Long non-coding RNA HOXA-AS2 may serve as a new therapeutic target and promising prognostic market for most of cancers

Backgroud: To elucidate the relationship between the expression level of HOXA-AS2 and it’s prognostic value of cancer by meta-analysis. Methods: Databases of PubMed, Cochrane Library, Embase, Web of Science, Google Scholar, CNKI and some others were searched systematically. Comprehensively screen according to the inclusion criteria and the exclusion criteria was conducted. The connection between HOXA-AS2 and the prognosis characteristics of patients with various types of cancer was screened by combining odds ratio (OR), Hazard ratios (HR) and 95% confidence interval (CI) for the studies collected in this meta-analysis. In addition, we further analyzed the expression of the gene and its potential clinical value in Gene Expression Profiling Interactive Analysis (GEPIA) and the lnCAR database. Results: A total of 12 studies consisting 796 patients were included in this research. Compared low HOXA-AS2 expression group, patients with high HOXA-AS2 expression were more likely to get poor overall survival (OS). Moreover, high HOXA-AS2 expression demonstrates advanced TNM stage, earlier lymph node metastasis, distant metastasis and bigger tumor size. But there was no correlation or the correlation was not statistically significant between the expression and the age, sex or the pathological differentiation. In addition, data from the GEPIA and LnCAR databases revealed that increasing HOXA-AS2 expression means bad prognosis in most of cancers. Conclusions: High HOXA-AS2 expression shows worse cancer prognosis in cancer patients, and HOXA-AS2 may be acted as therapeutic target and promising prognostic marker.

of most patients have reached the middle or the late stage at the time of diagnosis, as the result that the prognosis of the middle and late stage patients is still not satisfactory even though they have been treated with active operation, radiotherapy ,chemotherapy, endocrine therapy and so on. In recent years, targeted therapy has brought new hope to patients with advanced cancer, and is expected to improve the prognosis of cancer patients. For example, the interaction between opioid binding protein/cell adhesion molecule-like (OPCML) and HER2 destroys the formation of HER2-EGFR heterodimer, which leads to enhanced response of HER2 positive ovarian and breast cancer cells to rapatinib and erlotinib. In addition, increasing the expression of OPCML gene could strengthen the effect of rapatinib in breast cancer patients [1]. Epidermal growth factor receptor variant III (EGFRvIII) is a tumor specific mutation expressed in various types of tumors and it can prevent postoperative recurrence and metastasis through EGFRvIII targeted by CAR-T in patients with lung cancer [2]. Tetra nucleotide targeting regulation of VEGF could also inhibits the growth of non-small cell lung cancer cells [3]. What's more, WM-127, the inhibitor of Survivin, can inhibit the proliferation of hepatocellular carcinoma cells, induce cycle arrest and apoptosis of cancer cells, and at last delay the tumorigenesis of nude mice [4]. Therefore, it is necessary to explore new target molecules and biomarkers as targets for cancer treatment in order to improve the prognosis of tumor patients.
Long-chain non-coding RNA, which does not have the function of protein coding, is a class of RNA with a length of more than 200 nucleotides. Studies had shown that LncRNA was abnormally expressed in a variety of human cancers, and its abnormal expression was significantly related to the proliferation, migration, invasion of cancer cell and so on. For example, the expression of lncRNA PSMG3-AS1 was increased in breast cancer tissues and cells and Interference of PSMG3-AS1 expression can promote the expression of miR-143-3p, resulting in the weakness of proliferation and migration of breast cancer cells [5]. The expression of LINC00467 was up-regulated in HCC tissues and cells., and it could inhibit the proliferation and metastasis of HCC cells and promote apoptosis if LINC00467 was silenced [6]. In addition, the lncRNA TNRC6C-AS1 could increases methylation of STK4 promoter, thus activating the Hippo signaling pathway and promoting the development of thyroid cancer (TC) [7]. It shows that lncRNAs plays a vital role in the occurrence and progression of cancer and is expected to become a new target molecule and biomarker for cancer treatment. The long non-coding RNA HOXA-AS2 is located between the genes of HOXA3 and HOXA4, which is the antisense chain of the homeobox gene A (HOXA). It was found that HOXA-AS2 was expressed in human NB4 promyelocytes and peripheral blood neutrophils, which could be increased after treatment with all-trans retinoic acid (ATRA).
And further studies found that HOXA-AS2 could negatively regulated the apoptosis of NB4 cells induced by ATRA [8]. In recent years, it had been reported that HOXA-AS2 was abnormally expressed in colorectal cancer [9][10][11], acute lymphoblastic leukemia (ALL) and hepatocellular carcinoma [12][13][14], and was involved in the occurrence and development of tumor. The expression of HOXA-AS2 in colorectal cancer (CRC) was increased and high expression of HOXA-AS2 indicated larger tumor size, later stage, and worse prognosis of patients [9,10]. Disturbing the expression of HOXA-AS2 could inhibit the cell cycle G1/S, the proliferation of CRC cells, and induce apoptosis. On the contrary, overexpression of HOXA-AS2 would promote cell migration and invasion by regulating epithelial-interstitial transformation (EMT) [10,11]. Moreover, Knocking down HOXA-AS2 can inhibit the proliferation of acute myeloid leukemia (AML) ADR cells and induce apoptosis [12]. And the expression of HOXA-AS2 was also increased in the cell line and patients' samples of prednisone insensitive ALL. HOXA-AS2 can activate the EGFR/Ras/Raf/MEK/ERK signaling pathway and enhance the proliferation ability and inhibit the apoptosis rate of cancer cells by promoting the expression of HOXA3 gene, and then enhance the resistance of glucocorticoid (GC) [13]. The expression of HOXA-AS2 was increased in the tissues and cell lines of hepatic cellular cancer (HCC), knocking down HOXA-AS2 can block the G1 phase, promote the apoptosis, and then inhibit the growth of HCC cells. In addition, HOXA-AS2 could leads to migration and invasion of HCC cells by promoting the change of EMT characteristics. These results suggest that HOXA-AS2 plays an important role in the progression of cancer and can be used as a new biomarker and target for tumor therapy [14]. However, the potential clinical value of HOXA-AS2 in the prognosis of tumors is still controversial. Therefore, in this meta-analysis, we will explore the value of HOXA-AS2 in the prognosis and clinicopathological features of cancer for the first time, and also provide new targets and biomarkers for the diagnosis and treatment of cancer patients.

B i b l i o g r a p h y r e t r i e v a l
Search the studies which were published in Pubmed, CochraneLibrary, Embase, WebofScience, GoogleScholar, CNKI and Wanfang databases before February 15, 2019 at home or abroad, and were related to the relationship between HOXA-AS2 and the prognosis and clinicopathological characteristics of tumor patients. The Chinese keywords include: "antisense chain of HOXA gene, antisense HOXA gene, tumor, cancer". English search terms include: "Longnon-codingRNA HOXA-AS2" or "LincRNA HOXA-AS2" or "HOXA-AS2" AND "tumor" or "cancer".

Inclusion criteria and exclusion criteria
Inclusion criteria: 1) The subjects were cancer patients; 2) the expression level of HOXA-AS2 in tissues was detected by quantitative PCR or RT-PCR; 3) there were definite analyses for the relationship between HOXA-AS2 and the clinicopathological features or the OS of the tumor; 4) the patients could be divided into two groups according to the expression level of HOXA-AS2; 5) data on HR, OR and 95%CI were provided; 6) the literature was published in English or Chinese. Exclusion criteria: 1) the research species were animals; 2) the data for statistic are incomplete; 3) lack of clinical research data.

Data extraction and quality control
The literature information was extracted by two researchers independently according to the established screening criteria. The contents of data extraction included: 1) The name of the first author, the years published, the country and region of the researched objects; 2) the characteristics of the objects, including the type of cancer, the total number of cases and the number of patients in different subtypes; 3) outcome indicators, including overall survival time, TNM stage, lymph node metastasis, distant metastasis and so on.
The HR value and 95% CI were obtained by software EngaugeDigitizerversion10.1 (Http://Digiti-zer. source-forge.net/) for the literatures whose data only include Kaplan-Meier survival curves and lack of HR value, and the estimated HR value and its 95%CI were compared with the survival curve P value of the original literature. If the information of extracted data was inconsistent, it should be extracted by a third one. 4) the quality evaluation of the literature was independently evaluated by the two researchers according to NOS (The Newcastle-Ottawa Scale), including population selection (the highest score of Selection was 4), comparability (the highest score of Comparability was 2), exposure evaluation or result evaluation. NOS adopted the semi-quantitative principle of star system to evaluate the quality of literature. the full score was 9 stars and more than 5 stars were high quality literature.
Validating HOXA-AS2 expression in GEPIA and lnCAR databases GEPIA is an open database for in-depth analysis of TCGA gene expression data [15,16] LnCAR is a comprehensive and open resource, which has in-depth analysed the expression profile and the prognostic value of the lncRNAs in microarray data. At present, there are 52300 samples in lnCAR for differential expression analysis and 12883 samples for survival analysis. The expression of HOXA-AS2 in tumor and its normal samples was analyzed by GEPIA and lnCAR databases, and its relationship with staging and prognosis was analyzed at the same time.

Statistical analysis
The software of Stata 12 and RevMan 5.35 were used for statistical analysis. The HR value and its 95%CI were used as the effect index of survival data, the odds ratio (OR) and it's 95%CI were used as the effect index of clinicopathological characteristics of patients. The HR and its 95%CI should be estimated from the data extracted from the survival curve by EngaugeDigitizer 10.1 if only the Kaplan-Meier survival curve is given in the literature.
The heterogeneity of the included results should be judged by I 2 quantification. When Pvalue ≥ 0.05, I 2 ≤ 50%, the fixed effect model (Mantel-Haenszel method, M-H method) was used for Meta analysis; and when P-value < 0.05 or I 2 > 50%, the heterogeneity was significant, and the random effect model would be used for Meta analysis. The publication bias of the literature was evaluated by Begg's test, and the stability of the results was tested by sensitivity analysis. The difference was thought to be statistically significant if the P-value < 0.05.

Results
Incorporate the results of the study According to the retrieval strategy, 65 relevant articles were obtained. 16 of them were excluded because of repetitiveness and 19 articles were excluded after reading the title and abstract. Then, through detailed reading of the full text, it is found that 18 articles do not meet the inclusion criteria of this study. Among them, 8 articles did not explore the correlation between the expression of HOXA-AS2 and the prognosis of patients and 10 articles were excluded for incomplete data (Fig. 1). In the end, 12 articles with a total of 796 patients (Table 1) were included in this study. There were 8 types of cancer in these 12 literatures, including 2 cases of lung cancer [17,18], 2 of thyroid cancer [19,20], 1 of bladder cancer [21], 1 of breast cancer [22], 1 of gastric cancer [23], 2 of colorectal cancer [9,10], 1 of osteosarcoma [24] and 2 cases of liver cancer [14,25]. All the 12 articles come from China ( Table 1). The Lymph node metastasis, distant metastasis and tumor staging had been confirmed by pathological examination. And all the 12 literature were of high quality according to NOS. Table 1 Characteristics of the studies enrolled in the meta-analysis.

Study
Origin of Association between the expression level of HOXA-AS2 and overall survival time of the patients 7 literature were enrolled to explore the relationship between HOXA-AS2 expression and the OS in cancer patients. It was found that there was a significant correlation between high expression of HOXA-AS2 and poor OS in tumor patients (HR = 2.12, 95%CI = 1.61-2.80) after combining the effect index ( Fig. 2 and Table 2). Because of the small heterogeneity (I 2 = 8%, P = 0.36), we adopted the fixed effect model. In this study, we also considered the difference expression of HOXA-AS2 in different tumor tissues. Finally, we found that the HR = 2.56, 95%CI = 1.74-3.77, P = 0.19,I 2 = 40% for digestive system and HR = 1.73, 95%CI = 1.16-2.59, P = 0.72, I 2 = 0% for non-digestive system by subgroup analysis according to whether it was the source of digestive system. Association between expression level of HOXA-AS2 and TNM staging The relationship between the expression of HOXA-AS2 and TNM staging was reported in 11 of the 12 literature ( Fig. 3A and Table 3). Because of the small heterogeneity (P = 0.167, I 2 = 29.2%), we adopted the fixed effect model in the same way, and the combined OR value and its 95%CI were 4.40 and 3.16-6.14 (P = 0.22, I 2 = 32%), respectively. In addition, Subgroup analysis based on cancer type showed that high expression of HOXA-AS2 was significantly correlated with TNM staging in respiratory system (OR = 6.81, 95%CI  Association between expression level of HOXA-AS2 and lymph node metastasis The relationship between the expression of HOXA-AS2 and lymph node metastasis was reported in 7 of the 12 literature ( Fig. 3B and Table 3). Because of the small heterogeneity (P = 0.164, I 2 = 34.5%), the fixed effect model was used again, and the combination of OR and 95%CI showed that the high expression group was more likely to have metastasis (OR = 6.2, 95%CI = 4.01-9.59) than the low expression group. According to the subgroup analysis of cancer types, it was found that high expression of HOXA-AS2 was significantly correlated with lymph node metastasis in tumors of respiratory system (OR = 5. Association between expression level of HOXA-AS2 and distant metastasis The relationship between HOXA-AS2 expression and distant metastasis (DM) of tumors was reported in 3 of the 12 literature ( Fig. 3C and Table 3). Because of the low heterogeneity

Discussion
With the continuous breakthrough of the genome, high-throughput sequencing and the rapid development of cancer genome map, growing evidences have manifested that lncRNA is involved in the process of a variety of human diseases. At present, lncRNA has been found to express abnormally in a variety of cancers and is involved in the occurrence and development of cancer as an oncogene or tumor suppressor gene. For example, the expression of LINC00271 was down-regulated in adrenocortical carcinoma, and it tend to develop a poor prognosis for patients [26]. The expression of lncRNA FALEC in plasma of patients with cervical cancer was significantly up-regulated, and was significantly correlated with the tumor size, FIGO stage and lymph node metastasis. What's more, overexpression of FALEC could promote the proliferation and invasion of Hela cells [27].
These results suggest that lnCRNA provides a new prospect for the treatment of human cancer.
This meta-analysis found that increased HOXA-AS2 expression was significantly correlated with poor OS in tumor patients without significant publication bias. After further analyses of the association between HOXA-AS2 expression and the clinicopathological features of tumor patients, we found that high expression of HOXA-AS2 was correlated with lymph node metastasis, distant metastasis, advanced staging and larger tumor diameter, the same as the correlation between the prognosis and HOXA-AS2. Unfortunately, there is no evidence showed that high expression level of HOXA-AS2 was associated with the age, sex and pathological grade of tumor patients in this study. These results suggested that HOXA-AS2 is involved in the progression of cancer as an oncogene and is expected to be a new target and prognostic biomarker for cancer therapy. In addition, we confirmed the abnormal expression of HOXA-AS2 in LUAD, LUSC, THCA and other cancers in GEPIA data set, and that the expression level of HOXA-AS2 was correlated with the clinical stage and adverse OS of human cancers. Therefore, we speculate that HOXA-AS2 is an independent risk factor for cancer prognosis and is expected to be a new target and biomarker for cancer treatment.

Conclusions
The increased expression of HOXA-AS2 is associated with the poor prognosis of cancer patients. HOXA-AS2 can be used as a new target and biomarker for cancer treatment, which is beneficial to the early diagnosis for cancer patients and to the identification of the patients with poor prognosis. Supplementary File Legend Fig. S1 The targeted regulatory relationship of HOXA-AS2-miRNA-mRNA in various cancers. Figure 1 Flow diagram of the study search and selection process.

Figure 2
Forest plot for the association between SNHG1 expression levels with overall survival (OS).

Supplementary Files
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