Long noncoding RNA (lncRNA) H19: An essential developmental regulator with expanding roles in cancer, stem cell differentiation, and metabolic diseases

Abstract

Recent advances in deep sequencing technologies have revealed that, while less than 2% of the human genome is transcribed into mRNA for protein synthesis, over 80% of the genome is transcribed, leading to the production of large amounts of noncoding RNAs (ncRNAs). It has been shown that ncRNAs, especially long non-coding RNAs (lncRNAs), may play crucial regulatory roles in gene expression. As one of the first isolated and reported lncRNAs, H19 has gained much attention due to its essential roles in regulating many physiological and/or pathological processes including embryogenesis, development, tumorigenesis, osteogenesis, and metabolism. Mechanistically, H19 mediates diverse regulatory functions by serving as competing endogenous RNAs (CeRNAs), Igf2/H19 imprinted tandem gene, modular scaffold, cooperating with H19 antisense, and acting directly with other mRNAs or lncRNAs. Here, we summarized the current understanding of H19 in embryogenesis and development, cancer development and progression, mesenchymal stem cell lineage-specific differentiation, and metabolic diseases. We discussed the potential regulatory mechanisms underlying H19's functions in those processes although more in-depth studies are warranted to delineate the exact molecular, cellular, epigenetic, and genomic regulatory mechanisms underlying the physiological and pathological roles of H19. Ultimately, these lines of investigation may lead to the development of novel therapeutics for human diseases by exploiting H19 functions.

Introduction

Gene expression is the core process of all aspects of life. The process begins in the cell nucleus with genomic DNA, the template for transcription of messenger RNAs (mRNAs), after transcription, mRNAs translocate to the cytoplasm and act as blueprints for translation into proteins. In addition to mRNAs, there are many non-protein coding RNAs (ncRNAs), such as transfer RNAs, ribosomal RNAs, and small nuclear RNAs that are also essential for gene expression. Since the completion of the Human Genomic Project, it has been estimated that while over 80% of human genomic DNA is transcribed, only about 2% of the human genome is transcribed into mRNA and translation to proteins, indicating the pervasiveness of ncRNAs.1, 2, 3, 4, 5 Recently, ncRNAs have been characterized as having regulatory functions in many physiological and/or pathological processes and are regarded as the therapeutic targets for disease.^2,6, 7, 8, 9, 10, 11 In particular, long non-coding RNAs (lncRNAs), non-protein coding transcripts longer than 200 nucleotides, are rapidly being identified as having crucial roles in regulatory control of gene expression,^2,3,7,10,12 such as promoter-specific gene regulation,13, 14, 15 acting as competing endogenous RNAs (CeRNAs),^15,16 X-chromosome inactivation,17, 18, 19, 20 imprinting,21, 22, 23, 24 maintenance of nuclear architecture,25, 26, 27 etc.

LncRNA H19, first isolated and reported in the 1980s by 4 different laboratories, is one of the first identified imprinted genes and lncRNAs.21, 22, 23^,28, 29, 30, 31 In 1990, Brannan et al found that the H19 gene did not code for any protein even though it is transcribed by RNA polymerase II, spliced, and polyadenylated.²⁹ In mice, H19 contains three reading frames, however, multiple translation termination signals were found in all three reading frames.²⁹ In 1991, Bartolomei et al confirmed that H19 is only transcribed from the maternally inherited allele, the paternal H19 allele is not expressed in mice. Meanwhile, H19 and Igf2 genes are tightly linked and imprinted in opposite directions.²¹ The following year, Zhang et al identified that H19 is also maternally expressed in humans.³² Then, H19 was certified as a lncRNA since it does not contain the short introns that are characteristic of imprinted coding genes, complete absence of transcribed peptide, form secondary RNA structures and hairpin loops, localize in a cytoplasmic ribonucleoprotein particle, and may function as a riboregulator.^23,29,33,34 In the past few decades, the regulatory functions of H19 have been increasingly clarified. The expression patterns of H19, H19 encoded-microRNAs and anti-sense, the interlinking expression of H19 and Igf2, etc. are characterized. Here, we summarize these diverse regulatory functions in numerous physiological and pathological processes.

Critical roles of H19 in embryogenesis and development

H19 is highly expressed in developing embryos, especially in mesoderm- and endoderm-derived tissues. However, H19 is strongly down-regulated after birth in all tissues including skeletal and cardiac muscle tissues,^28,30 although not entirely turned off in skeletal and cardiac muscle tissues.³⁵ Loss of H19 function in the liver and a few other endodermal tissues did not influence the survival of mice and instead resulted in an overgrowth phenotype similar to babies with Beckwith-Wiedemann syndrome, which may have been caused by overexpression of Igf2.³⁴ However, the overexpression of H19 in the mouse zygote causes lethal effects between embryonic day 14 and birth, which indicates the importance of strict control of H19 during embryogenesis.³⁶ On the other hand, mice lacking H19 showed an overgrowth phenotype, while mice with H19 enhancer overexpression had reduced postnatal growth.³⁷ In addition, human imprinting control region 1 (IC1) can functionally replace mouse IC1on the maternal allele, but not paternally, which indicated the paternal imprinting at IC1 between mice and humans.³⁸ These results indicated the fine-tuning role of H19 during both embryogenesis and development.

As a maternally imprinted gene, H19 expression is regulated by methylation during embryogenesis. H19 methylation regions differ according to parental inheritance, the paternal copy of H19 is methylated and silent while the maternal copy is either hypomethylated or unmethylated, resulting in different maternal H19 allele expression in offspring.39, 40, 41, 42, 43 Meanwhile, methylation of the H19 promoter is negatively correlated with H19 expression and H19 expression is negatively correlated with the neighboring gene Igf2 expression.42, 43, 44, 45 Han et al recently characterized that disruption of Igf2/H19 expression in parthenogenetic fetuses and placentas contributes to implantation failure and/or abortion in swine parthenogenesis, which might be associated with differential methylation patterns in the imprinting control region of imprinted genes.⁴⁶ H19 and Igf2 shared several enhancers, which are tissue-specific.⁴⁷ In other words, the mutual regulation of Igf2 and H19 could not be ignored during embryogenesis.^41,48

Although H19 is strongly down-regulated after birth in most tissues, it still plays an important role in development. Since it was known that H19 was highly expressed in muscles after birth, the biological function of H19 was first explored in motor system development.^37,49,50 H19-deficient mice displayed hyperplasia in the late fetal stage, hypertrophy at the postnatal stage in skeletal muscles, and had decreased number of mutant satellite cells.⁵¹ On the other hand, Martinet et al found that H19 deficiency decreased the number of mutant satellite cells; however, the self-renewing capacity of these cells was not affected, which may be due to the up-regulation of other genes in the imprinted gene network.⁵¹ During the myogenic differentiation process, H19 is highly activated by the key transcription factor MyoD, which is highly expressed during myoblast differentiation and muscle regeneration. What is more, H19 and Igf2 misexpression work separately, cooperatively, and antagonistically to establish the developmental phenotype through different signaling pathways.⁵² These results indicated the essential role of H19 in muscle development.⁴⁹

As for osteogenic differentiation, H19 showed an increased expression pattern in mesenchymal stem cells (MSCs) during the initial period. H19 regulates several signaling pathways involved in MSC osteogenic differentiation.^49,53, 54, 55, 56, 57 Similar to the function of H19 in embryogenesis, we found that adenovirus-mediated overexpression of H19 inhibited osteogenic differentiation of MSCs, while down-regulation of H19 alleviates MSC osteogenic differentiation.^58,59 Meanwhile, it has been reported that H19 regulated chondrogenic differentiation by potentiating Sox9-mediated collagen type II formation, which indicates the regulatory function of H19 in cartilage formation.^49,60 In addition, H19 was found to promote tenogenic differentiation and suppress adipogenic differentiation of MSCs.61, 62, 63 Overall, these results suggest that H19 plays an important role in motor system development.

Molecular mechanisms underlying H19 functions

Imprinted tandem gene Igf2-H19

As one of the firstly identified maternal imprinted genes, H19 and Igf2 belong to the same locus and are separated only by 90 kb which is located on chromosome 7 in mice and chromosome 11 in human.^21,29,64,65 Most imprinted genes are methylated on maternally derived chromosomes; however, Igf2-H19 is methylated on paternally derived chromosomes. The expression balance of Igf2-H19 is one of the most important mechanisms underlying H19 functions.66, 67, 68 Loss of function of H19 and overexpression of Igf2 were associated with overgrowth syndrome called Beckwith-Wiedemann syndrome, on the contrary, will result in fetal and postnatal growth failure called Silver-Russell syndrome.^52,69,70

The imprinted tandem gene Igf2-H19 participates in several physiological and pathological processes, such as embryogenesis, tumorigenesis, and guardian of the proliferation of pluripotent stem cells, etc.^48,71,72 The regulation of Igf2-H19 gene expression is one of the essential mechanisms. When the 3′ gene enhancer shared between H19 and Igf2 is deleted, both H19 and lfg2 expression are down-regulated. The 3′ enhancer has a more robust effect on the expression of H19 since H19 has a stronger promoter in this area than Igf2 and is physically closer to the 3′ enhancer.^41,48,67,73 On the other hand, tissue-specific and parent-of-origin enhancers were also identified and proved to regulate the expression of Igf2-H19⁴⁷ (Fig. 1A).

Figure 1.

Current models of action for H19. (A) The imprinted Igf2-H19 tandem gene participates in several physiological and pathological processes. (B) The antisense RNA 91H can regulate H19-Igf2 expression and hence impacts cell growth. (C)H19 can encode miR-675 which regulates gene expression. (D)H19 can act as CeRNA and regulate gene expressions. (E)H19 can act as modular scaffolds for epigenetic modifications.

The gap between H19 and Ifg2 contains differentially methylated regions (DMRs) in the chromosome, whose methylation state regulates the expression of H19 and Ifg2. Generally, DMRs are methylated in paternal chromosomes and unmethylated in the maternal chromosome. Methylated DMRs block the binding of DNA-binding zinc finger insulator protein, CTCF, which prevents the transcription of downstream H19 in the paternal chromosome.66, 67, 68 On the contrary, unmethylated DMRs bind with CTCF and prevent transcription of Igf2, and in this situation, only H19 is transcribed to RNA. This process ensures the maternal imprinted expression of H19 and maintains a proper balance of H19 and Igf2 genes.^48,66, 67, 68^,73 When the methylation of DMRs within the Igf2-H19 locus is erased, the expression of H19 increased and blocked the expression of Igf2, which prevents the uncontrolled proliferation and teratoma formation of cells.73, 74, 75 In contrast, loss of imprinting induces hypermethylation of DMRs within Igf2-H19 on both maternally and paternally derived chromosomes, which is associated with several malignancies and results in high expression of Igf2^48,73 (Fig. 1A).

H19 antisense RNA

Since noncoding antisense transcripts have been suggested to constitute a new epigenetic regulatory system, H19 antisense (91H RNA) was identified with varying levels of expression in both the estrogen-sensitive T47D and the estrogen-insensitive BT20 breast cancer cell lines.⁷⁶ These results indicate the existence and regulatory function of 91H. In addition, through nucleic acid analysis, Berteaux et al⁷⁶ confirmed 91H is a single 120-kb transcript generated from the H19/Igf2 locus. Next, it was confirmed that, rather than affecting H19 expression, 91H influence Igf2 expression.⁷⁶ Moreover, although 91H, H19, and Igf2 are overexpressed in breast tumors, 91H was demonstrated to promote breast tumor cell growth, migration, and invasion by preventing histone and DNA methylation on the maternal allele at the H19/Igf2 locus.⁷⁷

In most tumor cells except breast tumor cells, 91H was identified to promote Ifg2 transcription by activating the Igf2 promoter in mouse myoblast.⁷⁸ Gao et al⁷⁹ found that 91H was down-regulated in esophageal squamous cell carcinoma with higher depth of invasion, neoplastic grading, and TNM. Lower 91H expression was associated with an increased risk for the occurrence, progression, and prognosis of esophageal squamous cell carcinoma. Recently, the regulatory function of 91H was also reported in colorectal cancer, osteosarcoma, and hepatocellular carcinoma, etc.80, 81, 82, 83 On the other hand, H19 antisense encodes a protein, H19 opposite tumor suppressor, and could inhibit Wilms, rhabdoid, rhabdomyosarcoma, and choriocarcinoma tumor cell growth, which indicates that H19 locus harbors an imprinted gene encoding a tumor suppressor protein.⁸⁴ Taken together, these results suggested a new mechanism of H19/Igf2-associated regulation (Fig. 1B).

H19: encoding miR-675

Exon 1 of H19 encodes two distinct miRNAs, miR-675-5p and miR-675-3p, which are highly conserved and confer functionality to H19^85,86. MiR-675-5p and miR-675-3p could synergistically regulate the same or different physiological or pathologic processes with different mechanisms87, 88, 89 (Fig. 1C). To prove H19 encodes miR-675, Cai et al⁹⁰ first identified the sequence in H19 located from 1014 to 1036 that encoded miR-675. They then identified that miR-675 was generated from longer pri-miRNA precursors by the RNase III enzymes Drosha and Dicer in the 293 T cell line, which does not normally express H19. In addition, the 293 T cell line readily expressed miR-675 at high levels after being transfected with human or mouse H19 plasmid, but not in mock-transfected cells. These results indicated that miR-675 is encoded by H19 and positively correlated with the expression of H19.^41,49,90,91

As an indispensable part of the H19-miR-675 axis, miR-675 has been reported to regulate myogenesis, chondrogenesis, adipogenesis, tenogenesis, and osteogenesis, etc.^41,49,60,92 Dudek et al identified that miR675 expression level was positively correlated with the expression level of Sox9 and regulated type II collagen expression in human articular chondrocytes.⁶⁰ Dey⁹³ et al found that H19 encoded-miR-675-3p and miR-675-5p, which promoted skeletal muscle differentiation and regeneration by directly targeting and down-regulating the anti-differentiation Smad transcription factors. Similarly, miR-675 was reported to down-regulate some functional genes and regulate signaling pathways during tumor progression.^86,94,95

H19: competing endogenous RNA (CeRNA)

MicroRNAs (miRNAs) have important regulatory functions in many physiological and pathological processes. One of the most essential roles of miRNAs is post-transcriptional regulation, in which miRNAs bind to specific recognition sequences (“seed sequences”) located in the 3′ untranslated regions (3′ UTRs) of target mRNAs. In this event, miRNAs inhibit the expression of target proteins by enhancing mRNA degradation and/or by directly interfering with protein translation.^7,96,97 As mentioned above, lncRNA can generate miRNAs, simultaneously, lncRNA can act as CeRNA, in which specific lncRNA can impair miRNA activity through sequestration and finally up-regulate miRNA target gene expression.^7,98, 99, 100

It is reported that H19 contains functional miRNAs let-7 interaction sites and can inhibit let-7 function by acting as a molecular sponge. In addition, this competitive binding can accelerate muscle differentiation and regulate muscle glucose metabolism in muscle cells, which indicates that H19's function as CeRNA is one of its essential mechanisms of the regulatory function of H19.^101,102 By preventing H19 from interacting with miR-let-7, Park et al identified cardiac phenotypes were associated with the interaction between H19 and miR-let-7.³⁵ At the same time, H19 can bind with miR-17–5p,¹⁰³ miR-93–5p,¹⁰⁴ miRNA-106 b-5p,¹⁰⁵ miR-130a,¹⁰⁶ miR-22–3p,¹⁰⁷ etc. respectively, and regulates the specific biological process. Significantly, H19 may regulate a specific biological process by simultaneously binding with several miRNAs. These results indicate the flexibility and diversity of H19 in regulating gene expression by acting as a CeRNA^58,108,109 (Fig. 1D).

H19 acts as a modular scaffold

It was known that lncRNAs regulate chromatin states and epigenetic inheritance. Tsai et al identified that lnRNA HOTAIR serves as a modular scaffold for two histone modification complexes, in which the 5′ domain of HOTAIR binds to the polycomb repressive complex 2 and the 3′ domain binds to the LSD1/CoREST/REST complex.¹¹⁰ Different from HOTAIR, it is reported that hypoxia regulates H19 expression and function by regulating allele-specific histone modification.¹¹¹ On the other hand, we found that H19 could act as a modular scaffold in the cytoplasm and promote specific protein phosphorylation, then it can regulate the activation of the protein and downstream signaling pathway^112,113 (Fig. 1E). The main mechanisms of H19 mediated regulation are shown in Figure 1.

Complex roles of H19 in cancer development and progression

Abnormal expression of H19 has been demonstrated in a variety of different types of cancer cells.⁸⁵ As H19-mediated regulatory function can occur through several different mechanisms, H19 was found to be associated with different types of cancer and played different roles in cancer development and cancer progression.^85,114

H19 in osteosarcoma (OS)

The disruption of H19 and Igf2 expression was associated with the development of rhabdomyosarcoma, which was identified in 1997, and H19 was regarded as the tumor suppressor of rhabdomyosarcoma.^115,116 Recently, defective osteogenic differentiation has been characterized to play an important role in the development of OS.¹¹⁷ With the use of induced pluripotent stem cells, Lee¹¹⁸ et al found that impaired osteoblast osteogenic differentiation resulted in Li-Fraumeni syndrome -associated OS. The mechanism underlying this is the low expression of H19 and dysregulation of H19 IGN. Aside from participating in the development of OS, H19 also regulates the migration and invasion of OS. It was reported that H19 has higher expression in OS tumor tissue than in adjacent healthy tissue.¹¹⁹ Furthermore, H19 expression level in OS is positively associated with tumor migration and invasion, which is mediated by the down-regulation of the nuclear factor-κB pathway (NF κB).^120,121 By acting as a CeRNA, H19 can regulate OS development, invasion, and migration by suppressing functional miRNA expression.^85,122 Taken together, the down-regulation of H19 increases the risk of OS development and up-regulated H19 is associated with a poor prognosis of OS.

H19 in breast cancer

Breast cancer is one of the most common cancers among women worldwide. Since H19 is an estrogen-regulated transcript and regulates cell proliferation and differentiation, several studies have been carried out to clarify the effects and mechanisms of H19 in regulating breast cancer development, progression, and metastasis.^85,123 Firstly, H19 expression levels were positively associated with breast cancer cell proliferation, invasion, and angiogenesis, and ectopic overexpression of H19 promotes tumorigenic properties of breast cancer cells.124, 125, 126 Secondly, a recent clinical case-control study showed that H19-associated single nucleotide polymorphisms were associated with breast cancer risk and have the potential for breast cancer diagnosis and estimating prognosis.^127,128 In addition, it was identified that H19 can lead to breast tumorigenesis by altering DNA methylation.¹²⁶ Simultaneously, as mentioned above, H19 antisense 91H also regulates breast cancer development by epigenetic modifications.76, 77, 78 In summary, it can be concluded that H19 is an oncogene in breast tumorigenesis.

Additionally, as the precursor of miR-675, it was reported that miR-675 expression was significantly higher in breast cancer tissue compared with the control group, which demonstrates regulation of H19-miR576 axis.^123,129 Further studies identified that ectopic expression of H19-miR675 reinforced breast cancer cell proliferation, migration, metastasis, and drug resistance.^{85,95,123,125,129} As for the ceRNA network of H19, by differently sponging miR-200b/c and let-7b, H19 regulates the epithelial-to-mesenchymal transition in breast cancer cells. Similarly, H19 can bind with miR-152, miR-93-5p, let-7a/b, miR-324-5p, miR-29b-3p, etc. in breast cancer progression.^85,123,130

H19 in gastrointestinal cancers

The digestive system includes the gastrointestinal tract (the esophagus, stomach, intestine, and colorectum) and the accessory organs of digestion (the pancreas, liver, and gallbladder). Recently, H19 has been reported to regulate digestive system cancers on multiple levels, both as an oncogene or regulating tumor-suppressing gene expression.^93,129,131, 132, 133, 134

Firstly, H19 is found to be up-regulated in gastric cancers,135, 136, 137 pancreatic cancers,138, 139, 140 hepatocellular carcinoma,141, 142, 143 esophageal squamous cell carcinoma,^144,145 colorectal cancer,^132,146, 147, 148 etc., which indicates that H19 may act as an oncogene in digestive system cancers. Simultaneously, H19 polymorphisms were identified to be a potential marker for the diagnosis of digestive system cancers,149, 150, 151 and H19 expression levels were reported to have a positive correlation with the proliferation, invasion, and metastasis of digestive cancer cells.^131,137, 138, 139^,152 In addition, Wang et al found that H19 could function as CeRNA, bind with miR-194–5p and influence SIRT1-mediated autophagy, and finally promote 5-Fu resistance in colorectal cancer.¹⁵³ Wu et al demonstrated that H19 promotes methotrexate resistance in colorectal cancer by activating Wnt/beta-catenin signaling.¹⁵⁴ In hepatocellular carcinoma, Xu et al¹⁵⁵ identified that H19 participated in sorafenib resistance by the H19-miR675 axis. In summary, H19 may act as an oncogene in digestive system cancers and hold the potential for early diagnosis, acting as a target of treatment and working as prognosis markers.^{135,140,146,156,157}

H19 in lung cancer

There are two main types of lung cancer, non-small cell lung cancer, which occupies 80%–84% of all lung cancer cases, and small cell lung cancer, which occupies 10%–15%. Although these two types of lung cancer are treated very differently, H19 was found to be up-regulated in both non-small and small cell lung cancer with different mechanisms.158, 159, 160, 161 Studies conducted on ethnic Chinese populations showed that the polymorphism rs2107425 in the H19 gene was associated with the risk of lung cancer among females whom never smoked,¹⁶² and the single nucleotide polymorphism rs217727 in lncRNA H19 was significantly associated with susceptibility to lung cancer, particularly in squamous cell carcinoma and adenocarcinoma in smokers.¹⁶³ In non-small cell lung cancer, Zheng et al¹⁶⁴ identified that up-regulated H19/miR675 promoted hypoxia-induced cancer progression by inhibiting p53 signaling. Ren et al¹⁶⁵ reported that H19 sponged miR-196b, which targeted a conserved RNA binding protein LIN28B, and accelerated lung cancer growth. Simultaneously, by acting as a CeRNA, H19 was found to sponge miR-200a,¹⁶⁶ miR-107,¹⁶¹ miR-17,¹⁶⁷ miR-6515-3p,¹⁶⁸ miR-138,¹⁶⁹ miR-203, etc, and promoted lung cancer cell proliferation, invasion, metastasis, or epithelial-mesenchymal transition. In small cell lung cancer, Li et al¹⁷⁰ found that H19 promoted small cell lung cancer tumorigenesis by regulating the miR-140-5p/FGF9 axis by acting as CeRNA.

In addition, several studies identified that H19 expression was associated with lung cancer drug resistance.171, 172, 173, 174 Zhou et al¹⁷⁴ characterized that inhibition of H19 could enhance gefitinib sensitivity and the effectiveness of chemotherapy, which indicated the function of H19 in lung cancer drug resistance. Pan et al¹⁷³ found that H19 was highly expressed in erlotinib resistance lung cancer cell lines via exosomes and regulated drug resistance via targeting miR-615-3p to regulate ATG7 expression. Lei et al¹⁷¹ also proved that H19 packaged into exosomes and mediated lung cancer cell resistance to gefitinib. In human lung adenocarcinoma cells, H19-mediated cisplatin resistance potentially serves as a molecular marker to predict the prognosis of lung adenocarcinoma.¹⁷²

In summary, H19 gene acts as an oncogene in lung cancer and participates in tumorigenesis, progression, metastasis, and drug resistance, which indicates that H19 can be a molecular marker or target for lung cancer diagnosis and treatment.

H19 in genitourinary cancer

By the function of H19 in other systems, H19 participates in tumorigenesis in genitourinary cancer.^{71,129,175,176} As a rare kidney cancer, Wilms tumor primarily affects children, H19 was identified to be an imprinted tumor-suppressor gene of Wilms tumor.¹⁷⁷ Meanwhile, H19 could interact with another anti-oncogene to suppress Wilms tumor development.177, 178, 179, 180 Except for breast cancer,¹⁸¹ genomic variants with H19 were identified to hold the potential to be diagnosis markers in bladder cancer,^182,183 epithelial ovarian cancer,¹⁸⁴ Wilms tumor,¹⁸⁵ etc. In prostate cancer, H19 is regarded as the essential regulator of cell fate, plasticity, and treatment resistance and holds the potential to be a diagnosis marker.¹⁸⁶ Meanwhile, H19 acts as an estrogen- and hypoxia-response regulator and promotes prostate cancer invasion through the epithelial-to-mesenchymal transition to a β integrin pathway.¹⁸⁷ By acting as CeRNA, H19 was reported to regulate prostate cancer, bladder cancer,¹⁸⁸ ovarian cancer,¹⁸⁹ seminoma,¹⁰⁵ etc. In addition, impaired Igf2-H19 imprinting was found in prostate cancer,¹⁹⁰ ovarian cancer,^184,191 bladder cancer,¹⁹² Wilm's disease,¹⁹³ malignant endometrium,¹⁹⁴ etc. Simultaneously, H19 regulates several genitourinary cancer cell proliferation, invasion, metastasis, and drug resistance through different mechanisms.^{184,186,188,189}

It is worth mentioning that, as an essential oncogene, targeting H19--mediated tumor treatment has been explored in various studies.¹⁹⁵ Sidi et al¹⁹⁶ investigated the effects of intravesical injection of a DNA plasmid that contains H19 gene regulatory sequences which drive the expression of an intracellular toxin in a phase I/II marker lesion study. They found an ablation effect on the bladder, which indicated the potential of this method. A fragment of diphtheria toxin (DTA) or herpes simplex virus thymidine kinase (HSV-tk), under the control of an 814-bp 5′-flanking region of the H19 gene, was applied for regulating the expression of H19.¹⁹⁵ A phase 1/2a clinical study found that intraperitoneal use of H19-DTA was a safe and effective method for recurrent ovarian/peritoneal cancer.¹⁹⁵ A phase 2 b marker lesion trial also confirmed the effect of H19-DTA in nonmusical invasive bladder cancer. These results indicated the treatment potential of targeting H19.

H19 in MSC lineage-specific differentiation

H19 in osteogenic differentiation

As we mentioned before, H19 plays an important role in motor system development. As the key process of bone development and regeneration, osteogenic differentiation of MSCs is closely involved in the regulation of H19.^53,197, 198, 199, 200

Like the function of H19 in development, we identified that the expression balance of H19 is essential for the osteogenic differentiation of MSCs. Our in vitro and in vivo studies found that overexpression of H19 blocked BMP9-induced osteogenic differentiation of MSCs and kept MSCs in undifferentiation status. However, down-regulation of H19 in BMP9 induced osteogenic differentiation of MSCs inhibited osteogenic differentiation and relatively promoted adipogenic differentiation.^58,109 Wang et al further found that H19 regulates osteogenic and adipogenic differentiation of BMSCs through ligand-dependent corepressor by sponging miR-188.²⁰¹ Furthermore, Huang et al proved that the H19-miR 675 axis regulated the balance between osteogenesis and adipogenesis of MSCs by targeting the 3′ UTRs of the histone deacetylase (HDAC) 4–6 transcripts.⁶² Simultaneously, H19 was reported to hold the potential to induce stem cell osteogenic differentiation. Li et al²⁰² found that H19 promoted osteogenic differentiation of bone marrow MSCs by sponging miR-149, which increased the expression of stromal cell-derived factor-1. Huang et al⁵⁴ identified that both H19 and miR-675 promoted osteogenic differentiation of MSCs by activating the TGF-beta1/Smad3/HDAC signaling pathways. In addition, H19 mediated CeRNA mechanism was proved to regulate several signaling, including the ERK1/2 and p38 MAPK signaling pathways,²⁰³ Runx2,²⁰⁴ focal adhesion protein-tyrosine kinases,¹⁹⁹ etc. Meanwhile, as an imprinted gene, H19-IFG2 also regulated osteogenic differentiation.²⁰⁵ Loss of imprinting of Igf2 could improve the osteogenic potential of human deciduous teeth MSCs, which could be the main difference compared with human adipose tissues.²⁰⁶

Crosstalk between H19 and Wnt signaling was identified recently. Wnt signaling is essential for the osteogenic differentiation of MSCs.²⁰⁷ As an enhancer of Wnt4, forkhead box C2 (Foxc2) binds to the promoter area of Wnt4 and then activates the Wnt-β-catenin pathway, and H19 synergistically promotes this process by directly binding with Foxc2.²⁰⁸ On the other hand, H19-encoded miR675-5p was proved to be a negative regulator of BMSC osteogenic differentiation, as the H19-miR675-5p axis regulates osteogenic differentiation through Wnt-β-catenin pathway.²⁰⁹ However, Ma et al found that H19-derived miR-675 targeted the adenomatous polyposis coli protein and promoted osteogenic differentiation of MSCs by activating Wnt signaling.²¹⁰ In addition, Liang et al⁵⁶ found that H19 acted as CeRNA and sponged miR-22 and miR-141 which targeted Wnt/beta-catenin signaling, and then regulated osteogenic differentiation of human MSCs. Similar results were detected by Gong et al²¹¹ in ectomesenchyme stem cells.

Moreover, H19 was reported to be activated by estrogen and regulated osteogenic differentiation of BMSCs, which could reduce osteoporosis via the miR-532-30/SIRT1 axis.¹⁹⁷ Simultaneously, H19 was also identified to regulate different types of body cells including osteoblasts, osteoclasts, and osteocytes in the osteogenic differentiation process which hold the potential to be a target for osteoporosis treatment.^203,212, 213, 214

H19 in endochondral ossification and angiogenesis

H19 was identified to regulate chondrogenic differentiation and maintain the anabolic and catabolic activities of chondrocytes and endochondral ossification.^60,92,215 In mouse limbs, we detected that the expression level of H19 is high in the proliferation zone and decreases gradually from the prehypertrophic zone to the hypertrophic zone. In addition, we characterized that H19 is essential for chondrogenic differentiation and maintaining the proliferation of chondrocytes, and down-regulation of H19 promoted hypertrophic differentiation of chondrocytes and followed endochondral ossification. In mechanism, we proved that H19 facilitated Runx2 phosphorylation.¹¹² In chondrocytes, Steck et al found that H19 expression levels were not correlated with Igf2 expression levels, but positively correlated with miR-675 expression levels; a further study confirmed that H19 could be an attractive marker for chondrocyte anabolism and be a potential target for cartilage recovery.⁹²

Angiogenesis is a key process of endochondral ossification and osteogenesis. Notch signaling regulates both endothelial cell generation and angiogenesis during both bone development and regeneration.216, 217, 218, 219, 220, 221, 222 We characterized that H19 functions as CeRNA that sponges miRNAs targeted to Notch signaling, which indicated the regulatory function of H19 in Notch signaling activation.⁵⁸ As reported by Shang et al,²²³ Notch signaling promoted the hypertrophic differentiation process. Our previous work also proved that the down-regulation of H19 promoted hypertrophic differentiation of MSCs.¹¹² Furthermore, our recent work found that H19 binds with p53 and promotes the phosphorylation of p53, phosphorylated p53 promoted Notch1 transcription by binding on the promoter area of Notch1, and then the activated Notch1 promoted endothelial cell generation.¹¹³ These results indicated the regulation axis of H19-Notch in endochondral ossification.

Roles of H19 in metabolic cardiovascular diseases

H19 and its variant expression alteration were associated with cardiovascular diseases, endocrine system disease, liver disease, nephrotic syndrome, etc., which indicated the functional effects of H19 in metabolism.224, 225, 226

H19 in atherosclerosis and cardiovascular diseases

Atherosclerosis is one of the main cardiometabolic diseases and drives cardiovascular disease, which is one of the main causes of mortality worldwide.²²⁵ Atherosclerosis is mechanistically involved in numerous biological processes including lipid metabolism, adipogenesis, angiogenesis, inflammatory response, cellular proliferation, and apoptosis, etc.^225,227 Foam cell-mediated lipid accumulation and inflammatory response occupy an important position in the progression of atherosclerosis. It was reported that H19 was highly expressed in atherosclerosis foam cells, and down-regulation of H19 counteracted the increase of triglycerides/total cholesterol/low-density lipoprotein-cholesterol and down-regulated the level of high-density lipoprotein-cholesterol.²²⁸ Meanwhile, down-regulation of H19 decreased the expression of pro-inflammatory factors (TNF-α and IL-β) and increased the expression of anti-inflammatory factors (IL-4 and IL-10).²²⁸ In mechanism, these effects were mediated by the H19-miR-130 b axis. In addition, the expression level of miR-130b was correlated with the expression levels of triglycerides, LDL, serum creatinine, and blood urea nitrogen and inflammatory response in diabetic nephropathy, which also indicated the regulation of miR-130b in lipid metabolism.²²⁹

H19 has also been identified to regulate the proliferation and apoptosis of vascular smooth muscle cells and endothelial cells, which contribute to the development of atherosclerosis. Park et al identified that H19 interacting with miR-let-7 and regulating the expression of Igf2 were associated with cardiac phenotypes.³⁵ Huang et al found that H19 promoted the proliferation of vascular smooth muscle cells and endothelial cells and suppressed apoptosis through acid phosphatase 5, which promoted the development of atherosclerosis.²³⁰ Sun et al identified that H19 sponging miR-let-7a, which targets cyclin D1, promoted vascular remodeling by regulating the cell cycle.²³¹ Meanwhile, it is reported that H19 regulates vascular smooth muscle cell proliferation by miR-148b/Wnt/beta-catenin singling. Overall, H19 regulates atherosclerosis development through lipid metabolism, inflammatory response, cell proliferation, apoptosis, etc. Meanwhile, in myocardial infarction, H19 was found to ameliorate myocardial infarction-induced myocardial injury and maladaptive cardiac remodeling by acting as CeRNA, which sponges miRNA-22-3p and promotes the expression of KDM3A.²³² Moreover, by acting as CeRNA, H19 was reported to inhibit myocardial infarction and myocardial ischemia/reperfusion by sponging different miRs.²²⁵ Recently, Viereck et al found that H19 is an anti-hypertrophic lncRNA and represents a promising therapeutic target to combat pathological cardiac remodelling.²³³

H19 in hepatic lipid metabolism, non-alcoholic fatty liver disease, and liver fibrosis

H19 was also proved to play an important role in liver diseases.^234,235 In hepatic stellate cells, H19 was up-regulated by hypoxia-inducible factor-1alpha through binding to the lncRNA-H19 promoter at two hypoxia response element sites located at 492–499 and 515–522 bp. Up-regulated H19 activated hepatic stellate cells and promoted the development of liver fibrosis through the AMPKα signaling pathway.²³⁶ Inhibition of H19-AMPKα signaling by dihydroartemisinin holds the potential for improving lipid droplet metabolism and the prevention and treatment of liver fibrosis.²³⁷ The liver plays an essential role in lipid metabolism. It was reported that up-regulated H19 was associated with non-alcoholic fatty liver disease. Mechanistically, H19 induced lipid accumulation and up-regulated the expression of lipid synthesis-, storage-, and breakdown-associated genes by up-regulating both the mTORC1 signaling axis and MLXIPL transcriptional network.²²⁶ As a lncRNA, H19 was also identified to promote lipogenesis and triglyceride secretion in hepatocytes by sponging miR130a, which targets PPARγ directly.¹⁰⁶ In addition, the expression level of H19 was positively associated with the expression levels of lipids, which served as a lipid sensor. By synergizing with the RNA-binding polypyrimidine tract-binding protein 1, H19 regulated hepatic metabolic homeostasis and exacerbated the development of fatty liver.²³⁸

H19 in insulin sensitivity, obesity, and diabetes

As H19 is highly expressed in skeletal muscles, which is essential for glucose and lipid metabolism. It was reported that H19 was down-regulated in the muscle of both db/db mice and high-fat diet-induced obese mice and that over-expression of H19 ameliorated glucose intolerance and insulin resistance in db/db mice and decreased ectopic lipid accumulation in the skeletal muscle and liver. In terms of mechanism, H19 promotes the oxidation of fatty acids by targeting heterogeneous nuclear ribonucleoprotein.²³⁹ As a cellular energy sensor, AMPK enhanced muscle insulin sensitivity and whole-body energy metabolism by regulating glucose uptake, lipid oxidation, and mitochondrial biogenesis. Geng et al²⁴⁰ found that H19 sponged miR-let7 and increased the expression of DUSP27, which activated AMPK signaling and enhanced muscle insulin sensitivity. Meanwhile, H19 variants were identified to have a significant association with T2MD.²⁴¹

In addition, an intrauterine hyperinsulinemia environment may cause hepatic Igf2/H19 epigenetic alteration and induce glucose intolerance in gestational diabetes mellitus. Mechanistically, an intrauterine hyperinsulinemia environment could increase hepatic FoxO1 levels, which subsequently increases the expression of DNMT3A and epigenetic alterations on Igf2/H19 DMRs.²⁴² In other words, H19 may be a potential target for the treatment of type 2 diabetes mellitus.^243,244 The function of H19 in different diseases, physiological processes, and pathological processes are summarized in Figure 2.

Figure 2.

Summary of the functions of H19 in different physiological and/or pathological processes.

Conclusions and future directions

As one of the first identified noncoding RNAs, H19 was identified to play essential roles in several physiological and pathological processes. Dandolo et al found that H19-Igf2 locus interact with Myod and regulated diaphragm formation,²⁴⁵ and they also identified that H19 acted as a trans regulator on imprinted gene network and controlling growth and development.^37,41,51,246, 247, 248 On the other hand, Hochberg and Adriaenssens et al found that H19 was associated with cancer development, invasion, and metastasis, and implied that H19 was a potential target for cancer treatment.^77,129,194,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260 These studies indicated the multiple functions of H19 in different cell types. In mechanism, it has been shown that H19 mediates diverse regulatory functions by serving as CeRNA, Igf2/H19 imprinted tandem gene, modular scaffold, cooperating with H19 antisense, acting directly with other mRNAs or lncRNAs, etc. Biologically, H19 participates in embryogenesis, development, tumorigenesis, osteogenesis, and metabolism, which are associated with the development of many diseases. Therefore, H19 holds the potential for disease diagnosis, tissue regeneration, and the development of targeted therapeutics. Recently, a plasmid DNA encoding the A chain of the diphtheria toxin (DTA-H19) driven by the transcriptional regulatory sequences of human H19, was tested as a monotherapy or in combinations with other chemotherapeutics in treating some cancers, such as pancreatic cancer and ovarian cancer, while more clinical trials are being conducted.261, 262, 263, 264, 265 However, H19 was regarded as a double-edged sword in cancer therapy.²⁶⁶ Thus, more in-depth studies are warranted to delineate the exact molecular, cellular, and genomic mechanisms underlying the physiological and pathological roles of H19. Ultimately, these lines of investigation may lead to the development of novel therapeutics for human diseases by exploiting H19 functions.

Conflict of interests

The authors declare no conflict of interests.