Differential microRNA Expression and Regulation in the Rat Model of Post-Infarction Heart Failure

Abstract

Background: Heart failure is a complex end stage of various cardiovascular diseases with a poor prognosis, and the mechanisms for development and progression of heart failure have always been a hot point. However, the molecular mechanisms underlying the post transcriptional regulation of heart failure have not been fully elucidated. Current data suggest that microRNAs (miRNAs) are involved in the pathogenesis of heart failure and could serve as a new biomarker, but the precise regulatory mechanisms are still unclear.

Methods: The differential miRNA profile in a rat model of post-infarction heart failure was determined using high throughout sequencing and analyzed through bioinformatics approaches. The results were validated using qRT-PCR for 8 selected miRNAs. Then the expression patterns of 4 miRNAs were analyzed in different periods after myocardial infarction. Finally, gain- and loss-of-function experiments of rno-miR-122-5p and rno-miR-184 were analyzed in H2O2 treated H9c2 cells.

Results: In the heart failure sample, 78 miRNAs were significantly upregulated and 28 were downregulated compared to the controls. GO and KEGG pathway analysis further indicated the likely roles of these miRNAs in heart failure. Time-course analysis revealed different expression patterns of 4 miRNAs: rno-miR-122-5p, rno-miR-199a-5p, rno-miR-184 and rno-miR-208a-3p. Additionally, rno-miR-122-5p and rno-miR-184 were proved to promote apoptosis in vitro.

Conclusions: Differential profile and expression patterns of miRNAs in the rats model of post-infarction heart failure were found, and the pro-apoptotic roles of rno-miR-122-5p and rno-miR-184 were revealed. These findings may provide a novel way that may assist in heart failure diagnosis and treatment.

Fig 1. Evaluation of the heart functions of HF and control rats.

(A) Representative images of HF and control hearts. (B) Echocardiographic index, including EF, FS, IVSd, IVSs, LVIDd, LVIDs, LVPWd, LVPWs and HR. EF, ejection fraction; FS, fractional shortening; IVSd, interventricular septal thickness in diastole; IVSs, interventricular septal thickness in systole; LVIDd, left ventricular internal dimension in diastole; LVIDs, left ventricular internal dimension in systole; LVPWd, left ventricular posterior wall thickness in diastole; LVPWs, left ventricular posterior wall thickness in systole; HR, heart rate.(C) The levels of plasma nt-proBNP in HF and control rats. (D) Hematoxylin and eosin (HE) staining of left ventricular (LV) tissue showed pathological and morphological changes in control, MI-4, MI-8 and HF group (magnification, ×200). (E) Masson’s trichrome staining of LV tissure showed progressively cardiac interstitial and perivascular fibrosis in MI-4, MI-8 and HF group compared to control group (magnification, ×200). (F) Immunohistochemical staining of Caspase 3 in LV tissue showed apoptosis in MI-4, MI-8 and HF group (magnification, ×200). Data were presented as means ± SD. **P<0.01 and *P<0.05.

Fig 2. Summary of data cleaning and length distribution of tags.

(A) Venn chart for total sRNA distribution. (B) Venn chart for unique sRNA distribution.

Fig 3. Scatter plot of the differential expression.

Each plot represents a miRNA. Ratio: standard expression level(HF/C). Red plots: ratio>2; green plots: ratio<1/2; blue plots: 1/2≤ratio≤2.

Fig 4. Prospective validation and time course analysis of miRNA sequencing analysis by qRT-PCR.

(A)4 up-regulated miRNAs and 4 down-regulated miRNAs were selected for validation. The fold changes of selected miRNAs measured by qRT-PCR were statistically significant(p<0.05). miRNA expression difference was considered to be valid if the direction of change was the same. Time course analysis of miR-208a-3p (B), miR-184 (C), miR-122-5p (D) and miR-199a-5p (E) were studied. *p<0.05 and **p<0.01 vs the control group; ##p<0.01 vs the MI-4 group.

Fig 5. The apoptosis ratio detected by flow cytometry after transfection.

(A) Relative expression levels of miR-122-5p and miR-184 after transfection. (B) Percentage of apoptotic cells after H₂O₂ treatment. (C) Percentage of apoptotic cells without H₂O₂ treatment. BC, blank control group; NC, negative control miRNA group.