Long noncoding RNA HOXA11-AS promotes gastric cancer cell proliferation and invasion via S

World Journal ofGastroenterologyWorld J Gastroenterol 2019 June 14; 25(22): 2763-2775

ISSN 1007-9327 (print) ISSN 2219-2840 (online)

Submit a Manuscript: https://www.f6publishing.com

ORIGINAL ARTICLE

Basic Study

Long noncoding RNA HOXA11-AS promotes gastric cancer cell

proliferation and invasion via SRSF1 and functions as a biomarker ingastric cancer

Yun Liu, Yu-Mei Zhang, Feng-Bo Ma, Su-Rong Pan, Bao-Zhen Liu

ORCID number: Yun Liu

(0000-0003-1622-0078); Yu-MeiZhang (0000-0003-2087-4816); Feng-Bo Ma (0000-0002-2275-073X); Su-Rong Pan (0000-0002-4891-1941);Bao-Zhen Liu

(0000-0002-2453-4643).

Yun Liu, Department of Operating Room, Binzhou People's Hospital, Binzhou 256610,Shandong Province, China

Yu-Mei Zhang, Department of Return Visit, Binzhou People's Hospital, Binzhou 256610,Shandong Province, China

Feng-Bo Ma, Su-Rong Pan, Bao-Zhen Liu, Department of Gastroenterology, Binzhou People'sHospital, Binzhou 256610, Shandong Province, China

Corresponding author: Bao-Zhen Liu, PhD, Doctor, Department of Gastroenterology, BinzhouPeople's Hospital, Binzhou 256610, Shandong Province, China. zhebaonnt3411@163.comTelephone: +86-543-3282396

Author contributions: Liu BZ

designed the research; Liu Y,Zhang YM, Ma FB, and Pan SRperformed the research; Liu Yanalyzed the data; Liu Y and LiuBZ wrote the paper.

Institutional review boardstatement: This study was

reviewed and approved by theInstitutional Review BoardCommittee of Binzhou People'sHospital.

Abstract

BACKGROUND

Gastric cancer (GC) is the fourth most frequent malignancy all over the world.The diagnosis of GC is challenging and the prognosis of GC is very unfavorable.Accumulating evidence reveals that serum long noncoding RNAs (lncRNAs) canfunction as biomarkers in various types of cancers, including GC.

AIM

To explore the level and molecular mechanism of the lncRNA HOXA11-AS in GCand the diagnostic and prognostic significance of serum HOXA11-AS in GC.METHODS

HOXA11-AS levels in GC tissue, cell lines, and serum samples were measured.The correlation between HOXA11-AS expression and clinicopathologicalcharacteristics was analyzed. The role of HOXA11-AS in the diagnosis andprognosis of GC was evaluated. Cell function assays were performed for

exploration of the roles of HOXA11-AS in GC cells. Moreover, Western blot wasperformed to explore the target regulated by HOXA11-AS in GC cells.

RESULTS

Up-regulation of HOXA11-AS was found in GC tissues, cell lines, and serumsamples. In GC patients, decreased serum HOXA11-AS levels were negativelyrelated with tumor size, TNM stage, and lymph node metastasis. The area underthe receiver operating characteristic curve of serum HOXA11-AS in the diagnosisof GC was 0.924 (95%CI: 0.881-0.967; sensitivity, 0.787; specificity 0.978). Resultsof the Kaplan-Meier survival curves suggested the GC patients with a lower

2763

June 14, 2019

Volume 25

Issue 22

Conflict-of-interest statement: We

declare no conflict of interest.

Data sharing statement: No

additional data are available.

Open-Access: This article is an

open-access article which wasselected by an in-house editor andfully peer-reviewed by externalreviewers. It is distributed inaccordance with the CreativeCommons Attribution NonCommercial (CC BY-NC 4.0)license, which permits others todistribute, remix, adapt, build

upon this work non-commercially,and license their derivative workson different terms, provided theoriginal work is properly cited andthe use is non-commercial. See:http://creativecommons.org/licenses/by-nc/4.0/

WJGhttps://www.wjgnet.com

Liu Y et al. LncRNA HOXA11-AS promotes GC

Manuscript source: Unsolicited

manuscript

Received: March 28, 2019

Peer-review started: March 28, 2019First decision: April 11, 2019Revised: April 15, 2019Accepted: May 3, 2019

Article in press: May 3, 2019Published online: June 14, 2019P-Reviewer: Lazăr DC, Tanabe SS-Editor: Gong ZML-Editor: Wang TQE-Editor: Zhang YL

HOXA11-AS level having a better overall survival rate. HOXA11-AS promotedGC cell proliferation and invasion. SRSF1 may be the target regulated byHOXA11-AS in GC cells.

CONCLUSION

HOXA11-AS promotes GC cell proliferation and invasion via SRSF1 and mayfunction as a promising marker in GC.

Key words: Long noncoding RNA; HOXA11-AS; SRSF1; Gastric cancer; Biomarker©The Author(s) 2019. Published by Baishideng Publishing Group Inc. All rights reserved.Core tip: The long noncoding RNA (lncRNA) HOXA11-AS is up-regulated in gastriccancer (GC) tissues, cell lines, and serum samples. In GC patients, increased serumHOXA11-AS levels were positively related with tumor size, TNM stage, and lymphnode metastasis. HOXA11-AS functions as a diagnostic and prognostic biomarker inGC, and promotes GC cell proliferation and invasion. SRSF1 may be the target regulatedby HOXA11-AS in gastric cells.

Citation: Liu Y, Zhang YM, Ma FB, Pan SR, Liu BZ. Long noncoding RNA HOXA11-ASpromotes gastric cancer cell proliferation and invasion via SRSF1 and functions as abiomarker in gastric cancer. World J Gastroenterol 2019; 25(22): 2763-2775URL: https://www.wjgnet.com/1007-9327/full/v25/i22/2763.htmDOI: https://dx.doi.org/10.3748/wjg.v25.i22.2763

INTRODUCTION

Gastric cancer (GC) is a global health problem, holding the fourth most frequentmalignancy and the second most common cause of death from cancer all over theworld[1]. Estimated 27510 cases diagnosed with GC and 11140 deaths from GC willoccur in the United States in 2019[2]. The diagnosis of GC is challenging owing to thefact that patients often present with vague and non-specific symptoms[3], which causesmany patients being at the progressive stage at first diagnosis. However, patients withmetastatic GC carry a very poor prognosis and their median survival ranges from 4mo to around 12 mo[4,5]. Although GC is associated with Helicobacter pylori infection,the molecular mechanisms of its occurrence and progression remain unclear.Consequently, it is ineluctably necessary to probe into the molecular mechanism ofthe occurrence as well as progression of GC and finding a new marker applying to thedetection, therapy, and prognosis of GC.

Long noncoding RNAs (lncRNAs), a class of transcripts > 200 nucleotides (nts) inlength, exert their significant functions in the progression and metastasis ofmalignancy[6]. LncRNAs typically exhibit tissue-specific expression patterns and arereadily detectable in body fluids because of their high stability, in comparison withother protein biomarkers expressed in various types of tissues, making them idealbiomarkers[7]. Accumulating evidence reveals that serum lncRNAs function asbiomarkers in various types of cancers, such as HOTTIP in GC[8], MALAT1 inepithelial ovarian cancer[9], GIHCG in cervical cancer[10], and LRB1 in hepatocellularcarcinoma[11].

Previous studies suggested that aberrantly expressed lncRNA HOXA11-AS playssignificant roles in the development and progression of malignancies[12]. HOXA11-ASfunctions as an oncogene and promotes cell proliferation, invasion, and metastasis inGC[13,14]. However, the molecular mechanism of HOXA11-AS in GC is far from fullyelucidated and the diagnostic and prognostic role of HOXA11-AS in GC is stillunclear. In this study, we thoroughly investigated the molecular mechanism ofHOXA11-AS and the diagnostic and prognostic roles of serum HOXA11-AS in GC.

MATERIALS AND METHODS

Tissue and serum specimens

GC tissue specimens and corresponding paracancerous gastric tissue specimens were

WJGhttps://www.wjgnet.com

2764

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

obtained from 25 GC patients from Binzhou People's Hospital. Each patient wasdiagnosed with GC pathologically and none of the patients received any treatmentbefore operation. The tissue samples were processed within 1 hour and submerged inRNAlater reagent from Qiagen GmbH (Hilden, Germany) for half an hour. After that,the samples were stored at -80 ˚C till RNA extraction. All tissue samples wereexamined and classified under the management of experienced pathologists.

As for blood samples, the present study totaled 134 participants, consisting of 94patients with GC and 40 healthy controls. Postoperative blood specimens were alsoobtained from 25 patients with GC. All the blood samples were disposed within 2 h.Serum was disposed at 4 °C and recruited by centrifugation (1200 × g , 10 min).Another centrifugation (10000 × g, 10 mins, at 4 °C) was performed for completelyremoving residual cellular debris. The serum specimens were kept in liquid nitrogentill RNA extraction.

Our research was managed by the ethics committee of Binzhou People's Hospital.Consent statement was obtained from each participant. Table 1 lists the clinicalcharacteristics of the GC patients.

Cell culture and transfection

A culture medium consisting of 88% of Dulbecco's modified Eagle's medium (DMEM;Gibco; Thermo Fisher Scientific. Inc., Waltham, MA, United States), 10% of fetalbovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc.), 1% of antibiotics (100μL/mL penicillin and 100 mg/mL streptomycin sulfate), and 1% of glutamine wasused to cultivate normal gastric mucosal epithelial cells (GES-1) and GC cells (MKN-45). MKN-45 and GES-1 cells were maintained in an incubator (37 °C, 5% CO2). MKN-45 cells were pre-seeded in the 6-well plates (3.0 × 105 cells/per well) and maintaineduntil the confluence of 60%. HOXA11-AS primers, siRNA, and negative control (si-NC) were provided by Ribobio (Guangzhou, China); pcDNA-HOXA11-AS andpcDNA3.1 were constructed by Generay (Shanghai, China). siRNAs or pcDNAs weretransfected into GES-1 cells with Lipofectamine RNAiMAX Reagent (LifeTechnologies, Carlsbad, CA, United States).

RNA isolation

The extraction of total RNA from tissue and cell samples was done using TRIzolreagent from Invitrogen (Thermo Fisher Scientific) following the manufacturer’sprocedures. The isolation of total RNA from serum samples was done with QiagenmiRNeasy Serum/Plasma Kit from Qiagen GmbH (Hilden, Germany) following themanufacturers’ procedures. The quantity and purity of the extracted RNA sampleswere detected with NanoDrop 2000c (Thermo Fisher Scientific, Inc.). The RNAsamples with an optical density ratio (260/280) of 1.8-2.0 were accepted into laterstudy. RNA samples were either kept at -80 °C or used for direct synthesis of cDNA.

Reverse transcription and quantitative real-time polymerase chain reaction (qRT-PCR)

The PrimerScript RT Master from Takara Biotechnology (Dalian, China) was used forcDNA synthesis. The level of HOXA11-AS was measured by RT-qPCR with SYBR-Green PCR Master mix (Roche, Mannheim, Germany) on the Roche Lightcycler 480Real-Time PCR system (Roche Diagnostics, Basel, Switzerland). The qRT-PCRreactions were performed in triplicate. Changes in HOXA11-AS level were calculatedusing the 2−ΔCt method. Gene expression values were normalized to the expression ofGADPH. The sequences used in qRT-PCR are as follows: HOXA11-AS-F: 5′-GATTTCTCCAGCCTCCCTTC-3′ and HOXA11-AS-R: 5′-AGAAATTGGACGAGACTGCG-3′; GAPDH-F: 5′-TGGTATCGTGGAAGGACTCAT-3′ andGAPDH-R: 5′- GTGGGTGTCGCTGTTGAAGTC-3′.

Cell counting kit 8 (CCK-8) assay

CCK-8 assay was conducted to explore cell proliferation ability. In this assay, a 96-well plate was used and transfected MKN-45 cells were seeded in each well at adensity of 3.0 × 103 cells/well. The cells were maintained in a cell incubator. Tenmicroliters of CCK-8 reagent (US Everbright Inc.) were added to each well at 24, 48,and 72 h after the cells were incubated, respectively. The optical density of each wellof 96-well plates was detected using an enzyme immunoassay instrument from BioRad Laboratories (Hercules, CA, USA).

Transwell assay

Transwell assay was conducted to explore cell invasive capability. Transfected MKN-45 cells (4 × 104) with 200 μL of DMEM medium without serum were added into atranswell chamber (BD Biosciences, New York, NJ, United States) with Matrigel,which was inserted into a 24-well plate. Each well of the 24-well plate was added with

WJGhttps://www.wjgnet.com

2765

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Table 1 Clinicopathological characteristics of the study population

Tissue samplesSerum samples(n = 25)GenderMaleFemaleAgeMean ± SDMedian (range)Tumor size (cm)<5≥5TNM stageIIIIIIIVDifferentiationWell and moderatePoorInvasion depthT1T2T3T4Lymph node metastasisPositiveNegative1875044371239323518131250441393018214213196593560 ± 761 (45-72)59 ± 760 (40-74)59 ± 860 (41-74)151057372614Healthy controls450 μL of DMEM medium and 50 μL of FBS. MKN-45 cells were cultured for another36 hours. The MKN-45 cells that did not invade through the membrane were washedand cleaned. Crystal violet (1%) was used and the invaded MKN-45 cells invadingthrough the membrane were stained for 25 min. After washing cells with PBS threetimes, the number of MKN-45 cells invading through the membrane was calculated inthree random visual places using a light microscope.

Protein extraction and Western blot analysis

After rinsing with pre-cooling PBS, MKN-45 cells were dissociated with RIPA buffer(Thermo Fisher Scientific) mixed with protease and phosphatase inhibitor cocktail(Roche). The total protein of MKN-45 cells was isolated on the basis of instructions.The Bio-Rad assay system (Bio-Rad Laboratories, Hercules, CA, United States) wasused to detect the protein concentration. In Western blot analysis, 12% sodiumdodecyl sulfate-polyacrylamide gels were prepared and the same amounts of proteinextract were separated by electrophoresis and then transferred to polyvinylidenedifluoride (PVDF) membranes (Millipore, Billerica, MA, USA). After blocking in 5%skim milk, the blotted membranes were immersed in primary antibodies (SRSF1,1:1000; GAPDH, 1:2000, Proteintech, Wuhan, China) for 24 h. The membranes wererinsed with TBST three times (10 min/time). Subsequently, the membranes wereimmersed in a secondary antibody (HRP conjugated goat anti-rabbit antibody, 1:1000,Sigma) at room temperature for 1 h. GAPDH was used as the internal control. Kodakfilm (Kodak, Rochester, NY, United States) was used to detect the blots with anenhanced chemiluminescence kit (Merck Millipore).

Statistical analysis

SPSS 21.0 software and Graphpad prism 7 software were used for statistical analyses.The Student’s t-test was conducted to evaluate the differences between subgroups.The Chi-square test and Fisher’s exact test was conducted to explore the associationsbetween HOXA11-AS expression and clinicopathological features. ROC curve

WJGhttps://www.wjgnet.com

2766

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

analysis was performed for the assessment of the possibility of serum HOXA11-AS forGC detection. The significance of serum HOXA11-AS for prognosis evaluation of GCwas evaluated by the Kaplan-Meier method. Cox regression analysis was conductedfor evaluating the possible factors that can predict the prognosis of GC. P < 0.05 wasconsidered statistically significant.

RESULTS

HOXA11-AS levels in tissue and serum samples

We first explored the levels of HOXA11-AS in tissue and serum samples. Aspresented in Table 1, there was no significant difference in age or gender between GCpatients and healthy controls. A significantly increased level of HOXA11-AS wasfound in GC tissues vs the paired normal gastric tissues (Figure 1A, P < 0.001).Furthermore, a similar result was obtained in serum samples. Significant up-regulation of HOXA11-AS was found in the serum of GC patients vs healthyindividuals (Figure 1B, P < 0.001). As expected, the same result was obtained in GCcell lines, which revealed that HOXA11-AS was up-regulated in MKN-45 cellscompared with GES-1 cells (Figure 1C, P < 0.01).

Association between HOXA11-AS level and clinicopathologic features of GCpatients.

We next thoroughly investigated the association between HOXA11-AS level and GCpatients’ clinicopathologic features. The 94 GC patients were split into two parts usingmedian serum HOXA11-AS l as the cutoff (47 with a high serum HOXA11-AS leveland 47 with a low serum HOXA11-AS level). As shown in Table 2, there was nosignificant correlation between serum HOXA11-AS and gender, age, differentiation,or invasion depth (P > 0.05), while a remarkable association was obtained betweenserum HOXA11-AS and TNM stage (P = 0.001), tumor size (P = 0.001), as well aslymph node metastasis (P = 0.011). We found that the GC patients with decreasedserum HOXA11-AS more probably had smaller tumor, earlier TNM stage, andnegative lymph node metastasis.

HOXA11-AS functions as a potential diagnostic marker in GC

We further tested HOXA11-AS expression in serum samples from postoperative GCpatients. The data demonstrated that compared with preoperative samples, serumHOXA11-A levels showed no significant difference in the samples obtained 1 d aftersurgery (P > 0.05), but they were greatly decreased in the samples obtained 1 mo aftersurgery (P = 0.001) (Figure 2A). Furthermore, the area under the ROC curve (AUC) ofserum HOXA11-AS in the diagnosis of GC was 0.924 (95%CI: 0.881-0.967, sensitivity0.787, specificity 0.978) (Figure 2A). These data demonstrated that HOXA11-ASpossibly acts as a potential marker in the diagnosis of GC.

HOXA11-AS may function as a potential prognostic biomarker in GC

Subsequently, we analyzed the role of HOXA11-AS expression in the prognosisevaluation of GC patients. As shown in Table 3, the univariate Cox proportionalhazard regression analysis revealed that tumor size (HR = 0.527, 95%CI: 0.301-0.924, P= 0.025), TNM stage (HR = 0.195, 95%CI: 0.092-0.417, P = 0.001), and serum HOXA11-AS (HR = 0.201, 95%CI: 0.104-0.0.835, P = 0.001) were potential factors affecting theoverall survival of GC patients, and the multivariate Cox proportional hazardregression analysis demonstrated that serum HOXA11-AS (HR = 0.338, 95%CI: 0.115-0.989, P = 0.048) was a potential factor affecting the overall survival of GC patients.What's more, the Kaplan-Meier survival curve was drawn, which demonstrated thatGC individuals with lower expression of HOXA11-AS had a better overall survivalrate (P = 0.001, Figure 3). These data demonstrated that HOXA11-AS may act as apotential marker for the prognosis of GC.

Cell transfection efficiency

The down-regulation or up-regulation of HOXA11-AS in MKN-45 cells transfectedwith pcDNA HOXA11-AS or siRNA was authenticated by RT-qPCR. The amount ofHOXA11-AS in MKN-45 cells transfected with pc-DNA-HOXA11-AS wasnoteworthily upregulated vs those transfected with pcDNA3.1 (P < 0.01, Figure 4A).Moreover, MKN-45 cells transfected with HOXA11-AS-siRNA1 (P < 0.001) orHOXA11-AS -siRNA2 (P < 0.01) showed a higher level of HOXA11-AS than thosetransfected with si-NC (Figure 4B).

HOXA11-AS promotes MKN-45 cell proliferation

In our study, CCK8 assay was conducted to evaluate cell proliferative capability. As

2767

WJGhttps://www.wjgnet.comJune 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Figure 1Figure 1 HOXA11-AS expression in GC tissues, cell lines, and serum samples. A: Relative expression of HOXA11-AS in gastric cancer and normal tissues; B:Relative levels of serum HOXA11-A in gastric cancer patients and healthy controls; C: Relative levels of HOXA11-A in MNK-45 and GES-1 cells. The assays wererepeated three times. bP < 0.01, cP < 0.001. GC: Gastric cancer.

expected, cell proliferation was faster in MKN-45 cells transfected with pcDNAHOXA11-AS compared with those transfected with pcDNA 3.1 (Figure 4C). On thecontrary, cell proliferation was slower in MKN-45 cell transfected with siRNA1 orsiRNA2 in comparison with those transfected with si-NC (Figure 4D).

HOXA11-AS promotes MKN-45 cell invasion

Transwell assay was conducted to evaluate cell invasion capability, and the results arepresented in Figure 4E and F, which revealed that the quantity of invaded cells wassignificantly higher in MKN-45 cells transfected with pcDNA HOXA11-AS than inthose transfected with pcDNA 3.1 (P < 0.01, Figure 4E). However, the quantity ofinvaded cells was remarkably lower in MKN-45 cells transfected with siRNA1 (P <0.05) or siRNA2 (P < 0.05) than in those transfected with si-NC (Figure 4F).

HOXA11-AS regulates expression of SRSF1 in MKN-45 cells

The result of Western blot indicated that the protein expression of SRSF1 in MKN-45cells was upregulated when the expression of HOXA11-AS was increased (P < 0.001,Figure 5A). On the contrary, the protein expression of SRSF1 in MKN-45 cells wasdownregulated when the expression of HOXA11-AS was decreased (P < 0.01, Figure5B).

DISCUSSION

GC is one of the most frequent and fatal malignancies, seriously threatening humanhealth. Because of the position of the stomach and atypical clinical features of GC, it isquite difficult to identify GC patients from the healthy control at an early curablestage. Therefore, over half of GC patients are already in the advanced stage when theyare initially diagnosed with GC and the 5-year overall survival (OS) rate of GC is lessthan 30%[15]. However, there are no biomarkers that are accurate and easy to detect forthe diagnosis, prognosis evaluation, and treatment of GC clinically, which may beattributed to the mystery of the molecular mechanism about the genesis andprogression of GC.

HOXA11-AS, located at the 5′ region of the HOXA cluster, is one of frequentlystudied lncRNAs[12]. The level of HOXA11-AS was significantly increased in lungcancer[16], breast cancer[17], renal cancer[18], and osteosarcoma[19]. Previous studies havefound that HOXA11-AS served as a biomarker in head and head-neck carcinoma[20]and esophageal carcinoma[21]. Moreover, a meta-analysis revealed that HOXA11-ASfunctioned as a prognostic marker in human solid tumors[22]. As for non-small celllung cancer (NSCLC), an increased level of HOXA11-AS had a promotive role in cellepithelial–mesenchymal transition, which was achieved through suppressing miR-200b[23]. Moreover, the levels of HOXA11-AS in hepatocellular carcinoma tissues andcell lines were upregulated. HOXA11-AS promoted cell proliferation by regulatingcell cycle and apoptosis via DUSP5 in hepatocellular carcinoma[24]. These resultsdemonstrated that HOXA11-AS may play a significant role in various types ofcancers, including GC. In GC, HOXA11-AS was reported to be act as an oncogene andpromote GC cell proliferation, invasion, and metastasis[13,14]. However, the roles ofHOXA11-AS in the diagnosis and prognosis of GC have yet to be fully elucidated.In our study, the levels of HOXA11-AS in GC tissue samples, cell lines, and serum

WJGhttps://www.wjgnet.com

2768

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Table 2 Association of serum HOXA11-AS with clinicopathological characteristics in patientswith gastric cancerClinical featureGenderMaleFemaleAge (yr)<60≥60Tumor size (cm)<5≥5TNM stageI and IIIII and IVDifferentiationWell and moderatePoorInvasion depthT1 and T2T3 and T4Lymph node metastasisPositiveNegative50440.377 ± 0.0880.312 ± 0.10041530.348 ± 0.1000.345 ± 0.0990.01150440.346 ± 0.1120.347 ± 0.0840.86739550.250 ± 0.0730.415 ± 0.0400.93459350.320 ± 0.0940.391 ± 0.0920.00138560.337 ± 0.1110.355 ± 0.0890.00157370.345 ± 0.1040.349 ± 0.0910.304CasesSerum HOXA11-ASP-value0.824samples were detected, which revealed increased levels of HOXA11-AS in thesesamples and cell lines. Moreover, in GC patients, serum HOXA11-AS was negativelyrelated to tumor size, TNM stage, and lymph node metastasis. The role of HOXA11-AS in the diagnosis of GC was explored, which revealed that the AUC of serumHOXA11-AS in the diagnosis of GC was 0.924 (95%CI: 0.881-0.967, sensitivity 0.787,specificity 0.978). The Kaplan-Meier survival curve was drawn to evaluate HOXA11-AS in the prognosis of GC and the data revealed that the GC patients with lowHOXA11-AS expression has a better overall survival rate. Further functional assayswere conducted to probe into how HOXA11-AS affects the biological behavior of GCcells, which revealed that HOXA11-AS promoted GC cell proliferation and invasion.Serine/arginine splicing factor 1 (SRSF1), an important family member of SRproteins, was reported to participate in multiple biological functions, includingsplicing regulation, translation control, RNA transport, cell proliferation, invasion,and senescence[25-29]. SRSF1 has emerged as a key oncodriver in numerous solidtumors, such as GC. In our research, SRSF1 was found to be positively co-expressedwith HOXA11-AS in GC by StarBase 3.0 (http://starbase.sysu.edu.cn/index.php).Therefore, we assume that SRSF1 may be a target of HOXA11-AS in GC cells. Westernblot analysis was performed, and as expected, the results showed that SRSF1 may bethe target of HOXA11-AS in GC cells.

LncRNAs play a significant role in the genesis and progression of malignanttumors. Previous research found that downregulation of HOXA11-AS caused positivechanges in NSCLC cell proliferation and migration, reversed the epithelialmesenchymal transition process, and induced cell apoptosis[30]. HOXA11-AS wasincreased in breast cancer and promoted cell invasion and metastasis via EMT,providing a theoretical basis and important molecular target for the therapy of breastcancer[17]. Results of previous research verified that HOXA11-AS promoted livermetastasis in colorectal cancer by acting as a miR-125a-5p sponge[31]. HOXA11-AS alsoexerted its functions in glioblastoma, uveal melanoma, renal cancer, and cervicalcancer[18,32-34].

Previous studies revealed that lncRNAs can function as diagnostic biomarkers inGC. It was found that serum lncRNA CTC-497E21.4 may function as a potentiallydiagnostic marker for GC[35]. A lower level of circulating LINC00086 expression wasidentified in GC patients than in normal individuals. LINC00086 distinguished GCpatients from healthy controls with a high sensitivity and specificity, suggested thatLINC00086 is a potential biomarker for the diagnosis of GC[36]. Moreover, circulating

WJGhttps://www.wjgnet.com

2769

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Figure 2Figure 2 Performance of HOXA11-AS in the diagnosis of gastric cancer. A: Serum HOXA11-AS levels in the samples obtained 1 d and 1 mo after surgery; B:The ROC curve demonstrates that serum HOXA11-AS can discriminate gastric cancer patients from healthy controls (AUC = 0.924, 95%CI: 0.881-0.967, P = 0.001)with a sensitivity of 78.7% and specificity of 97.8%. cP < 0.001. The assays were repeated three times. PreOp: Pre-operation; 1 d: The first day after surgical removalof the tumor; 1 mo: The 30th day after surgical removal of the tumor; ROC: Receiver operating characteristic curve; AUC: Area under the curve.

H19 was upregulated in GC patients in comparison with healthy individuals and H19expression declined significantly upon surgical removal of the tumors, suggestingthat H19 can act as a diagnostic biomarker of GC[37].

Previous studies also revealed that lncRNAs can function as prognostic biomarkersin GC. It was found that serum lncRNA HULC was upregulated in GC patients andKaplan-Meier curve analysis suggested that HULC was a good predictor of theprognosis of GC[7]. Circulating GACAT2 level was noteworthily up-regulated inpreoperative GC patients than in the postoperative group. Moreover, the expressionof GACAT2 were associated with the clinicopathologic features (lymphatic metastasis,distal metastasis, and perineural invasion) of patients with GC, which suggested thatGACAT2 may be a potential tumor marker for the prediction of GC patientprognosis[16]. It was reported that up-regulation of plasma MALAT1 was inde-pendently correlated with a poor prognosis in GC, demonstrating MALAT1 as a novelmarker for the distant metastasis of GC patients[38].

In conclusion, serum HOXA11-AS significantly increases in GC patients. HOXA11-AS promotes GC cell proliferation and invasion via SRSF1 and may function as adiagnostic and prognostic biomarker in GC.

WJGhttps://www.wjgnet.com

2770

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Table 3 Univariate and multivariate survival analyses of 94 gastric cancer patientsUnivariate analysisVariableHRGender (Male vs Female)Age (yr) (<60 vs ≥60)Tumor size (cm) (<5 vs ≥5)TNM stage (I + II vs III + IV)Differentiation (Well and moderate vs Poor)Invasion depth (T1 + T2 vs T3 vs T4)Lymph node metastasis (Positive vs Negative)Serum HOXA11-AS (High vs Low)1.3100.9870.5270.1950.9231.0500.6670.201Multivariate analysisP-value0.3440.9630.0250.0010.7790.8630.1620.00195%CI0.749-2.2910.560-1.7390.301-0.9240.092-0.4170.529-1.610.601-1.8360.379-1.1760.104-0.385HR- - -- - -0.8160.437- - -- - -- - -0.33895%CI- - -- - -0.442-1.5080.125-1.522- - -- - -- - -0.115-0.989P-value- - -- - -0.5160.193- - -- - -- - -0.048Figure 3Figure 3 Kaplan-Meier survival curves demonstrating that gastric cancer patients with low HOXA11-AS expression has a better overall survival rate. Theassays were repeated three times.

WJGhttps://www.wjgnet.com

2771

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Figure 4Figure 4 Transfection efficiencies and cell function assays. A and B: Transfection efficiencies of pcDNA-HOXA11-AS (A) and HOXA11-AS-siRNA (B) in gastriccancer cells; C and D: Migration of MNK-45 cells after HOXA11-AS overexpression (C) or knockdown (D); E and F: Invasion of MNK-45 cells after HOXA11-ASoverexpression (E) or knockdown (F). The assays were repeated three times. aP < 0.05, bP < 0.01, cP < 0.001. OD: Optical density; NC: Normal control.

WJGhttps://www.wjgnet.com

2772

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

Figure 5Figure 5 HOXA11-AS regulates expression of SFSR1 in gastric cancer cells. A: Protein expression of SFSR1 after overexpression of HOXA11-AS in gastriccancer (GC) cells. B: Protein expression of SFSR1 after knockdown of HOXA11-AS in GC cells. The assays were repeated three times. aP < 0.05, cP < 0.001. NC:Normal control.

ARTICLE HIGHLIGHTS

Research background

As one of the most frequent cancers, gastric cancer (GC) caused more than 700000 deaths in just2012 worldwide. Many recent studies have demonstrated the molecular mechanisms involved intranscriptional regulation in GC, and long noncoding RNAs (lncRNAs) play an irreplaceable rolein the initiation and progression of GC, such as maintaining cell growth, evasion of apoptosis,promotion of invasion and metastasis, stemness maintenance, and EMT.

Research motivation

To identify more biomarkers for the diagnosis and treatment of GC.

Research objectives

This study aimed to investigate the underlying mechanisms of HOXA11-AS in GC.

Research methods

HOXA11-AS expression was detected by qRT-PCR assay in GC tissues, cell lines, and serumsamples. Clinicopathological characteristics were collected and expression analysis of HOXA11-AS was performed to evaluate the role of HOXA11-AS. Cell function assays were performed toexplore the functions of HOXA11-AS in GC cell lines. Moreover, Western blot was performed toexplore the target regulated by HOXA11-AS in GC cell lines.

Research results

We found that HOXA11-AS was upregulated in GC tissues, cell lines, and serum samples, andexhibited a significant negative correlation with tumor size, TNM stage, and lymph nodemetastasis. Cell experiments showed that HOXA11-AS promoted the proliferation and invasioncapacity of GC cell lines, and SRSF1 may be the target regulated by HOXA11-AS in GC cells.Especially, GC patients with a lower HOXA11-AS level had a better overall survival rate.

Research conclusions

Our study demonstrated that HOXA11-AS can significantly promote GC cell growth, migration,and invasion. Furthermore, it can work through SRSF1. Therefore, our study provides thepossible molecular mechanism and two new biomarkers for GC.

Research perspectives

In the future, research may reveal the important role of HOXA11-AS that enhances thesensitivity of GC detection and facilitate its application in anti-cancer treatments. Theidentification of the HOXA11-AS/SRSF1 molecular axis may further explain the underlyingmechanism.

WJGhttps://www.wjgnet.com

2773

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

REFERENCES

123456789

Van Cutsem E, Sagaert X, Topal B, Haustermans K, Prenen H. Gastric cancer. Lancet 2016; 388: 2654-2664 [PMID: 27156933 DOI: 10.1016/S0140-6736(16)30354-3]

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019; 69: 7-34 [PMID:30620402 DOI: 10.3322/caac.21551]

Thrumurthy SG, Chaudry MA, Hochhauser D, Mughal M. The diagnosis and management of gastriccancer. BMJ 2013; 347: f6367 [PMID: 24191271 DOI: 10.1136/bmj.f6367]

Murad AM, Santiago FF, Petroianu A, Rocha PR, Rodrigues MA, Rausch M. Modified therapy with 5-fluorouracil, doxorubicin, and methotrexate in advanced gastric cancer. Cancer 1993; 72: 37-41 [PMID:8508427 DOI: 10.1002/1097-0142(19930701)72:1<37::AID-CNCR2820720109>3.0.CO;2-P]

Wagner AD, Unverzagt S, Grothe W, Kleber G, Grothey A, Haerting J, Fleig WE. Chemotherapy foradvanced gastric cancer. Cochrane Database Syst Rev 2010; CD004064 [PMID: 20238327 DOI:10.1002/14651858.CD004064.pub3]

Lee YR, Kim G, Tak WY, Jang SY, Kweon YO, Park JG, Lee HW, Han YS, Chun JM, Park SY, Hur K.Circulating exosomal noncoding RNAs as prognostic biomarkers in human hepatocellular carcinoma. Int JCancer 2019; 144: 1444-1452 [PMID: 30338850 DOI: 10.1002/ijc.31931]

Jin C, Shi W, Wang F, Shen X, Qi J, Cong H, Yuan J, Shi L, Zhu B, Luo X, Zhang Y, Ju S. Long non-coding RNA HULC as a novel serum biomarker for diagnosis and prognosis prediction of gastric cancer.Oncotarget 2016; 7: 51763-51772 [PMID: 27322075 DOI: 10.18632/oncotarget.10107]

Zhao R, Zhang Y, Zhang X, Yang Y, Zheng X, Li X, Liu Y, Zhang Y. Exosomal long noncoding RNAHOTTIP as potential novel diagnostic and prognostic biomarker test for gastric cancer. Mol Cancer 2018;17: 68 [PMID: 29486794 DOI: 10.1186/s12943-018-0817-x]

Chen Q, Su Y, He X, Zhao W, Wu C, Zhang W, Si X, Dong B, Zhao L, Gao Y, Yang X, Chen J, Lu J,Qiao X, Zhang Y. Plasma long non-coding RNA MALAT1 is associated with distant metastasis in patientswith epithelial ovarian cancer. Oncol Lett 2016; 12: 1361-1366 [PMID: 27446438 DOI:10.3892/ol.2016.4800]

Zhang X, Mao L, Li L, He Z, Wang N, Song Y. Long noncoding RNA GIHCG functions as an oncogeneand serves as a serum diagnostic biomarker for cervical cancer. J Cancer 2019; 10: 672-681 [PMID:30719165 DOI: 10.7150/jca.28525]

Wang ZF, Hu R, Pang JM, Zhang GZ, Yan W, Li ZN. Serum long noncoding RNA LRB1 as a potentialbiomarker for predicting the diagnosis and prognosis of human hepatocellular carcinoma. Oncol Lett 2018;16: 1593-1601 [PMID: 30008842 DOI: 10.3892/ol.2018.8825]

Lu CW, Zhou DD, Xie T, Hao JL, Pant OP, Lu CB, Liu XF. HOXA11 antisense long noncoding RNA(HOXA11-AS): A promising lncRNA in human cancers. Cancer Med 2018; 7: 3792-3799 [PMID:29992790 DOI: 10.1002/cam4.1571]

Liu Z, Chen Z, Fan R, Jiang B, Chen X, Chen Q, Nie F, Lu K, Sun M. Over-expressed long noncodingRNA HOXA11-AS promotes cell cycle progression and metastasis in gastric cancer. Mol Cancer 2017;16: 82 [PMID: 28441948 DOI: 10.1186/s12943-017-0651-6]

Sun M, Nie F, Wang Y, Zhang Z, Hou J, He D, Xie M, Xu L, De W, Wang Z, Wang J. LncRNAHOXA11-AS Promotes Proliferation and Invasion of Gastric Cancer by Scaffolding the ChromatinModification Factors PRC2, LSD1, and DNMT1. Cancer Res 2016; 76: 6299-6310 [PMID: 27651312DOI: 10.1158/0008-5472.CAN-16-0356]

Siegel RL, Miller KD, Jemal A. Cancer statistics, 2015. CA Cancer J Clin 2015; 65: 5-29 [PMID:25559415 DOI: 10.3322/caac.21254]

Tan L, Yang Y, Shao Y, Zhang H, Guo J. Plasma lncRNA-GACAT2 is a valuable marker for thescreening of gastric cancer. Oncol Lett 2016; 12: 4845-4849 [PMID: 28105191 DOI:10.3892/ol.2016.5297]

Li W, Jia G, Qu Y, Du Q, Liu B, Liu B. Long Non-Coding RNA (LncRNA) HOXA11-AS PromotesBreast Cancer Invasion and Metastasis by Regulating Epithelial-Mesenchymal Transition. Med Sci Monit2017; 23: 3393-3403 [PMID: 28701685]

Yang FQ, Zhang JQ, Jin JJ, Yang CY, Zhang WJ, Zhang HM, Zheng JH, Weng ZM. HOXA11-ASpromotes the growth and invasion of renal cancer by sponging miR-146b-5p to upregulate MMP16expression. J Cell Physiol 2018; 233: 9611-9619 [PMID: 29953617 DOI: 10.1002/jcp.26864]Cui M, Wang J, Li Q, Zhang J, Jia J, Zhan X. Long non-coding RNA HOXA11-AS functions as a

competing endogenous RNA to regulate ROCK1 expression by sponging miR-124-3p in osteosarcoma.Biomed Pharmacother 2017; 92: 437-444 [PMID: 28558357 DOI: 10.1016/j.biopha.2017.05.081]

Yao Y, Chen X, Lu S, Zhou C, Xu G, Yan Z, Yang J, Yu T, Chen W, Qian Y, Ding S, Tang J, Chen Y,Zhang Y. Circulating Long Noncoding RNAs as Biomarkers for Predicting Head and Neck Squamous CellCarcinoma. Cell Physiol Biochem 2018; 50: 1429-1440 [PMID: 30355951 DOI: 10.1159/000494605]Sun XY, Wang XF, Cui YB, Cao XG, Zhao RH, Wei HY, Cao W, Wu W. [Expression level and clinicalsignificance of LncRNA HOXA11-AS in esophageal squamous cell carcinoma patients]. Zhonghua ZhongLiu Za Zhi 2018; 40: 186-190 [PMID: 29575836 DOI: 10.3760/cma.j.issn.0253-3766.2018.03.005]

Mu S, Ai L, Fan F, Sun C, Hu Y. Prognostic and clinicopathological significance of long noncoding RNAHOXA11-AS expression in human solid tumors: a meta-analysis. Cancer Cell Int 2018; 18: 1 [PMID:29308050 DOI: 10.1186/s12935-017-0498-3]

Chen JH, Zhou LY, Xu S, Zheng YL, Wan YF, Hu CP. Overexpression of lncRNA HOXA11-ASpromotes cell epithelial-mesenchymal transition by repressing miR-200b in non-small cell lung cancer.Cancer Cell Int 2017; 17: 64 [PMID: 28615992 DOI: 10.1186/s12935-017-0433-7]

Liu B, Li J, Liu X, Zheng M, Yang Y, Lyu Q, Jin L. Long non-coding RNA HOXA11-AS promotes theproliferation HCC cells by epigenetically silencing DUSP5. Oncotarget 2017; 8: 109509-109521 [PMID:29312625 DOI: 10.18632/oncotarget.22723]

Erkelenz S, Mueller WF, Evans MS, Busch A, Schöneweis K, Hertel KJ, Schaal H. Position-dependentsplicing activation and repression by SR and hnRNP proteins rely on common mechanisms. RNA 2013; 19:96-102 [PMID: 23175589 DOI: 10.1261/rna.037044.112]

Wang Y, Xiao X, Zhang J, Choudhury R, Robertson A, Li K, Ma M, Burge CB, Wang Z. A complexnetwork of factors with overlapping affinities represses splicing through intronic elements. Nat Struct MolBiol 2013; 20: 36-45 [PMID: 23241926 DOI: 10.1038/nsmb.2459]

Das R, Yu J, Zhang Z, Gygi MP, Krainer AR, Gygi SP, Reed R. SR proteins function in coupling RNAPII transcription to pre-mRNA splicing. Mol Cell 2007; 26: 867-881 [PMID: 17588520 DOI: 10.1016/j.mol-cel.2007.05.036]

1011121314

15161718192021222324252627

WJGhttps://www.wjgnet.com

2774

June 14, 2019Volume 25Issue 22

Liu Y et al. LncRNA HOXA11-AS promotes GC

28293031

Fregoso OI, Das S, Akerman M, Krainer AR. Splicing-factor oncoprotein SRSF1 stabilizes p53 via RPL5and induces cellular senescence. Mol Cell 2013; 50: 56-66 [PMID: 23478443 DOI:10.1016/j.molcel.2013.02.001]

Zhou X, Wang R, Li X, Yu L, Hua D, Sun C, Shi C, Luo W, Rao C, Jiang Z, Feng Y, Wang Q, Yu S.Splicing factor SRSF1 promotes gliomagenesis via oncogenic splice-switching of MYO1B. J Clin Invest2019; 129: 676-693 [PMID: 30481162 DOI: 10.1172/jci120279]

Zhao X, Li X, Zhou L, Ni J, Yan W, Ma R, Wu J, Feng J, Chen P. LncRNA HOXA11-AS drives cisplatinresistance of human LUAD cells via modulating miR-454-3p/Stat3. Cancer Sci 2018; 109: 3068-3079[PMID: 30099826 DOI: 10.1111/cas.13764]

Chen D, Sun Q, Zhang L, Zhou X, Cheng X, Zhou D, Ye F, Lin J, Wang W. The lncRNA HOXA11-ASfunctions as a competing endogenous RNA to regulate PADI2 expression by sponging miR-125a-5p inliver metastasis of colorectal cancer. Oncotarget 2017; 8: 70642-70652 [PMID: 29050308 DOI:10.18632/oncotarget.19956]

Se YB, Kim SH, Kim JY, Kim JE, Dho YS, Kim JW, Kim YH, Woo HG, Kim SH, Kang SH, Kim HJ,Kim TM, Lee ST, Choi SH, Park SH, Kim IH, Kim DG, Park CK. Underexpression of HOXA11 IsAssociated with Treatment Resistance and Poor Prognosis in Glioblastoma. Cancer Res Treat 2017; 49:387-398 [PMID: 27456940 DOI: 10.4143/crt.2016.106]

Lu Q, Zhao N, Zha G, Wang H, Tong Q, Xin S. LncRNA HOXA11-AS Exerts Oncogenic Functions byRepressing p21 and miR-124 in Uveal Melanoma. DNA Cell Biol 2017; 36: 837-844 [PMID: 28749709DOI: 10.1089/dna.2017.3808]

Kim HJ, Eoh KJ, Kim LK, Nam EJ, Yoon SO, Kim KH, Lee JK, Kim SW, Kim YT. The long noncodingRNA HOXA11 antisense induces tumor progression and stemness maintenance in cervical cancer.Oncotarget 2016; 7: 83001-83016 [PMID: 27792998 DOI: 10.18632/oncotarget.12863]

Zong W, Feng W, Jiang Y, Ju S, Cui M, Jing R. Evaluating the diagnostic and prognostic value of serumlong non-coding RNA CTC-497E21.4 in gastric cancer. Clin Chem Lab Med 2019; Epub ahead of print[PMID: 30763257 DOI: 10.1515/cclm-2018-0929]

Ji B, Huang Y, Gu T, Zhang L, Li G, Zhang C. Potential diagnostic and prognostic value of plasma longnoncoding RNA LINC00086 and miR-214 expression in gastric cancer. Cancer Biomark 2019; 24: 249-255 [PMID: 30689553 DOI: 10.3233/cbm-181486]

Yörüker EE, Keskin M, Kulle CB, Holdenrieder S, Gezer U. Diagnostic and prognostic value ofcirculating lncRNA H19 in gastric cancer. Biomed Rep 2018; 9: 181-186 [PMID: 30083318 DOI:10.3892/br.2018.1116]

Xia H, Chen Q, Chen Y, Ge X, Leng W, Tang Q, Ren M, Chen L, Yuan D, Zhang Y, Liu M, Gong Q, BiF. The lncRNA MALAT1 is a novel biomarker for gastric cancer metastasis. Oncotarget 2016; 7: 56209-56218 [PMID: 27486823 DOI: 10.18632/oncotarget.10941]

32

333435363738

WJGhttps://www.wjgnet.com

2775

June 14, 2019Volume 25Issue 22