control U6 and no expression of the negative scramble control. 3 Figure S6. The expression of miR-4423 is highly correlated with that of pri-miR- 4423 and WDR63. A) Expression of miR-4423 (3p and 5p), pri-miR-4423 and WDR63 was measured in the human tissue panel (n=24). The expression of pri-miR-4423 and WDR63 is strongly correlated across these tissues (R=0.97). In addition, the expression of miR-4423-3p and 5p is positively correlated to that of WDR63 (R= 0.71 and R=0.71, respectively). The red diamonds correspond to tissues in which pri-miR-4423, WDR63 and both mature forms of miR-4423 are highly expressed (i.e. bronchial epithelium, lung, trachea, nasal epithelium, fallopian tube epithelium and ovary). The black diamonds correspond to tissues in which pri-miR-4423 and WDR63 are highly expressed but the mature forms of miR-4423 are not highly expressed (i.e. testis, placenta, kidney and brain). The blue diamonds correspond to the remaining 14 tissues that do not express any of the transcripts. B) Expression of miR-4423 (3p and 5p), pri-miR-4423 and WDR63 4 was measured at different time points of the differentiation of the airway epithelium at an ALI (days 0,2,4,6,9,11 and 13). Expression of all three transcripts was highly correlated during this process (WDR63 vs. pri-miR-4423; R=0.97, WDR63 vs. miR-4423-3p; R=0.99, WDR63 vs. miR-4423-50; R=0.98). Figure S7. MiR-4423 expression is highly correlated to FOXJ1. A) NHBE cells isolated from three different lung donors were differentiated at an ALI for 13 days. Expression of the 3p and 5p forms of miR-4423 are first detected at day 6 and expression increases through day 13. Expression of the ciliated cell markers, FOXJ1 and WDR63 is highly correlated with both miR-4423-3p (R=0.92, R=0.94) and miR-4423-5p (R=0.9, R=0.89). B) The expression of miR-4423 (3p and 5p) and that of the airway epithelial cell type markers, FOXJ1, MUC5AC, MUC5B, CC10 and ASCL1 were measured during differentiation of airway epithelium at an ALI. Pairwise spearman correlations between all marker genes, WDR63 and miR-4423 were clustered showing that the pattern of miR- 4423 expression during differentiation is more similar to the expression pattern of the 5 ciliated markers FOXJ1 and WDR63 than the other cell type markers. Red indicates a more positive correlation coefficient. Figure S8. MiR-4423 overexpression results in an increase in the number of FOXJ1- expressing cells. Expression of FOXJ1 was assayed via qRT-PCR in NHBE cells overexpressing miR-4423 or control at different time points of differentiation into mucociliary epithelium at an ALI (Days 1,5,7,9,11,13, 15 and 17). Using a linear model that had FOXJ1 expression as the response and time point and treatment as predictors we found that the expression of FOXJ1 is significantly increased in cells overexpressing miR-4423 compared to controls (p=0.025). Expression of FOXJ1 was normalized using both eGFP and GAPDH. 6 Figure S9. MiR-4423 knockdown results in a modest decrease in the number of cells expressing ciliated cell markers. A) NHBE cells were transduced with either miRZip™ anti-microRNA expression lentivectors to inhibit both mature miR-4423-3p and -5p or a scramble control and were differentiated into mucociliary epithelium at an ALI. miR- 7 4423 knocked-down cells show a modest reduction in FOXJ1 and β-tubulin staining (top) compared to control (bottom). Representative images shown were taken at days 13 (FOXJ1) and 16 (β-tubulin). Arrows are pointing to regions of positive staining. B) Expression of FOXJ1 was assayed via qRT-PCR in miR-4423 knocked-down NHBE or control at different time points of differentiation into mucociliary epithelium at an ALI (Days 3,7,13 and 17). FOXJ1 showed a modest trend to downregulation. 8 Figure S10. MiR-4423 expression is associated with lung cancer. A) The expression of miR-4423-3p was measured in matched tumor and adjacent normal tissue derived from SCC (SCC) (n=15), ADC (ADC) from non-smokers (n=10) and ADC (ADC) from current and former smokers (n=10). The log2 ratio of tumor/adjacent normal tissue was calculated for each matched sample. mir-4423-3p expression decreases by at least 2-fold 9 in 80% of SCC, 70% of ADC from current and former smokers and 70% of ADC from non-smokers. B) Expression of WDR63 (n=10), pri-miR-4423 (n=10), miR-4423-3p (n=10) and miR-4423-5p (n=8) was measured via qRT-PCR in lung tumors and matched adjacent normal tissues. There is a strong positive correlation between WDR63 and pri- miR-4423 (R=0.86), between pri-miR-4423 and miR-4423-3p (R=0.86) and between WDR63 and miR-4423-3p (R=0.78). Similar results were obtained when calculating the correlations between WDR63 and miR-4423-5p (R=0.74) and between pri-miR-4423 and miR-4423-5p (R=0.80). The blue diamonds correspond to the adjacent normal tissues and the red diamonds to the tumors. C) We examined WDR63 expression in a larger set of tumor and matched adjacent normal tissue pairs using RNA-seq data generated by the TCGA(2). Similar to the previous findings, WDR63 was downregulated in 81.4% of SCC (n=43), 71% of ADC from current and former smokers (n=7) and 73% of ADC from non-smokers (n=42). 10 Figure S11. MiR-4423 expression decreases in squamous metaplasia. The expression of miR-4423-3p and -5p was measured in laser captured normal airway epithelium (n=2), squamous metaplasia (n=1) and SCC (n=2) from resected lung tissue of a single individual with lung cancer. MiR-4423 expression decreases in squamous metaplasia and even further in SCC compared to the normal airway epithelium. 11 Figure S12. Copy number variation (CNV) for the region of miR-4423 and WDR63 in lung tumors. A) In the TCGA(2), CNVs were measured in ADC and adjacent normal pairs (n=129) and in a larger number of ADC with corresponding RNA-seq (n=196). B) CNVs were also measured in SCC and adjacent normal tissue (n=133) and in a larger number of SCC with corresponding RNA-seq (n=172). CNVs were measured using the Affymetrix Genome-Wide Human SNP Array 6.0. A positive segmentation mean indicates amplification while a negative segmentation means indicates deletion. Expression was measured by Fragments per Kilobase per Million (FPKM) using the 12 Illumina HiSeq 2000. We did not observe a significant CNV in the region overlapping with miR-4423 and WDR63 compared to adjacent normal or a significant correlation between a CNV in that region and WDR63 expression in both ADC and SCC tumors. These results suggest that a CNV in the region of miR-4423 is not responsible for the loss of miR-4423 expression in lung tumors. 13 Figure S13. Methylation for the region of miR-4423 and WDR63 in lung ADC. In the TCGA (2), matched methylation and gene expression was measured in lung ADC (n=325). Methylation was measured using the Infinium HumanMethylation450 BeadChip Kit. Ten probes on this platform overlapped with the region of miR-4423 and WDR63. Expression was measured by Fragments per Kilobase per Million (FPKM) using the 14 Illumina HiSeq 2000. The x-axis indicates the level of methylation at that CpG dinucleotide (Beta value) with zero corresponding to no methylation and one corresponding to complete methylation. The y-axis shows log2 transformed FPKM values for WDR63 after adding a pseudocount of one.
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