ZEB1‐AS1: A crucial cancer‐related long non‐coding RNA

Long non‐coding RNAs (lncRNAs) recently emerge as a novel class of non‐coding RNAs (ncRNAs) with larger than 200 nucleotides in length. Due to lack an obvious open reading frame, lncRNAs have no or limited protein‐coding potential. To date, accumulating evidence indicates the vital regulatory function of lncRNAs in pathological processes of human diseases, especially in carcinogenesis and development. Deregulation of lncRNAs not only alters cellular biological behavior, such as proliferation, migration and invasion, but also represents the poor clinical outcomes. Zinc finger E‐box binding homeobox 1 antisense 1 (ZEB1‐AS1), an outstanding cancer‐related lncRNA, is identified as an oncogenic regulator in diverse malignancies. Dysregulation of ZEB1‐AS1 has been demonstrated to exhibit a pivotal role in tumorigenesis and progression, suggesting its potential clinical value as a promising biomarker or therapeutic target for cancers. In this review, we make a summary on the current findings regarding the biological functions, underlying mechanisms and clinical significance of ZEB1‐AS1 in cancer progression.


| INTRODUCTION
The genome-wide studies showed that greater than 70% of the human genome is transcribed into RNAs, while only approximately 2% of the sequences have the capacity to encode proteins. 1,2 The non-coding RNAs (ncRNAs), which were long assumed to be transcriptional noise, comprise the most proportion of the transcripts. 3 The ncRNAs are divided into diverse types according to their size, such as microRNAs (miR-NAs), small nucleolar RNAs (snoRNAs), PIWI-interacting RNAs (piRNAs), and long non-coding RNAs (lncRNAs). [4][5][6][7] Among them, miRNAs have been extensively investigated to understand their properties for clinical application, and the focus of research is recently shifting to lncRNAs.
LncRNAs mainly transcribed by RNA polymerase II are generally defined as a group of non-protein-coding RNAs, which are more than 200 nucleotides in length. [8][9][10] LncRNAs have been confirmed to regulate gene expression and chromatin structure at epigenetic, transcriptional and post-transcriptional levels, and participate in physiological and pathological processes generally through gene imprinting, histone modification, chromatin remodelling, transcriptional activation, transcriptional interference, alternative splicing and cell cycle control. [11][12][13][14][15][16][17][18] Increasing studies indicate that numerous lncRNAs, which may function as either oncogenes or tumor suppressor genes, are aberrantly expressed in a variety of human cancers, and contribute to the initiation and progression of malignancies. 19,20 LncRNAs dysregulation can promote proliferation, invasion and metastasis of tumor cells and inhibit cellular senescence and apoptosis by multiple mechanisms, including working as miRNA sponges, protein scaffolds, regulatory signals or transcript decoys. [21][22][23][24][25] Notably, lncRNAs may be used as feasible tumor biomarkers and potential therapeutic targets for diagnosis and treatment of cancers due to high tissue specificity and elevated efficiency. 26,27 Zinc finger E-box binding homeobox 1 antisense 1 (ZEB1-AS1) is a well-characterized oncogenic regulator among all tumor-associated lncRNAs, and is overexpressed in hepatocellular carcinoma, esophageal squamous cell carcinoma, glioma, osteosarcoma, bladder cancer, colorectal cancer, prostate cancer and B-lymphoblastic leukemia.
The present review summarizes current evidence concerning the expression signature, functional feature, regulatory mechanism and clinical significance of ZEB1-AS1 during the occurrence and development of tumors (Tables 1 and 2).

| DISCOVERYANDCHARACTERIZATION OFZEB1-AS1
ZEB1-AS1, which is mainly located in the nucleus, was originally discovered as the most efficient lncRNA to boost cellular proliferation in human HCC by Li in 2015. Both GSE55191 and GSE58043, which are two human lncRNA microarray datasets, were used to analyze aberrantly expressed lncRNAs in HCC, and the results showed that 573 lncRNAs were consistently upregulated and 19 lncRNAs were consistently downregulated in both microarray datasets. Among them, ZEB1-AS1 was identified as a central lncRNA by co-expression network analysis, and exhibited excellent characteristics. 28 ZEB1-AS1 is located on chromosome 10p11.22 with approximately 2 535 nucleotides in length, including two exons and one intron between the two exons. 29 In addition, ZEB1-AS1 is an antisense transcript deriving from the promoter region of ZEB1, which is a critical transcription factor in tumor development. 30 EMT, epithelial-mesenchymal transition; ZEB1, zinc finger E-box binding homeobox 1; MLL1, mixed-lineage leukemia 1; BMI1, B cell-specific Moloney murine leukemia virus insertion site 1; CDK2, cyclin dependent kinase 2; MMP2/9, matrix metalloproteinase 2/9; IL-11, interleukin-11; STAT3, signal transducer and activator of transcription 3.

OverexpressionofZEB1-AS1andclinicalfeatures References
Hepatocellular carcinoma Positive microvascular invasion, shorter overall survival and higher recurrence rate [28] Esophageal squamous cell carcinoma Higher tumor grade, greater depth of invasion, positive lymph node metastasis, poorer overall survival and disease-free survival [36] Prostate cancer Later clinical stage and positive perineural invasion [29] Glioma Advanced clinical stage and reduced overall survival [41] Osteosarcoma Larger tumor size, advanced Enneking stage, positive tumor metastasis, poorer recurrence-free survival and overall survival [44] Bladder cancer Higher histological grade and advanced tumor stage [47] Colorectal cancer Greater depth of tumor invasion, positive microvascular invasion, positive lymph node metastasis, poorer overall survival and lower recurrence-free survival rate [49] B-lymphoblastic leukemia Poorer survival [51] ZEB1-AS1, zinc finger E-box binding homeobox 1 antisense 1.
T A B L E 2 Clinical significance of ZEB1-AS1 in diverse cancers rate is still low due to the high recurrence rate and distant metastasis. 33 Therefore, finding competent biomarkers for early diagnosis and prognosis evaluation will contribute to reduce the mortality of HCC patients.
Li et al 28

| Esophagealsquamouscellcarcinoma
Esophageal squamous cell carcinoma (ESCC) is the most common category of esophageal cancer, which is the sixth main cause of cancer deaths worldwide, and its incidence rates remain quite high in China. 34 ESCC patients often reach the advanced stage when diagnosed, and the overall 5-year survival rate is less than 10%. 35 The unfavorable prognosis is mainly due to the lake of sensitive and specific indicator for early detection. As a result, the identification of the biomarkers that contribute to early diagnosis of ESCC is extremely meaningful.
Wang et al 36

| Prostatecancer
Prostate cancer, which leads to approximately 1-2% of deaths in men annually, is the most commonly diagnosed tumor in males worldwide. 37 The incidence rates of prostate cancer are gradually increasing largely due to the high-fat diet and prolongation of life expectancy. 38 The management of prostate cancer remains a tremendous medical challenge as a result of the lack of efficient therapeutic methods. Thus, elucidating novel molecular mechanisms involved in the development of prostate cancer is an indispensable step to overcome this refractory malignancy.
Su et al 29 reported that ZEB1-AS1 expression levels were significantly enforced in prostate cancer tissues and cell lines compared with those in non-tumor controls. Moreover, upregulated ZEB1-AS1 was remarkably associated with later clinical stage and perineural invasion.
In addition, ZEB1-AS1 knockdown inhibited proliferation and migration of prostate cancer cells. Accordingly, ZEB1-AS1 plays an important role in the initiation and progression of prostate cancer, implying that ZEB1-AS1 may function as a prospective biomarker to improve early diagnosis rate of prostate cancer.

| Glioma
Glioma is the most serious type of primary tumor in central nervous system, and causes exceedingly high death rates each year. 39

| Osteosarcoma
Osteosarcoma, a highly aggressive cancer, is the most common group

| Bladdercancer
Bladder cancer is the ninth most frequent malignancy worldwide, leading to approximately 52 395 cancer deaths annually. 45 The effectiveness of chemotherapy in advanced bladder cancer is quite limited, and the recurrence rate of bladder cancer remains greatly high. 46 Lin et al 47

| Colorectalcancer
Colorectal cancer (CRC) is the fourth main cause of tumor-related mortality and the third most commonly diagnosed cancer worldwide. 48 Gong et al 49

| B-lymphoblasticleukemia
B-lymphoblastic leukemia is one of the most lethal hematological malignancies. 50

| ZEB1-AS1promotesZEB1expression
ZEB1-AS1 is located in physical contiguity with ZEB1, which is a momentous regulatory factor involved in the EMT. To our knowledge, the antisense transcripts may function as a positive regulator for corresponding gene expression. 52 For example, fibroblast growth factor

| ZEB1-AS1activatesIL-11/STAT3signalling
The tumor microenvironment is composed of extracellular matrix, diverse non-tumor cells around cancer cells and a variety of surrounding biological active molecules. 56 These cells and their secretory molecules interact with each other and establish an exceedingly complicated signal network to impact cancer progression. 57 Signal transducer and activator of transcription 3 (STAT3) as an oncogene can be constitutively activated in diverse tumors, and elevated STAT3 expression has been shown to be promoted by upregulated interleukin-11 (IL-11), which is a well-known component of tumor microenvironment. 58

| Otherregulatorymechanisms
DNA methylation is one of the most important epigenetic modifications, and DNA methylation in lncRNAs promoters likely represents an epigenetic modulator of lncRNAs expression. 60 The methylation frequency of ZEB1-AS1 gene is found dramatically decreased during HCC progression, and hypomethylation of ZEB1-AS1 promoter is significantly associated with ZEB1-AS1 overexpression, implying that aberrant methylation of ZEB1-AS1 promoter may exhibit a vital role in ZEB1-AS1 expression and HCC development. 28 Both apoptosis-related proteins (e.g., Bcl-2 and Bax) and cell cycle regulators (e.g., Cyclin D1, CDK2 and p15) play a key role in tumorigenesis and progression. 61 Gong et al 49

| CONCLUSIONANDFUTURE DIRECTIONS
An increasing number of lncRNAs are found to exert important regulatory functions in gene expression, and the aberrant expression of lncRNAs is gradually recognized as a hallmark feature in cancer. 62 Investigating these molecules as biomarkers or therapeutic targets will be a promising field for cancer treatment. At present, some wellcharacterized lncRNAs have been detected not only in tumor tissues but also in body fluids, such as blood plasma and urine. 63 67 The cis regulating mechanism of lncRNAs indicates a viable research direction in this field.
ZEB1-AS1 is identified as an oncogenic lncRNA in diverse malignancies, and upregulated ZEB1-AS1 promotes malignant phenotypes, such as proliferation, invasion and migration by inducing the EMT, activating signalling pathways and regulating cell cycle ( Figure 2). However, the precise molecular mechanisms upstream and downstream of ZEB1-AS1 are not thoroughly understood, and remain to be systematically investigated. ZEB1-AS1 dysregulation is significantly associated with multiple poorer clinicopathological characteristics such as distant metastasis and reduced survival rate, suggesting its potential clinical utility as a favorable biomarker for diagnosis and prognosis. Nevertheless, the expression and function of ZEB1-AS1 in body fluids remains quite obscure, and needs to be further validated. Furthermore, ZEB1-AS1 is extremely prospective to be a feasible drug target due to forceful tumor specificity and reduced systemic toxicity. Despite recent efforts to explore the functions and mechanisms of ZEB1-AS1, its research is still at the preliminary stage. Further systematic studies in larger patient cohorts are warranted to accelerate its clinical application.