Long Non-Coding RNA-Cardiac-Inducing RNA 6 Mediates Repair of Infarcted Hearts by Inducing Mesenchymal Stem Cell Differentiation into Cardiogenic Cells through Cyclin-Dependent Kinase 1

Abstract

This study aims to investigate the induction effect of LncRNA-CIR6 on MSC differentiation into cardiogenic cells in vitro and in vivo. In addition to pretreatment with Ro-3306 (a CDK1 inhibitor), LncRNA-CIR6 was transfected into BMSCs and hUCMSCs using jetPRIME. LncRNA-CIR6 was further transfected into the hearts of C57BL/6 mice via 100 μL of AAV9-cTnT-LncRNA-CIR6-ZsGreen intravenous injection. After three weeks of transfection followed by AMI surgery, hUCMSCs (5 × 10⁵/100 μL) were injected intravenously one week later. Cardiac function was evaluated using VEVO 2100 and electric mapping nine days after cell injection. Immunofluorescence, Evans blue-TTC, Masson staining, FACS, and Western blotting were employed to determine relevant indicators. LncRNA-CIR6 induced a significant percentage of differentiation in BMSCs (83.00 ± 0.58)% and hUCMSCs (95.43 ± 2.13)% into cardiogenic cells, as determined by the expression of cTnT using immunofluorescence and FACS. High cTNT expression was observed in MSCs after transfection with LncRNA-CIR6 by Western blotting. Compared with the MI group, cardiac contraction and conduction function in MI hearts treated with LncRNA-CIR6 or combined with MSCs injection groups were significantly increased, and the areas of MI and fibrosis were significantly lower. The transcriptional expression region of LncRNA-CIR6 was on Chr17 from 80209290 to 80209536. The functional region of LncRNA-CIR6 was located at nucleotides 0-50/190-255 in the sequence. CDK1, a protein found to be related to the proliferation and differentiation of cardiomyocytes, was located in the functional region of the LncRNA-CIR6 secondary structure (from 0 to 17). Ro-3306 impeded the differentiation of MSCs into cardiogenic cells, while MSCs transfected with LncRNA-CIR6 showed a high expression of CDK1. LncRNA-CIR6 mediates the repair of infarcted hearts by inducing MSC differentiation into cardiogenic cells through CDK1.

Keywords: BMSCs; CDK1; LncRNA-CIR6; hUMSCs; myocardial infarction.

Figure 1

Effect of LncRNA-CIR6 on the induced differentiation of BMSCs and hUCMSCs into Ccardiomyogenic cells. (A) Inverted optical microscope images of MSCs transfected with empty plasmid or LncRNA-CIR6 plasmid on the 10th day (n = 3 per group); scale bar: 1000 μm. (B) Immunofluorescence map of MSCs transfected with empty plasmid or LncRNA-CIR6 plasmid on the 10th day (n = 3 per group); scale bar: 50 μm. (C) Western blot and quantification of cTNT in MSCs transfected with empty plasmid or LncRNA-CIR6 plasmid on the 10th day (n = 3 per group). (D) The fluorescence intensity of APC in MSCs transfected with empty plasmid or LncRNA-CIR6 plasmid on the 10th day (n = 3 per group). For all statistical plots, the data are presented as mean ± SD. ** p < 0.01, **** p < 0.0001 vs. Vehicle group (C,D). (Means ± SD, n = 3).

Figure 2

The effects of LncRNA-CIR6 or combined hUMSCs on heart function and cardioprotection in vivo in mice with myocardial infarction. (A) Representative echo image of M-mode after 17 days of MI was treated with LncRNA-CIR6 or combined hUMSCs (n = 3 per group). (B) Left ventricular EF (LVEF) and FS (LVFS), LVID,s and LVID,d assessed by echocardiography in mice (n = 3 per group). (C) Maps of ventricular conduction time in mice after 17 days of MI treated with LncRNA-CIR6 or combined hUMSCs (n = 3 per group). (D) Quantification of illustrated conduction time (n = 3 per group). (E) Quantification of average ventricular conduction velocity (n = 3 per group). (F) Quantification of dispersion absolute of ventricular conduction (n = 3 per group). (G) Fluorescence imaging of frozen mouse heart slices (scale bar: 1000 μm for upper panel; 75 μm for lower panel) for measuring infarct size after 17 days of MI was treated with LncRNA-CIR6 or combined hUMSCs (n = 3 per group). (H) Heart sections were stained with Evans blue-TTC, staining for infarct size; after 17 days of MI, they were treated with LncRNA-CIR6 or combined hUMSCs (n = 3 per group). (I) Heart sections were stained with Masson staining; after 17 days of MI, they were treated with LncRNA-CIR6 or combined hUMSCs (Scale bar: 500 μm) (n = 3 per group). For all statistical plots, the data are presented as mean ± SD. ^&&&& p < 0.0001 vs. Before MI group; * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 vs. MI group; ^# p < 0.05, ^## p < 0.01, ^### p < 0.001, vs. LncRNA-CIR6 group (Means ± SD, n = 3).

Figure 3

Prediction of LncRNA-CIR6 secondary structure and its protein interactions. (A) Transcription gene location of LncRNA-CIR6. (B) Predicted LncRNA-CIR6 secondary structure. (C) Mountain chart of LncRNA-CIR6 (LncRNA-CIR6 MFE (minimum free energy) structure), thermodynamic set of RNA structures, and centroid structure. (D) The Venn diagram below illustrates the interaction of 28 proteins with LncRNA-CIR6 (Area 1: Proteins binding to more than 10 binding sites on the secondary structure of LncRNA-CIR6. Area 2: Proteins with binding sites within the functional regions of LncRNA-CIR6. Area 3: Proteins with both more than 10 binding sites and located within the functional regions of LncRNA-CIR6. Area 4: Proteins associated with cardiovascular system diseases. Area 5: Proteins with binding sites within the functional region of LncRNA-CIR6 and associated with cardiovascular diseases. Area 6: Proteins with more than 10 binding sites, all in the functional region of LncRNA-CIR6, and these proteins are related to cardiovascular diseases). (E) CDK1 binding site on LncRNA-CIR6. (F) Western blot and quantification of CDK1 in MSCs transfected with empty plasmid or LncRNA-CIR6 plasmid on the 10th day (n = 3 per group). (G) Immunofluorescence map of MSCs transfected with LncRNA-CIR6 pretreated with/without Ro-3306 on the 10th day (n = 3 per group); Scale bar: 50 μm. For all statistical plots, the data are presented as mean ± SD.* p < 0.05, ** p < 0.01 vs. Vehicle group. (means ± SD, n = 3).

Figure 4

The cDNA sequence of LncRNA-CIR6.

Figure 5

Construction of LncRNA-CIR6 plasmid.

Figure 6

Experimental protocols and treatment reagents in vitro.

Figure 7

Experimental protocols and treatment reagents in vivo.