miR-373 induces gene expression by targeting a complementary sequence in the E-cadherin promoter. (A) Schematic representation of the E-cadherin promoter and its CpG island. Indicated is the location of the miR-373 target site. (B) Sequence of the miR-373 target site located at nucleotide −645 relative to the transcription start site. Bases in boldface indicate the target site in the sense strand of promoter DNA. The sequence in red corresponds to those bases in miR-373 that are complementary to the target site, including G:U/T wobble base-pairing. (C) Sequence and structure of miR-373 and dsEcad-640. Lowercase letters in both strands of miR-373 correspond to the native two-base 3′ overhangs. Sequences in red correspond to those bases that are complementary to the target site. (D) PC-3 cells were transfected with 50 nM concentrations of the indicated dsRNAs for 72 h. Combination treatment of miR-373 and dsEcad-640 (miR-373 + dsEcad-640) was performed by transfecting at 50 nM each dsRNA. Mock samples were transfected in the absence of dsRNA. E-cadherin and GAPDH mRNA expression levels were assessed by standard RT-PCR. GAPDH served as a loading control. (E) Relative expression of E-cadherin was determined by real-time PCR (mean ± standard error from four independent experiments). Values of E-cadherin were normalized to GAPDH. (F) Induction of E-cadherin protein was confirmed by immunoblot analysis. GAPDH was also detected and served as a loading control.

Dicer is required for E-cadherin induction by pre-miR-373. (A) PC-3 cells were treated with Dicer-PMO or Con-PMO for 48 h. No PMO treatments were performed in absence of PMO molecules. Cells cotreated with miR-373 or pre-miR-373 were transfected 24 h after initial PMO treatments. Total protein was extracted and levels of Dicer and GAPDH were determined by immunoblot analysis. GAPDH served as a loading control. (B) PC-3 cells were treated with or without Dicer-PMO or Con-PMO. The following day, cells were transfected with 50 nM pre-miR-Con or pre-miR-373 for 72 h. E-cadherin and GAPDH mRNA expression levels were assessed by standard RT-PCR. (C) PC-3 cells were treated with the indicated PMO molecules. The following day, cells were transfected at 50 nM dsControl or miR-373 for 72 h. E-cadherin and GAPDH mRNA expression levels were assessed by standard RT-PCR.

Enrichment of RNApII at miR-373-targeted promoters. PC-3 cells were transfected with mock, dsControl, or miR-373 for 72 h. ChIP assays were performed by using an RNApII-specific antibody to immunoprecipitate transcriptionally active regions of DNA. The purified DNA was amplified by PCR, using primer sets specific to the transcriptional start sites of E-cadherin, CSDC2, or GAPDH. DNA pulled down in the absence of antibody (No AB) served to identify background amplification. Input DNA was amplified as a loading control.

Pre-miR-373 induces E-cadherin expression. (A) Sequence of the miR-373 precursor hairpin RNA (pre-miR-373). Bases in red indicate the mature sequence of the miR-373 duplex. (B) PC-3 cells were transfected at 50 nM pre-miR-Con, pre-miR-373, or miR-373 for 72 h. E-cadherin and GAPDH mRNA expression levels were assessed by standard RT-PCR. (C) Relative expression was determined by real-time PCR (mean ± standard error from four independent experiments). Values of E-cadherin were normalized to GAPDH. (D) E-cadherin and GAPDH protein levels were detected by immunoblot analysis. GAPDH served as a loading control.

miR-373 induces the expression of CSDC2. (A) Sequence of the miR-373 target site located at nucleotide −787 relative to the transcription start site in the CSDC2 promoter. Bases in boldface indicate the putative target site in the sense strand of promoter DNA. The sequence in red corresponds to those bases in miR-373 that are complementary to the target site, including G:U/T wobble base-pairing. (B) PC-3 cells were transfected at 50 nM dsControl or miR-373 for 72 h. Expression of CSDC2 and GAPDH was determined by standard RT-PCR. GAPDH served as a loading control. (C) CSDC2 and GAPDH expression levels were also determined in PC-3 cells after mock, pre-miR-Con, or pre-miR-373 transfections. (D) Relative expression of CSDC2 was determined by real-time PCR (mean ± standard error from four independent experiments). Values of CSDC2 were normalized to GAPDH.

Sequence specificity for miR-373. (A) Mutations to four of the first or last base pairs in miR-373 resulted in miR-373–5MM and miR-373–3MM, respectively. The mutated bases are shown in blue. (B) PC-3 cells were transfected at 50 nM each indicated miRNA duplex. Expression of E-cadherin, CSDC2, and GAPDH were assessed by standard RT-PCR. GAPDH served as a loading control. (C) dsRNAs targeting divergent sites in either the E-cadherin or CSDC2 promoter specifically induce the expression of only the target gene. PC-3 cells were transfected with dsControl, miR-373, dsEcad-215, or dsCSDC2–670 for 72 h, as indicated. E-cadherin, CSDC2, and GAPDH mRNA expression levels were determined by standard RT-PCR.