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Increased MALAT1 expression predicts poor prognosis in esophageal cancer patients
Biomedicine & Pharmacotherapy 83 (2016) 8–13
Available online at
ScienceDirect www.sciencedirect.com
Increased MALAT1 expression predicts poor prognosis in esophageal cancer patients Congxiu Huang, Zhilong Yu, Hao Yang, Yu Lin* Department of Oncology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 December 2015 Received in revised form 25 May 2016 Accepted 26 May 2016
Aim: This study was designed to determine the expression of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in patients with esophageal cancer (EC). In addition, we attempted to seek the prognostic value of MALAT1 in EC based on its expression. Methods: The expression of MALAT1 in EC tissues and cell lines were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). The association between MALAT1 expression and the clinical characteristics was analyzed using Chi-square test. Kaplan-Meier survival curves were plotted to describe the overall survival of EC patients with different expression of MALAT1. Cox regression analysis was per formed to evaluate the prognostic value of MALAT1 for EC patients. Results: Expression levels of MALAT1 were significantly higher in EC tissues and cells than the controls (P < 0.05). MALAT1 expression was tightly related to lymphatic invasion (P = 0.018), distant metastasis (P = 0.033) and tumor differentiation (P = 0.025), but shared no association with age, gender and tumor location (P > 0.05). In addition, patients with high MALAT1 expression had a shorter overall survival than those with low MALAT1 (P < 0.001). The results of Cox analysis shown that MALAT1 was significantly linked with the prognosis of EC patients (HR = 6.638; P = 0.000; 95% CI = 2.948-14.947). Conclusion: Taken together, the expression of MALAT1 could be a predictor for prognosis of EC patients. ã 2016 Published by Elsevier Masson SAS.
Keywords: MALAT1 LncRNA Esophageal cancer Prognosis Overall survival
1. Introduction Esophageal cancer (EC) is the eighth most common cancer in the world and ranks sixth among all cancers in mortality [1,2]. It is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer related death in China [3]. EC consists of two major subtypes, squamous cell carcinoma and adenocarcinomas histologically [4,5]. EC possesses an aggressive tumor growth and is often diagnosed at an advanced stage with the occurrence of dysphagia [6,7]. Over recent decades, numerous types and combinations of treatments, including surgery, radiotherapy and chemotherapy as well as other therapeutic strategies, have been used for the EC patients [8,9]. However, despite these treatments, patients usually suffer local recurrence or distant metastasis because of the aggressive nature of this disease. Currently, the overall survival rate of EC patients is less than 50%, even is between 15 and 25% [10]. Therefore, we attempted to identify more effective molecules for prognosis of EC patients.
* Corresponding author. E-mail address: [email protected] (Y. Lin). http://dx.doi.org/10.1016/j.biopha.2016.05.044 0753-3322/ã 2016 Published by Elsevier Masson SAS.
Long non-coding RNAs (lncRNAs) are defined as a group of RNAs that have limited or no coding capacity with the length of more than 200 nt [11,12]. lncRNAs are first thought to be spurious transcriptional noise and now are novel regulators in the cancer progressions, including chromatin modification, gene transcription and splicing [13]. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1, also NEAT2) is a highly abundant lncRNA with nucleus-restriction [14]. MALAT1 is a well-studied lnc-RNA and locates on chromosome 11q13.1 of about 8 kb [15,16]. Emerging evidence has confirmed that MALAT1 was involved in multiple biological functions, such as modifications of primary transcripts post-transcriptionally and regulation of gene expression [17,18]. Aberrant MALAT1 expression has been observed in several cancers and has been regarded as a prognostic factor for these diseases, including gallbladder cancer and non-small cell lung cancer [19,20]. However, the relationship between MALAT1 expression and prognosis of EC patients remains unclear. To address this problem, the aim of the present study was to determine the prognostic value of MALAT1 expression for EC patients.
C. Huang et al. / Biomedicine & Pharmacotherapy 83 (2016) 8–13
2. Materials and methods 2.1. Cell lines and cell culture Two EC cell lines (EC109 and KYSE150) and a human esophageal epithelial cell line (HEF-1A) were obtained from Cell Bank of the Chinese Academy of Sciences (Shang hai, China). Cells were cultivated in RPMI-1640 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco) at a modified atmosphere with 5% CO2. 2.2. Patients and specimens A total of 132 patients, who were pathologically and histologically diagnosed as EC in The Affiliated Hospital of Inner Mongolia Medical University from April 2009 to May 2014, were enrolled in the study. All the patients were treated with the same strategy and received no chemotherapy or radiotherapy before surgery. The
9
clinical characteristics of patients were recorded at the time of diagnosis. In addition, 50 patients suffered esophagectomy due to benign esophageal disease were recruited as controls in the study. Tissues were immediately stored at 80 C until use. The present study was approved by the Ethic Committee of The Affiliated Hospital of Inner Mongolia Medical University. All the participants were asked to sign the informed consents. 2.3. RNA extraction and quantitative real-time reverse transcriptasepolymerase chain reaction (qRT-PCR) Total RNA was extracted from EC tissues and cell lines using Trizol reagent (Invitrogen, Carisbad, CA, USA). Then the reverse transcription and qRT-PCR were conducted successively. Each sample was detected with Applied Biosystem 7500 Step One RealTime PCR System in triplicate and analyzed with Applied Biosystem 7500 software. The expression of MALAT1 was determined by the 2 DDct method and normalized with GAPDH.
Fig. 1. Expression of MALAT1 in EC tissues and cell lines was determined by qRT-PCR. The relative expression of MALAT1 was normalized to GAPDH. The results showed that MALAT1 was highly expressed in EC tissues and cells. A: Relative expression of MALAT1 in EC tissues. *P < 0.05. B: Relative expression of MALAT1 in EC cells.
10
C. Huang et al. / Biomedicine & Pharmacotherapy 83 (2016) 8–13
2.4. Plasmid construction and stable transfection Recombinant expression vector pEGFP-C1-MALAT1 was constructed by inserting the MALAT1 gene into pEGFP-C1 (Invitrogen). EC109 cells were transfected with pEGFP-C1-MALAT1 or pEGFP-C1 (control) using ScreenFectA Transfection reagent (InCellA, Germany) according to the manufacturer’s protocol. 2.5. Cell proliferation assay The EC cells were first planted into 96-well plates and then transfected with the mentioned vectors. After incubation for 0 h, 12 h, 24 h, 36 h, 48 h and 60 h, 10 mL Cell Counting Kit-8 reagents (Dojindo Laboratories, Japan) was added to each well to examine the viability of EC cells. The absorbance was measured at 450 nm in a spectrophotometer (Thermo, USA). 2.6. Transwell assay The migration and invasion assays were performed in a 24-well transwell chamber. Cells with a concentration of 1 105 per well were added to the upper compartment and incubated for 24 h in the serum-free RPMI-1640 medium. A total of 300 mL medium containing 20% FBS was added to the lower compartment as the chemotactic factor. After incubation, migrated cells in the lower compartment were stained with 0.1% crystal violet and counted in seven randomly selected fields under a microscope. For invasion assay, the membrane was pre-coated with matrigel (Corning, Acton, MA).
Table 1 The relationship between clinicopathologic characteristics and the MALAT1 expression. Clinical features
Case NO.
Expression High
Low
Age 245 >45
63 70
45 54
18 16
Gender Male Female
73 60
58 41
15 19
Tumor location Upper Lower
72 61
55 44
17 17
Lymphatic invasion N0-N1 N2-N3
61 72
39 60
22 12
Distant metastasis Yes No
62 71
52 47
10 24
Tumor differentiation Well, Moderate Poor
66 67
43 56
23 11
x2
P value
0.308
0.579
1.595
0.207
0.131
0.718
5.551
0.018
4.544
0.033
5.006
0.025
tumor differentiation (P = 0.025), whereas it had no relationship with age, gender and tumor location (all P > 0.05). 3.3. Promotive effects of MALAT1 on progression of EC cells
2.7. Statistical analysis All the statistical analyses were performed by Sigmaplot software (Systat Software Inc, CA, USA). The difference of MALAT1 expression between EC samples and the normal controls was examined by using Student’s t-test. Associations between MALAT1 expression and clinicopathological characteristics were analyzed via Chi-square test. Kaplan-Meier analysis was performed to describe the overall survival rate of EC patients with different expression level of MALAT1. Cox regression analysis was adopted to evaluate the prognostic value of MALAT1 for EC patients. 3. Results 3.1. Up-regulated expression of MALAT1 in EC tissues and cell lines The relative expression of MALAT1 in EC tissues and cells was determined by qRT-PCR. The level of MALAT1 in EC tissues was 2.67 0.33, and that in normal esophageal tissues was 1.50 0.21. As shown in Fig. 1A, high expression of MALAT1 was obviously higher in EC tissues compared to the controls (P < 0.05). Besides, we also found that the expression of MALAT1 was significantly higher in EC cells than that in the esophageal epithelial cells (Fig. 1B, P < 0.05). 3.2. Relationship between MALAT1 expression and clinical features of EC patients We manually grouped the 132 specimens into two groups (MALAT1-high group and MALAT1-low group) according to the MALAT1 expression levels from the qRT-PCR analysis. The relationship between MALAT1 expression and clinical features of EC patients was exhibited in Table 1. The result demonstrated that high MALAT1 expression was significantly associated with lymphatic invasion (P = 0.018), distant metastasis (P = 0.033) and
To study the biological functions of MALAT1 in EC cells, we applied pEGFP-C1- MALAT1 to up-regulate MALAT1 in EC cells. The CCK-8 results revealed that the overexpression of MALAT1 significantly promoted the proliferation of EC cells (Fig. 2A, P < 0.05). In addition, the results of transwell assay result suggested that the upregulated expression of MALAT1 could promote the migration and invasion of EC cells (Fig. 2B and C, P < 0.05). 3.4. Prognostic value of MALAT1 in EC The overall survival of EC patients was assessed by a 5-year follow-up. During the follow up time, 80 out of 133 patients died, including 73 (73.7%) cases with high MALAT1 expression. KaplanMeier curves showed that patients with high MALAT1 expression had a relatively shorter survival time than those with low MALAT1 expression (Fig. 3, P < 0.001). Multivariate Cox analysis demonstrated that the expression of MALAT1 was an independent predictor for survival of EC patients (Table 2, HR = 6.638; P = 0.000; 95% CI = 2.948 14.947). 4. Discussion EC is a serious malignancy with high mortality and poor prognosis. It has been a growing health concern because of the increased incidence in recent years. Currently, a wide of tumor biomarkers have recieved more and more attentions on their diagnostic and prognostic value in patients with EC. Chen et al. suggested that positive expression of B7-H1 could predict poor survival for EC patients [21]. Wang et al. [22] reported that overexpression of Stathmin promoted cell growth and was a marker for poor prognosis of EC patients. In this study, we explored the expression of MALAT1 in EC patients and evaluated its roles in progression and prognosis of EC.
C. Huang et al. / Biomedicine & Pharmacotherapy 83 (2016) 8–13
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Fig. 2. The effects of MALAT1 on the progression of EC cells in vitro. A: The promotive effects of MALAT1 overexpression on the growth of EC cells. B: MALAT1 overexpression significantly promoted the migration of EC cells. C: MALAT1 overexpression significantly promoted the invasion of EC cells. *P < 0.05.
Abundant reports have demonstrated that the expression of MALAT1 is increased in various cancers. Fan et al. illustrated the up-regulation of MALAT1 in bladder cancer [23]. Xie et al. demonstrated that MALAT1 was overexpressed in nasopharyngeal carcinoma [24]. Zhang et al. suggested that MALAT1 was upregulated in both brain cortex and liver [25]. In the present study, we determined the MALAT1 expression in EC patients and EC cells. The qRT-PCR results showed that the expression of MALAT1 in EC
tissues and cells was significantly higher than that in controls, which was in accordance with the previous studies. Thess results demonstrated MALAT1 might be an oncogene in EC. Due to the aberrant expression of MALAT1 in EC tissues and cells, we subsequent tested the relationship between MALAT1 expression and clinical features of EC patients. The result showed that MALAT1 expression was tightly associated with lymphatic invasion, tumor differentiation and distant metastasis. Tumor
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C. Huang et al. / Biomedicine & Pharmacotherapy 83 (2016) 8–13
Conflict of interest None. Acknowledgement None. References
Fig. 3. Overall survival curves for low and high MALAT1 expression in EC patients. Kaplan-Meier analysis suggested that patients with high MALAT1 expression had a shorter survival time than those with low MALAT1 expression (Log-rank test, P < 0.001).
Table 2 The multivariate analysis of prognostic factors in EC. Clinical features
P value
HR
95%CI
Gender Age Lymphatic metastasis Tumor differentiation MALAT1 expression
0.490 0.788 0.275 0.334 0.000
0.807 0.927 1.360 1.270 6.638
0.438–1.484 0.536–1.606 0.783–2.362 0.782–2.062 2.948–14.947
metastatic dissemination and growth develop via a complex molecular dysregulation, including cell migration, invasion immune escape mechanisms and resistance to apoptosis [26]. With this regard, we detected the influence of MALAT1 on the pathological processes of EC cells. In our study, we found that the overexpression of MALAT1 could significantly promote the migration, invasion and proliferation of EC cells in vitro. The above results revealed that MALAT1 might be involved in the progression of EC. As the overexpression of MALAT1 was reported to correlate with unfavorable prognosis in pancreatic cancer in the previous study [27], we hypothesized MALAT1 might be related to the prognosis of EC. Hence, we conducted Kaplan-Meier analysis and cox regression analysis, the result indicated that the patients with high MALAT1 expression owned a lower overall survival rate than those with low MALAT1 expression, and MALAT1 possessed high prognostic value for EC patients. Though we determined the expression of MALAT1 in EC patients and estimated its prognostic value, the precise mechanisms of MALAT1 on EC progression were still unclear, which need more and further investigations in the future. In conclusion, MALAT1 expression may play an important role in the progression of EC and can be used as an independent factor to determine EC prognosis.
[1] Y. Chen, Y. Tong, C. Yang, Y. Gan, H. Sun, H. Bi, S. Cao, X. Yin, Z. Lu, Consumption of hot beverages and foods and the risk of esophageal cancer: a meta-analysis of observational studies, BMC Cancer 15 (2015) 449. [2] E. He, F. Pan, G. Li, J. Li, Fractionated ionizing radiation promotes epithelialMesenchymal transition in human esophageal cancer cells through PTEN deficiency-Mediated Akt activation, PLoS One 10 (2015) e0126149. [3] F. Lordick, A.H. Holscher, K. Haustermans, C. Wittekind, Multimodal treatment of esophageal cancer, Langenbecks Arch. Surg. 398 (2013) 177–187. [4] L. Chen, G. Luo, Y. Tan, J. Wei, C. Wu, L. Zheng, X. Zhang, N. Xu, Immunolocalisation of tissue factor in esophageal cancer is correlated with intratumoral angiogenesis and prognosis of the patient, Acta Histochem. 112 (2010) 233–239. [5] L.J. Chen, J. Sun, H.Y. Wu, S.M. Zhou, Y. Tan, M. Tan, B.E. Shan, B.F. Lu, X.G. Zhang, B7-H4 expression associates with cancer progression and predicts patient's survival in human esophageal squamous cell carcinoma, Cancer Immunol. Immunother. 60 (2011) 1047–1055. [6] J.R. Siewert, K. Ott, Are squamous and adenocarcinomas of the esophagus the same disease, Semin. Radiat. Oncol. 17 (2007) 38–44. [7] L. Dreikhausen, S. Blank, L. Sisic, U. Heger, W. Weichert, D. Jager, T. Bruckner, N. Giese, L. Grenacher, C. Falk, K. Ott, T. Schmidt, Association of angiogenic factors with prognosis in esophageal cancer, BMC Cancer 15 (2015) 121. [8] L. Chen, D. Di, G. Luo, L. Zheng, Y. Tan, X. Zhang, N. Xu, Immunochemical staining of MT2-MMP correlates positively to angiogenesis of human esophageal cancer, Anticancer Res. 30 (2010) 4363–4368. [9] L. Chen, Y. Xu, B. Xu, H. Deng, X. Zheng, C. Wu, J. Jiang, Over-expression of lysine-specific demethylase 1 predicts tumor progression and poor prognosis in human esophageal cancer, Int. J. Clin. Exp. Pathol. 7 (2014) 8929–8934. [10] G. Wang, H. Chen, J. Liu, Y. Ma, H. Jia, A comparison of postoperative early enteral nutrition with delayed enteral nutrition in patients with esophageal cancer, Nutrients 7 (2015) 4308–4317. [11] L. Hu, Y. Wu, D. Tan, H. Meng, K. Wang, Y. Bai, K. Yang, Up-regulation of long noncoding RNA MALAT1 contributes to proliferation and metastasis in esophageal squamous cell carcinoma, J. Exp. Clin. Cancer Res. 34 (2015) 7. [12] Y. Jiang, Y. Li, S. Fang, B. Jiang, C. Qin, P. Xie, G. Zhou, G. Li, The role of MALAT1 correlates with HPV in cervical cancer, Oncol. Lett. 7 (2014) 2135–2141. [13] T. Thum, J. Fiedler, LINCing MALAT1 and angiogenesis, Circ. Res. 114 (2014) 1366–1368. [14] H.T. Zheng, D.B. Shi, Y.W. Wang, X.X. Li, Y. Xu, P. Tripathi, W.L. Gu, G.X. Cai, S.J. Cai, High expression of lncRNA MALAT1 suggests a biomarker of poor prognosis in colorectal cancer, Int. J. Clin. Exp. Pathol. 7 (2014) 3174–3181. [15] S.F. Cho, Y.C. Chang, C.S. Chang, S.F. Lin, Y.C. Liu, H.H. Hsiao, J.G. Chang, T.C. Liu, MALAT1 long non-coding RNA is overexpressed in multiple myeloma and may serve as a marker to predict disease progression, BMC Cancer 14 (2014) 809. [16] J.A. Brown, D. Bulkley, J. Wang, M.L. Valenstein, T.A. Yario, T.A. Steitz, J.A. Steitz, Structural insights into the stabilization of MALAT1 noncoding RNA by a bipartite triple helix, Nat. Struct. Mol. Biol. 21 (2014) 633–640. [17] J.H. Liu, G. Chen, Y.W. Dang, C.J. Li, D.Z. Luo, Expression and prognostic significance of lncRNA MALAT1 in pancreatic cancer tissues, Asian Pac. J. Cancer Prev. 15 (2014) 2971–2977. [18] P. Schorderet, D. Duboule, Structural and functional differences in the long non-coding RNA hotair in mouse and human, PLoS Genet. 7 (2011) e1002071. [19] X.S. Wu, X.A. Wang, W.G. Wu, Y.P. Hu, M.L. Li, Q. Ding, H. Weng, Y.J. Shu, T.Y. Liu, L. Jiang, Y. Cao, R.F. Bao, J.S. Mu, Z.J. Tan, F. Tao, Y.B. Liu, MALAT1 promotes the proliferation and metastasis of gallbladder cancer cells by activating the ERK/ MAPK pathway, Cancer Biol. Ther. 15 (2014) 806–814. [20] A.E. Tee, D. Ling, C. Nelson, B. Atmadibrata, M.E. Dinger, N. Xu, T. Mizukami, P.Y. Liu, B. Liu, B. Cheung, E. Pasquier, M. Haber, M.D. Norris, T. Suzuki, G.M. Marshall, T. Liu, The histone demethylase JMJD1A induces cell migration and invasion by up-regulating the expression of the long noncoding RNA MALAT1, Oncotarget 5 (2014) 1793–1804. [21] L. Chen, H. Deng, M. Lu, B. Xu, Q. Wang, J. Jiang, C. Wu, B7-H1 expression associates with tumor invasion and predicts patient's survival in human esophageal cancer, Int. J. Clin. Exp. Pathol. 7 (2014) 6015–6023. [22] F. Wang, X.Y. Xuan, X. Yang, L. Cao, L.N. Pang, R. Zhou, Q.X. Fan, L.X. Wang, Stathmin is a marker of progression and poor prognosis in esophageal carcinoma, Asian Pac. J. Cancer Prev. 15 (2014) 3613–3618. [23] Y. Fan, B. Shen, M. Tan, X. Mu, Y. Qin, F. Zhang, Y. 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Available online at
ScienceDirect www.sciencedirect.com
Increased MALAT1 expression predicts poor prognosis in esophageal cancer patients Congxiu Huang, Zhilong Yu, Hao Yang, Yu Lin* Department of Oncology, The Affiliated Hospital of Inner Mongolia Medical University, Hohhot 010050, Inner Mongolia, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 23 December 2015 Received in revised form 25 May 2016 Accepted 26 May 2016
Aim: This study was designed to determine the expression of metastasis associated lung adenocarcinoma transcript 1 (MALAT1) in patients with esophageal cancer (EC). In addition, we attempted to seek the prognostic value of MALAT1 in EC based on its expression. Methods: The expression of MALAT1 in EC tissues and cell lines were measured by quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). The association between MALAT1 expression and the clinical characteristics was analyzed using Chi-square test. Kaplan-Meier survival curves were plotted to describe the overall survival of EC patients with different expression of MALAT1. Cox regression analysis was per formed to evaluate the prognostic value of MALAT1 for EC patients. Results: Expression levels of MALAT1 were significantly higher in EC tissues and cells than the controls (P < 0.05). MALAT1 expression was tightly related to lymphatic invasion (P = 0.018), distant metastasis (P = 0.033) and tumor differentiation (P = 0.025), but shared no association with age, gender and tumor location (P > 0.05). In addition, patients with high MALAT1 expression had a shorter overall survival than those with low MALAT1 (P < 0.001). The results of Cox analysis shown that MALAT1 was significantly linked with the prognosis of EC patients (HR = 6.638; P = 0.000; 95% CI = 2.948-14.947). Conclusion: Taken together, the expression of MALAT1 could be a predictor for prognosis of EC patients. ã 2016 Published by Elsevier Masson SAS.
Keywords: MALAT1 LncRNA Esophageal cancer Prognosis Overall survival
1. Introduction Esophageal cancer (EC) is the eighth most common cancer in the world and ranks sixth among all cancers in mortality [1,2]. It is the fourth most frequently diagnosed cancer and the fourth leading cause of cancer related death in China [3]. EC consists of two major subtypes, squamous cell carcinoma and adenocarcinomas histologically [4,5]. EC possesses an aggressive tumor growth and is often diagnosed at an advanced stage with the occurrence of dysphagia [6,7]. Over recent decades, numerous types and combinations of treatments, including surgery, radiotherapy and chemotherapy as well as other therapeutic strategies, have been used for the EC patients [8,9]. However, despite these treatments, patients usually suffer local recurrence or distant metastasis because of the aggressive nature of this disease. Currently, the overall survival rate of EC patients is less than 50%, even is between 15 and 25% [10]. Therefore, we attempted to identify more effective molecules for prognosis of EC patients.
* Corresponding author. E-mail address: [email protected] (Y. Lin). http://dx.doi.org/10.1016/j.biopha.2016.05.044 0753-3322/ã 2016 Published by Elsevier Masson SAS.
Long non-coding RNAs (lncRNAs) are defined as a group of RNAs that have limited or no coding capacity with the length of more than 200 nt [11,12]. lncRNAs are first thought to be spurious transcriptional noise and now are novel regulators in the cancer progressions, including chromatin modification, gene transcription and splicing [13]. Metastasis-associated lung adenocarcinoma transcript1 (MALAT1, also NEAT2) is a highly abundant lncRNA with nucleus-restriction [14]. MALAT1 is a well-studied lnc-RNA and locates on chromosome 11q13.1 of about 8 kb [15,16]. Emerging evidence has confirmed that MALAT1 was involved in multiple biological functions, such as modifications of primary transcripts post-transcriptionally and regulation of gene expression [17,18]. Aberrant MALAT1 expression has been observed in several cancers and has been regarded as a prognostic factor for these diseases, including gallbladder cancer and non-small cell lung cancer [19,20]. However, the relationship between MALAT1 expression and prognosis of EC patients remains unclear. To address this problem, the aim of the present study was to determine the prognostic value of MALAT1 expression for EC patients.
C. Huang et al. / Biomedicine & Pharmacotherapy 83 (2016) 8–13
2. Materials and methods 2.1. Cell lines and cell culture Two EC cell lines (EC109 and KYSE150) and a human esophageal epithelial cell line (HEF-1A) were obtained from Cell Bank of the Chinese Academy of Sciences (Shang hai, China). Cells were cultivated in RPMI-1640 (Gibco, Grand Island, NY, USA) with 10% fetal bovine serum (FBS, Gibco) at a modified atmosphere with 5% CO2. 2.2. Patients and specimens A total of 132 patients, who were pathologically and histologically diagnosed as EC in The Affiliated Hospital of Inner Mongolia Medical University from April 2009 to May 2014, were enrolled in the study. All the patients were treated with the same strategy and received no chemotherapy or radiotherapy before surgery. The
9
clinical characteristics of patients were recorded at the time of diagnosis. In addition, 50 patients suffered esophagectomy due to benign esophageal disease were recruited as controls in the study. Tissues were immediately stored at 80 C until use. The present study was approved by the Ethic Committee of The Affiliated Hospital of Inner Mongolia Medical University. All the participants were asked to sign the informed consents. 2.3. RNA extraction and quantitative real-time reverse transcriptasepolymerase chain reaction (qRT-PCR) Total RNA was extracted from EC tissues and cell lines using Trizol reagent (Invitrogen, Carisbad, CA, USA). Then the reverse transcription and qRT-PCR were conducted successively. Each sample was detected with Applied Biosystem 7500 Step One RealTime PCR System in triplicate and analyzed with Applied Biosystem 7500 software. The expression of MALAT1 was determined by the 2 DDct method and normalized with GAPDH.
Fig. 1. Expression of MALAT1 in EC tissues and cell lines was determined by qRT-PCR. The relative expression of MALAT1 was normalized to GAPDH. The results showed that MALAT1 was highly expressed in EC tissues and cells. A: Relative expression of MALAT1 in EC tissues. *P < 0.05. B: Relative expression of MALAT1 in EC cells.
10
C. Huang et al. / Biomedicine & Pharmacotherapy 83 (2016) 8–13
2.4. Plasmid construction and stable transfection Recombinant expression vector pEGFP-C1-MALAT1 was constructed by inserting the MALAT1 gene into pEGFP-C1 (Invitrogen). EC109 cells were transfected with pEGFP-C1-MALAT1 or pEGFP-C1 (control) using ScreenFectA Transfection reagent (InCellA, Germany) according to the manufacturer’s protocol. 2.5. Cell proliferation assay The EC cells were first planted into 96-well plates and then transfected with the mentioned vectors. After incubation for 0 h, 12 h, 24 h, 36 h, 48 h and 60 h, 10 mL Cell Counting Kit-8 reagents (Dojindo Laboratories, Japan) was added to each well to examine the viability of EC cells. The absorbance was measured at 450 nm in a spectrophotometer (Thermo, USA). 2.6. Transwell assay The migration and invasion assays were performed in a 24-well transwell chamber. Cells with a concentration of 1 105 per well were added to the upper compartment and incubated for 24 h in the serum-free RPMI-1640 medium. A total of 300 mL medium containing 20% FBS was added to the lower compartment as the chemotactic factor. After incubation, migrated cells in the lower compartment were stained with 0.1% crystal violet and counted in seven randomly selected fields under a microscope. For invasion assay, the membrane was pre-coated with matrigel (Corning, Acton, MA).
Table 1 The relationship between clinicopathologic characteristics and the MALAT1 expression. Clinical features
Case NO.
Expression High
Low
Age 245 >45
63 70
45 54
18 16
Gender Male Female
73 60
58 41
15 19
Tumor location Upper Lower
72 61
55 44
17 17
Lymphatic invasion N0-N1 N2-N3
61 72
39 60
22 12
Distant metastasis Yes No
62 71
52 47
10 24
Tumor differentiation Well, Moderate Poor
66 67
43 56
23 11
x2
P value
0.308
0.579
1.595
0.207
0.131
0.718
5.551
0.018
4.544
0.033
5.006
0.025
tumor differentiation (P = 0.025), whereas it had no relationship with age, gender and tumor location (all P > 0.05). 3.3. Promotive effects of MALAT1 on progression of EC cells
2.7. Statistical analysis All the statistical analyses were performed by Sigmaplot software (Systat Software Inc, CA, USA). The difference of MALAT1 expression between EC samples and the normal controls was examined by using Student’s t-test. Associations between MALAT1 expression and clinicopathological characteristics were analyzed via Chi-square test. Kaplan-Meier analysis was performed to describe the overall survival rate of EC patients with different expression level of MALAT1. Cox regression analysis was adopted to evaluate the prognostic value of MALAT1 for EC patients. 3. Results 3.1. Up-regulated expression of MALAT1 in EC tissues and cell lines The relative expression of MALAT1 in EC tissues and cells was determined by qRT-PCR. The level of MALAT1 in EC tissues was 2.67 0.33, and that in normal esophageal tissues was 1.50 0.21. As shown in Fig. 1A, high expression of MALAT1 was obviously higher in EC tissues compared to the controls (P < 0.05). Besides, we also found that the expression of MALAT1 was significantly higher in EC cells than that in the esophageal epithelial cells (Fig. 1B, P < 0.05). 3.2. Relationship between MALAT1 expression and clinical features of EC patients We manually grouped the 132 specimens into two groups (MALAT1-high group and MALAT1-low group) according to the MALAT1 expression levels from the qRT-PCR analysis. The relationship between MALAT1 expression and clinical features of EC patients was exhibited in Table 1. The result demonstrated that high MALAT1 expression was significantly associated with lymphatic invasion (P = 0.018), distant metastasis (P = 0.033) and
To study the biological functions of MALAT1 in EC cells, we applied pEGFP-C1- MALAT1 to up-regulate MALAT1 in EC cells. The CCK-8 results revealed that the overexpression of MALAT1 significantly promoted the proliferation of EC cells (Fig. 2A, P < 0.05). In addition, the results of transwell assay result suggested that the upregulated expression of MALAT1 could promote the migration and invasion of EC cells (Fig. 2B and C, P < 0.05). 3.4. Prognostic value of MALAT1 in EC The overall survival of EC patients was assessed by a 5-year follow-up. During the follow up time, 80 out of 133 patients died, including 73 (73.7%) cases with high MALAT1 expression. KaplanMeier curves showed that patients with high MALAT1 expression had a relatively shorter survival time than those with low MALAT1 expression (Fig. 3, P < 0.001). Multivariate Cox analysis demonstrated that the expression of MALAT1 was an independent predictor for survival of EC patients (Table 2, HR = 6.638; P = 0.000; 95% CI = 2.948 14.947). 4. Discussion EC is a serious malignancy with high mortality and poor prognosis. It has been a growing health concern because of the increased incidence in recent years. Currently, a wide of tumor biomarkers have recieved more and more attentions on their diagnostic and prognostic value in patients with EC. Chen et al. suggested that positive expression of B7-H1 could predict poor survival for EC patients [21]. Wang et al. [22] reported that overexpression of Stathmin promoted cell growth and was a marker for poor prognosis of EC patients. In this study, we explored the expression of MALAT1 in EC patients and evaluated its roles in progression and prognosis of EC.
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Fig. 2. The effects of MALAT1 on the progression of EC cells in vitro. A: The promotive effects of MALAT1 overexpression on the growth of EC cells. B: MALAT1 overexpression significantly promoted the migration of EC cells. C: MALAT1 overexpression significantly promoted the invasion of EC cells. *P < 0.05.
Abundant reports have demonstrated that the expression of MALAT1 is increased in various cancers. Fan et al. illustrated the up-regulation of MALAT1 in bladder cancer [23]. Xie et al. demonstrated that MALAT1 was overexpressed in nasopharyngeal carcinoma [24]. Zhang et al. suggested that MALAT1 was upregulated in both brain cortex and liver [25]. In the present study, we determined the MALAT1 expression in EC patients and EC cells. The qRT-PCR results showed that the expression of MALAT1 in EC
tissues and cells was significantly higher than that in controls, which was in accordance with the previous studies. Thess results demonstrated MALAT1 might be an oncogene in EC. Due to the aberrant expression of MALAT1 in EC tissues and cells, we subsequent tested the relationship between MALAT1 expression and clinical features of EC patients. The result showed that MALAT1 expression was tightly associated with lymphatic invasion, tumor differentiation and distant metastasis. Tumor
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Conflict of interest None. Acknowledgement None. References
Fig. 3. Overall survival curves for low and high MALAT1 expression in EC patients. Kaplan-Meier analysis suggested that patients with high MALAT1 expression had a shorter survival time than those with low MALAT1 expression (Log-rank test, P < 0.001).
Table 2 The multivariate analysis of prognostic factors in EC. Clinical features
P value
HR
95%CI
Gender Age Lymphatic metastasis Tumor differentiation MALAT1 expression
0.490 0.788 0.275 0.334 0.000
0.807 0.927 1.360 1.270 6.638
0.438–1.484 0.536–1.606 0.783–2.362 0.782–2.062 2.948–14.947
metastatic dissemination and growth develop via a complex molecular dysregulation, including cell migration, invasion immune escape mechanisms and resistance to apoptosis [26]. With this regard, we detected the influence of MALAT1 on the pathological processes of EC cells. In our study, we found that the overexpression of MALAT1 could significantly promote the migration, invasion and proliferation of EC cells in vitro. The above results revealed that MALAT1 might be involved in the progression of EC. As the overexpression of MALAT1 was reported to correlate with unfavorable prognosis in pancreatic cancer in the previous study [27], we hypothesized MALAT1 might be related to the prognosis of EC. Hence, we conducted Kaplan-Meier analysis and cox regression analysis, the result indicated that the patients with high MALAT1 expression owned a lower overall survival rate than those with low MALAT1 expression, and MALAT1 possessed high prognostic value for EC patients. Though we determined the expression of MALAT1 in EC patients and estimated its prognostic value, the precise mechanisms of MALAT1 on EC progression were still unclear, which need more and further investigations in the future. In conclusion, MALAT1 expression may play an important role in the progression of EC and can be used as an independent factor to determine EC prognosis.
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