Fig 1.
Long noncoding RNAs, including NATs show differential expression during breast cancer progression.
A) Total number of genes analyzed in M1 and M4 cells. B) Number of deregulated ncRNA and protein-coding RNA genes in M4 cells compared to M1 cells. C) Number of deregulated lncRNA genes in M4 compared to M1 cells. D) Different classes of aberrantly expressed lncRNA genes in M4 compared to M1 cells. E) Number of sense/antisense pairs that show concordant or discordant pattern of expression in M4 compared to M1 cells. F) Heatmap representing deregulated sense/antisense pairs in M4 compared to M1. G) Representative cancer-associated protein coding genes in the NAT/protein-coding gene pairs from the heatmap (Fig 1F). Please see S6 Table for the details of NAT/protein-coding gene pairs.
Fig 2.
PDCD4-AS1 is a stable nuclear-enriched lncRNA that shows concordant pattern of expression with its coding partner PDCD4.
A) Schematic representation of PDCD4/PDCD4-AS1 gene locus. B) PDCD4-AS1 RNA level measured by RT-qPCR in M1- M4 TNBC cells. C) PDCD4 mRNA level measured by RT-qPCR in M1- M4 TNBC cells. D) Immunoblot analysis shows the relative levels of PDCD4 protein in M1- M4 cells. E) Correlation analysis between PDCD4 and PDCD4-AS1 RNA in TCGA breast cancer dataset, analyzed by TANRIC platform. F) PDCD4-AS1 RNA level in different subclasses of breast cancer patients, analyzed by TANRIC platform. G) PDCD4-AS1 RNA level in different stages of breast cancer patients analyzed by TANRIC platform. H) Kaplan–Meier analysis to depict the survival rate in TCGA breast cancer patients with high and low levels of PDCD4-AS1, analyzed by TANRIC platform. I-J) RT-qPCR analyses in poly A+ and poly A- (I) and nuclear and cytoplasmic fractionated RNA (J) from M1 cells. K) RT-qPCR to quantify the stability of PDCD4-AS1 and PDCD4 mRNA using RNA from M1 cells treated with Flavopiridol (1M) for indicated time points (0, 1,2,4 and 6 hrs). Error bars in (B, C, J & K) represent mean ± SEM of N≥3 independent experiments (biological replicates). *P<0.05, ** P< 0.01 and ***P<0.001 using Student’s t test.
Fig 3.
PDCD4-AS1 negatively regulates cell proliferation and migration.
A-B) Transwell migration assay in control and PDCD4-AS1-depleted M1 cells. C-D) Wound healing assay in control and PDCD4-AS1 depleted M1 cells. Images were taken at Day 0, 1, 2 and 3 after wound creation; the magnified image of Day 3 in control and PDCD4-AS1 depleted cells is shown in the figure C. Ea,b) Transwell migration assay in M4 cells that are overexpressing PDCD4-AS1. Ec,d) Long-term anchorage-dependent plastic colony formation assay in M4 cells upon PDCD4-AS1 overexpression. F-G) Cell cycle flow cytometry in control and PDCD4-depleted M1 cells. H-I) Cell cycle flow cytometry in control and PDCD4-AS1-depleted M1 cells. J-K) Transwell migration assay in control and PDCD4-depleted M1 cells. L-M) Transwell migration assay in control and PDCD4-overexpressing M1 cells that are depleted of PDCD4-AS1 (AS-sh3). Error bars in (B, D, Eb, Ed, G, H, K & M) represent mean ± SEM of N≥3 independent experiments (biological replicates). *P<0.05, ** P< 0.01 and ***P<0.001 using Student’s t test. NS depicts not significant results.
Fig 4.
PDCD4-AS1 promotes the stability of PDCD4 mRNA.
A) PDCD4 and PDCD4-AS1 RNA level measured by RT-qPCR in control and PDCD4-AS1-depleted M1 cells. B) PDCD4 protein level in control and PDCD4-AS1-depleted M1 cells. C) PDCD4 and PDCD4-AS1 RNA level measured by qPCR in control and PDCD4-depleted M1 cells. D) PDCD4 protein level in control and PDCD4-depleted M1 cells. E) Nascent RNA assay in control and PDCD4-AS1-depleted M1 cells. F) RT-qPCR to quantify PDCD4 mRNA stability assay using RNA from control and PDCD4-AS1-depleted M1 cells treated with Flavopiridol (1M) for indicated time points (0, 1,2,3 and 6 hrs). Error bars in (A, D, E & F) represent mean ± SEM of N≥3 independent experiments (biological replicates). *P<0.05, ** P< 0.01 and ***P<0.001 using Student’s t test. N.S. represents not significant result.
Fig 5.
PDCD4-AS1 forms RNA duplex with PDCD4 mRNA and regulates the association of RNA decay factors to PDCD4 mRNA.
A) Schematic representation of PDCD4-AS1/PDCD4 gene locus, and PDCD4-AS1 full-length and mutants that are used for rescue assay. Red bars show regions of PDCD4-AS1 with potential complementarity to PDCD4 mRNA. Shaded region represents the minimum region within PDCD4-AS1 that is required for stabilizing the level of PDCD4 mRNA. B) RT-qPCR analyses followed by RNase protection assay. GAPDH is used as negative control where as BACE-AS1 is used as positive control. C) Affinity RNA pulldown assay followed by RT-qPCR to quantify the interaction between PDCD4 and biotin-PDCD4-AS1. D) RT-qPCR to quantify the relative levels of PDCD4 mRNA in PDCD4-AS1-depleted M1 cells overexpressing vector alone or other PDCD4-AS1 constructs. E) RT-qPCR to quantify the levels of PDCD4 mRNA post HuR-RIP in control and PDCD4-AS1-depleted M1 cells. F) RT-qPCR to detect PDCD4 mRNA level in HuR-depleted control and PDCD4-AS1-depleted cells. G) PDCD4 protein level in HuR-depleted control and PDCD4-AS1-depleted cells. H) RT-qPCR to detect PDCD4-AS1 RNA level in HuR-depleted control and PDCD4-AS1-depleted cells. I) Proposed model showing the mode of action of PDCD4-AS1 in promoting the stability of PDCD4 mRNA by attenuating the association of HuR to the 3UTR of PDCD4 mRNA. Error bars in (B, C, D, E, F & H) represent mean ± SEM of N≥3 independent experiments (biological replicates). *P<0.05, ** P< 0.01 and ***P<0.001 using Student’s t test. N.S. represents not significant change.