1. Volume 59, Issue 1, Pages 50-61 (July 2015)
Oxidative Modification of miR-184 Enables It to Target Bcl-xL and Bcl-w 
Jian-Xun Wang, Jie Gao, Su-Ling Ding, Kun Wang, Jian-Qin Jiao, Yin Wang, Teng Sun, Lu-Yu Zhou, Bo Long, Xiao-Jie Zhang, Qian Li, Jin-Ping Liu, Chang Feng, Jia Liu, Ying Gong, Zhixia Zhou, Pei-Feng Li 
Molecular Cell 
Volume 59, Issue 1, Pages (July 2015)
DOI: /j.molcel
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2. Molecular Cell 2015 59, 50-61DOI: (10.1016/j.molcel.2015.05.003)
Copyright © 2015 Elsevier Inc. Terms and Conditions

3. Figure 1 miRNAs Are Oxidatively Modified in Various Oxidation Systems
(A and B) H9c2 cells were treated with different concentrations of H2O2 (A) or 100 μM H2O2 for the indicated time (B). miRNA fraction was purified, and 8-OHG levels were determined by northwestern blot using the anti-8-OHG antibody.
(C) H9c2 cells were treated with H2O2. miRNAs were isolated for the analysis of 8-oxoG by HPLC. Standard, 8-oxoG standard; Control, untreated cells; H2O2, H9c2 cells treated with H2O2; UV, UV absorption spectra of the 8-oxoG peak.
(D) Determination of 8-OHG in miRNA duplexes in the in vitro model by HPLC. Control, chemically synthesized hsa-let-7a-1 without treatment; Fe3+-Asc, hsa-let-7a-1 was incubated with ferric citrate (Fe3+) and ascorbate; H2O2, hsa-let-7a-1 was incubated with H2O2; Cu2+-Asc, hsa-let-7a-1 was incubated with Cu2+ and Asc.
(E) Analysis of 8-OHG in the purified miR-184 in cells by HPLC. Control, untreated cells; H2O2, H9c2 cells treated with H2O2; miR-184 purified, miR-184 was purified by affinity purification. miR-NC was employed as negative control.
(F) Analysis of endogenous oxidized miR-184 copy number upon H2O2 treatment in H9c2 cells. Input, total miR-184; 8-OHG IP, miR-184 was analyzed after immunoprecipitation with anti-8-OHG antibody; IgG IP, miR-184 was analyzed after immunoprecipitation with anti-IgG antibody, undetectable (UD). ∗p < The results are expressed as means ± SEM of at least three independent experiments See also Figure S1 and Table S1.
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4. Figure 2 oxi-miR-184 Mismatches with the 3′ UTRs of Bcl-xL and Bcl-w
(A and B) H9c2 cells were transfected with oxi-miR-184, native miR-184 (n-miR-184), or its negative control (miR-NC), respectively. miR-184 and U6 were analyzed by northern blot. Bcl-xL (A) and Bcl-w (B) protein levels were analyzed by immunoblot. Quantitative analysis of Bcl-xL and Bcl-w levels is shown in the lower panels. ∗p < 0.05 versus Control.
(C and D) H9c2 cells were exposed to 100 μM H2O2 for the indicated times. Bcl-xL and Bcl-w mRNA levels were analyzed by qRT-PCR (C). Bcl-xL and Bcl-w protein levels were analyzed by immunoblot (D).
(E) H9c2 cells were transfected with miR-184 antagomir (Anti-miR-184) and exposed to H2O2. miR-184 and U6 were analyzed by northern blot. Bcl-xL and Bcl-w protein levels and mRNA levels were analyzed. ∗p < 0.05 versus H2O2 alone.
(F and G) Outline of luciferase reporter assay for validating the interaction of oxi-miR-184 with Bcl-xL 3′ UTR (F) or Bcl-w 3′ UTR (G). The miRNA response elements (MREs) of oxi-miR-184 in rat Bcl-xL or Bcl-w 3′ UTR were predicted by the bioinformatics program RNAhybrid. The red color indicates the potential mismatching sites in which 8-oxoG matches with A. M1, MRE1; M2, MRE2.
(H and I) Oxidized miR-184 reduces the luciferase activities of Bcl-xL 3′ UTR and Bcl-w 3′ UTR. HEK293 cells were transfected with the luciferase constructs of the wild-type Bcl-xL 3′ UTR (Bcl-xL-3′ UTR) (G) or the wild-type Bcl-w 3′ UTR (Bcl-w-3′ UTR) (H), along with n-miR-184, oxi-miR-184, or miR-NC. The empty vector pGL3 served as a control. ∗p < 0.05 versus Bcl-xL-3′ UTR or Bcl-w-3′ UTR alone. The results are expressed as means ± SEM of at least three independent experiments. See also Figure S2 and Table S2.
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5. Figure 3
Identification of oxi-miR-184-Targeting Sites in the 3′ UTRs of Bcl-xL and Bcl-w
(A) Verification of the MREs of oxi-miR-184 in Bcl-xL 3′ UTR. The mutations were introduced to MRE1 (Bcl-xL-m-MRE1), MRE2 (Bcl-xL-m-MRE2), or both MRE1 and MRE2 (Bcl-xL-m-MRE1-2) in the Bcl-xL-3′ UTR. ∗p < 0.05.
(B) The target protectors (TPs) of Bcl-xL 3′ UTR attenuate the effect of oxi-miR-184 on luciferase activity. Cells were cotransfected with the construct of Bcl-xL-3′ UTR and TP Bcl-xL-TP1oxi-miR-184, Bcl-xL-TP2oxi-miR-184, Bcl-xL-TPoxi-miR-184 (a mixture of both Bcl-xL-TP1oxi-miR-184 and Bcl-xL-TP2oxi-miR-184), or its negative control TPcontrol, and then transfected with oxi-miR-184. ∗p < 0.05.
(C) The mutated miR-184 and its oxidized form are unable to inhibit the luciferase activity. Cells were cotransfected with the luciferase constructs containing four Bcl-xL MRE1 repeat sequences of oxi-miR-184 (4 × Bcl-xL-MRE1), along with n-miR-184, oxi-miR-184, mutant miR-184 (m-miR-184), or oxidized mutant miR-184 (oxi-m-miR-184). ∗p < 0.05 versus m-miR-184 or oxi-m-miR-184.
(D and E) H2O2 induces a substantial reduction in the luciferase activity of wild-type Bcl-xL 3′ UTR and Bcl-w 3′ UTR. H9c2 cells were transfected with miR-184 antagomir (Anti-184) or antagomir control (Anti-NC), along with the luciferase constructs of wild-type Bcl-xL 3′ UTR or wild-type Bcl-w 3′ UTR, or their mutated forms (Bcl-xL-3′ UTR-mut and Bcl-w-3′ UTR-mut), and then treated with H2O2 for the indicated time. The data show the luciferase activities. The results are expressed as means ± SEM of at least three independent experiments. See also Figure S3.
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6. Figure 4
Endogenous miR-184 Can Directly Bind to Bcl-xL and Bcl-w in the Animal Model
(A) The schematic outline of the affinity purification procedures of miR-184 and Bcl-xL. The same procedures were used for Bcl-w.
(B) Purification of miR-184. The mice were subjected to sham or I/R. miR-184 were purified by affinity purification using the biotin-labeled probes of Bcl-xL, Bcl-w, GAPDH, and miR-184 as described in the Experimental Procedures. miR-184, Bcl-xL, Bcl-w, and GAPDH were detected in the indicated fractions of affinity purification by RT-PCR. U6 served as a control.
(C) Analysis of 8-OHG in the purified miR-184 by HPLC. Standard, the commercial 8-OHG; Control, the mice without treatment; Sham, the mice were subjected to sham; IR, the mice were subjected to ischemia/reperfusion; Anti-184, the mice were administered with miR-184 antagomir; miR-NC, the mice were administered with antagomir negative control; Bcl-xL, the samples were precipitated with Bcl-xL probe followed by miR-184 probe; Bcl-w, the samples were precipitated with Bcl-w probe followed by miR-184 probe. See also Figure S4.
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7. Figure 5
Oxidized miR-184 Promotes Apoptosis via Its Targets Bcl-xL and Bcl-w
(A) H9c2 cells were pretreated with 5 mM NAC followed by exposure to H2O2; 8-oxoG levels were determined by northwestern blot (upper panel). Apoptosis was detected by TUNEL assay (lower panel). ∗p < 0.05.
(B) NAC reduces miR-184 oxidation. H9c2 cells were treated as described in (A). Oxidized miRNAs were purified by immunoprecipitation using the anti-8-oxoG antibody. miR-184 was detected by northern blot.
(C) Bcl-xL and Bcl-w protein levels were analyzed in H9c2 cells treated as described in (A). The numbers show the ratio of Bcl-xL or Bcl-w to actin.
(D) H9c2 cells were pretreated with 5 mM NAC followed by transfection with oxi-miR-184; Bcl-xL and Bcl-w protein levels were analyzed.
(E) H2O2 and oxidized miR-184 synergistically suppress Bcl-xL and Bcl-w expression. Cells were transfected with miR-184, oxi-miR-184, or miR-NC, respectively. At 24 hr after transfection, they were treated with H2O2. miR-184 and U6 were detected by northern blot. Bcl-xL and Bcl-w were detected by immunoblot. The results were densitometrically scanned and shown in the lower panel. ∗p < 0.05.
(F) Bcl-xL with 3′ UTR exerts a differential effect on apoptosis in the presence of oxi-miR-184 and n-miR-184. H9c2 cells were infected with adenoviral Bcl-xL with 3′ UTR (Bcl-xL-W-3′ UTR), and then transfected with oxi-miR-184, n-miR-184, or miR-NC. At 24 hr after transfection cells were exposed to H2O2. miR-184 and U6 were detected by northern blot. Bcl-xL was analyzed by immunoblot. Apoptosis was assessed by the TUNEL assay. ∗p < 0.05.
(G and H) H9c2 cells were transfected with target protector TPoxi-miR-184 (Bcl-xL-TPoxi-miR-184 or Bcl-w-TPoxi-miR-184) or TPcontrol, and then exposed to H2O2. Bcl-xL (G) and Bcl-w (H) were analyzed by immunoblot. Apoptosis was detected by the TUNEL assay. ∗p < 0.05.
(I) H9c2 cells were transfected with miR-184 antagomir or antagomir control. At 24 hr after transfection, they were exposed to H2O2. Apoptotic cells were detected by TUNEL assay. ∗p < The results are expressed as means ± SEM of at least three independent experiments. See also Figure S5.
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8. Figure 6
Oxidized miR-184 Increases the Susceptibility of the Heart to I/R Injury
(A) Mice were injected with the oxi-miR-184, n-miR-184, or miR-NC. miR-184, Bcl-xL, and Bcl-w levels were analyzed 1 hr after I/R.
(B) Mice were continuously infused with NAC. Bcl-xL and Bcl-w protein levels were analyzed 1 hr after I/R.
(C) Oxidized miR-184 promotes apoptosis upon I/R. Apoptosis was determined by TUNEL assay 1 hr after I/R. Green, TUNEL-positive myocyte nuclei; blue, DAPI-stained nuclei; red, cardiomyocytes labeled with the antibody to α-actinin. Scale bar, 20 μm. Quantitative analysis of apoptosis is shown in the right panel. ∗p < 0.05.
(D) Oxidized miR-184 increases myocardial infarct sizes in I/R heart. Quantification of infarct sizes in each group with different treatment is shown. AAR/LV, ratio of area at risk (AAR) to left ventricular area; INF/AAR, ratio of infarct area to AAR; INF/LV, ratio of infarct area to left ventricular area. ∗p < 0.05.
(E) Mice were injected with miR-184 antagomir or antagomir negative control. miR-184 levels were analyzed 1 hr after I/R.
(F) Knockdown of miR-184 reduces apoptosis induced by I/R. Apoptosis was determined by TUNEL assay. ∗p < 0.05.
(G) Target protectors of Bcl-xL and Bcl-w reduce myocardial infarct sizes induced by I/R. Representative photos of mid-ventricular sections and quantification of infarct sizes are shown. Non-ischemic area (dark blue), infarct area (white), viable myocardium within area at risk (red). Scale bar, 2 mm. Quantification of infarct sizes in each group with different treatment is shown. ∗p < n = 5 or 6 mice per group.
(H) Target protectors of Bcl-xL and Bcl-w ameliorate cardiac function. Cardiac function was assessed by echocardiography at 7 days after I/R. LVIDd, diastolic left ventricular internal diameters; LVIDs, systolic left ventricular internal diameters; FS, fractional shortening. n = 8–10 mice per group. The results are expressed as means ± SEM. See also Figure S6.
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9. Figure 7 Oxidative Modification of miRNA Is a Comprehensive Phenomenon
(A and B) H9c2 cells were treated with doxorubicin (A) or Ang II (B). Oxidized miR-184 copy number was analyzed. ∗p < 0.05.
(C and D) Knockdown of miR-184 and NAC treatment both can inhibit apoptosis induced by Ang II. Cells were transfected with miR-184 antagomir and exposed to Ang II (C). Cells were simultaneously exposed to Ang II and NAC (D). Apoptotic cells were detected. ∗p < Levels of Bcl-xL and Bcl-w were analyzed.
(E) H9c2 cells were transfected with miR-184 antagomir, along with the luciferase constructs of wild-type Bcl-xL. After 24 hours, cells were exposed to Ang II for the indicated time. The data show the luciferase activities.
(F) Oxi-miR-204-3p reduces the luciferase activity of Pcif1 3′ UTR.
(G) H9c2 cells were transfected with oxi-miR-204-3p, native miR-204-3p, or miR-NC. Pcif1 levels were detected.
(H) Oxi-miR-139-3p reduces the luciferase activity of RNMT 3′ UTR.
(I) H9c2 cells were transfected with oxi-miR-139-3p, native miR-139-3p, or miR-NC. RNMT levels were detected. The results are expressed as means ± SEM of at least three independent experiments. See also Figure S7 and Tables S3 and S4.
Molecular Cell  , 50-61DOI: ( /j.molcel )
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