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Results: 6

1.
Figure 5

Figure 5. mRNA expression of miR-21 targets. From: Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach.

The expression of selected miR-21 targets was performed by real time RT-PCR. Data are shown as the mean ± SD of 3 replicates. * p<0.05 using two-tailed t-test (anti-miR-21 oligo vs control oligo).

Yi Yang, et al. Proteomics. ;9(5):1374-1384.
2.
Figure 2

Figure 2. The expression of miR-21 in MCF-7 cells. From: Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach.

MCF-7 cells were transfected with anti-miR-21 oligo or control oligo. After 72 or 96 hours transfection, total RNA were isolated from the transfections. The relative expression of miR-21 in MCF-7 was significantly decreased by anti-miR-21 oligo compared with control oligo as detected by Real Time RT-PCR. Data are shown as the mean ± SD of 3 replicates and are representative of 3 independent experiments. *: p<0.05 using two-tailed t-test (anti-miR-21 oligo vs control oligo).

Yi Yang, et al. Proteomics. ;9(5):1374-1384.
3.
Figure 3

Figure 3. MS/MS spectra of selected proteins identified by proteomics. From: Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach.

Panels A-D show the MS/MS spectra of representative peptides from programmed cell death 4 (PDCD4), chromosome condensation protein G (NCAPG), Oxidative-stress responsive 1 (OXSR1), and SEC23-related protein A (SEC23A), respectively. The inset in each case shows the corresponding relative intensity of reporter ions generated during MS/MS fragmentation and indicates upregulation of protein in samples with anti-miR-21 oligo compared to control oligo. (*: the spectrum of reporter ions)

Yi Yang, et al. Proteomics. ;9(5):1374-1384.
4.
Figure 1

Figure 1. Strategy for iTRAQ labeling. From: Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach.

MCF-7 cells were transfected with anti-miR-21 oligo or control oligo. After 72 or 96 hours post-transfection, total proteins were harvested and protein lysates (100qg from each sample) were digested with trypsin and labeled with iTRAQ reagents. Labeled peptides were combined and fractionated by strong cation exchange chromatography (SCX). Twenty-eight fractions were obtained and analyzed by LC-MS/MS. The fold changes were calculated from the ratio of intensity of iTRAQ reporter ions obtained from samples with anti-miR-21 oligos to those with control oligos.

Yi Yang, et al. Proteomics. ;9(5):1374-1384.
5.
Figure 6

Figure 6. Verification of direct miR-21 targets using luciferase assay. From: Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach.

A: 3’UTR fragments of known and selected novel targets of miR-21 were cloned downstream of the luciferase open reading frame at Bgl II restriction site of pGL3-control vector. B: The above constructs and anti-miR-21 oligo or control oligo were co-transfected along with Renilla luciferase plamid. Shown are relative luciferase activity normalized to corresponding transfections with control oligo. Data are shown as the mean ± SD of 3 replicates and are representative of 3 independent experiments. * p<0.05 using two-tailed t-test (pGL3-3’UTR construct vs pGL3-control). The protein and mRNA fold change for each gene are also indicated.

Yi Yang, et al. Proteomics. ;9(5):1374-1384.
6.
Figure 4

Figure 4. Sequence complementary analysis of miR-21 with 3’-UTR of candidate targets. From: Identification of miR-21 targets in breast cancer cells using a quantitative proteomic approach.

A: The transcripts of all genes identified by proteomics were searched for annotated 3’UTRs. The frequency of motifs in the 3’UTRs complementary to perfect 7 mer seed region of miR-21 (2 to 8 nucleotide at 5’ end) was evaluated. * p<0.05 using chi square statistic when upregulated genes (protein fold-change: >1.5) were compared with unchanged genes (protein fold-change: 1.5-0.67). 3’UTRs of 5 out of 53 upregulated genes (9.4%) bore the 7 mer miR-21 seed matches. Panels B-F show the complementarity of miR-21 sequence to the five upregulated genes bearing perfect seed matches. The seed sequence of miR-21 is shown in red. Vertical lines denote identity between miR-21 sequence and the corresponding 3’UTR sequence. Nucleotide accession numbers from RefSeq database are indicated.

Yi Yang, et al. Proteomics. ;9(5):1374-1384.

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