MiR-590-3p regulates cardiomyocyte P19CL6 proliferation, apoptosis and differentiation in vitro by targeting PTPN1 via JNK/STAT/NF-kB pathway

Abstract

Cardiomyocyte differentiation is a multi-step process which involves a number of signalling pathways. microRNAs exhibit regulatory functions in various diseases and are involved in the signalling pathways in multiple physiological processes, but the specific functions of particular mRNAs is often not fully understood. of an example of this is that the role of miR-590-3p in the differentiation of cardiomyocytes remains unclear. In the current study, RT-qPCR was used to determine the expression of miR-590-3p in cardiomyocytes differentiated from the embryonic carcinoma cell line P19CL6. MTT, EdU, caspase-3 activity and flow cytometry assays were performed to examine the influence of miR-590-3p on cell behaviour. A luciferase assay was used to confirm binding between miR-590-3p and PTPN1. Western blotting was used to determine the relationship between the JNK/STAT/NF-kB pathway and PTPN1. The results inferred that miR-590-3p became heavily expressed in differentiated P19CL6. Knockdown miR-590-3p suppressed the cell proliferation while at the same time, accelerated apoptosis. Moreover, PTPN1 was identified as the target of miR-590-3p. More importantly, PTPN1 overexpression activated the JNK/STAT/NF-kB pathway and limited the differentiation of P19CL6. Thus the conclusions from this study are that miR-590-3p has the potential to regulate the proliferation, apoptosis and differentiation of cardiomyocyte P19CL6 in vitro by targeting PTPN1 via the JNK/STAT/NF-kB pathway.

Keywords: JNK; MiR-590-3p; NF-kB pathway; P19CL6; PTPN1; STAT.

Figure 1

MiR‐590‐3p is highly expressed in differentiated P19CL6 cells. A, RT‐qPCR was utilized to test the expressions of GATA4, α‐MHC and miR‐590‐3p in P19CL6 cells. **P < .01

Figure 2

Silenced miR‐590‐3p inhibits proliferation and accelerates apoptosis of P19CL6 cells. A, RT‐qPCR was adopted to test the interference efficiency of miR‐590‐3p in P19CL6 cells. B‐C, MTT and EdU experiments were adopted to test the capability of cell proliferation after silencing miR‐590‐3p. D‐E, The cell apoptotic capability was estimated through flow cytometry and caspase‐3 activity detection assays, when miR‐590‐3p was subjected to inhibition. **P < .01

Figure 3

PTPN1 acts as the target of miR‐590‐3p in P19CL6 cells. A, The binding sites of miR‐590‐3p and PTPN1. B, Luciferase reporter assays were adopted to test the binding situation of miR‐590‐3p and PTPN1. C, RT‐qPCR was conducted to test the expression of PTPN1 in P19CL6 cells. D, RT‐qPCR was utilized to detect the relationship of miR‐590‐3p and PTPN1. E, RT‐qPCR was carried out to test the overexpression efficiency of PTPN1 in P19CL6 cells. F‐G, MTT and EdU experiments were done to test the cell proliferative capability after PTPN1 got expressed. H‐I, Flow cytometry and caspase‐3 activity detection assays were conducted to estimate cell apoptosis when PTPN1 got expressed. **P < .01

Figure 4

Upregulation of PTPN1 activates JNK/STAT/NF‐kB pathway and restrains differentiation of P19CL6 cells. A, Western blot experiments were carried out to test the protein levels of phosphorylated JNK, phosphorylated STAT and Bcl‐2. B‐C, RT‐qPCR was utilized to detect the expression of GATA4 and α‐MHC. **P < .01