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

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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 develop- ment 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 ex- pression 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 knock- down 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 tis- sue 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]. 0091-7370/16/0400-418. © 2016 by the Association of Clinical Scientists, Inc. MALAT1 expression in breast cancer 419 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 differen- tiation was graded according to the WHO grading sys- tem. 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 deter- mined 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 expres- sion 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-regulat- ed in 85.9% of (67/78) cancerous tissues com- *P<0.05 pared with normal counterparts (P<0.01) (Figure 1A).
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