miR‑320a in serum exosomes promotes myocardial fibroblast proliferation via regulating the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells

Figures

• 

  Figure 1. Bioinformatics prediction of miRNA. (A) Volcano plot displaying the differential expression of miRNAs in CHF and healthy control groups using the ggplot2 package in R. The x axis presents the mean fold-change in miRNA expression between CHF and healthy control groups [log2 (Fold Change) and P<0.05]. (B) Heat map of the expression levels of the top 30 differentially expressed miRNAs drawn using gplots and RColorBrewer packages. Red, green and black indicate upregulation, downregulation and no change, respectively (C) Venn diagram of the differentially expressed miRNAs in GEO and HMDD. (D) Differentially expressed miRNAs identified by GEO and HMDD. miRNA/miR, microRNA; CHF, chronic heart failure; GEO, Gene Expression Omnibus; HMDD, Human MicroRNA Disease Database.

• 

  Figure 2. Correlation analysis of the expression levels of serum miR-320a and clinical characteristics in patients with CHF. miR-320a expression levels and the content of sST2 in serum samples collected from patients with CHF and healthy controls were detected via reverse transcription-quantitative PCR and ELISA, respectively. LVEF and LVEDD parameters were measured using Color Doppler Ultrasonic Diagnostic Equipment. New York Heart Association classification of patients with CHF was based on their limitations during physical activity according to the 2017 Chinese CHF Diagnosis and Treatment guidelines. Serum levels of NT-proBNP were determined by performing an electrochemiluminescence immunoassay. (A) miR-320a expression levels and (B) the content of sST2 in serum samples collected from patients with CHF and healthy controls. Correlation between miR-320a and (C) LVEDD, (D) sST2, (E) NYHA classification, (F) NT-proBNP or (G) LVEF. Data are presented as the mean ± SEM from at least three independent experiments. Comparisons between two groups were analysed using one-way ANOVA followed by Tukey's post hoc test. Pearson's correlation coefficient was used to assess correlations. *P<0.05 and…

• 

  Figure 3. Characterization of exosomes extracted from serum samples, and the expression levels of miR-320a in exosomes and exosome-depleted supernatant. Exosomes were extracted from serum samples of patients with CHF and normal controls using the exoRNeasy Serum-Plasma Midi kit. (A) Exosomes from NOR control group were observed via transmission electron microscopy. (B) Exosome biomarkers, including CD63, CD9 and TSG101, were identified via western blotting in CHF and normal groups (two patients each group, CHF1-2 and Normal 1-2, respectively). (C) Particle sizes of extracted exosomes from CHF and normal groups (two patients each group) were confirmed via Nanoparticle Size Analyzer. (D) Relative expression levels of miR-320a in exosomes and exosome-depleted supernatants (residues) were determined via reverse transcription-quantitative PCR. Data are presented as the mean ± SEM from at least three independent experiments. Comparisons among multiple groups were analysed using one-way ANOVA followed by Tukey's post hoc test. ##P<0.01 vs. Normal exosomes; ††P<0.01 vs. CHF exosomes. miR, microRNA; CHF, chronic heart failure; TSG101, tumor susceptibility 101.

• 

  Figure 4. Transfection of exosomes into cardiac fibroblasts and the expression of miR-320a in HEH2 cells. (A) Following incubation with PKH67-stained exosomes for 12 h, cells were visualized via fluorescence microscopy. Scale bar, 100 µm. (B) Following incubation with NOR or CHF exosomes for 12 h, the expression levels of miR-320a were quantified via reverse transcription-quantitative PCR. Data are presented as the mean ± SEM from at least three independent experiments. Comparisons among multiple groups were analysed using one-way ANOVA followed by Tukey's post hoc test. **P<0.01 vs. control; ##P<0.01 vs. normal exosomes; miR, microRNA.

• 

  Figure 5. miR-320a knockdown decreases cell viability and intracellular sST2 content, as well as enhances apoptotic cell death in HEH2 cells. HEH2 cells were transfected with NC-mimics, NC-inhibitor, miR-320a mimics or miR-320a inhibitor for 72 h. (A) Cell viability was determined by performing the MTT assay. (B) sST2 content in HEH2 cells was assessed by performing ELISAs. Cell apoptosis (C) was determined via flow cytometry and quantified. (D) The apoptosis rate of cells including early and late stage of apoptotic cells. Data are presented as the mean ± SEM from at least three independent experiments. Comparisons between two groups were analysed using the unpaired Student's t-test. **P<0.01 vs. NC-mimics; #P<0.05 and ##P<0.01 vs. NC-inhibitor; ††P<0.01 vs. miR-320a mimics. miR, microRNA; sST2, soluble growth stimulation expressed gene 2; NC, negative control.

• 

  Figure 6. PIK3CA is a direct target of miR-320a. (A) miR-320a target genes were predicted using TargetScan, miRwalk and miRDIP bioinformatics tools. (B) Enriched KEGG pathways of target genes of miR-320a. (C) Predicted miR-320a binding site in the 3'-UTR region of PIK3CA. (D) Predicted genes of the PI3K/Akt pathway. PIK3CA, phosphoinositide-3-kinase catalytic α polypeptide gene; miR, microRNA; miRDIP, microRNA Data Integration Portal; KEGG, Kyoto Encyclopedia of Genes and Genomes; UTR, untranslated region.

• 

  Figure 7. Expression of miR-320a and PIK3CA in HEH2 cells. Cells were seeded into 6-well plates and transfected with NC-mimics, NC-inhibitor, miR-320a mimics or miR-320a inhibitor for 72 h. (A) miR-320a and (B) PIK3CA expression levels were quantified via reverse transcription-quantitative PCR. (C) Pearson's correlation analysis was performed to assess the correlation between miR-320a and PIK3CA expression levels. Data are presented as the mean ± SEM from at least three independent experiments. Comparisons between two groups were analyzed using the unpaired Student's t-test. **P<0.01 vs. NC-mimics; ##P<0.01 vs. NC-inhibitor. miR, microRNA; PIK3CA, phosphoinositide-3-kinase catalytic α polypeptide gene; NC, negative control.

• 

  Figure 8. miR-320a regulates the PIK3CA/Akt/mTOR signaling pathway in HEH2 cells. Cells were seeded into 6-well plates and transfected with NC-mimics, NC-inhibitor, miR-320a mimics or miR-320a inhibitor for 72 h. α-SMA, collagen I, collagen III, PIK3CA, p-Akt (ser 473), p-Akt (thr 308), Akt, p-mTOR (ser 2481), p-mTOR (ser 2488), mTOR and β-actin protein expression levels were (A) determined by western blotting and (B) semi-quantified. Data are presented as the mean ± SEM from three independent experiments. Comparisons between two groups were analyzed using the unpaired Student's t-test. **P<0.01 vs. NC-mimics; ##P<0.01 vs. NC-inhibitor. miR, microRNA; PIK3CA, phosphoinositide-3-kinase catalytic α polypeptide gene; NC, negative control; α-SMA, α-smooth muscle actin; p, phosphorylated.