Int J Stem Cells.  2021 Aug;14(3):298-309. 10.15283/ijsc21025.

Role of miRNA-324-5p-Modified Adipose-Derived Stem Cells in Post-Myocardial Infarction Repair

• Ji Z
• Wang C
• Tong Q

• KMID: 2519372
• DOI: http://doi.org/10.15283/ijsc21025

Abstract

Background and Objectives
To seek out the role of mircoRNA (miR)-324-5p-modified adipose-derived stem cells (ADSCs) in post-myocardial infarction (MI) myocardial repair.
Methods and Results
Rat ADSCs were cultivated and then identified by morphologic observation, osteogenesis and adipogenesis induction assays and flow cytometry. Afterwards, ADSCs were modified by miR-324-5p lentiviral vector, with ADSC proliferation and migration measured. Then, rat MI model was established, which was treated by ADSCs or miR-324-5p-modified ADSCs. Subsequently, the function of miR-324-5p-modified ADSCs in myocardial repair of MI rats was assessed through functional assays. Next, the binding relation of miR-324-5p and Toll-interacting protein (TOLLIP) was validated. Eventually, functional rescue assay of TOLLIP was performed to verify the role of TOLLIP in MI. First, rat ADSCs were harvested. Overexpressed miR-324-5p improved ADSC viability. ADSC transplantation moderately enhanced cardiac function of MI rats, reduced enzyme levels and decreased infarct size and apoptosis; while miR-324-5p-modified ADSCs could better promote post-MI repair. Mechanically, miR-324-5p targeted TOLLIP in myocardial tissues. Moreover, TOLLIP overexpression debilitated the promotive role of miR-324-5p-modified ADSCs in post-MI repair in rats.
Conclusions
miR-324-5p-modified ADSCs evidently strengthened post-MI myocardial repair by targeting TOLLIP in myocardial tissues.

Keyword

Myocardial infarction; Radiotherapy; Adipose-derived stem cells; mircoRNA-324-5p; Myocardial repair; Stem cell survival

Figure

• 

  Fig. 1 Identification of rat ADSCs. Rat ADSCs were cultivated. Note: A, morphology of ADSCs observed under an inverted microscope. B and C, alizarin red (B) and oil red O (C) staining of ADSCs after adipogenesis and osteogenesis induction. D, expressions of CD34, CD45, CD105 and CD90 on the surface of ADSCs were detected by flow cytometry. The cell experiment was repeated three times.

• 

  Fig. 2 miR-324-5p promotes ADSC proliferation and migration. Note: ADSCs were injected with LV-miR-324-5p, with LV-NC as control. A, miR-324-5p expression in ADSCs was determined via RT-qPCR (A). B and C, ADSC proliferation and migration were examined by CCK-8 method (B) and Transwell assay (C). The cell experiment was repeated three times; the results were shown as mean±standard deviation. One-way ANOVA was used to analyze data in panels A and C, and two-way ANOVA was used to analyze data in panel B. Tukey’s multiple comparisons test was applied for post hoc test. **p<0.01.

• 

  Fig. 3 miR-324-5p-modified ADSCs enhance post-MI repair in rats. Note: MI model was established in rats via LAD, with ADSCs or miR-324-5p-modified ADSCs injected into the peri-MI area at multiple sites, with PBS injection as control. A, 28 days after ADSCs transplantation, myocardial function of rats was tested using echocardiography, N=12. B, after ultrasound detections, levels of cTnT and CK-MB in serum of rats were measured, N=12. C, MI degree in rats observed by HE staining. D, apoptosis in MI rats verified through TUNEL staining. N=6, the results of panels B and D were shown as mean±standard deviation. One-way ANOVA was used to analyze data in panels A, B and D. Tukey’s multiple comparisons test was applied for post hoc test. **p<0.01. MI: myocardial infarction, NC-ADSCs: ADSCs after lentivirus infection with miR-324-5p negative control, miR-ADSCs: ADSCs after lentivirus infection with miR-324-5p overexpression vectors.

• 

  Fig. 4 miR-324-5p targets TOLLIP expression in myocardial tissues of MI rats. Note: A, downstream target gene of miR-324-5p predicted by miRDB (http://mirdb.org/index.html) and Targetscan (http://www.targetscan. org/vert_71/) databases. B, binding site between miR-324-5p and TOLLIP was predicted by Targetscan data-base. C, binding relation of miR-324-5p and TOLLIP was verified through dual-luciferase reporter gene assay. D and E, expressions of miR-324-5p (D) and TOLLIP (E) were measured by RT-qPCR. N=6, the cell experiment was repeated three times; the results in panel C were shown as mean±standard deviation. Two-way ANOVA was used to analyze data in panel C, and Sidak’s multiple comparisons test was applied for post hoc test. One-way ANOVA was used to analyze data in panels D and E, and Tukey’s multiple comparisons test was applied for post hoc test. **p<0.01. MI: myocardial infarction, NC-ADSCs: ADSCs after lentivirus infection with miR-324-5p negative control, miR-ADSCs: ADSCs after lentivirus infection with miR-324-5p overexpression vectors.

• 

  Fig. 5 TOLLIP overexpression spoils the promotive role of miR-324-5p-modified ADSCs in post-MI repair in rats. Note: Ad-TOLLIP was injected in rats from the miR-ADSCs group, with Ad-NC as control. A, silencing efficiency of Ad-TOLLIP was verified by RT-qPCR. B, 28 days after ADSCs transplantation, myocardial function of rats were tested by echocardiography, N=12. C, levels of cTnT and CK-MB in serum of rats were measured, N=12. D, MI degree in rats was observed by HE staining. E, apoptosis in MI rats was verified through TUNEL staining. N=6, the results of panels C and E were shown as mean±standard deviation. One-way ANOVA was used to analyze data in panels A, B, C and E. Tukey’s multiple comparisons test was applied for post hoc test. **p<0.01. MI: myocardial infarction, NC-ADSCs: ADSCs after lentivirus infection with miR-324-5p negative control, miR-ADSCs: ADSCs after lentivirus infection with miR-324-5p overexpression vectors, Ad-TOLLIP: TOLLIP adenovirus.

Reference

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