A review on the role of HAND2-AS1 in cancer

HAND2 antisense RNA 1 (HAND2-AS1) is a newly recognized lncRNA encoded by a gene on 4q34.1. This lncRNA has 10 exons and is predicted to have a positive effect on expression of certain genes. HAND2-AS1 is mainly considered as a tumor suppressive lncRNA in different tissues. Moreover, HAND2-AS1 has been shown to regulate expression of several targets with possible roles in the carcinogenesis through serving as a sponge for miRNAs. This lncRNA can also influence activity of BMP, TGF-β1, JAK/STAT and PI3K/Akt pathways. Down-regulation of HAND2-AS1 in tumor tissues has been associated with larger tumor size, higher tumor grade, higher chance of metastasis and poor clinical outcome. The present study aims at summarization of the impact of HAND2-AS1 in the carcinogenesis and its potential in cancer diagnosis or prediction of cancer prognosis.


Introduction
Long non-coding RNAs (lncRNAs) are a group of transcripts with established roles in the fundamental process of cell physiology.Abnormal expressions of these transcripts have been linked with the development of several disorders, particularly cancers [1,2].This is mainly attributed to the important effects of lncRNAs in the regulation of cell cycle transition, cell proliferation, apoptosis and differentiation as well as stemness [3,4].Through interacting with mRNAs, DNA molecules, proteins, and miRNAs, lncRNAs influence expression of genes at almost all possible levels [5].At epigenetic level, they mediate chromatin modification and DNA methylation.At transcriptional level, they mainly interact with transcription factors and DNA sequences.Moreover, they modulate mRNA processing; thus, they are involved in the regulation of gene expression at the post-transcriptional level.Finally, they have functional interactions with proteins to affect translational and post-translational mechanisms [5].
HAND2 antisense RNA 1 (HAND2-AS1) is a newly recognized lncRNA encoded by a gene on 4q34.1, in an opposite direction to HAND2 gene.Transcription of HAND2AS1 might regulate expression of HAND2 (class A basic helix-loop-helix protein 26), a regulator of heart development which is involved in the pathogenesis of cardiomyopathy [6].HAND2-AS1 has 10 exons and is predicted to have a positive effect on the expression of certain genes.Moreover, it has been mainly detected in the cytoplasm [6].Several alternative spliced variants have been identified for this lncRNA with different sets of exons (Fig. 1).
Although the main physiological role of HAND2-AS1 is regulation of cardiac development through influencing expression of HAND2 [6], most of studies conducted on the function of this lncRNA have been performed in the context of cancer.The present study aims at summarization of the impact of HAND2-AS1 in the carcinogenesis and its potential in cancer diagnosis or prediction of cancer prognosis.We have organized the obtained evidence from literature into three distinct subtitles, including cell line studies, studies in xenograft models of cancer and studies in clinical samples.

Search strategy
We searched PubMed and Google Scholar databases with key words "HAND2-AS1" AND "Cancer" OR "Tumor."Then, the relevance of the retrieved articles was assessed through reviewing the abstract and full text of the manuscript.After reviewing 50 articles, a total of 31 articles were selected for inclusion in the manuscript.The inclusion

In vitro studies
Function of HAND2-AS1 has been assessed in a variety of cancer cell lines mainly though knock-in studies.These studies have used some techniques to enhance expression of HAND2-AS1 in a variety of cancer cell lines.Then, they have assessed the functional consequences of over-expression of HAND2-AS1 in these cell lines.

Colorectal cancer
Forced overexpression of HAND2-AS1 in colorectal cancer cells has reduced the proliferation ability and invasive properties of these cells.Mechanistically, HAND2-AS1 can serve as a molecular sponge for miR-1275.This miRNA can target KLF14; therefore, HAND2-AS1 suppresses progression of colorectal cancer via enhancing KLF14 expression [7].This study has confirmed the tumor suppressor role of HAND2-AS1 in colorectal cancer.
HAND2-AS1 has also been found to be down-regulated in 5-fluororacil-resistant colorectal cancer cells, parallel with down-regulation of PDCD4 and up-regulation of miR-20a.HAND2-AS1 could suppress 5-fluororacil resistance, cell proliferation, migratory potential and invasive properties and promote cells apoptosis in 5-fluororacil-resistant colorectal cancer cells.Mechanistically, HAND2-AS1 acts as a sponge for miR-20a to modulate levels of PDCD4 [8].Thus, HAND2-AS1 is a target for modulating resistance of colorectal cancer cells to 5-fluororacil.

Esophageal cancer
Up-regulation of HAND2-AS1 in squamous cell carcinoma cells of esophagus has resulted in downregulation of miR-21 and inhibition of cell proliferation, migration, and invasive aptitude of these cells.miR-21 up-regulation could not influence expression level of HAND2-AS1, yet it could attenuate the suppressive impact of HAND2-AS1 up-regulation on these cells [9].Cumulatively, tumor suppressive roles of HAND2-AS1 in esophageal cancer are exerted through regulation of miR-21 expression.

Lung cancer
An experiment in lung cancer cells has shown that HAND2-AS1 over-expression blocks migration and invasive aptitude and stemness features of cells, while TGF-β1 has the opposite effects.Up-regulation of HAND2-AS1 in these cells has led to down-regulation of TGF-β1, while TGF-β1 has failed to affect expression of HAND2-AS1 (Fig. 2).However, TGF-β1 could attenuate the inhibitory effect of HAND2-AS1 up-regulation on invasive properties of lung cancer cells.Therefore, HAND2-AS1 has a role in the regulation of migratory potential, invasion, and stemness of lung cancer cells via interacting with TGF-β1 [10].

Hepatocellular carcinoma
In hepatocellular carcinoma, HAND2-AS1 reduces viability of cancer cells through modulation of miR-300/SOCS5 axis [11].Moreover, it can suppress cancer cell proliferation through down-regulation of RUNX2 [12] and ROCK2 [13].Besides, HAND2-AS1 suppresses proliferation of liver cancer cells and their migration through enhancing expression of SOCS5 and inactivating the JAK-STAT signaling [14].Thus, malignant feature of hepatocellular carcinoma cells is modulated by HAND2-AS1 via different mechanisms.
In spite of the observed down-regulation of HAND2-AS1 in most of cancer cells, this lncRNA has been found to be over-expressed in liver cancer stem cells (CSCs).Most notably, HAND2-AS1 has a fundamental role in the maintenance of self-renewal of these CSCs and is required for development of hepatocellular carcinoma.From a mechanistical point of view, HAND2-AS1 can recruit the INO80 chromatin-remodeling complex to the BMPR1A promoter to induce expression of this gene and subsequently activate BMP signaling [15].Table 1 shows the role of HAND2-AS1 in different cancer cell lines.

Animal studies
Up-regulation of HAND2-AS1 has been shown to inhibit propagation of colorectal cancer [7] and its in vivo growth [8].Similar effects have been reported for HAND2-As1 upregulation in cervical, breast, ovarian, bladder, and gastric cancers (Table 2).
However, experiments in the humanized models of hepatocellular carcinoma have shown that antisense oligonucleotidemediated suppression of HAND2-AS1 exerts anti-tumor activity in a synergic manner with siRNA-mediated inhibition of BMPR1A.Besides, silencing of lncHand2 or Bmpr1a in mice hepatocytes has been shown to impair BMP signaling and suppress development of liver cancer [15].Therefore, there is a discrepancy between the bulk of evidence from most types of cancers versus hepatocellular carcinoma.

Experiments in clinical samples
HAND2-AS1 has been demonstrated to be under-expressed in colorectal cancer tissues.Moreover, expression levels of HAND2-AS1 have been negatively correlated with metastasis and advanced stages.Besides, HAND2-AS1 down-regulation has been identified as a marker of poor prognosis [7].Another study in colorectal cancer tissues has demonstrated down-regulation of this lncRNA in 5-fluorurcil-resistant tissues [8].
In addition, HAND2-AS1 has been reported to be downregulated in esophagus squamous cell carcinoma tissues, while miR-21 has been upregulated in these tissues compared with paired adjacent healthy tissues.Transcript levels of HAND2-AS1 and miR-21 have been inversely correlated in tumor samples but not in non-tumoral samples of these patients.Notably, authors have reported down-regulation of plasma levels of HAND2-AS1 in esophagus squamous cell carcinoma patients compared with normal persons.Besides, downregulation of plasma levels of HAND2-AS1 could distinguish early stage of this cancer from healthy status [9].Thus, HAND2-AS1 can be regarded as a marker for early detection of cancer.
HAND2-AS1 expression has also been lower in lung cancer tissues compared with adjacent healthy tissues.Moreover, plasma levels of HAND2-AS1 have been lower in patients with lung cancer compared with controls, while TGF-β levels have been higher in these patients.Notably, plasma levels of HAND2-AS1 and TGF-β1 have been negatively correlated in patients but not in control subjects [10].This study further supports the potential of HAND2-AS1 in cancer diagnosis.
In chronic myeloid leukemia (CML), down-regulation of HAND2-AS1 has been much prominent in accelerated and blast phases compared with chronic phase [18], indicating a putative prognostic role for this transcript.Totally, downregulation of HAND2-AS1 has been associated with metastasis in colorectal cancer [7] and hepatocellular carcinoma [11].Moreover, its down-regulation has been associated with advanced stage in colorectal cancer [7] and with high pathological grade in hepatocellular carcinoma [11], endometrial cancer [24], bladder cancer [27], and glioma [37].Finally, levels of this lncRNA are associated with tumor size in non-small cell lung cancer [16], osteosarcoma [20], and melanoma [36].Table 3 shows the role of HAND2-AS1 as described by studies in clinical samples.

Discussion
This review provides a comprehensive summary of the expression level, function, and prognostic value of HAND2-AS1 in different cancers.Based on the  above-mentioned data, HAND2-AS1 is mainly regarded as a tumor suppressive lncRNA in different tissues.The most possible explanation for its down-regulation in cancer tissues is hypermethylation of its promoter as demonstrated in ovarian cancer tissues [39].Moreover, HAND2-AS1 has been shown to regulate expression of several targets with possible roles in the carcinogenesis through serving as a sponge for miRNAs.miR-1275, miR-20a, miR-21, miR-300, miR-590-3p, miR-184, miR-769-5p, miR-340-5p, miR-106a, miR-146, miR-330-5p, miR-106a-5p, and miR-3118 are the main miRNAs that are influenced by this lncRNA.The interaction between HAND2-AS1 and miR-1275 has been verified in different contexts, including colorectal cancer, CML, and breast cancer.Most notably, the rs2276941 polymorphism within HAND2-AS1 has been shown to affect its binding with hsa-miR-1275 and influence the susceptibility to colorectal cancer [40].
BMP, TGF-β1, JAK/STAT, and PI3K/Akt pathways are the main cancer-related pathways being influenced by HAND2-AS1.In addition to regulation of cell proliferation and apoptosis, it can affect cancer metabolism and angiogenesis through modulation of a number of targets including GLUT1, VEGFA, and HIF1α.Most importantly, HAND2-AS1 can modulate response of cancer cells to 5-fluouracil and cisplatin.
The impact of HAND2-AS1 on the maintenance of CSCs should be further investigated, since data regarding this function are contradictory [10,15].Based on the importance of this population of cells in tumor metastasis and recurrence, this issue has practical significance.
Experiments in clinical samples have shown association between dysregulation of HAND2-AS1 and a number of tumor characteristics such as level of differentiation, tumor size, lymph node metastasis, and most importantly overall survival of patients.Thus, evidence regarding the prognostic role of HAND2-AS1 is ample.On the other hand, diagnostic value of this lncRNA has not been investigated thoroughly.
Although the clinical application of HAND2-AS1 as a therapeutic target has not been assessed, based on the observed down-regulation of this lncRNA in a vast variety of tumors with different origins, up-regulation of HAND2-AS1 can be regarded as a therapeutic target for several types of cancer.This can be achieved by specific epigenetic regulators that affect epigenetic marks in the promoter of this lncRNA.This therapeutic modality can also affect response of patients to conventional chemotherapeutic agents.
Finally, the impact of polymorphisms within HAND2-AS1 gene on risk of cancer has not been investigated.These polymorphisms can affect binding of this lncRNA to certain miRNAs, thus influencing the functional roles of HAND2-AS1.

Future perspectives
Forced up-regulation of HAND2-AS1 in cancer cells is a possible route for reduction in malignant features.Thus, the applicability and safety of this method should be assessed in clinical settings to find a novel treatment option for cancer.

Fig. 1
Fig. 1 HAND2-AS1 is located on human chromosome 4.It has at least 11 transcripts (based on the NCBI database).HAND2-AS1 has various functions at different levels, such as DNA, mRNA, and pro-

Fig. 2
Fig. 2 Impacts of HAND2-AS1 on different types of cancer.HAND2-AS1 performs its function as a tumor suppressor by either sponging various types of miRNAs or by suppressing the activity of various proteins that are carcinogenic

Table 1
Effects of HAND2-AS1 in cancer based on cell line studies

Table 2
Effects of HAND2-AS1 on growth and metastasis of cancer xenograftsFunding Open Access funding enabled and organized by Projekt DEAL.