- Email: [email protected]
Expression of miRNA-130a in Nonsmall Cell Lung Cancer
BASIC INVESTIGATION
Expression of miRNA-130a in Nonsmall Cell Lung Cancer Xiao-Chun Wang, PhD, Li-Li Tian, MD, Hai-Liang Wu, MD, Xiao-Yan Jiang, MD, Li-Qing Du, PhD, Heng Zhang, MA, Yue-Ying Wang, BM, Hong-Ying Wu, BM, De-Guan Li, MA, Yi She, BM, Qing-Fen Liu, BM, Fei-Yue Fan, MA and Ai-Min Meng, MA
Abstract: MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the posttranscriptional level and are deeply involved in the pathogenesis of several types of cancer. The miRNA-130a has been shown to play a role in antagonizing the inhibitory effects of GAX on endothelial cell proliferation, migration and tube formation, and antagonizing the inhibitory effects of HoxA5 on tube formation in vitro. Here the authors show, for the first time, that miRNA-130a expression is increased in nonsmall cell lung cancer (NSCLC) tissues. Statistical analysis showed that overexpression of miRNA-130a was strongly associated with lymph node metastasis, stage of tumor node metastasis classification and poor prognosis. Moreover, there was a significant difference in miRNA-130a expression levels between smoking and nonsmoking patients. Multivariate Cox regression analysis showed that miRNA-130a was an independent prognostic factor for patients with NSCLC. Together, these data suggest that miRNA-130a may comprise a potential novel prognostic marker for this disease. Key Indexing Terms: miRNA-130a; Overexpression; Prognosis; NSCLC. [Am J Med Sci 2010;340(5):385–388.]
I
t is a well-known fact that lung cancer ranks as the single biggest cause of cancer deaths in developed countries. The number of new lung cancer cases and the number of deaths from lung cancer are very similar, and the cure rate is regarded to be approximately 15% in advanced countries and 7% to 8% in developing countries. Nonsmall cell lung cancer (NSCLC) accounts for approximately 80% of all cases.1 Accumulating evidence suggests that microRNAs (miRNAs) may be involved in controlling lung cancer development and play critical roles in its pathogenesis.2 The miRNAs are a species of small noncoding singlestranded RNAs of approximately 21 to 23 nucleotides, which function by binding to target mRNAs, resulting in their degradation or translational inhibition based on the degree of complementary with their target mRNA. Primary transcripts of miRNAs (pri-miRNA) are generated by RNA polymerase II,3 after which they are sequentially processed by RNase III class From the Tianjin Key Laboratory of Molecular Nuclear Medicine (X-CW, L-QD, HZ, Y-YW, H-YW, D-GL, YS, Q-FL, F-YF, A-MM), Institute of Radiation Medicine, Chinese Academy of Medical Science, Tianjin, China; Institute of Infectious and Endemic Disease Prevention (L-LT), Beijing Centers for Disease Control and Prevention, Beijing, China; Department of Tumor Medicine (H-LW), Shandong Provincial Chest Hospital, Jinan, China; and Department of Policy and Standard Research (X-YJ), The National Institute for Radiological Protection and Nuclear Safety, Chinese Centre for Disease Control and Prevention Beijing, China. Submitted February 24, 2010; accepted in revised form May 14, 2010. The first two authors contributed equally to this work. This study was supported by National Natural Science Foundation of China grant 30901723, Research Fund for the Doctoral Program of Higher Education of China grant 200800231052, CMB grant A2009002 and Institute fund of Radiation Medicine grant SR0815. Correspondence: Ai-Min Meng, Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science, Tianjin, 300192 China (E-mail: [email protected]).
enzymes, Drosha and Dicer, to first produce approximately 70 nt-long intermediate hairpin structures (premiRNAs) and finally the mature miRNAs.4 Human miRNAs have been reported, and a number of these have been shown to play normal physiologic roles in cell proliferation, apoptosis and differentiation.5 In addition, studies have showed that miRNAs contribute to oncogenesis by promoting the expression of oncogenes or by inhibiting tumor suppressor genes. As such, some miRNAs may be markers for cancer diagnosis and prognosis.6 In this study, the expression of miRNA-130a was investigated in NSCLC and analyzed for significance. The results indicate that miRNA-130a was overexpressed and was associated with lymph node metastasis and poor prognosis in NSCLC, suggesting that miRNA-130a may be a potential diagnosis and prognosis marker for NSCLC.
MATERIALS AND METHODS Tissue Specimens Fresh tissue samples, containing NSCLC and adjacent histologically normal tissue, were procured from surgical resection specimens collected by the Department of Tumor Medicine, Shandong Provincial Chest Hospital, from 2001 to 2007. Primary tumor regions and corresponding histologically normal tissues from the same patients were separated by experienced pathologists and immediately stored at ⫺70°C until use. All patients received no treatment before surgery and signed informed consent forms for sample collection. Use of samples of the patients comprising tumor and adjacent histologically normal tissues had been approved by our institutional ethics committee. RNA Extraction Total RNA was extracted from NSCLC tissue and its corresponding normal tissue using the Absolutely RNA RTPCR Miniprep kit (Stratagene, Austin, TX), according to the manufacturer’s instructions and quantification was done with the DUVR 800 UV/Vis Spectrophotometer (Beckman Coulter, Fullerton, CA). Quantitative Reverse Transcription-Polymerase Chain Reaction Taq Man miRNA assays (ABI PRISM, Foster City, CA) used the stem-loop method to detect expression levels of mature miRNA-130a. For reverse transcription (RT) reactions, 10 ng total RNA was used in each reaction and mixed with RT primer. RT reactions were performed at 16°C for 30 minutes, 42°C for 30 minutes and 85°C for 5 minutes, and then maintained at 4°C. After RT reactions, 1.5 l cDNA was used for a polymerase chain reaction (PCR) along with Taq Man primers (2 l). PCR was conducted at 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds and at 60°C for 60 seconds in the ABI 7500 real-time PCR system. Real-time PCR results were analyzed and expressed as relative miRNA expression of the quantification cycle (Cq) values. RT and PCR primers for miRNA-130a were
The American Journal of the Medical Sciences • Volume 340, Number 5, November 2010
385
Wang et al
TABLE 1. Relationship between miRNA-130a expression and tumor clinicopathologic features Clinicopathologic features
FIGURE 1. Expression of miRNA-130a in NSCLC. (A) Representative Q-PCR results of miRNA-130a expression in NSCLC tissue and adjacent noncancerous tissue. The miRNA-130a expression levels were significantly higher in NSCLC tissue than in corresponding noncancerous tissue. Triplicate assays were performed for each sample. (B) Agarose gel electrophoresis results of Q-PCR. (C) Northern blots show that expression of miRNA130a in NSCLC tissue was increased compared with corresponding noncancerous tissue, which was in accordance with Q-PCR results.
purchased from ABI PRISM. U6B was used for normalization. Relative expression levels between treatments were then calculated using the following equation: relative gene expression ⫽ 2⫺⌬⌬Ct, ⫺⌬⌬Ct ⫽ (Ct gene of interest ⫺ Ct internal control gene)Treated ⫺ (Ct gene of interest ⫺ Ct internal control gene)Untreated.7 miRNA Northern Blots For miRNA northern blots, 15 g of total RNA were separated on 15% denaturing polyacrylamide gels, electrotransferred onto GeneScreen Plus membranes (PerkinElmer, Waltham, MA), and hybridized using UltraHyb-Oligo buffer (Ambion, Austin, TX). Oligonucleotides complementary to mature miRNA-130a were end labeled with T4 Kinase (Invitrogen, Carlsbad, CA) and used as probes. Hybridization was performed at 42°C overnight, and membranes were washed twice in 0.1 ⫻ sodium chloride/sodium phosphate/EDTA and 0.1% sodium dodecyl sulfate at 42°C for 15 minutes each. Membranes were then exposed to a storage phosphor screen (GE Healthcare Bio-Sciences, Piscataway, NJ) for 8 hours and imaged using a Typhoon 9410 Variable Mode Imager (GE Healthcare Bio-Sciences). Saved images were cropped using Photoshop 6.0 (Adobe Systems, San Jose, CA). Statistical Analysis All statistical analyses were performed using SPSS13.0 software. Results were statistically evaluated using the Kruskal-Wallis test and Mann-Whitney test. Survival curves
386
miRNA-130a expression No. cases
Low
High
124 76
50 41
74 35
0.079
59 141
42 49
17 92
0.000a
60 69 71
26 39 26
34 30 45
0.056b
95 105
69 22
26 83
0.000a
44 40 52 64
30 18 21 22
14 22 31 42
0.005b
70 62 68
29 33 29
41 29 39
0.352b
Age (yr) ⱖ60 ⬍60 Smoking Nonsmokers Smokers TNM classification (pT) pT1 pT2 pT3 Lymph node metastasis N0 N1 Stage I II III IV Grade G1 G2 G3 a b
P
Kruskal-Wallis test. Mann-Whitney test.
were estimated by the Kaplan-Meier method, and P values ⬍0.05 were considered to be statistically significant.
RESULTS Expression of miRNA-130a in NSCLC Tissue In this study, expression of miRNA-130a was detected in 200 NSCLC samples and adjacent histologically normal tissue using RT-qPCR, and its expression was normalized to that of the control U6B small nuclear RNA (RNU6B) gene. Results showed that miRNA-130a expression levels were significantly higher in NSCLC tissues than in corresponding noncancerous tissues. These results were confirmed by agarose gel electrophoresis of RT-qPCR products (Figures 1A and 1B). To further confirm the RT-qPCR results, northern blot analysis for miRNA-130a was performed in the same 4 pair of samples that were examined using RT-qPCR. Increased miRNA-130a expression was observed in all NSCLC tissues (Figure 1C). Correlation Between miRNA-130a Expression and Clinicopathologic Features in NSCLC Statistical analysis showed that overexpression of miRNA-130a was associated with lymph node metastasis, stage of tumor node metastasis classification and smoking (Table 1). To determine the association between miRNA-130a expression and prognosis, we plotted Kaplan-Meier curves for overall survival. Significant differences were observed in survival of Volume 340, Number 5, November 2010
Role of miRNA-130a in Nonsmall Cell Lung Cancer
FIGURE 2. Kaplan-Meier survival curve of patients with NSCLC subgrouped as miRNA-130a low or high expression. The prognosis of miRNA-130a-positive cases was significantly shorter than that of miRNA-130a-negative cases (P ⫽ 0.008).
patients with NSCLC according to miRNA-130a expression in tumor tissues. The survival rate of patients with low miRNA130a expression (60 months) was higher than that of patients with high miRNA-130a expression (37 months, P ⫽ 0.008; Figure 2). Multivariate Cox regression analysis showed that miRNA-130a expression (P ⫽ 0.021), regional lymph node metastasis (P ⫽ 0.026) and smoking (P ⫽ 0.027) were independent prognostic factors for patients with NSCLC (Table 2).
DISCUSSION Increasing numbers of reports have revealed aberrant expression of certain miRNAs in tumors. Not only is the spectrum of miRNAs expressed in malignant cells significantly different from that of normal counterpart cells but also miRNA expression profiles can better classify poorly differentiated tumors compared with the mRNA (EST)-based classifier.8 A potential role for miRNAs in cancer has been suggested by the location of genes encoding many miRNAs in, or close to, minimal regions of amplicons, loss of heterozygosity or breakpoint cluster regions.9 Amplification or overexpression of an oncogenic miRNA could eliminate the expression of a miRNAtarget tumor suppressor gene and result in cancer progression.9 The miRNA-130a is located at chromosome position 11q12, close to a region (11q13) that often amplified in cancers.10 –13 The miRNA-130a has been identified as a proangiogenic miRNA and is able to downregulate the homeobox genes HOXA5 and GAX (growth arrest-specific homeobox), which are inhibitors of tumor angiogenesis.14 Mandrup et al found miRNA-130a was upregulated in nodal diffuse large B-cell lymphomas when compared with the extranodal diffuse large B-cell lymphoma. However, the expression status of miRNA130a in NSCLC has not been reported until recently. In this study, expression of miRNA-130a was detected for the first
TABLE 2. Multivariate analysis of prognostic factors by the Cox proportional hazards model in patients with NSCLC Variables Lymph nodes metastasis Tumor stage Smoking miRNA-130a expression
Relative risk
95% Confidence interval
P
3.221
1.131–4.462
0.026
0.841 3.647 4.271
0.240–2.431 1.382–4.052 1.249–4.260
0.674 0.027 0.021
© 2010 Lippincott Williams & Wilkins
time in NSCLC samples, and the results showed that miRNA130a level was increased. Studies have shown that miRNA expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggression, response to therapy and prognosis.15 A large amount of diagnostic information was encoded in a relatively small number of miRNAs. For lung cancer, it has been shown that miRNA-let-7 expression was frequently reduced both in vivo and in vitro and that this was significantly associated with shortened postoperative survival, independent of disease stage.16 High mRNA-155 and low miRNA-let-7a-2 expression levels were shown to correlate with poor survival.17 Markou et al18 found overexpression of mature miRNA-21 was an independent negative prognostic factor for overall survival in patients with NSCLC. Given these results, it was decided to investigate whether miRNA-130a expression also correlated with the clinicopathologic features and prognosis of patients with NSCLC. So, the relationship between miRNA-130a expression and patient clinicopathologic factors was analyzed using 200 NSCLC samples. Statistical analysis showed that overexpression of miRNA-130A was significantly positively associated with lymph node metastasis and stage of tumor node metastasis classification. An interesting result was that miRNA-130a expression was significantly associated with the smoking status of patients with NSCLC. It is well reported that epidemic studies have shown smoking as a high risk factor for lung cancer. The exact effects of smoking on miRNA-130a expression in patients with NSCLC require further study. In conclusion, our results revealed for the first time that miRNA-130a was overexpressed in NSCLC tumors. Increased expression of miRNA-130a was strongly associated with lymph node metastasis and poor prognosis. Moreover, miRNA130a expression was an independent prognostic factor for patients with NSCLC. Our findings provide a new role of miRNA-130a in NSCLC, and it may be considered as a potential novel prognostic marker and a target for future development of specific therapeutic interventions in NSCLC. ACKNOWLEDGMENTS We thank Dr. Wu for the collection of tumor samples. REFERENCES 1. Ramalingam S, Pawlish K, Gadgeel S, et al. Lung cancer in young patients: analysis of a Surveillance, Epidemiology, and End Results database. J Clin Oncol 1998;16:651–7.
387
Wang et al
2. Yu SL, Chen HY, Chang GC, et al. MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell 2008;13:48 –57.
FRA11F and 11q13 gene amplification in oral cancer. Genes Chromosomes Cancer 2007;46:143–54.
3. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97.
12. Cheng CK, Chow LW, Loo WT, et al. The cell cycle checkpoint gene Rad9 is a novel oncogene activated by 11q13 amplification and DNA methylation in breast cancer. Cancer Res 2005;65:8646 –54.
4. Kennedy LJ, Barnes A, Happ GM, et al. Extensive interbreed, but minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. Tissue Antigens 2002;59:194 –204. 5. Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell 2005;122:6 –7.
13. Zheng SL, Stevens VL, Wiklund F, et al. Two independent prostate cancer risk-associated Loci at 11q13. Cancer Epidemiol Biomarkers Prev 2009;18:1815–20.
6. He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828 –33.
14. Chen Y, Gorski DH. Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5. Blood 2008;111:1217–26.
7. Schmittgen TD, Lee EJ, Jiang J. High-throughput real-time PCR. Methods Mol Biol 2008;429:89 –98.
15. Garzon R, Fabbri M, Cimmino A, et al. MicroRNA expression and function in cancer. Trends Mol Med 2006;12:580 –7.
8. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834 – 8.
16. Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 2004;64:3753– 6.
9. Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A 2004;101:11755– 60. 10. Gibcus JH, Menkema L, Mastik MF, et al. Amplicon mapping and expression profiling identify the Fas-associated death domain gene as a new driver in the 11q13.3 amplicon in laryngeal/pharyngeal cancer. Clin Cancer Res 2007;13:6257– 66. 11. Reshmi SC, Huang X, Schoppy DW, et al. Relationship between
388
17. Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006;9:189 –98. 18. Markou A, Tsaroucha EG, Kaklamanis L, et al. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR. Clin Chem 2008; 54:1696 –704.
Volume 340, Number 5, November 2010
Expression of miRNA-130a in Nonsmall Cell Lung Cancer Xiao-Chun Wang, PhD, Li-Li Tian, MD, Hai-Liang Wu, MD, Xiao-Yan Jiang, MD, Li-Qing Du, PhD, Heng Zhang, MA, Yue-Ying Wang, BM, Hong-Ying Wu, BM, De-Guan Li, MA, Yi She, BM, Qing-Fen Liu, BM, Fei-Yue Fan, MA and Ai-Min Meng, MA
Abstract: MicroRNAs are short regulatory RNAs that negatively modulate gene expression at the posttranscriptional level and are deeply involved in the pathogenesis of several types of cancer. The miRNA-130a has been shown to play a role in antagonizing the inhibitory effects of GAX on endothelial cell proliferation, migration and tube formation, and antagonizing the inhibitory effects of HoxA5 on tube formation in vitro. Here the authors show, for the first time, that miRNA-130a expression is increased in nonsmall cell lung cancer (NSCLC) tissues. Statistical analysis showed that overexpression of miRNA-130a was strongly associated with lymph node metastasis, stage of tumor node metastasis classification and poor prognosis. Moreover, there was a significant difference in miRNA-130a expression levels between smoking and nonsmoking patients. Multivariate Cox regression analysis showed that miRNA-130a was an independent prognostic factor for patients with NSCLC. Together, these data suggest that miRNA-130a may comprise a potential novel prognostic marker for this disease. Key Indexing Terms: miRNA-130a; Overexpression; Prognosis; NSCLC. [Am J Med Sci 2010;340(5):385–388.]
I
t is a well-known fact that lung cancer ranks as the single biggest cause of cancer deaths in developed countries. The number of new lung cancer cases and the number of deaths from lung cancer are very similar, and the cure rate is regarded to be approximately 15% in advanced countries and 7% to 8% in developing countries. Nonsmall cell lung cancer (NSCLC) accounts for approximately 80% of all cases.1 Accumulating evidence suggests that microRNAs (miRNAs) may be involved in controlling lung cancer development and play critical roles in its pathogenesis.2 The miRNAs are a species of small noncoding singlestranded RNAs of approximately 21 to 23 nucleotides, which function by binding to target mRNAs, resulting in their degradation or translational inhibition based on the degree of complementary with their target mRNA. Primary transcripts of miRNAs (pri-miRNA) are generated by RNA polymerase II,3 after which they are sequentially processed by RNase III class From the Tianjin Key Laboratory of Molecular Nuclear Medicine (X-CW, L-QD, HZ, Y-YW, H-YW, D-GL, YS, Q-FL, F-YF, A-MM), Institute of Radiation Medicine, Chinese Academy of Medical Science, Tianjin, China; Institute of Infectious and Endemic Disease Prevention (L-LT), Beijing Centers for Disease Control and Prevention, Beijing, China; Department of Tumor Medicine (H-LW), Shandong Provincial Chest Hospital, Jinan, China; and Department of Policy and Standard Research (X-YJ), The National Institute for Radiological Protection and Nuclear Safety, Chinese Centre for Disease Control and Prevention Beijing, China. Submitted February 24, 2010; accepted in revised form May 14, 2010. The first two authors contributed equally to this work. This study was supported by National Natural Science Foundation of China grant 30901723, Research Fund for the Doctoral Program of Higher Education of China grant 200800231052, CMB grant A2009002 and Institute fund of Radiation Medicine grant SR0815. Correspondence: Ai-Min Meng, Tianjin Key Laboratory of Molecular Nuclear Medicine, Institute of Radiation Medicine, Chinese Academy of Medical Science, Tianjin, 300192 China (E-mail: [email protected]).
enzymes, Drosha and Dicer, to first produce approximately 70 nt-long intermediate hairpin structures (premiRNAs) and finally the mature miRNAs.4 Human miRNAs have been reported, and a number of these have been shown to play normal physiologic roles in cell proliferation, apoptosis and differentiation.5 In addition, studies have showed that miRNAs contribute to oncogenesis by promoting the expression of oncogenes or by inhibiting tumor suppressor genes. As such, some miRNAs may be markers for cancer diagnosis and prognosis.6 In this study, the expression of miRNA-130a was investigated in NSCLC and analyzed for significance. The results indicate that miRNA-130a was overexpressed and was associated with lymph node metastasis and poor prognosis in NSCLC, suggesting that miRNA-130a may be a potential diagnosis and prognosis marker for NSCLC.
MATERIALS AND METHODS Tissue Specimens Fresh tissue samples, containing NSCLC and adjacent histologically normal tissue, were procured from surgical resection specimens collected by the Department of Tumor Medicine, Shandong Provincial Chest Hospital, from 2001 to 2007. Primary tumor regions and corresponding histologically normal tissues from the same patients were separated by experienced pathologists and immediately stored at ⫺70°C until use. All patients received no treatment before surgery and signed informed consent forms for sample collection. Use of samples of the patients comprising tumor and adjacent histologically normal tissues had been approved by our institutional ethics committee. RNA Extraction Total RNA was extracted from NSCLC tissue and its corresponding normal tissue using the Absolutely RNA RTPCR Miniprep kit (Stratagene, Austin, TX), according to the manufacturer’s instructions and quantification was done with the DUVR 800 UV/Vis Spectrophotometer (Beckman Coulter, Fullerton, CA). Quantitative Reverse Transcription-Polymerase Chain Reaction Taq Man miRNA assays (ABI PRISM, Foster City, CA) used the stem-loop method to detect expression levels of mature miRNA-130a. For reverse transcription (RT) reactions, 10 ng total RNA was used in each reaction and mixed with RT primer. RT reactions were performed at 16°C for 30 minutes, 42°C for 30 minutes and 85°C for 5 minutes, and then maintained at 4°C. After RT reactions, 1.5 l cDNA was used for a polymerase chain reaction (PCR) along with Taq Man primers (2 l). PCR was conducted at 95°C for 10 minutes followed by 40 cycles at 95°C for 15 seconds and at 60°C for 60 seconds in the ABI 7500 real-time PCR system. Real-time PCR results were analyzed and expressed as relative miRNA expression of the quantification cycle (Cq) values. RT and PCR primers for miRNA-130a were
The American Journal of the Medical Sciences • Volume 340, Number 5, November 2010
385
Wang et al
TABLE 1. Relationship between miRNA-130a expression and tumor clinicopathologic features Clinicopathologic features
FIGURE 1. Expression of miRNA-130a in NSCLC. (A) Representative Q-PCR results of miRNA-130a expression in NSCLC tissue and adjacent noncancerous tissue. The miRNA-130a expression levels were significantly higher in NSCLC tissue than in corresponding noncancerous tissue. Triplicate assays were performed for each sample. (B) Agarose gel electrophoresis results of Q-PCR. (C) Northern blots show that expression of miRNA130a in NSCLC tissue was increased compared with corresponding noncancerous tissue, which was in accordance with Q-PCR results.
purchased from ABI PRISM. U6B was used for normalization. Relative expression levels between treatments were then calculated using the following equation: relative gene expression ⫽ 2⫺⌬⌬Ct, ⫺⌬⌬Ct ⫽ (Ct gene of interest ⫺ Ct internal control gene)Treated ⫺ (Ct gene of interest ⫺ Ct internal control gene)Untreated.7 miRNA Northern Blots For miRNA northern blots, 15 g of total RNA were separated on 15% denaturing polyacrylamide gels, electrotransferred onto GeneScreen Plus membranes (PerkinElmer, Waltham, MA), and hybridized using UltraHyb-Oligo buffer (Ambion, Austin, TX). Oligonucleotides complementary to mature miRNA-130a were end labeled with T4 Kinase (Invitrogen, Carlsbad, CA) and used as probes. Hybridization was performed at 42°C overnight, and membranes were washed twice in 0.1 ⫻ sodium chloride/sodium phosphate/EDTA and 0.1% sodium dodecyl sulfate at 42°C for 15 minutes each. Membranes were then exposed to a storage phosphor screen (GE Healthcare Bio-Sciences, Piscataway, NJ) for 8 hours and imaged using a Typhoon 9410 Variable Mode Imager (GE Healthcare Bio-Sciences). Saved images were cropped using Photoshop 6.0 (Adobe Systems, San Jose, CA). Statistical Analysis All statistical analyses were performed using SPSS13.0 software. Results were statistically evaluated using the Kruskal-Wallis test and Mann-Whitney test. Survival curves
386
miRNA-130a expression No. cases
Low
High
124 76
50 41
74 35
0.079
59 141
42 49
17 92
0.000a
60 69 71
26 39 26
34 30 45
0.056b
95 105
69 22
26 83
0.000a
44 40 52 64
30 18 21 22
14 22 31 42
0.005b
70 62 68
29 33 29
41 29 39
0.352b
Age (yr) ⱖ60 ⬍60 Smoking Nonsmokers Smokers TNM classification (pT) pT1 pT2 pT3 Lymph node metastasis N0 N1 Stage I II III IV Grade G1 G2 G3 a b
P
Kruskal-Wallis test. Mann-Whitney test.
were estimated by the Kaplan-Meier method, and P values ⬍0.05 were considered to be statistically significant.
RESULTS Expression of miRNA-130a in NSCLC Tissue In this study, expression of miRNA-130a was detected in 200 NSCLC samples and adjacent histologically normal tissue using RT-qPCR, and its expression was normalized to that of the control U6B small nuclear RNA (RNU6B) gene. Results showed that miRNA-130a expression levels were significantly higher in NSCLC tissues than in corresponding noncancerous tissues. These results were confirmed by agarose gel electrophoresis of RT-qPCR products (Figures 1A and 1B). To further confirm the RT-qPCR results, northern blot analysis for miRNA-130a was performed in the same 4 pair of samples that were examined using RT-qPCR. Increased miRNA-130a expression was observed in all NSCLC tissues (Figure 1C). Correlation Between miRNA-130a Expression and Clinicopathologic Features in NSCLC Statistical analysis showed that overexpression of miRNA-130a was associated with lymph node metastasis, stage of tumor node metastasis classification and smoking (Table 1). To determine the association between miRNA-130a expression and prognosis, we plotted Kaplan-Meier curves for overall survival. Significant differences were observed in survival of Volume 340, Number 5, November 2010
Role of miRNA-130a in Nonsmall Cell Lung Cancer
FIGURE 2. Kaplan-Meier survival curve of patients with NSCLC subgrouped as miRNA-130a low or high expression. The prognosis of miRNA-130a-positive cases was significantly shorter than that of miRNA-130a-negative cases (P ⫽ 0.008).
patients with NSCLC according to miRNA-130a expression in tumor tissues. The survival rate of patients with low miRNA130a expression (60 months) was higher than that of patients with high miRNA-130a expression (37 months, P ⫽ 0.008; Figure 2). Multivariate Cox regression analysis showed that miRNA-130a expression (P ⫽ 0.021), regional lymph node metastasis (P ⫽ 0.026) and smoking (P ⫽ 0.027) were independent prognostic factors for patients with NSCLC (Table 2).
DISCUSSION Increasing numbers of reports have revealed aberrant expression of certain miRNAs in tumors. Not only is the spectrum of miRNAs expressed in malignant cells significantly different from that of normal counterpart cells but also miRNA expression profiles can better classify poorly differentiated tumors compared with the mRNA (EST)-based classifier.8 A potential role for miRNAs in cancer has been suggested by the location of genes encoding many miRNAs in, or close to, minimal regions of amplicons, loss of heterozygosity or breakpoint cluster regions.9 Amplification or overexpression of an oncogenic miRNA could eliminate the expression of a miRNAtarget tumor suppressor gene and result in cancer progression.9 The miRNA-130a is located at chromosome position 11q12, close to a region (11q13) that often amplified in cancers.10 –13 The miRNA-130a has been identified as a proangiogenic miRNA and is able to downregulate the homeobox genes HOXA5 and GAX (growth arrest-specific homeobox), which are inhibitors of tumor angiogenesis.14 Mandrup et al found miRNA-130a was upregulated in nodal diffuse large B-cell lymphomas when compared with the extranodal diffuse large B-cell lymphoma. However, the expression status of miRNA130a in NSCLC has not been reported until recently. In this study, expression of miRNA-130a was detected for the first
TABLE 2. Multivariate analysis of prognostic factors by the Cox proportional hazards model in patients with NSCLC Variables Lymph nodes metastasis Tumor stage Smoking miRNA-130a expression
Relative risk
95% Confidence interval
P
3.221
1.131–4.462
0.026
0.841 3.647 4.271
0.240–2.431 1.382–4.052 1.249–4.260
0.674 0.027 0.021
© 2010 Lippincott Williams & Wilkins
time in NSCLC samples, and the results showed that miRNA130a level was increased. Studies have shown that miRNA expression fingerprints correlate with clinical and biological characteristics of tumors, including tissue type, differentiation, aggression, response to therapy and prognosis.15 A large amount of diagnostic information was encoded in a relatively small number of miRNAs. For lung cancer, it has been shown that miRNA-let-7 expression was frequently reduced both in vivo and in vitro and that this was significantly associated with shortened postoperative survival, independent of disease stage.16 High mRNA-155 and low miRNA-let-7a-2 expression levels were shown to correlate with poor survival.17 Markou et al18 found overexpression of mature miRNA-21 was an independent negative prognostic factor for overall survival in patients with NSCLC. Given these results, it was decided to investigate whether miRNA-130a expression also correlated with the clinicopathologic features and prognosis of patients with NSCLC. So, the relationship between miRNA-130a expression and patient clinicopathologic factors was analyzed using 200 NSCLC samples. Statistical analysis showed that overexpression of miRNA-130A was significantly positively associated with lymph node metastasis and stage of tumor node metastasis classification. An interesting result was that miRNA-130a expression was significantly associated with the smoking status of patients with NSCLC. It is well reported that epidemic studies have shown smoking as a high risk factor for lung cancer. The exact effects of smoking on miRNA-130a expression in patients with NSCLC require further study. In conclusion, our results revealed for the first time that miRNA-130a was overexpressed in NSCLC tumors. Increased expression of miRNA-130a was strongly associated with lymph node metastasis and poor prognosis. Moreover, miRNA130a expression was an independent prognostic factor for patients with NSCLC. Our findings provide a new role of miRNA-130a in NSCLC, and it may be considered as a potential novel prognostic marker and a target for future development of specific therapeutic interventions in NSCLC. ACKNOWLEDGMENTS We thank Dr. Wu for the collection of tumor samples. REFERENCES 1. Ramalingam S, Pawlish K, Gadgeel S, et al. Lung cancer in young patients: analysis of a Surveillance, Epidemiology, and End Results database. J Clin Oncol 1998;16:651–7.
387
Wang et al
2. Yu SL, Chen HY, Chang GC, et al. MicroRNA signature predicts survival and relapse in lung cancer. Cancer Cell 2008;13:48 –57.
FRA11F and 11q13 gene amplification in oral cancer. Genes Chromosomes Cancer 2007;46:143–54.
3. Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116:281–97.
12. Cheng CK, Chow LW, Loo WT, et al. The cell cycle checkpoint gene Rad9 is a novel oncogene activated by 11q13 amplification and DNA methylation in breast cancer. Cancer Res 2005;65:8646 –54.
4. Kennedy LJ, Barnes A, Happ GM, et al. Extensive interbreed, but minimal intrabreed, variation of DLA class II alleles and haplotypes in dogs. Tissue Antigens 2002;59:194 –204. 5. Croce CM, Calin GA. miRNAs, cancer, and stem cell division. Cell 2005;122:6 –7.
13. Zheng SL, Stevens VL, Wiklund F, et al. Two independent prostate cancer risk-associated Loci at 11q13. Cancer Epidemiol Biomarkers Prev 2009;18:1815–20.
6. He L, Thomson JM, Hemann MT, et al. A microRNA polycistron as a potential human oncogene. Nature 2005;435:828 –33.
14. Chen Y, Gorski DH. Regulation of angiogenesis through a microRNA (miR-130a) that down-regulates antiangiogenic homeobox genes GAX and HOXA5. Blood 2008;111:1217–26.
7. Schmittgen TD, Lee EJ, Jiang J. High-throughput real-time PCR. Methods Mol Biol 2008;429:89 –98.
15. Garzon R, Fabbri M, Cimmino A, et al. MicroRNA expression and function in cancer. Trends Mol Med 2006;12:580 –7.
8. Lu J, Getz G, Miska EA, et al. MicroRNA expression profiles classify human cancers. Nature 2005;435:834 – 8.
16. Takamizawa J, Konishi H, Yanagisawa K, et al. Reduced expression of the let-7 microRNAs in human lung cancers in association with shortened postoperative survival. Cancer Res 2004;64:3753– 6.
9. Calin GA, Liu CG, Sevignani C, et al. MicroRNA profiling reveals distinct signatures in B cell chronic lymphocytic leukemias. Proc Natl Acad Sci U S A 2004;101:11755– 60. 10. Gibcus JH, Menkema L, Mastik MF, et al. Amplicon mapping and expression profiling identify the Fas-associated death domain gene as a new driver in the 11q13.3 amplicon in laryngeal/pharyngeal cancer. Clin Cancer Res 2007;13:6257– 66. 11. Reshmi SC, Huang X, Schoppy DW, et al. Relationship between
388
17. Yanaihara N, Caplen N, Bowman E, et al. Unique microRNA molecular profiles in lung cancer diagnosis and prognosis. Cancer Cell 2006;9:189 –98. 18. Markou A, Tsaroucha EG, Kaklamanis L, et al. Prognostic value of mature microRNA-21 and microRNA-205 overexpression in non-small cell lung cancer by quantitative real-time RT-PCR. Clin Chem 2008; 54:1696 –704.
Volume 340, Number 5, November 2010