Clinical Significance of Long Non-Coding RNA MALAT1 Expression in Tissue and Serum of Breast Cancer

Available online at www.annclinlabsci.org 418 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016 Clinical Significance of Long Non-coding RNA MALAT1 Expression in Tissue and Serum of Breast Cancer

Yufeng Miao1,2,*, Rengen Fan3,*, Lige Chen4, and Haixin Qian1

1Department of General Surgery, The First Affiliated Hospital of Soochow University, 2Department of Medical Oncol- ogy, Yancheng City No.1 People's Hospital, Yancheng, 3Department of General Surgery, Yancheng City No.1 People's Hospital, Yancheng, and 4Department of Pharmacy, Yancheng City No.1 People's Hospital, Yancheng, China

Abstract. Long non-coding RNAs (lncRNAs) have been proven to serve a critical role in cancer development and progression. The aim of this study was to elucidate clinical significance of lncRNA MALAT1 expression in breast cancer (BC). A total of 78 BC patients treated with radical resection were enrolled in this study. Quantitative reverse transcription-polymerase chain reaction was used to detect MALAT1 expression in tissues and serum samples. The receiver operating characteristics (ROC) curve was constructed to describe diagnostic speciﬁcity and sensitivity. Lentivirus-mediated RNA interference was used to knockdown MALAT1 in the MDA-MB-231 cell line, and then cell proliferation and invasion were explored. Results showed that MALAT1 expression was significantly up-regulated in 85.9% (67/78) of cancerous tissues compared with normal counterparts (P<0.01). Further, an elevated MALAT1 expression in BC tissue was significantly associated with lymph metastasis (P=0.037) and adverse 5-year disease-free survival (mean 48.5 months vs 62.7 months, P=0.012). Suppression of lncRNA MALAT1 significantly inhibited BC cells proliferation, migration and invasion, induced apoptosis and cell cycle G1 arrest. In addition, serum MALAT1 levels in BC patients were much higher than levels in patients with benign breast disease (P<0.001), its diagnostic efficacy was satisfactory, area under the curve (AUC) was 0.833. In conclusion, MALAT1 upregulation plays an important rolein BC development, and serum MALAT1 level may be a potential tumor marker for BC diagnosis.

Key words: breast cancer, lncRNA, MALAT1, prognosis, diagnosis.

Introduction Long non-coding RNAs (lncRNAs) are kinds of transcriptional products of the eukaryotic genome Breast cancer (BC) is the most common type of tu- that are composed of more than 200 nucleotides in mor in women. Nowadays, the incidence of BC is length. For a long time these lncRNAs have been still increasing in China and many other develop- considered as transcriptional noise; however, grow- ing countries [1]. Although the molecular mecha- ing evidence suggest that lncRNAs are key regula- nisms of BC have been extensively researched over tors which govern various biological processes [4]. the past decades, there are still many unresolved is- Dysregulation of lncRNAs is associated with vari- sues in clinic, such as delayed diagnosis, recurrence ous types of cancers [5]. More recently, many ln- and metastasis. Therefore, it is crucial to develop cRNAs have been shown to exert oncogenic or tu- more effective screening methods and novel thera- mor suppressor properties in BC [6,7]. Metastasis peutic targets to better treat the disease. Currently, associated lung adenocarcinoma transcript 1 molecular markers including circulating tumor (MALAT1), one of the first found cancer-associat- cells, microRNAs, and DNA methylation have ed lncRNAs, is highly expressed in metastasizing contributed to improving the diagnosis, prognosis, non-small cell lung cancer [8]. Subsequent studies and guidance of adjuvant treatments for BC [2,3]. have found that MALAT1 was also upregulated and played a pivotal role in the progression of other *Those authors equally contributed to this work. Address cancers, including hepatocellular carcinoma, correspondence to Prof. Haixin Qian, Department of General Surgery, The First Affiliated Hospital of Soochow University, Suzhou 215006, esophageal squamous cell carcinoma, colorectal China; e mail: [email protected] cancer, pancreatic cancer and osteosarcoma [9-13].

However, the clinical significance of MALAT1 in BC research remains contradictory [14]. Therefore, we studied MALAT1 expression levels in tissue and serum samples from BC patients treated with radical resection, and the functional roles of MALAT1 in BC cell line were explored.

Materials and Methods

Study population. BC tissues and paired adjacent non- tumor tissues were obtained from 78 patients treated with radical resection at our institutes from June 2008 to June 2010. Tu mor stage was conducted according to the 7th edition of the TNM staging system of the International Union Against Cancer. Cellular differentiation was graded according to the WHO grading system. Clinical follow-up data were available for all the patients. For each patient, 5 mL of peripheral blood pre- operation was collected by promoting coagulation tubes, and then serum was isolated and stored at -80˚C. Serum samples from 40 patients with benign breast disease were also collected. Ethical approval was obtained from the hospital, and informed consent was obtained from all patients prior to sample examination.

RNA isolation and qRT-PCR. Total RNA was extracted from tissues and serum samples using Tr izol reagent (Invitrogent) according to the manufacturer’s instruc- tions. The expression levels of MALAT1 were determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) using the SYBR®Green (TaKaRa) dye detection method on ABI StepOne PCR Figure 1. LncRNA MALAT1 expression in human breast instrument, with GAPDH as an internal control. The cancer tissues. (A) Relative expression of MALAT1 in BC primers are as follows: MALAT1 forward: 5’-GGA TCC tissues (n=78) compared with corresponding non-tumor tissues (n=78). MALAT1 expression was examined by TAG ACC AGC ATG CC-3’; reverse: 5’-AAA GGT qPCR and normalized to GAPDH expression. Results are TAC CAT AAG TAA GTT CCA GAA AA-3’; GAPDH presented as the fold-change in tumor tissues relative to forward: 5’-GCT CTC TGC TCC TCC TGT TC-3’; normal tissues. (B) LncRNA MALAT1 expression was reverse: 5’-ACG ACC AAA TCC GTT GAC TC-3’. For classified into two groups, according the expression level in quantification of lncRNAs, the ΔΔCt method was used BC tissues. (C) The correlation between MALAT1 expression and prognosis. 5 year disease-free survival was ana- by co-amplifying GAPDH for each sample and by com- lyzed by Kaplan–Meier survival curve. paring the Ct values.

Cell line and lentivirus-mediated RNA interference. negative control shRNA was 5′-TTCTCCGAAC BC cell line MDA-MB-231 was cultured in RPMI- GTGTCACGT-3′. These shRNAs were synthesized and 1640 medium supplemented with 10% calf serum, 0.1 inserted into the pFH1UGW lentivirus core vector con- mM non-essential amino acids, 1 mM sodium-pyruvate taining a cytomegalovirus (CMV)-driven enhanced and 1% penicillinstreptomycin in a 37˚C humidified green fluorescent protein (EGFP) reporter gene; expres- incubator with 5% CO2. The following short hairpin sion of the shRNA was driven by the H1 promoter. RNA (shRNA) was used to target human MALAT1: Recombinant lentivirus expressing MALAT1 small in- sense: 5-CACAGGGAAAGCGA GTGGTTGGTAA-3′ terfering RNA (siRNA) or control siRNA (siMALAT1 and antisense: 5′-TTACCAACCA or siCON) was designed and produced by GeneChem CTCGCTTTCCCTGTG-3′. The sequence of the (Shanghai, China). 420 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016

Transwell assay. Tr answell (24-well) chambers Table 1. Association between MALAT1 expression in BC (Costar, Cambridge, MA, USA) were used to evaluate tissues and clinicopathological features. cell invasion. Initially, fibronectin (2 µg/filter) was dis- solved in 100 µl of MEM and poured into the upper Characteristics No. High P value part of the polyethylene filter (pore size, 8 µm). The expression wells were coated overnight in a laminar flow hood. of MALAT1 (%) Then, 105 cells (in 100 µL of growth medium) were added to the top of the filter in the upper well. The Age chamber was incubated for 24 h in 5% CO2 at 37°C. < 50 42 23 (54.8) 0.364 Finally, attached cells in the lower section were stained ≥ 50 36 16 (41.0) with H&E, and counted using light microscopy. Tumor size ≤ 2cm 32 15 (46.9) 0.645 Statistical analysis. Statistical tests were carried out > 2cm 46 24 (52.2) using SPSS version 16.0 (SPSS Inc., Chicago, IL, Differentiation USA). The differences between groups were analyzed Well 15 6 (40.0) 0.651 using a Student’s t test when only 2 groups or 1-way Moderate 28 14 (50.0) analysis of variance when more than 2 groups were Poor 35 19 (54.3) compared. Differences in frequency were assessed by Lymph metastasis Chi-square test. Disease-free survival (DFS) curves Yes 47 28 (59.6) 0.037* were calculated using the Kaplan-Meier method and No 31 11 (35.5) compared by log-rank testing. The receiver operating Clinical stage characteristics (ROC) curve was constructed to de- I/II 45 19 (42.2) 0.109 scribe diagnostic speciﬁcity and sensitivity. P<0.05 was III 33 20 (60.6) taken as statistically significant. Histological type Ductal 48 22 (45.8) 0.352 Results Lobular 30 17 (56.7) ER status Negative 49 24 (49.0) 0.815 Expression of MALAT1 is up-regulated in BC Positive 29 15 (51.7) tissues. The levels of MALAT1 were detected in PR status 78 paired BC tissues and adjacent normal tissues Negative 47 23 (48.9) 0.817 by qRT-PCR, and normalized to GAPDH. Positive 31 16 (51.6) MALAT1 expression was significantly up-regulated in 85.9% of (67/78) cancerous tissues com- *P<0.05 pared with normal counterparts (P<0.01) (Figure 1A). Further, according to the relative MALAT1 MTT assay. The cells were seeded onto 96-well plates at expression in tumor tissues, the 78 BC patients a density of 2×103/well in a final volume of 100 µl. After were classified into two groups: relatively high incubation for 24, 48 and 72 h, 20 µl MTT (Merck group (n=39) and relatively low group (n=39) Millipore, Billerica, MA, USA) (5 mg/ml) in phosphate- (Figure 1B). As shown in Table 1, an elevated buffered saline was added to each well, and the cells were incubated at 37°C for a further 4 h. A total of 150 µl MALAT1 expression in BC tissue was significant- dimethyl sulfoxide was added to the cells in each well ly associated with lymph metastasis, but not cor- after the supernatants were discarded. The absorbance of related with patients' age, tumor size, cellular dif- each well was measured at 490 nm using a microplate ferentiation, histological type, clinical stage, reader. estrogen receptor (ER) and progesterone receptor (PR) status. Flow cytometric analysis. Cells were harvested directly or 48 h after siRNA transient transfection and washed Kaplan-Meier analysis and log-rank test were used with ice-cold phosphate-buffered saline (PBS). The PI/ to evaluate the correlation of MALAT1 expression RNase staining kits (Multisciences, Hangzhou, China) and prognosis, as shown in Figure 1C, the 5-year and annexin V-fluorescein isothiocyanate (FITC) apoptosis detection kits (KeyGEN Biotech, Nanjing, China) DFS in BC patients with high expression of were used to detect cell cycle and apoptosis in a FACScan MALAT1 was inferior to that with low expression instrument (Becton Dickinson,, Mountain View, CA, [mean 48.5 months (95%CI: 42.012-22.005) vs USA), respectively. 62.7 months (95%CI: 55.445-69.888), P=0.012]. MALAT1 expression in breast cancer 421

Figure 2. Knockdown of MALAT1 inhibits MDA-MB-231 cells proliferation and invasion in vitro. (A) Knockdown efficiency was determined by qRT-PCR. (B) Knockdown of MALAT1 in MDA-MB-231 cells significantly reduced their proliferative capacities, as determined by cell number counting assay. Knockdown of MALAT1 resulted in cell arrest in G1 phase of cell cycle (C) and a dramatic increase of apoptosis (D), determined by flow cytometric analysis. Invasion and metastasis capacities determined by transwell assays (E). 422 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016

Discussion

Numerous reports of dys- regulated lncRNA expression across numerous cancer types suggest that abnormal lncRNA expression may be a major contributor to tumori- genesis [15]. The aberrant expressions of specific ln- cRNAs in cancer can mark the spectrum of disease progression and these lncRNAs may serve as in- Figure 3. Serum MALAT1 levels in breast cancer patients and controls. (A) Serum dependent biomarkers for MALAT1 levels determined by qPCR. The data are expressed as mean ± SD, tumor, pre- operative BC patients (n=78); benign, patients with benign breast disease as controls diagnosis and prognosis (n=40). (B) ROC curve was constructed to describe the diagnostic specificity and sensitiv- [16]. At present, not ity of serum MALAT1 in BC patients and controls. many lncRNAs have been shown to be directly in- Knockdown of MALAT1 inhibits BC cells prolif- volved in BC initiation and development. The dis- eration and invasion. To investigate the functional covery of HOTAIR (HOX antisense intergenic role of MALAT1 in BC cells, MALAT1 siRNA was RNA) and the understanding of its function was an transfected in to MDA-MB-231 cell line, and fol- important milestone. HOTAIR has been shown to lowing this, qPCR assays revealed that MALAT1 be highly expressed both in primary and metastatic expression was significantly reduced (Figure 2A). BC [17]. Further, HOTAIR expression in primary MTT assay showed that knockdown of MALAT1 tumors was a major indicator of eventual metastasis expression significantly inhibited cell proliferation and death. Other lncRNAs implicated and overex- in MDA-MB-231 cell line compared with the con- pressed in BC include BCYRN1 (Brain Cytoplasmic trols (Figure 2B). Next, flow cytometric analysis RNA 1), H19 and LSINCT5 [18]. The latest ln- revealed that the knockdown of MALAT1 resulted cRNA to be implicated in BC modulation and pro- in cell arrest in G1 phase of cell cycle (Figure 2C) gression is UCA 1 (urothelial carcinoma associated and a dramatic increase of apoptosis (Figure 2D) in 1) [19], which forms functional ribonucleoprotein MDA-MB-231 cell line. Finally, transwell assays complex with hnRNP l (heterogeneous nuclear ri- showed the number of MDA-MB-231 cells in low- bonucleoprotein) and promotes tumor growth by er section was significantly reduced in the MALAT1 suppression of p27 (Kip1). knockdown group compared with the control groups (Figure 2E), which indicated that expres- MALAT1, also known as non-coding nuclearen- sion of lncRNA MALAT1 promoted cell invasion riched abundant transcript 2 (NEAT2), is a ~8000- and metastasis . nt long, spliced non-coding RNA [20]. The expression of MALAT1 is significantly increased in tumor Serum MALAT1 levels in BC patients. As shown tissues and associated with large tumor size, ad- in Figure 3A, serum MALAT1 levels in BC pa- vanced tumor stage, and poor prognosis in various tients were signiﬁcantly higher than that in patients human cancers. Functional studies have shown that with benign breast disease (1.17±0.54 vs 0.56±0.39, transient overexpression of MALAT1 enhanced cel- P<0.001). Then ROC curve was constructed to de- lular proliferation in vitro and tumor formation in scribe the diagnostic speciﬁcity and sensitivity of nude mice, while depletion of MALAT1 in tumor serum MALAT1. Obviously, its detectability to dis- cells repressed migration and invasion cell process- tinguish BC from benign diseases is satisfactory be- es, induced cell apoptosis and reduced tumorige- cause the area under the curve (AUC) is 0.833 nicity [21]. Further studies about mechanisms sug- (95%CI: 0.756-0.910, P<0.001) (Figure 3B). gest the involvement of MALAT1 in regulating the MALAT1 expression in breast cancer 423

E2F1 transcription factor activity, activating the MDA-MB-231 cell line indicated that RNAi- ERK/MAPK and PI3K/Akt pathway [13,22,23]. mediated suppression of lncRNA MALAT1 signifi- MALAT1 has also been found to localize to sub- cantly inhibited cells’ proliferation, migration, and nuclear structures, areas of active or silent gene ex- invasion, and induced apoptosis and cell cycle G1 pression, and thus may be able to activate expres- arrest. Thus, lncRNA MALAT1 plays an oncogenic sion by interfering with the assembly of coactivator role in BC and represents a potential prognostic complexes [24]. biomarker for BC treatment.

However, the clinical significance of MALAT1 in Blood-based testing is an ideal method for bio- BC research remains contradictory. Guffanti et al markers in cancer care due to its ease and lower in- [25] have analyzed over 132,000 Roche 454 high- vasiveness. Contrary to the conventional belief, confidence deep sequencing reads from a primary cell-free RNA is present and relatively stable in cir- human lobular breast cancer tissue specimen, and culation [28]. It has been suggested that RNA in detected a range of unusual transcriptional events plasma or serum is associated with particulate mat- that were subsequently validated by RT-PCR in ad- ter such as apoptotic bodies or lipid vesicles and ditional eight primary human breast cancer sam- thus is protected from degradation by nucleases ples. They found MALAT1 was abundantly ex- [29]. Although a variety of RNA molecules includ- pressed in BC. Recently, Feng et al [26] have ing mRNA and miRNA have been investigated in showed that MALAT1 was aberrantly increased in the circulation so far [20], no data is available on breast cancer tissues and cells. MALAT1-siRNA in- the circulating lncRNAs and their relevance in BC. hibited breast cancer cell proliferation and cell cycle Here, we firstly reported that serum MALAT1 lev- progression in vitro and in vivo. Furthermore, els in BC patients were signiﬁcantly higher than MALAT1 acted as an endogenous potent regulator those in patients with benign breast disease, and its by directly binding to miR-124 and reversed the diagnostic efficacy was satisfactory according to inhibitory effect of miR-124 on breast cancer pro- AUC curve. liferation. Similarly, Jin et al [27] also reported that MALAT1 was upregulated in triple-negative breast In conclusion, we confirm an oncogenic role of cancer (TNBC) tissues. Knockdown of MALAT1 MALAT1 in BC, that MALAT1 upregulation plays inhibited proliferation, motility, and increased important roles in BC development, and that circu- apoptosis in vitro. In vivo study indicated that lating MALAT1 level may be a potential tumor knockdown of MALAT1 inhibited tumor growth marker for BC diagnosis. and metastasis. Patients with high MALAT1 expression had poorer overall survival time than those References with low MALAT1 expression. These data suggest- 1. Youlden DR, Cramb SM, Yip CH, Baade PD. Incidence and ed oncogene function of MALAT1 in BC. mortality of female breast cancer in the Asia-Pacific region. Conversely, Xu et al [14] demonstrated that Cancer Biol Med. 2014; 11:101–15. 2. Chimonidou M, Kallergi G, Georgoulias V, Welch DR, MALAT1 was downregulated in breast tumor cell Lianidou ES. Breast cancer metastasis suppressor-1 promoter lines and cancer tissue, and showed that knock- methylation in primary breast tumors and corresponding circulating tumor cells. Mol Cancer Res. 2013;11:1248–57. down of MALAT1 in breast cancer cell lines in- 3. Lee YH, Liu X, Qiu F, O'Connor TR, Yen Y, Ann DK. HP1β duced an epithelial-to-mesenchymal transition Is a Biomarker for Breast Cancer Prognosis and PARP Inhibitor (EMT) program via phosphatidylinositide-3 ki- Therapy. PLoS One. 2015;10:e0121207. 4. Ponting CP, Oliver PL, Reik W. Evolution and functions of nase-AKT pathways. long non-coding RNAs. Cell. 2009; 136(4):629–41. 5. Cheetham SW, Gruhl F, Mattick JS, Dinger ME. Long non- In the present study, MALAT1 expression levels coding RNAs and the genetics of cancer. Br J Cancer. 2013 Jun 25;108(12):2419–25. were determined in 78 pairs of BC tissues and adja- 6. Silva JM, Boczek NJ, Berres MW, Ma X, Smith DI. LSINCT5 cent normal tissues by qRT-PCR, our results also is over expressed in breast and ovarian cancer and affects cellular proliferation. RNA Biol. 2011;8:496–505. confirmed that MALAT1 expression was signifi- 7. Xu N, Wang F, Lv M, Cheng L. Microarray expression profile cantly up-regulated in BC tissues. Furthermore, an analysis of long non-coding RNAs in human breast cancer: a elevated MALAT1 expression in BC tissue was sig- study of Chinese women. Biomed Pharmacother. 2015; 69:221–7. nificantly associated with lymph metastasis and ad- 8. Ji P, Diederichs S, Wang W, Boing S, Metzger R, Schneider verse 5-year DFS. The effects of loss of function on PM, et al. MALAT-1, a novel noncoding RNA, and thymosin 424 Annals of Clinical & Laboratory Science, vol. 46, no. 4, 2016

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