H19 potentiates let-7 family expression through reducing PTBP1 binding to their precursors in cholestasis

Cholestasis induces the hepatic long non-coding RNA H19, which promotes the progression of cholestatic liver fibrosis. However, microRNAs that are dysregulated by H19 during cholestasis remain elusive. Using miRNA-sequencing analysis followed by qPCR validation, we identified marked upregulation of eight members of the let-7 family in cholestatic livers by bile duct ligation (BDL) and H19 overexpression. In particular, the expression of let-7a-1/7d/7f-1 was highly induced in H19-BDL livers but decreased in H19KO-BDL livers. Interestingly, H19 decreased the nuclear let-7 precursors as well as the primary transcripts of let-7a-1/7d/7f-1 levels in BDL mouse livers. Bioinformatics, RNA pull-down, and RNA immunoprecipitation (RIP) assays revealed that the crucial RNA-binding protein polypyrimidine tract-binding protein 1 (PTBP1), an H19 interaction partner, interacted with the precursors of let-7a-1 and let-7d and suppressed their maturation. Both PTBP1 and let-7 expression was differentially regulated by different bile acid species in hepatocyte and cholangiocyte cells. Further, H19 negatively regulated PTBP1’s mRNA and protein levels but did not affect its subcellular distribution in BDL mouse livers. Moreover, we found that H19 restrained but PTBP1 facilitated the bioavailability of let-7 miRNAs to their targets. Taken together, this study revealed for the first time that H19 promoted let-7 expression by decreasing PTBP1’s expression level and its binding to the let-7 precursors in cholestasis.


Introduction
The imprinted oncofetal long non-coding RNA (lncRNA) H19 is one of the first identified imprinted lncRNAs and is predominantly distributed in the cytoplasm of cells 1,2 . Due to the methylation modifications within the differentially methylated region (DMR) of H19 promoters, H19 is only transcribed from the maternally inherited allele while the paternal H19 allele is not expressed 3 . The aberrant H19 expression has been frequently linked to human Beckwith-Wiedemann syndrome and Silver-Russell syndrome 4,5 . Intriguingly, H19 maintains a high expression level in embryogenesis but is barely detectable in most of the tissues after birth except muscle and heart, implying a crucial role in mammal development and growth 3 . Although extensive studies have revealed important roles of H19 in various cancers 6 , the regulation of H19 in human liver diseases is largely uncovered. Emerging evidence shows that reactivation of H19 expression exacerbates cholestatic liver fibrosis 7 and the development of fatty liver 8 . Phenotypically, the high induction of H19 expression is observed in human cirrhotic livers 9 . Despite these recent advances, the downstream molecular networks of H19 in liver pathogenesis remain elusive.
The polypyrimidine tract-binding protein 1 (PTBP1, also known as PTB or heteronuclear ribonucleoprotein (hnRNP) I) is an RNA-binding protein and regulates precursor mRNA (pre-mRNA) splicing, alternative splicing events, and mRNA stability 10 . PTBP1 has been implicated in different liver diseases 8 . PTBP1 complexes with heterogeneous nuclear RNA in the nucleus to regulate pre-mRNA processing and other aspects of mRNA metabolism and transport. PTBP1 has been reported to associate with multiple lncRNAs. For instance, maternally expressed 3 (MEG3), another lncRNA, binds to PTBP1 to control small heterodimer partner mRNA stability and cholestatic liver injury 11 , whereas H19 binds PTBP1 and reprograms hepatic lipid homeostasis 8 . In most mammals, there are two tissue-specific isoforms of PTBP. PTBP1 is widely expressed, while PTBP2 (also called nPTB or brPTB) is mainly expressed in neurons and testis 12 . The PTBP proteins preferentially bind CU tracts (e.g., UCUUC and CUCUCU, located within a polypyrimidine-rich context in RNAs). Because all four repeats of quasi-RNA recognition motif domains in PTBP can bind RNAs, it is difficult to define one RNA consensus sequence and to identify RNA targets of PTBP 13 . The interaction between PTPB1 with miRNAs has been noticed 14 , but the exact role of how PTPB1 regulates miRNA expression remains to be determined. Let-7 belongs to a family of miRNAs required for development timing, tumor suppression, and metabolism regulation 15 . To generate a let-7 miRNA, a primary transcript (pri-let-7) is transcribed by RNA polymerase II and then subsequently processed. Pri-let-7 is cleaved by the microprocessor complex, composed of Drosha and its cofactor DGCR8, to produce precursor let-7 (pre-let-7) in the nucleus. Pre-let-7 is then exported into the cytoplasm and cleaved into an~22-nucleotide duplex by Dicer complex, followed by unloading into argonaute (AGO) proteins that are essential components of the RNAinduced silencing complex (RISC) 15,16 . In addition to these basic processing factors, the biogenesis of let-7 is also tightly regulated by other cellular factors, such as the RNA-binding proteins (RBPs) LIN28A/B and DIS3L2 17,18 . Dysregulation of let-7 processing contributes to multiple pathological processes including cholestatic liver diseases 19 .
The goal of this study is to identify aberrant miRNAs that are regulated by H19 in cholestatic liver fibrosis. In this study, we determined the role of H19 and its binding protein PTPB1 in the expression and bioavailability of a cluster of let-7 miRNAs in cholestasis. The results show that H19 represses PTPB1 expression in cholestatic mouse livers, which is permissive to let-7 maturation from precursors. We also reveal that the bioavailability of let-7 miRNAs is suppressed by H19 but facilitated by PTPB1.

Mouse models
H19-deficient mice were described previously 7 . Because H19 is a paternally imprinted gene, maternal H19-deleted mice were used for experiments and paternal H19-deleted mice were used as negative controls. Mice were fed a standard rodent chow diet (#2918, Teklad global 18% protein rodent diets; Envigo Bioproducts, Inc) with free access to water and maintained in a 12 h light/dark cycle (light on 6 a.m. to 6 p.m.), temperature-controlled (23°C), and pathogen-free facility. In vivo experiments were performed on mice (n = 3-5) at the age of 6 weeks unless stated otherwise. Considering sex as a biological variable does not affect the induction of cholestasis after bile duct ligation (BDL), only male mice were involved in the study to reduce animal use. For in vivo viral transduction 20 , mice (C57BL/6J, Jackson Laboratory, Bar Harbor, ME) were injected via tail vein with purified adeno-associated viral vector serotype 8 (AAV8) containing a liver-specific thyroxine-binding globulin promoter driving H19 gene expression (5 × 10 10 virus particles/mouse). The operation of common BDL has been described previously 21 . All experiments were performed in accordance with the guidelines and regulations approved by the Institutional Animal Care and Use Committee at the University of Connecticut.

MicroRNA-sequencing analysis
MicroRNA-sequencing (miR-seq) was conducted at the Division of Experimental and Translational Genetics, Children's Mercy Hospital (Kansas City, MO). A total of 14 livers were subjected to miR-seq analysis (Null-sham, n = 3; Null-BDL, n = 4; H19-sham, n = 3; and H19-BDL, n = 4). The read counts of miR-seq were used as criteria to compare miRNA levels. Quantitative PCR (qPCR) validation of miRNA expression was conducted in a new cohort of male mice subjected to sham or BDL (n = 5/ group).

Cell culture
Cell culture procedures for human hepatocellular carcinoma (HCC) cell line Huh7 and mouse HCC cell line Hepa1 have been reported previously and maintained in Dulbecco's modified Eagle's medium with 100 μg/ml streptomycin, 100 U/ml penicillin, and 10% fetal bovine serum 22,23 . Cell culture procedures for mouse small cholangiocytes (MSCs) and mouse large cholangiocytes (MLCs) were described previously 7 . Mouse primary hepatocytes were isolated and cultured as described previously 24 .

Preparation of RNAs from nuclear and cytoplasmic extracts
As described previously, the nuclear and cytoplasmic fractions of mouse liver tissues were prepared using a hypotonic buffer and a high-salt buffer that were supplemented with RNase inhibitor (1 U/µl) 22 . The fractions were immediately extracted for RNAs using Trizol reagent.
Biotinylated RNA pull-down assay and RNA immunoprecipitation assay A biotinylated RNA pull-down assay was performed as described previously 11 . Briefly, whole-cell lysates were incubated fully with purified biotinylated RNA probes that were synthesized via in vitro transcription. RNA-protein complexes were further isolated by Streptavidin Sepharose High-Performance beads (GE Healthcare, Marlborough, MA). The recruited proteins were detected by western blotting with antibodies. For RNA immunoprecipitation (RIP), anti-PTBP1 antibody or mouse immunoglobulin G (Sigma) was incubated with ultravioletcrosslinked cell lysate for 2 h with gentle shaking. Protein A/G agarose beads were added to recruit RNA-protein complexes. RNAs associated with PTBP1 were recovered with Trizol-chloroform and analyzed by reverse transcription-qPCR.

Other standard methods
Western blotting, RNA extraction from cells and liver tissues, qPCR, transient transfection, and luciferase reporter assays were performed as described previously 22,[28][29][30][31] . The primers were listed in Supplementary  Table 2. Additional results were included in the Supplementary Figure files.

Statistical analysis
All cell-based in vitro experiments were performed in triplicate and repeated at least two times. All animalbased in vivo experiments were performed with various animals (n = 3-5) per group based on an estimated statistical power for over 80% possibility to find significant difference. The data were displayed as the mean values ± standard error of the mean 32 . Statistical analysis was carried out using the Student's t test. P < 0.05 was considered statistically significant.
Taken together, the above results demonstrated that H19 positively regulated hepatic let-7 family expression in cholestatic mice.
Bile acid species exhibit differential potency to regulate let-7 expression BDL-induced cholestasis causes the disruption of bile flow thus the accumulation of bile acids (BAs) in the liver, which deteriorates biliary epithelia (cholangiocytes) to promote the development of biliary hyperplasia, intrahepatic inflammatory response, and biliary liver fibrosis. In order to determine whether BAs regulate let-7 family expression, mouse primary hepatocytes were isolated from WT mice and treated with various BAs. Since let-7a-5p/7d-5p/7f-5p were significantly upregulated by H19 in cholestatic mouse livers, we focused on the expression of these three let-7 family members. Overall, the expression of let-7a-5p, let-7d-5p, and let-7f-5p was induced by different BAs to various extents (Fig. 2a). UDCA showed the highest potency to induce all three let-7 members, whereas LCA appeared to moderately repress let-7a-5p and let-7d-5p expression.
We also examined the effects of BAs on let-7 expression in MSCs and MLCs. In MSC cells, CA exhibited the highest potency to induce let-7a-5p, let-7d-5p, and let-7f-5p expression, whereas CDCA inhibited all three let-7 expression (Fig. 2b). In MLC cells, TCA and LCA induced let-7a-5p; CA and TCA induced let-7d-5p; CA and LCA induced let-7f-5p, whereas CDCA inhibited all three let-7 members (Fig. 2c). Because these BAs showed no effects on H19 promoter luciferase reporter activities in MSC and MLC cells (Fig. S2A), we postulated that the induction of let-7a-5p/7d-5p/7f-5p in H19-BDL mice might be presumably not due to a direct activation of H19 transcription by BAs.

H19 promotes maturation of let-7 miRNAs in BDL-induced cholestasis
Let-7 family members are expressed either independently or as clusters from different genomic location. The biogenesis of let-7 miRNA is a complex process involving two key processing steps, i.e. from primary-miRNA (pri-miR) to the pre-miR and from pre-miR to the miR 15 . The murine let-7a-5p/7d-5p/7f-5p are derived from the let-7a-1/7d/7f-1 cluster in chromosome 13 (Supplementary  Table 1); the pri-miRNAs from this cluster occupy a substantial portion of all let-7 primary transcripts (~24% in human) 34 .

PTBP1 physically interacts with pre-let-7a-1 and pre-let-7d
The biogenesis of let-7 is subjected to the regulations at genomic, transcriptional, and posttranscriptional levels 35 . To date, several RBPs, which recognize let-7 precursors and affect their processing have been characterized. For instance, Lin-28 homology B (Lin-28B) serves as a classic suppressor of let-7 biogenesis via binding to their precursors 17 . PTPB1 has been identified as an interacting partner of H19 8 and is involved in RNA processing, transport, and metabolism through a direct association with target RNAs 12,36 .
To determine whether PTBP1 participates in the biogenesis of let-7 miRNAs, online bioinformatics tools (RBPmap and starBase v2.0) were employed to predict PTBP1-binding motifs in the stem-loop sequences of both mouse and human let-7a-1, let-7f-1, and let-7d (they are clustered in the same chromosome). RBPmap analysis revealed that there were multiple potential PTBP1binding motifs within pre-let-7a-1 and pre-let-7d but not within pre-let-7f-1 in both humans and mice ( Fig. 4a  and Fig. S3).

H19 negatively regulates PTBP1 expression in mouse cholestatic livers
Next, we examined whether H19 affected PTBP1 expression in BDL-induced cholestasis. qPCR results demonstrated that PTPB1 mRNA was significantly decreased in H19-BDL livers vs Null-BDL livers, and increased in HKO-BDL livers vs Con-BDL livers (Fig. 5a). Similarly, PTBP1 protein levels were downregulated by H19 overexpression and upregulated by H19 depletion (Fig. 5b). Corresponding to the induction of H19 in cholestasis, we examined PTBP1 mRNA expression in BA-insulted cells in vitro and found that it was significantly decreased by the treatments with several BAs in MSC cells (Fig. S4). In addition, CDCA reduced PTBP1 mRNA in hepatocytes, whereas UDCA decreased PTBP1 mRNA in MLC cells.
The subcellular shuttling between the cytoplasm and nucleus is critical to PTBP1's function 38 . In H19-BDL livers, PTBP1 protein was reduced in both cytoplasmic and nuclear fractions in comparison with Null-BDL livers (Fig. 5c). The simultaneous increase of PTBP1 protein in both subcellular fractions was found in H19-deficient cholestatic (HKO-BDL) livers (Fig. 5d). Interestingly, the indispensable miRNA processing factor protein AGO2 (also named as RISC catalytic component) decreased in cytoplasmic fractions but accumulated in nuclear fractions of H19-BDL livers (Fig. 5c); in addition, AGO2 was remarkably increased in the cytoplasmic fractions of HKO-BDL livers (Fig. 5d). These suggest that H19 might dampen the function of RISC through inhibiting AGO2 to regulate miRNA bioavailability. Overall, these findings indicated that H19 suppressed PTBP1 expression without altering its subcellular localization in cholestatic mouse livers.
Therefore, these findings suggested that H19 increased let-7a-5p/7d-5p production, with an inhibitory effect on the bioavailability to their targets, and that PTBP1 decreased let-7a-5p/7d-5p production and promoted their bioavailability. We examined the expression levels of several let-7 targets to interrogate the functional significance of H19 regulation of let-7 in BDL-induced cholestatic livers, considering the opposite effects of H19 on let-7 expression and bioavailability. The hepatic mRNA expression of several inflammation-related target genes of let-7, including Stat3 39 , Hmga2 40,41 , Tlr4 42 , Il6 43 , and Il10 43 significantly increased in H19-BDL vs Null- Fig. 4 Polypyrimidine tract-binding protein 1 (PTBP1) physically interacts with pre-let-7a-1/7d. a Predicated PTBP1-binding sites in the human pre-let-7a-1/7d using RBPmap (http://rbpmap.technion.ac.il). The position, genomic motifs, occurrence, z-score, and P-value were shown. b RNA pulldown followed by western blotting. Either cell lysates of Hepa1 or Huh7 or cell lysates prepared from Hepa1 or Huh7 cells transfected without (−) or with (+) H19 overexpression plasmid were incubated with biotin-labeled pre-let-7d (sense or antisense) or pre-let-7a-1 (sense or antisense) probes. After pull down, the recruited PTBP1 to probes was examined by western blotting with anti-PTBP1 antibody. c RNA-immunoprecipitation (RIP) assay to detect the interactions between PTBP1 and pre-let-7a-1 or pre-let-7d using anti-PTBP1 or immunoglobulin GIgG (negative control) in Huh7 cells. Data were shown as mean ± SEM from triplicate assays. ***P < 0.001 BDL livers (Fig. 6c), suggesting H19 might decrease let-7 bioavailability to exacerbate BDL-induced hepatic inflammatory response, which is in concordance with the previous finding that H19-BDL livers develop more severe liver injury and fibrosis than Null-BDL livers 7 . In contrast, the hepatic mRNA expression of metabolism-related let-7 target genes Lpl 44 , Igf1r 45 , and Insr 45 did not show significant change after H19 overexpression (Fig. 6d). H19-BDL livers displayed more protein expression of STAT3 and TLR4, with an undetectable level of HMGA2 revealed by western blotting (Fig. 6e). Further, ELISA revealed that IL-6 but not IL-10 increased in H19-BDL vs Null-BDL livers (Fig. 6f).

Discussion
The dysregulation of let-7 miRNAs is found in cholestatic liver disorders 19,46 . In this study, we reveal that lncRNA H19 promotes the biogenesis and expression of a cluster of let-7 miRNAs, including let-7a-5p, let-7d-5p, and let-7f-5p, in cholestatic mouse livers, and functionally suppresses their bioavailability. We also find that H19 antagonizes the expression of PTBP1, an H19-interacting protein, which associates with pre-let-7d and pre-let-7a-1 and inhibits let-7 biogenesis but promotes their bioavailability (Fig. 7).
Because of the hydrophobic or hydrophilic nature as well as the different affinity to BA receptors, BAs act as signaling molecules but display differential potency to regulate gene expression 47 . We find that the hydrophilic UDCA significantly and to the maximal extent increases the expression of let-7a-5p/7d-5p/7f-5p in primary hepatocytes but almost has no effect in MLC and MSC cells (Fig. 2). A previous study shows that H19 is only induced by LCA among several BAs in primary hepatocytes 7 . The inconsistent induction of H19 and let-7 miRNAs by UDCA in primary hepatocytes suggest that UDCA may upregulate let-7a-5p/7d-5p/7f-5p in hepatocytes independent of H19. Indeed, H19 is mainly expressed in cholangiocytes and cholangiocyte-derived exosomes, which are the sources of H19 in hepatocytes during cholestatic disease progression 48 . We show that LCA moderately decreases let-7a-5p and let-7d-5p in primary hepatocytes, which apparently disputes a positive correlation between H19 and let-7 miRNAs expression. Thus, isolating primary hepatocytes from cholestatic livers may help to establish a positive expression correlation between let-7 and H19 in hepatocytes. Indeed, in primary hepatocytes isolated from cholestatic mouse livers, the expression of let-7a-5p/7d-5p/7f-5p was enhanced by H19 overexpression (Fig. S1C). Furthermore, CA, TCA, or both induces the expression of let-7a-5p/7d-5p/7f-5p in MSC and MLC cells (Fig. 2b, c), which supports a positive correlation between H19 and let-7 miRNA expression.
We show that the hydrophobic BA CDCA, the most efficacious endogenous FXR ligand, which is in low level or absent in cholangiocytes 49 , decreases the expression of let-7a-5p/7d-5p/7f-5p in MSC and MLC cells, suggesting Data were shown as mean ± SEM from triplicate assays (n = 5 mice/group). ***P < 0.001. b Western blotting of PTBP1 in mouse livers as a. c, d Western blotting of the expression of indicated proteins in cytoplasm and nucleus fractions prepared from BDL mouse livers. Cyclophilin A was used as a cytoplasmic fraction maker and Lamin A/C was used as a nuclear fraction marker. c, d Protein samples were pooled from five individual mice per group that let-7 expression can be induced independent of FXR. In vitro cultured MSC and MLC cells might not well reflect the precise regulation of let-7 expression by H19 in vivo. Therefore, isolating primary cholangiocytes from cholestatic livers to examine let-7 and H19 levels will be the future directions. Further, the dysregulated levels of let-7a-5p/7d-5p/7f-5p in H19-modulated cholestatic mouse livers is a consequence of multiple additive/ synergistic effects of H19 not only on BA components but also on other biological processes, such as intrahepatic inflammation and biliary proliferation.
It has been shown that PTBP1 is in complex with AGO2 and miRNAs and is involved in let-7-loaded miRNAinduced silencing complex in human cells, indicating potential roles of PTBP1 in miRNA-mediated gene regulation 50 . In this study, for the first time, we identify PTBP1 as a novel pre-let-7d/pre-let-7a-1 interacting partner, which expands the regulatory network of let-7 biogenesis. The binding of PTBP1 to pre-let-7 might reduce their accessibility to Dicer and/or degrade pre-let-7 directly by recruiting certain RNases or miRNAs, which have been used as mechanisms by other RBPs, such as LIN28A/B and hnRNP A1, to post-transcriptionally regulate let-7 biogenesis 17,25,51 . The exact mechanisms by which PTPB1 suppresses let-7 biogenesis need further investigation.
Under pathological conditions, the association between RBPs and RNAs can be dynamically enhanced or Fig. 6 H19/PTBP1 modulates the expression and bioavailability of let-7a-5p/7d-5p in vitro. a Quantitative PCR (qPCR) of let-7a-5p and let-7d-5p in Huh7 cells transfected with pcDNA3, H19, or polypyrimidine tract-binding protein 1 (PTBP1) plasmids for 48 h. b Luciferase reporter assays using psiCHECK2-let-7 4× (top) in Huh7 (left) and Hepa1 (right) cells transfected with pcDNA3, PTBP1, and H19 plasmids. NC none transfected. The renillar luciferase (RLuc) activity was determined 24 h after transfection. c, d qPCR of let-7 target genes in Null-BDL and H19-BDL mouse livers as Fig. 1c. e Western blotting of indicated proteins in Null-BDL and H19-BDL mouse livers (left). Protein samples were from three individual mice per group. The band densitometry was quantified using ImageJ and relative protein expression was graphed (right). f Enzyme-linked immunosorbent assay of interleukin (IL)-6 and IL-10 in Null-BDL and H19-BDL mouse livers (n = 5 mice/group). N.S. no significance; *P < 0.05, **P < 0.01 disturbed. We find that H19 significantly induces let-7a-5p/7d-5p/7f-5p in cholestatic mouse livers and facilitates the interactions between PTBP1 and pre-let-7d/pre-let-7-7a-1 in vitro, suggesting that the PTBP1/let-7 miRNAs may be involved in the progression of cholestasis. A handful of studies show that let-7 family members are engaged in several biliary diseases. For example, let-7i is able to modulate lipopolysaccharide receptor Toll-like receptor (TLR) expression in inflammation processes in cholangiocytes 52 . Inhibition of let-7a in BDL mice increases intrahepatic bile duct mass and the expression of nerve growth factor 53 . Let-7d is downregulated in primary biliary cirrhosis (PBC) liver tissues compared with normal tissues in human 54 . Let-7b is significantly decreased in the peripheral blood cells of PBC patients 55 . Therefore, we will continue to determine the association between PTBP1 and pre-let-7d/pre-let-7a-1 in the BDL mouse model and reveal their expression correlation in human cholestatic liver diseases, which will shed light on the molecular mechanisms of pathogenesis and progression of human cholestatic liver diseases.
There are still some open questions: (1) are H19, PTBP1, and pre-let-7 integrated into the same complex? (2) If so, what are other functional factors including proteins and RNAs? (3) Although H19 associates with PTBP1 and reduces its mRNA and protein expression, it is unknown whether H19 regulates PTBP1 transcriptionally or post-transcriptionally. (4) The interaction between H19 and pre-let-7 needs to be elucidated. (5) The intriguing part of this study is that H19 potentiates let-7 expression but decreases its bioavailability. The "Sponge" function of H19 to restrain let-7 bioavailability has been revealed 15,26,44 . We find that H19 decreases the cytoplasmic level of AGO2, an essential component of the RISC to silence gene expression; this may also partially explain the upregulation of let-7 target genes in H19-BDL livers (Fig. 6). The biological significance of let-7 miRNA upregulation in cholestatic livers needs further investigation. It is unknown whether their upregulation by H19 is a feedback to dampen H19' pathological effects through inhibiting let-7 target genes, which are upregulated by H19.
H19 functions via physically associating with its partners. Nuclear H19 controls a series of Imprinted Gene Network genes to modulate embryo growth through functional interaction with methyl-CpG-binding domain protein 1 to establish H3K9me3 repressive markers at their DMRs 1 . Cytoplasmic H19 can bind to a RBP, human antigen R, and prohibit miR-675 processing to limit the growth of placenta before birth 56 . The implication of H19/PTBP1 in regulating let-7 expression and bioavailability may help to delineate the molecular mechanisms of let-7 dysregulation in various human diseases.
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