Alu-mediated p21 transcriptional regulator and long intergenic non-coding RNA-p21 regulate the p21-mediated progression of liver fibrosis

Animal cells synthesize and secrete ECM, which is distributed along the cell surface or between cells. An array of primary and/or secondary causes of ECM synthesis and an imbalance between ECM synthesis and degradation may induce fibrosis. Hepatic stellate cells (HSCs) are the predominant producers of ECM during liver fibrogenesis.^{20, 21} Current studies suggest that the activation of HSCs, such as by liver injury, and the subsequent secretion of ECM are key mechanisms in the initiation and progression of liver fibrosis.^{22, 23, 24} However, this process reverts to the original (non-fibrotic) state after removal of the liver damage stimulus.^{25, 26}

Collagen is the main constituent of a fibrotic scar, and TGF-β promotes its expression in HSC myofibroblasts.^{27, 28, 29} Negishi et al.³⁰ recently performed a small-interfering (si)RNA screen of human lncRNAs to identify the molecules that contribute to cell proliferation. Alu-mediated p21 transcriptional regulator (APTR) was identified as an lncRNA target, and it acted in trans to repress the promoter of the CDKN1A/p21 tumor suppressor gene and promote cell proliferation independently of p53. A detailed molecular analysis revealed that APTR recruited polycomb repressive complex 2 (PRC2) to repress the activity of the p21 promoter and inhibit p21 expression³⁰ (Figure 1). Another study demonstrated increased APTR levels in serum and liver biopsy samples and activated HSCs from patients with liver cirrhosis.³¹ Knockdown of APTR inhibited HSC activation, mitigated the accumulation of collagen, and abrogated the TGF-β1-induced upregulation of α-smooth muscle actin (α-SMA) in HSCs. However, treatment with p21 siRNA attenuated the inhibition of cell cycling and cell proliferation that occurred in primary HSCs following APTR knockdown.³¹

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Figure 1

Schematic diagram of the effects and underlying mechanisms of lncRNAs in various fibroses. Green: LncRNAs involved in liver fibrosis. APTR promotes HSC activation via recruitment of the PRC2 complex to and subsequent inhibition of the p21 promoter. The inhibited expression of the tumor repressor gene p21 promotes ECM synthesis and liver fibrosis. LincRNA-p21 also promotes liver fibrosis via a direct decrease in p21 expression. MEG3 inhibits cell proliferation and induces apoptosis, but TGF-β1 decreases MEG3 expression via mediation of the methylation of the MEG3 promoter, which induces liver fibrosis. MiRNA-222 is involved in liver fibrosis via targeting of the GAS5 gene. However, GAS5 is a competing endogenous RNA (ceRNA) that directly binds to miR-222 and increases the level of the tumor suppressor p27, which inhibits HSC activation and proliferation. GAS5 is downregulated in activated HSCs and fibrotic liver tissues. Yellow: LncRNAs involved in cardiac fibrosis. TGF-β1-induced upregulation of lncRNA H19 correlates with a gradual decrease in DUSP5 expression, which represses the DUSP5/ERK1/2 signaling pathway in primary rat cardiac fibroblasts. Ang II treatment decreases the levels of lncRNA-NR024118 and Cdkn1c in cardiac fibroblasts via its cognate receptor, Ang II receptor type I (AT1). MIAT functions as a ceRNA for miR-24 and upregulates TGF-β1 expression to induce the subsequent activation of CFs. Blue: LncRNAs involved in kidney fibrosis. H19 is upregulated in TGF-β2-induced HK-2 cell fibrosis via sponging of miR-17 (artificial miRNA decoy), and np_5318/np_17856 level is upregulated in TGF-β-induced cells, which promotes the fibrotic process. CYP4B1-PS1-001/ ENSMUST0000 0147869 is downregulated in kidney tissues from db/db mice and promotes the proliferation of MCs. Brown: LncRNAs involved in lung fibrosis. MRAK088388 may be regulated in lung myofibroblast growth and collagen deposition via sponging of miR-29, which binds to N4bp2. MRAK081523 may compete with the let-7i pool to regulate the expression of Plxna4. MiR-489 may function as an anti-fibrotic miRNA via targeting MyD88 and Smad3, and the lncRNA CHRF inhibits miR-489 expression. Upregulated uc.77 and 27000086A05Rik target Zeb2 and Hoxa3, respectively. Downexpression of BGas induces CFTR overexpression and results in LEC proliferation and ECM production. ‘T’ (blunt) ends of connectors represent negative regulation, and pointed (arrowhead) ends represent positive regulation. Red-colored bold font indicates lncRNAs. CFs, cardiac fibroblasts; ECM, extracellular matrix; HSCs, hepatic stellate cells; LECs, lung epithelial cells; MCs, mesangial cells.

Other reports indicated that long intergenic non-coding RNA (lincRNA)-p21 inhibited liver fibrosis via promotion of p21 expression.³² Studies demonstrated that lincRNA-p21 induced apoptosis in mouse cell models,^{31, 33} and its expression is decreased in tumors.³⁴ Zheng et al.³² recently reported that the targeting the tumor suppressor gene p21 markedly reduced lincRNA-p21 expression in mouse liver fibrosis models and human cirrhotic liver (Figure 1). Liver cirrhosis patients, especially patients with decompensated cirrhosis, exhibited significantly lower serum lincRNA-p21 levels compared to healthy subjects. Overexpression of lincRNA-p21 in vitro upregulated p21 mRNA and protein levels, inhibited cell cycle progression and the proliferation of primary HSCs and reversed the activation of HSCs to their quiescent phenotype. Lentivirus-mediated lincRNA-p21 transfer into these mice decreased the severity of liver fibrosis.³² These studies collectively revealed a new biological role of APTR and lincRNA-p21 in liver fibrogenesis and suggest the potential of these molecules as biomarkers or therapeutic targets to clinically address liver fibrosis via the targeting of a common tumor suppressor gene, p21^{31, 32} (Figure 1; Table 1).

Inhibitory effects of lncRNA MEG3 in liver fibrosis

Maternally expressed gene 3 (MEG3) is an imprinted gene located at 14q32 that encodes an lncRNA correlated with several human cancers.³⁵ MEG3 does not encode any protein, and it functions at the RNA level.^{36, 37} A methylation-dependent downregulation of MEG3 was described in liver cancers.³⁵ Braconi et al.³⁸ reported that the forced expression of MEG3 in hepatocellular carcinoma cells significantly decreased anchorage-dependent and anchorage-independent cell growth and induced apoptosis. He et al.¹³ recently investigated the role of MEG3 in the development of liver fibrosis and TGF-β1-induced HSC activation. These authors demonstrated that MEG3 levels were remarkably decreased in carbon tetrachloride (CCl₄)-induced mouse liver fibrosis models and human fibrotic livers. MEG3 expression was downregulated in the human HSC line LX-2 in response to TGF-β1 stimulation, and this effect was dose- and time-dependent. This downregulation of MEG3 expression was likely mediated by hypermethylation of the MEG3 promoter, which was identified using methylation-specific PCR. Inhibition of methylation by treatment with 5-aza-2-deoxycytidine or siRNA targeted to DNA methyltransferase 1 (DNMT1) robustly increased MEG3 expression in TGF-β1-induced LX-2 cells, which activated p53, mediated cytochrome c release, and induced caspase-3-dependent apoptosis¹³ (Figure 1; Table 1).

LncRNA H19 control of the dual-specificity phosphatase 5/ERK1/2 axis contributes to cardiac fibrosis

LncRNA H19 is a 3-kb ncRNA that is expressed in the nucleus and cytoplasm. H19 is activated in embryonic cells, and it is highly expressed during embryogenesis. H19 expression is significantly reduced after birth, but it may significantly increase in disease conditions.⁶³ The primary function of H19 is the upregulation of cell activation.⁶⁴ The downregulation of dual-specificity phosphatase 5 (DUSP5) increased cell proliferation,⁶⁵ and this effect was mediated via epigenetic events involving lncRNA H19 in a human choriocarcinoma cell line.⁶⁶ Tao et al.¹⁰ recently demonstrated the ability of H19 to negatively regulate DUSP5 gene expression in cardiac fibroblast and fibrosis tissues. These authors also demonstrated an upregulation of H19 expression in response to TGF-β1 treatment in freshly isolated rat cardiac fibroblasts, which correlated with the gradual decrease in DUSP5 expression. Decreased DUSP5 expression was the consequence of at least a partial repression of DUSP5/ERK1/2. H19 ectopic overexpression reduced DUSP5 abundance and increased the proliferation of cardiac fibroblasts, and H19 silencing induced opposite effects. Therefore, the collective literature suggests a broader perspective for H19 beyond its roles in tumor cells, with functional contributions to cardiac fibroblast proliferation and fibrosis (Figure 1; Table 1).

LncRNA-NR024118 and Cdkn1c in adult rat cardiac fibroblasts

Angiotensin II (Ang II) increases blood pressure via stimulation of the Gq protein in vascular smooth muscle cells (VSMCs), which activates an IP3-dependent mechanism to increase intracellular calcium levels, induce blood vessel contraction, and increase blood volume and pressure.⁶⁷ Ang II also plays numerous roles in cardiac fibroblasts, including the stimulation of cardiac fibroblasts proliferation and ECM synthesis and the promotion of cytokine secretion, which eventually leads to cardiac fibrosis.^{68, 69}

Recent research indicates that Ang II regulates specific lncRNAs in the pathogenesis of cardiac fibrosis. Jiang et al.⁷⁰ analyzed the expression profile of lncRNAs in Ang II-treated cardiac fibroblasts using lncRNAs arrays to investigate the role of lncRNAs in cardiac fibrosis. These authors found that 282 of the 4376 detected lncRNAs exhibited a >2-fold differential expression in response to 24-h Ang II (100nm) treatment. Twenty-two of the lncRNAs exhibited a greater than four-fold change. Ang II also induced broad expression changes in protein-coding genes in cardiac fibroblasts. Quantitative real-time PCR confirmed the changes of six lncRNAs (AF159100, BC086588, MRNR026574, MRAK134679, NR024118 and AX765700) and nine mRNAs (IL6, RGS2, PRG4, TIMP1, Cdkn1c, TIMP3, Col I, Col III and fibronectin) in cardiac fibroblasts. The Ang II-induced decrease in lncRNA-NR024118 and Cdkn1c was mediated by an Ang II receptor type I (AT1)-dependent pathway and not an AT2 receptor-dependent pathway, as demonstrated using AT1 and AT2 blockers, respectively⁷⁰ (Figure 1; Table 1). These results provide a foundational understanding of the molecular mechanisms of Ang II receptors in adult rat cardiac fibroblasts.

LncRNAs associate with TGF-β/Smad3-mediated renal fibrosis

TGF-β/Smad signaling plays a critical role in renal fibrosis and inflammation in chronic kidney diseases, and Smad3 is a key mediator of downstream TGF-β/Smad signaling, which mediates renal inflammation and fibrosis via several miRNAs.^{78, 79, 80} Zhou et al.⁸¹ recently identified the Smad3-dependent lncRNAs related to renal inflammation and fibrosis in Smad3 knockout mouse models of unilateral ureteral obstructive (UUO) nephropathy and immunologically induced anti-glomerular basement membrane glomerulonephritis (anti-GBM GN). A total of 151 lncRNAs in UUO kidney and 413 lncRNAs in anti-GBM GN kidneys were significantly altered in the Smad3 knockout mice compared to wild-type mice. Twenty-one novel lncRNAs were co-expressed in both disease models. Twenty-one lncRNAs were upregulated in wild type but downregulated in Smad3 knockout mice. The kidneys in both disease models exhibited progressive renal inflammation and fibrosis and beneficial effects following Smad3 gene deletion or suppression. The pool of lncRNAs that are differentially expressed in the UUO kidney may be related to fibrogenesis because progressive renal fibrosis in the UUO kidney is Smad3-dependent.^{80, 82, 83} Real-time PCR confirmed these findings and revealed a functional link between the Smad3-dependent lncRNA np_5318/np_17856 and progressive kidney injury (Figure 1; Table 1). This study suggested that the identification and characterization of functional lncRNAs associated with kidney disease represent a promising research direction for renal disorders and may lead to the development of new lncRNA-based therapies.

LncRNAs MRAK088388 and MRAK081523 as ceRNAs in pulmonary fibrosis

Song et al.⁹³ evaluated the functions of lncRNAs in pulmonary fibrosis by searching portions of lncRNAs that were adjacent or homologous to protein-coding genes in the UCSC genome bioinformatics database (https://genome.ucsc.edu/). The authors selected two differentially expressed lncRNAs, MRAK088388 and MRAK081523, for detailed examination of their regulatory mechanisms. Both lncRNAs were analyzed as lincRNAs and identified as orthologues of mouse lncRNAs AK088388 and AK081523, respectively, which are significantly upregulated in the bleomycin-induced pulmonary fibrosis mouse model, which was evidenced using quantitative reverse transcription-PCR and in situ hybridization assays. These authors also found that MRAK088388 and N4bp2 (a Bcl-3 binding protein) shared miRNA response elements for miR-200, miR-429, miR-29 and miR-30, and MRAK081523 and Plxna4 (a tumor-promoting protein) shared miRNA response elements for miR-218, miR-141, miR-98 and let-7.⁹³ The expression levels of N4bp2 and Plxna4 increased significantly in fibrotic rats and highly correlated with MRAK088388 and MRAK081523 expression, respectively. MiR-29b-3p and let-7i-5p were decreased in the model group and negatively correlated with MRAK088388 and MRAK081523 expression, respectively. MRAK088388 and MRAK081523 regulate N4bp2 and Plxna4 expression via sponging miR-29b-3p and let-7i-5p, respectively, and exhibited regulatory functions as ceRNAs.⁹³ The resulting insights of this study into the functional interactions of lncRNAs, miRNAs and mRNAs may lead to new theories on the pathogenesis and treatment of pulmonary fibrosis (Figure 1; Table 1).

LncRNA CHRF promotes silica-induced pulmonary fibrosis via targeting of miR-489

Silicosis is an incurable occupational disease associated with inflammation, fibroblast proliferation and an accumulation of ECM in lung tissues.⁹⁴ Ji et al.⁹⁵ reported decreased expression levels of miR-489 in lung tissues of silica-induced pulmonary fibrosis detected using miRNA microassay screening. Wu et al.⁹⁶ examined the role of miR-489 in a mouse model of silicosis and found reduced miR-489 levels in silica-exposed macrophages and TGF-β1-exposed fibroblasts. The results of in vivo miR-489 overexpression were consistent with its anti-fibrotic role of attenuating inflammation and fibrotic progression. The underlying molecular mechanisms include miR-489 inhibition of silica-induced pulmonary fibrosis, which occurs primarily via repression of its target genes MyD88 and Smad3. However, upregulation of the lncRNA cardiac hypertrophy-related factor (CHRF) in the mouse model reversed the inhibitory effect of miR-489 on MyD88 and Smad3 to trigger the inflammation and fibrotic signaling pathways⁹⁶ (Figure 1; Table 1). Therefore, the CHRF-miR-489-MyD88 Smad3 signaling axis appears to exert key functions in silica-induced pulmonary fibrosis and may represent a novel therapeutic target for silicosis.

LncRNA BGas regulates cystic fibrosis

Cystic fibrosis is a lethal multisystem, autosomal recessive disorder, but the detailed mechanisms of this disease are not known. McKiernan et al.⁹² defined the expression profile of 30586 lncRNAs using a microarray assessment of bronchial cells isolated from endobronchial brushings of cystic fibrosis and non-cystic fibrosis individuals. A total of 1063 lncRNAs exhibited differential expression, and gene ontology bioinformatics analysis highlighted that the numerous over-represented processes in the cystic fibrosis bronchial epithelium were related to inflammation. TLR8 natural antisense lncRNA (the 1349-bp TLR8-AS1 transcript) was downregulated in cystic fibrosis bronchial epithelium and exhibited low-level expression in three of the nine non-cystic fibrosis controls and only one of seven cystic fibrosis bronchial epithelial samples (absent in all other samples). However, this study did not further examine how TLR8-AS1 contributed to the molecular pathogenic processes of cystic fibrosis.

The root cause of cystic fibrosis is heritable recessive mutations that affect the cystic fibrosis transmembrane conductance regulator (CFTR) gene and the subsequent expression and activity of encoded ion channels at the cell surface.⁹⁷ Saayman et al.⁹ confirmed that lncRNA BGas contributed to cystic fibrosis pathogenesis via regulation of CFTR gene expression. These authors demonstrated that CFTR was regulated transcriptionally by the actions of lncRNA BGas, which emanates from intron 11 of the CFTR gene and is expressed in the antisense orientation relative to the protein-coding sense strand.⁹ BGas functions in concert with several proteins, including HMGA1, HMGB1 and WIBG, to modulate the local chromatin and DNA architecture of intron 11 of the CFTR gene, which affects transcription. Suppression of BGas or its associated proteins results in a gain of CFTR expression and chloride ion function⁹ (Figure 1; Table 1). These observations highlighted the possible effects of certain lncRNAs on cystic fibrosis pathogenesis, such as the involvement of TLR8-AS1 in the chronic infection and inflammation that exist in the lungs of people with cystic fibrosis and the involvement of BGas in the normal function of ion channels. The collective data also suggest that these lncRNAs represent novel therapeutic targets for cystic fibrosis.

This work was supported by grants from the Major State Basic Research Development Program of China (no. 2013CB531503), the National Foundation of China (no. 81502728), the Anhui Provincial Natural Science Foundation (no. 1408085MH149) and the Research Program of Foundation Science and Application Technology of Chongqing (nos. cstc2015jcyjA10105 and cstc2015jcyjA10119). The funders had no role in the study design, data analysis, or decision to publish.