A natural antisense lncRNA controls breast cancer progression by promoting tumor suppressor gene mRNA stability
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.