Hindawi BioMed Research International Volume 2018, Article ID 2850657, 7 pages https://doi.org/10.1155/2018/2850657

Research Article HOTAIR Is a Potential Novel Biomarker in Patients with Congenital Heart Diseases

Yu Jiang,1,2 Hongdan Mo,1,2 Jing Luo,1,2 Suhong Zhao,1,2 Shuang Liang,1,2 Maomao Zhang,1,2 and Jie Yuan 1,2

1 Department of Cardiology, Te Second Afliated Hospital of Harbin Medical University, Harbin 150001, China 2Te Key Laboratory of Myocardial Ischemia, Harbin Medical University, Ministry of Education, Heilongjiang Province 150001, China

Correspondence should be addressed to Jie Yuan; [email protected]

Received 5 October 2017; Revised 27 January 2018; Accepted 7 February 2018; Published 7 March 2018

Academic Editor: Diego Franco

Copyright © 2018 Yu Jiang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Objective. To investigate the expression of HOX transcript antisense RNA (HOTAIR) in cardiac tissues and plasma of patients with congenital heart diseases (CHDs). Methods. qRT-PCR was used to detect the expression of HOTAIR in right atrial appendage tissues of 16 patients with CHDs and 14 patients with rheumatic valvular heart diseases (RVHDs), as well as in plasma of 36 normal people and 90 patients with CHDs including 36 cases of ASD, 23 cases of VSD, and 31 cases of PDA. Besides, the proteins interacting with HOTAIR were obtained from databases. Results. Te HOTAIR expression in cardiac tissues of CHDs group was signifcantly higher than that of the RVHDs group (� < 0.01). Compared with the control group, the expression of plasma HOTAIR in the ASD group, the VSD group, and the PDA group was all remarkably upregulated (� < 0.01), whereas there was no relationship between HOTAIR and pulmonary arterial hypertension and defects size. Databases show that HOTAIR is associated with polycomb repressive complex 2 (PRC2) which contributes to heart development. Conclusion.TelevelsofHOTAIRwereincreasedincardiac tissues and plasma of patients with CHDs. HOTAIR is a potential novel diagnostic biomarker in patients with CHDs.

1. Introduction HOX transcript antisense RNA (HOTAIR) is the frst transregulation lncRNA to be found. HOTAIR, which has Congenital heart disease (CHD) is the most common type a function of epigenetic regulation, has been shown to of congenital deformity and still represents an important be relevant to many diseases, such as multiple cancers, noninfectious cause of death in children [1]. CHD is widely osteoarthritis, and cardiovascular diseases [6]. Lots of stud- perceived to be a polygene heredity disease as a result of the ies have illustrated an important role of HOTAIR in car- complex interplay of genetic factors and environmental fac- diac diseases, including aortic valve calcifcation, cardiac tors in the early development of embryos, but its pathogenic hypertrophy, heart failure, and cardiac-associated sepsis [6– mechanism is still under research. A host of studies have 9]. However, little is known about the potential roles of shown that abnormal development of heart and vessels is HOTAIR in embryonic heart development. Terefore, in the associated with the mutation of many factors present study we sought to determine the expression levels including Nkx2.5, GATA4, and Tbx5 [2]. Long noncoding of HOTAIR in cardiac tissues and plasma of patients with RNAs (lncRNAs) are defned as being noncoding RNA CHDs, establishing some bases for further research about the sequences of >200 nucleotides that can regulate target- function of HOTAIR on the embryonic heart development. expression, and they are closely related to malignant tumors, cardiovascular diseases, embryonic development, organism 2. Materials and Methods aging, and so on [3]. Mounting studies indicate that lncRNAs are new regulatory molecules involved in embryonic heart 2.1. Study Population. Te all-included cases with CHDs and development afer miRNAs [4, 5]. rheumatic valvular heart diseases (RVHDs) were derived 2 BioMed Research International from patients who had been diagnosed at the Second recentrifuged at 12000 rpm for 10 minutes. All steps were ∘ Afliated Hospital of Harbin Medical University, China, performed at 4 C. Plasma was then collected, divided into ∘ from November 2015 to June 2016. Human cardiac tissues aliquots, and stored frozen at −80 Cbeforeuse. were collected from 16 patients with CHDs and 14 patients with RVHDs who all required surgical treatment. Blood 2.5. RNA Isolation. Total plasma RNA was extracted from samples were collected from 90 patients with CHDs and 500 �l plasma using the Trizol (Invitrogen) according to the 36normalpeople.CHDsgroupsofbloodsampleswere manufacturer’s instruction and dissolved in 15 �lofdiethyl composed of 36 cases of atrial septal defect (ASD), 23 cases pyrocarbonate (DEPC) water. (i) Mix 500 �lplasmaand of ventricular septal defect (VSD), and 31 cases of patent 800 �lTrizol,andthenletitsitfor5minutesatroom ductus arteriosus (PDA). Te inclusion criteria of CHDs are temperature. (ii) Ten 160 �l chloroform was added. (iii) as follows: all cases were diagnosed with lef-to-right shunt Te mixture was centrifuged at 12000 rpm for 15 minutes CHDs (ASD, VSD, and PDA), the diagnosis was verifed by afer 15 minutes’ standing at room temperature. Ten 800 �l physical examination, X-ray, ECG, three-dimensional color supernatant was collected. (iv) Mix the supernatant with ∘ Doppler echocardiography, and the corrective operation. Te 800 �l isopropanol and let it sit for 4 hrs at −20 C. (v) Te healthy individuals were from center of health examination, mixture was centrifuged at 12000 rpm for 15 minutes, then the without a family history of CHDs and other congenital supernatant was discarded, and 1 ml 75% anhydrous ethanol malformation. Patients with CHDs were diagnosed by two (DEPC water preparation) was added to the centrifuge tube. cardiology experts with three-dimensional color Doppler. (vi) Repeat step (v). (vii) Te mixture was centrifuged at Exclusion criteria were as follows: (i) complex CHDs; (ii) 12000 rpm for 10 minutes, discarded the supernatant, dried patients with acute infection, serious heart valve disease, the white RNA precipitation at the bottom of the tube at room stroke, malignant tumor, severe liver and kidney disease, temperature for 5 minutes, and dissolved it in 16 �lDEPC and other severe systemic diseases; (iii) patients with use of water. Te concentration and purity of RNA were detected by immunosuppressant; (iv) patients with a history of recent ultraviolet spectrophotometer. Total RNA of cardiac tissues surgery and muscle damage which change the levels of was extracted from 50 mg tissues that were ground on ice lncRNAs; (v) patients who provided incomplete information; and the following steps were also carried out according to the (vi) patients who refused to participate in the study. Te manufacturer’s instruction. protocol was approved by the Ethics Committee of the Second Afliated Hospital of Harbin Medical University (Number 2.6. Primer Design and Quantitative Real-Time PCR Analysis � 246 Xuefu Road, Harbin, China) and informed consent was (qRT-PCR). HOTAIR forward primer: 5 -GGTAGAAAA- � � obtained from the parents or guardians. Tis study complies AGCAACCACGAAGC-3 , reverse primer: 5 -ACATAA- � with the Declaration of Helsinki. ACCTCTGTCTGTGAGTGCC-3 ,productlengthis170bp. � Reference GAPDH forward primer: 5 -CCGGGAAACTGT- � � 2.2. Defnition of Pulmonary Arterial Hypertension (PAH). GGCGTGATGG-3 , reverse primer: 5 -AGGTGGAGG- � PAH is evaluated according to the pulmonary artery systolic AGTGGGTGTCGCTGTT-3 , product length is 309 bp [13]. pressure (PASP) [10]: no pulmonary hypertension: PASP < According to the manufacturer’s protocol of the reverse 30 mmHg; pulmonary hypertension: PASP > 30 mmHg. transcription reagent kit (Bioneer company), 15 �ltotal plasma RNA was used for cDNA synthesis. Next, we used 2.3. Dividing Standard of Defect Size (DS). Defect size is the AccuPower5 2×Greenstar6 qPCR Master Mix kit (Bioneer maximum value of each section. According to the ultrasonic company) to detecte cDNA by CFX96TM real-time quanti- measurement DS defne [11, 12], for adult, small defect of tative PCR detect system (Bio-Rad company). Te reaction VSDissmallerthan1cmindiameter,smalldefectofASD was performed in 20 �lvolumewith10�l2×Greenstar Master is smaller than 2 cm in diameter, medium to large defect of Mix, 0.4 �lRoxdye,0.5�leachofforwardandreverse VSD is 1 cm or more in diameter, and medium to large defect primers, 7.2 �l DEPC-treated water, and 2.0 �lcDNA(2.0�l of ASD is 2 cm or more in diameter. For child, small defect of cDNA was replaced with 2.0 �l DEPC-treated water in nega- 2 ∘ VSDissmallerthan1cm/m in diameter, small defect of ASD tive controls). Cycling parameters were as follows: 95 Cfor10 2 ∘ is smaller than 2 cm/m in diameter, medium to large defect minutes for initial denaturation followed by 40 cycles of 95 C 2 ∘ of VSD is 1 cm/m or more in diameter, and medium to large for 15 sec and 61 Cfor1minute,thenfuorescencesignals 2 defect of ASD is 2 cm/m or more in diameter (defect size and were detected. Te Ct value was defned as the cycle number the ratio of surface area). at which the fuorescence (ΔRn) exceeded the threshold. A threshold of 0.20 was used as the default setting. Each 2.4. Tissue and Plasma Samples. Cardiac tissue specimens sample’s experiment was replicated 3 times; the average of the were acquired from right atrial appendage tissues of patients 3 results was the experimental result. Te relative expression with CHDs and patients with RVHDs during surgical resec- of target lncRNAs was determined by using the comparative −ΔΔCT tion. Ten the cardiac tissues were snap-frozen in liquid cycle threshold (Ct) method (2 ); the melt curve analysis ∘ nitrogen and stored at −80 C immediately. Blood samples was used to confrm the specifcity of amplifcation and the were collected from CHDs patients and healthy individuals absence of primer dimers. and put into a test tube containing EDTA. Blood samples were centrifuged at 3000 rpm for 10 minutes immediately. 2.7. Statistical Analysis. Te data was analyzed by SPSS 21.0 Te supernatant was transferred to new centrifuge tubes and statistical sofware package. Te normal distribution data BioMed Research International 3

Table 1: General conditions of patients with CHDs group and patients with RVHDs group.

Characteristics CHDs (�=16)RVHDs(�=14)Statistic � 2 Gender (male/female) 9/7 6/8 � = 0.536 0.464 Age (�±�,years) 11.94 ± 1.42 45.50 ± 1.58 � = 0.068 <0.05 ∗ Expression of HOTAIR 0.111 ± 0.015 0.055 ± 0.007 � = 3.171 0.002 ∗ Compared with RVHDs group, � < 0.05; CHDs: congenital heart diseases; RVHDs: rheumatic valvular heart diseases.

Table 2: General conditions of patients with CHDs group and control group (CN).

Characteristics CN (�=36)ASD(�=36)VSD(�=23)PDA(�=31)Statistic � 2 Gender (male/female) 17/19 16/20 10/13 9/22 � = 2.638 0.451 Age (�±�,years) 23.08 ± 14.75 24.03 ± 16.52 15.30 ± 16.31 21.48 ± 19.77 � = 6.050 0.109 DS (�±�, cm) — 2.27 ± 0.90 1.17 ± 0.76 0.69 ± 0.44 —— PASP (�±�, mmHg) — 34.21 ± 9.11 38.75 ± 17.03 50.90 ± 27.25 � = 4.341 0.114 LEVF (�±�,%) — 62.56 ± 1.93 62.87 ± 1.14 61.70 ± 4.87 � = 1.047 0.355 ∗∗ ∗ ∗ Expression of HOTAIR 1.00 ± 0.33 1.37 ± 0.42 1.39 ± 0.60 1.35 ± 0.57 � = 15.665 0.001 ∗ ∗∗ Compared with CN group, � < 0.05; compared with CN group � < 0.01; LEVF: lef ventricular ejection fraction; ASD: atrial septal defect; VSD: ventricular septal defect; PDA: patent ductus arteriosus. was expressed by �±�. Te nonnormal distribution data were consisted of 17 males and 19 females. Tere were no was expressed by median (M) and quartile (QR). And the statistical diferences between composition of gender and age 2 2 data from categorical variables was analyzed by � text. (� = 2.638, � = 0.451 and; � = 6.050, � = 0.109)(Table2). Distribution of continuous variables was tested by the single sample K-S test in nonparametric test. If the variables 3.2. Results of Real-Time Quantitative PCR. Te light density obey the normal distribution and homogeneity of variance, was detected afer total RNA was isolated from cardiac the two groups were compared by independent samples � tissues and plasma. Te purity was good; meanwhile, the � test and multiple groups were compared by single factor (260 nm)/� (280 nm) was between 1.8∼2.0.Atthesametime, analysis of variance. Otherwise, the mean was compared by there was no amplifcation in negative control. 2% agarose nonparametric K-W test. Te correlation test was performed gel electrophoresis was used to detect the PCR products, by the Spearman correlation analysis. Images were rendered which showed that specifc purpose strips of HOTAIR and by GraphPad Prism 5.0 sofware. Bilateral � < 0.05 was GAPDH separately appeared in 170 bp and 309 bp (Figure 1). considered statistically signifcant. Te average levels of HOTAIR expression in cardiac tissues of patients with CHDs and patients with RVHDs as well as in 2.8. Bioinformatic Analysis. Proteins interacting with plasma of the ASD group, the VSD group, the PDA group, and HOTAIR were obtained from LncRNA and Disease Database the normal group were (0.111±0.015), (0.055±0.007), (1.37± (LncRNADisease) (http://www.cuilab.cn/lncrnadisease) up 0.42), (1.39±0.60), (1.35±0.57), and (1.00±0.33), respectively. until July 2016 with “HOTAIR” as the search term and the Te HOTAIR expression in cardiac tissues of patients with interaction between proteins was downloaded from Human CHDs was signifcantly higher than that of patients with Protein Reference Database (HPRD) (http://www.hprd.org/) RVHDs (� = 0.002). Compared with the normal group, the up until July 2016. expressionofplasmaHOTAIRinCHDsgroupswasmarked increased (� = 0.001). Among the CHDs groups, the expres- �= 3. Results sion levels of plasma HOTAIR in the ASD group ( 0.002), the VSD group (� = 0.040), and the PDA group 3.1. General Conditions. 16 patients with CHDs were enrolled (� = 0.020) were all remarkable upregulated. Among these in tissue research including 9 males and 7 females, and groups, the ASD group increased predominantly (Figure 2). 14 patients with RVHDs were composed of 6 males and 8 In addition, we also analyzed the relevance between age and females.Terewasnostatisticaldiferenceincomposition the expression of plasma HOTAIR in the ASD group. Te 2 of gender (� = 0.536, � = 0.464). However, there was a age range was from 1 to 53 and the average age was 24.03 ± signifcant diference in composition of age as it was difcult 16.53. Result showed that there was no statistically signifcant to match the age between patients with CHDs and patients diference between age and the expression of HOTAIR (�> with RVHDs (� = 0.068, � < 0.05)(Table1).90patientswith 0.05). CHDs were enrolled in plasma research including 36 cases of ASD consisted of 16 males and 20 females, 23 cases of VSD 3.3. Comparison of HOTAIR Levels in ASD and VSD Patients composed of 10 males and 13 females, and 31 cases of PDA with Diferent DS. According to the defect size of ASD and made up of 9 males and 22 females. 36 cases of healthy group VSD measured via ultrasound, patients were divided into two 4 BioMed Research International

∗∗ ∗∗ bp M 1234— bp 3

309 GAPDH 300 200 2 170 HOTAIR 150 HOTAIR Figure 1: Electrophoresis results of real-time quantitative PCR 1

HOTAIR and GAPDH products.1:normalgroup.2:ASDgroup.3: of level Expression VSDgroup.4:PDAgroup.—:negativecontrols.M:DNAladder.

0 CN ASD VSD PDA groups. In ASD group, HOTAIR expression in plasma was � = 1.876 � = 0.069 not statistically signifcant ( , ) between 14 Figure 2: HOTAIR were detectable in plasma from CHDs patients ∗ patients with small defect (1.53 ± 0.43)and22patientswith and healthy controls. Compared with control group, � < 0.05; ∗∗ moderate to severe (1.27 ± 0.39). In VSD group, HOTAIR compared with control group, � < 0.01. expression in plasma was not statistically signifcant (�= 1.907, � = 0.070) between 9 patients with small defect (1.11± 0.41) and 14 patients with moderate to severe (1.57 ± 0.65) (Figure 3). physiological and pathological processes by epigenetic regu- lation. 3.4. Comparison of the Expression of HOTAIR in CHDs Because the age range of patients with CHDs is widely Patients with or without PAH. In ASD group, HOTAIR levels distributed, including all ages from newborn to the survived were (1.27±0.47)in13casesofwithoutPAHand(1.43±0.39) crowd, and it is difcult to obtain myocardial tissue samples in 23 cases of with PAH. Te diference revealed no statistical from normal population, we chose to detect the expression signifcance (� = 1.128, � = 0.267). In VSD group, HOTAIR of HOTAIR in right atrial appendage tissues of patients with levels were (1.26 ± 0.77)in12casesofwithoutPAHand CHDs and patients with RVHDs. Te results of the present (1.54 ± 0.32) in 11 cases of with PAH. Te diference revealed study indicated that the HOTAIR expression in cardiac tis- no statistical signifcance (� = 1.113, � = 0.278). In PDA sues of patients with CHDs was signifcantly higher than that group, HOTAIR levels were (1.12 ± 0.51)in7casesofsmall of patients with RVHDs. In order to match the composition defect without PAH (1.42 ± 0.57) in 24 cases of small defect of age and expand the number of samples, we also chose with PAH. Te diference revealed no statistical signifcance to determine the expression of plasma HOTAIR between (� = 1.254, � = 0.220) (Figure 4). patients with CHDs and healthy individuals. In our study, plasma HOTAIR expression was also detected to be upreg- 3.5. Proteins Interacting with HOTAIR and the Interaction ulatedinCHDs.Notably,ASDgroupwasmostsignifcantly between Proteins. Te interacting proteins contain EZH1, increased. Besides, there was no correlation between age and EZH2, SUZ12, REST, KDM1A (LSD1), RCOR1 (COREST), HOTAIR expression. Tere was no signifcant relationship and HOXD. Ten a network of HOTAIR and its interacting between plasma HOTAIR and defects size and PAH to be proteins as well as a network of the interaction between found in CHDs patients. proteinscanbeconstructed(Figure5). PRC2 is mainly composed of three core subunits EED, SUZ12, and EZH2 [19]. Te biological analysis shows that 4. Discussion proteins interacting with HOTAIR include EZH2, SUZ12, and KDM1A (LSD1), so HOTAIR has the function of acting TehumanHOTAIRlocatesintheHOXCclusteronchro- as a scafold for methylase (PRC2) and histone mosome12.TeHOTAIRgeneistranscribedinanantisense (LSD1). Many malignant tumor experiments direction relative to its fanking HOXC11 and HOXC12 . have demonstrated that HOTAIR altered histone H3 lysine Its principal transcript is a 2364 bp RNA transcribed from a 27 methylation by recruiting PRC2 to suppress target genes 6449 bp gene locus and composed of 6 exons. HOTAIR was and control Wnt/�-catenin [13, 20]. Previous studies have coexpressed with HOXC [14]. Human shown that Wnt signaling pathway played a vital role in cluster contains four subfamilies: HOXA, HOXB, HOXC, and regulating cell proliferation, migration, and heart devel- HOXD that the proteins they encode have a conserved home- opment. Abnormal activation of Wnt signaling pathway odomain, which is related to human embryonic development caused by genetic and epigenetic changes was associated and plays a pivotal role in regulating tissue diferentiation and with the pathogenetic mechanism of CHDs [21]. Tus, as a organ sculpting [15]. Studies have shown that HOTAIR was key Wnt signaling pathway regulating factor, HOTAIR may involved in cancer invasion and metastasis [16, 17]. A previous play an important role in cardiac development. Moreover, study has proved that HOTAIR was important to modulate PRC2 has methyltransferase activity that can regulate the proliferation and diferentiation of mesenchymal stem cells expression of noncardiac genes such as INK4a/b, ISL1, and [18]. Tus, HOTAIR plays an indispensable part in various Six1 and cardiac transcription factors such as GATA4 by BioMed Research International 5

2.5 2.0

2.0 1.5

1.5 1.0 HOTAIR HOTAIR 1.0 Expression level of Expression level of level Expression 0.5 0.5

0.0 0.0 Small DS Moderate-to-severe DS Small DS Moderate-to-severe DS (a) (b)

Figure 3: Comparison of the expression of HOTAIR in ASD (a) and VSD (b) patients with diferent DS.

2.0 2.0 2.0

1.5 1.5 1.5

1.0 1.0 1.0 HOTAIR HOTAIR HOTAIR 0.5 0.5 0.5 Expression level of of level Expression Expression level of

0.0 0.0 0.0 Without PAH With PAH Without PAH With PAH Without PAH With PAH (a) (b) (c)

Figure 4: Comparison of the expression of HOTAIR in ASD (a), VSD (b), and PDA (c) patients with or without PAH.

HOXD SUZ12 DNMT3A

EZH2

DNMT3B EZH1 HOTAIR

RCOR1

DNMT1 KDM1A

REST

Figure 5: Te proteins interacting with HOTAIR and the interaction between proteins. 6 BioMed Research International catalyzing histone H3K27 and DNA methylation, thereby [4] C. A. Klattenhof, J. C. Scheuermann, L. E. Surface et al., “Brave- having an impact on embryonic heart development [19, 22]. heart, a long noncoding RNA required for cardiovascular line- On the other hand, HOTAIR might adjust the level of the age commitment,” Cell,vol.152,no.3,pp.570–583,2013. intracellular calcium by repressing the expression of CaV1.2 [5] P. Grote, L. Wittler, D. Hendrix et al., “Te tissue-specifc and interfere with the occurrence and development of many lncRNA Fendrr is an essential regulator of heart and body wall diseases [23]. Recent studies have found that calcium channel development in the mouse,” Developmental Cell,vol.24,no.2, blockade agent (nifedipine) inhibits the early embryonic pp.206–214,2013. mesoderm multipotent stem cells which diferentiate into [6] A. Bhan and S. S. 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