LncRNA PCFL promotes cardiac fibrosis via miR-378/GRB2 pathway following myocardial infarction

doi:10.1016/j.yjmcc.2019.06.011

Journal of Molecular and Cellular Cardiology

Volume 133, August 2019, Pages 188-198

https://doi.org/10.1016/j.yjmcc.2019.06.011 Get rights and content

Abstract

Long noncoding RNAs (lncRNAs) are a class of novel molecular regulators in cardiac development and diseases. However, the role of specific lncRNAs in cardiac fibrosis remains to be fully explored. The aim of the present study was to investigate the effects and underlying mechanisms of lncRNA PCFL (pro-cardiac fibrotic lncRNA) on cardiac fibrosis after myocardial infarction (MI).

Cardiac fibroblasts (CFs) with gain and loss of function of PCFL and mice with global knockout or overexpression of PCFL were used to explore the effects of PCFL on cardiac fibrosis. The data showed that PCFL was significantly increased in hearts of mice subjected to MI and CFs treated with transforming growth factor-β1 (TGF-β1). Overexpression of PCFL promoted collagen production and CF proliferation, while silencing PCFL exhibited the opposite effects. Compared with wild type MI mice, heterozygous knockout of PCFL (PCFL^+/−) in mice significantly improved heart function and reduced cardiac fibrosis after MI. While overexpression of PCFL impaired cardiac function and aggravated MI-induced cardiac fibrosis. The mechanistic data demonstrated that PCFL functioned as a sponge of miR-378. Luciferase reporter assay confirmed the interaction of PCFL with miR-378. MiR-378 inhibited collagen production by suppressing its target gene, GRB2 (growth factor receptor bound protein 2). Knockdown of PCFL led to an increase of miR-378. Silencing of miR-378 reserved the inhibitory effects of PCFL knockdown on collagen production, cell proliferation and GRB2 expression. In conclusion, the study identifies a novel pro-fibrotic lncRNA, PCFL, and the mechanism involves the direct interaction of PCFL with miR-378, which in turn relieves the inhibition effect of miR-378 on GRB2 and promotes cardiac fibrosis.

Introduction

Heart disease is a leading cause of death worldwide and cardiac lesion is frequently accompanied with cardiac hypertrophy and fibrosis [1]. Cardiac fibrosis is characterized by a series of pathological changes, including aberrant cardiac fibroblast activity, proliferation and collagen deposition, which is associated with impairment of heart function [2]. Identification and characterization of novel molecules and pathways involved in cardiac fibrosis are indispensable.

Long noncoding RNAs (lncRNAs) are a class of transcripts longer than 200 nucleotides without protein-translation ability [3]. Studies demonstrate that lncRNAs are correlated with a vast array of cell biological functions, for instance, chromatin modification, RNA splicing and protein localization [4,5]. Furthermore, deregulation of lncRNAs is associated with various cardiac diseases. It was reported that CHRF (cardiac hypertrophy related factor) is upregulated in mouse heart subjected to angiotensin II infusion or transaortic constriction (TAC) and it regulates cardiac hypertrophy via repressing miR-489 activity [6]. Han et al. demonstrated that the cardiac-specific myosin heavy chain associated RNA transcripts (Mhrt) is downregulated in TAC hearts, and gain-of-function of Mhrt blocks the recognition of Brg1 to its genomic DNA targets, which inhibits cardiac hypertrophy [7]. LncRNAs have gained a lot of attention for their function in tissue fibrosis. Studies have revealed the roles of lncRNAs in pulmonary, liver or renal fibrosis [[8], [9], [10]]. LncRNAs have also been demonstrated to play crucial roles in cardiac fibrosis. Recently, we demonstrated significant increase of myocardial infarction associated transcript (MIAT) in mice one week after myocardial infarction (MI) and identified this lncRNA as an inducer of cardiac fibrosis [11]. Liang et al. revealed that lncRNA PFL (pro-fibrotic lncRNA) contributes to cardiac fibrosis by acting as a ceRNA of let-7d [12]. However, considering the large number of lncRNAs and their functional complexity, the published studies on lncRNA control of cardiac fibrosis are still rather sparse and there is a necessity to identify and characterize novel lncRNAs that participate in the regulation of cardiac fibrosis.

Our previous study showed that lncRNA KnowTID_00006395 is upregulated in cardiac fibrotic tissue and potentially involved in the process of cardiac fibrosis [13]. In the present study, we investigated the effects of KnowTID_00006395 on cardiac fibrosis. For convenience, here we named this lncRNA pro-cardiac fibrotic lncRNA (PCFL). LncRNA PCFL possesses 2956 bp in length and it is located on mouse chromosome 6 (strand: +, chr6:95,039,797-95,042,894). Our data showed that PCFL promoted cardiac fibroblast proliferation and collagen production, and heterozygous knockout of PCFL (PCFL^+/−) alleviated cardiac fibrosis and improved heart function in mice with MI, and the underlying mechanism likely involves the binding between microRNA-378 (miR-378) and PCFL.

Section snippets

Animals

In this study healthy male C57BL/6 mice (20–25 g) were purchased from the Animal Center of the Second Affiliated Hospital of Harbin Medical University. Mice were kept under standard conditions for animals (Temperature, 21 ± 1 °C; Humidity, 55–60%) and received food and water ad libitum. All experimental procedures were in accordance with and approved by the Institutional Animal Care and Use Committee of the Harbin Medical University.

Mouse model of myocardial infarction

Mice were anesthetized with Avertin (160 mg/kg, Sigma-Aldrich,

Overexpression of PCFL promoted collagen production and proliferation of CFs

Our previous microarray data showed that lncRNA PCFL is significantly increased in MI mice compared with sham controls and has potential association with the fibrosis-related factors in fibrotic tissue of MI mouse hearts [13]. In the present study, the qRT-PCR data further demonstrated that the expression level of PCFL was increased by about 1.2 folds in the border regions of MI heart compared with that in the sham group (Fig. 1A). Consistently, PCFL was also significantly upregulated in

Discussion

In this study, we characterized PCFL as a positive regulator of cardiac fibrosis through directly interacting with miR-378 and mediating its target GRB2. We observed that PCFL was dramatically increased in mice hearts subjected to MI, and in CFs stimulated with TGF-β1. PCFL overexpression enhanced collagen production and cardiac fibrosis both in vitro and in vivo, and knockdown of PCFL exerts anti-fibrotic effects. Furthermore, the effect of PCFL on cardiac fibrosis is dependent on its direct

Conclusion

Collectively, we proposed a novel fibrotic signaling pathway composed of PCFL, miR-378 and GRB2. Although PCFL conservation is low between human and mouse, our study leads to a step forward to better understanding the lncRNA involvement in fibrosis disease and lncRNA may serve as novel target for prevention and treatment of cardiac fibrosis.

Funding sources

The work was supported partly by the National Natural Science Foundation of China (No 81530010, 81872871, 81700219 and 81703510), National Key R&D Program of China (2017YFC1307404).

Declaration of competing interests

None.

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¹: With equal contributions to the work.

View full text

LncRNA PCFL promotes cardiac fibrosis via miR-378/GRB2 pathway following myocardial infarction

Abstract

Introduction

Section snippets

Animals

Mouse model of myocardial infarction

Overexpression of PCFL promoted collagen production and proliferation of CFs

Discussion

Conclusion

Funding sources

Declaration of competing interests

J. Mol. Cell. Cardiol.

Cell

Nat. Commun.

Toxicology

Mol. Cell

Biochim. Biophys. Acta

The cardiac fibroblast: therapeutic target in myocardial remodeling and failure

Annu. Rev. Pharmacol. Toxicol.

Epigenetic coordination of embryonic heart transcription by dynamically regulated long noncoding RNAs

Proc. Natl. Acad. Sci. U. S. A.

RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts

Nat. Rev. Mol. Cell Biol.

The long noncoding RNA CHRF regulates cardiac hypertrophy by targeting miR-489

Circ. Res.

A long noncoding RNA protects the heart from pathological hypertrophy

Nature

LincRNA-Gm4419 knockdown ameliorates NF-kappaB/NLRP3 inflammasome-mediated inflammation in diabetic nephropathy

Cell Death Dis.

A novel lnc-PCF promotes the proliferation of TGF-beta1-activated epithelial cells by targeting miR-344a-5p to regulate map3k11 in pulmonary fibrosis

Cell Death Dis.

MIAT is a pro-fibrotic Long non-coding RNA governing cardiac fibrosis in post-infarct myocardium

Sci. Rep.

LncRNA PFL contributes to cardiac fibrosis by acting as a competing endogenous RNA of let-7d

Theranostics

Expression signature of lncRNAs and their potential roles in cardiac fibrosis of post-infarct mice

Biosci. Rep.