|Year : 2016 | Volume
| Issue : 3 | Page : 69-74
High expression of mir-25 predicting poor prognosis in gastric cancer
Chuanli Ren1, Wenshu Wang2, Chongxu Han3, Hui Chen4, Deyuan Fu5, Daxin Wang3, Ming Shen2
1 Department of Clinical Medical Laboratory, Clinical Medical Testing Laboratory, Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University, Yangzhou; Department of Epidemiology and Biostatistics, Ministry of Education, Key Laboratory of Modern Toxicology, School of Public Health, Nanjing Medical University, Nanjing, China
2 Department of Chemistry, College of Chemistry and Chemical Engineering, Yangzhou University, Yangzhou, China
3 Department of Clinical Medical Laboratory, Clinical Medical Testing Laboratory, Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University, Yangzhou, China
4 Division of Geriatric Medicine, Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University, Yangzhou, China
5 Department of Breast Oncology Surgery, Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University, Yangzhou, China
|Date of Submission||20-Jul-2016|
|Date of Acceptance||01-Sep-2016|
|Date of Web Publication||30-Sep-2016|
Clinical Medical Testing Laboratory, Northern Jiangsu People's Hospital and Clinical Medical College of Yangzhou University, No. 98 Western Nantong Road, Yangzhou 225001
Source of Support: None, Conflict of Interest: None
Aim: The aim of this study was to explore miR-25 expression pattern and its prognostic value in gastric carcinoma (GC). Methods: MiR-25 expression was detected using miRNA-locked nucleic acid in situ hybridization in 180 patients with GC undergoing surgery. Correlation with clinicopathological features and overall survival (OS) was analyzed. Results: MiR-25 expression was decreased in 10.0% (18/180) of GC, increased in 62.2% (112/180), and unchanged in 27.8% (50/180), compared to samples of morphologically normal tissue taken from the same patient (P < 0.001). Univariate analysis showed that high miR-25 expression, tumor stage, tumor status, node status, and tumor size were significant negative prognostic predictors for OS in patients with GC, and the results were shown as P < 0.001, P < 0.001, P = 0.002, P> 0.001, and P = 0.001, respectively. High miR-25 expression remained a significant predictor of shorter survival in stage II (n = 56, P = 0.015) and stage III (n = 92, P < 0.001) GC. Multivariate regression analysis demonstrated that tumor status (hazard ratio [HR]: 1.91; 95% confidence interval [CI]: 0.79-4.62; P = 0.151), stage (HR: 2.26; 95% CI: 1.30-3.94; P = 0.004), lymph node metastasis (HR: 1.40; 95% CI: 0.73-2.68; P = 0.309), high expression of miR-25 (HR: 2.39; 95% CI: 1.53-3.72; P < 0.001), and tumor size (HR: 2.29; 95% CI: 1.40-3.74; P = 0.001) predicted shorter OS. Conclusion: High expression of miR-25 was associated with decreased OS. Thus, miR-25 may be useful for prognosis evaluation and may provide a novel treatment target in patients with GC.
Keywords: Gastric cancer, miR-25, prognosis, target therapy
|How to cite this article:|
Ren C, Wang W, Han C, Chen H, Fu D, Wang D, Shen M. High expression of mir-25 predicting poor prognosis in gastric cancer. Transl Surg 2016;1:69-74
|How to cite this URL:|
Ren C, Wang W, Han C, Chen H, Fu D, Wang D, Shen M. High expression of mir-25 predicting poor prognosis in gastric cancer. Transl Surg [serial online] 2016 [cited 2021 Sep 26];1:69-74. Available from: http://www.translsurg.com/text.asp?2016/1/3/69/191487
| Introduction|| |
Gastric cancer (GC) is the fifth most common malignant disease globally, with 50% of the total cancer burden occurring in Eastern Asia, predominantly in China.  GC is also associated with tumor relapse and poor overall survival (OS) time. Oncogenic activation (β-catenin) and tumor-suppressor gene (p53) mutations are associated with the histologic and genetic alterations of GC.  The molecular mechanisms depicting the prognosis of GC remain unclear.
Since the discovery of the small RNA molecules, miRNAs have been found to play an important role in regulating cellular biological functions by targeting different genes. ,, In general, miRNAs regulate normal cell function and homeostasis. Moreover, miRNAs are usually an aberrant expression in cancer cells and the tumor microenvironment, and can induce epithelial-to-mesenchymal transition via downregulation of E-cadherin and other proteins. ,, Aberrant miRNA expression in tissue or serum of patients with GC has the potential to be a diagnostic or prognostic biomarker. ,,,,,,,,, However, it is unclear whether these miRNAs are reliable for the role of early diagnosis or prognostic prediction of GC.
MiR-25 is known to be an essential oncogene influencing proliferation and invasion by targeting TOB1,  FBXW7,  and ERBB2.  A single nucleotide polymorphism located on the site of miR-25 (rs41274221) may protect the patients from distant metastasis of GC.  In our previous work, we found that circulating miR-25 was consistently increased in plasma of the GC patients compared with controls, indicating a potential role as a noninvasive biomarker for diagnosis of the early-stage GC.  As the miR-25 expression pattern and its prognostic value are unknown in GC, the goal of this study is to explore the prognostic value of miR-25 in 180 GC patients.
| Methods|| |
Patients and tissue samples
Paraffin-embedded tissue samples were acquired retrospectively from the Biobank Center at the National Engineering Center for Biochip at Shanghai (Shanghai Outdo Biotech Co., Ltd., Shanghai, China). Tumor tissues and corresponding normal tissues were obtained from 180 patients with histologically confirmed GC between 2006 and 2008. Written informed consent was required, and the Ethics Committee of the National Engineering Center approved the protocol for Biochip at Shanghai. OS time was calculated from the date of pathological diagnosis to the time of death or the date of final follow-up. The median follow-up time was 7.1 years, with the range of 6.6-8.1 years [Table 1].
MiRNA-locked nucleic acid in situ hybridization
Tissue microarrays were evaluated using tissue cores from paraffin-embedded GC samples as described by our group and others. ,, MiRNA-locked nucleic acid in situ hybridization was carried out using antisense oligonucleotide probes for miR-25 (Exiqon Inc., Woburn, MA, USA) with a scramble-miRNA serving as a negative control. The method and the definition of the positive and negative miR-25 staining are described in our previous publication. 
Associations between clinicopathological parameters and miR-25 expression were evaluated using Chi-square test. Multivariate Cox proportional hazards regression models were used to analyze the independent prognostic values. All analyses were performed using the SPSS 17.0 statistical software (SPSS Inc., Chicago, IL, USA). All tests were two-sided and P < 0.05 was considered statistically significant.
| Results|| |
Expression of miR-25 in gastric carcinoma
MiR-25 was mainly expressed in the cytoplasm of cells of GC and normal gastric tissue demonstrated by in situ hybridization [Figure 1]. MiR-25 expression was increased in 62.2% (112/180), decreased in 10.0% (18/180) of GC, and unchanged in 27.8% (50/180), compared to normal tissue (P < 0.001).
|Figure 1. MiR-25 levels stained using in situ hybridization. (a) MiR-25 staining using in situ hybridization in gastric carcinoma. (b) MiR-25 staining in adjacent normal tissue; increased miR-25 expression in gastric carcinoma compared with neighboring normal tissue. (c) MiR-25 staining using in situ hybridization in gastric carcinoma. (d) MiR-25 staining in adjacent normal tissue; increased miR-25 expression in gastric carcinoma compared to adjacent normal tissue|
Click here to view
MiR-25 expression and its clinicopathological features in gastric carcinoma
No significant correlations were found between miR-25 expression levels and other clinicopathological variables, including age (P = 0.891), gender (P = 0.760), tumor site (P = 0.938), tumor size (P = 0.185), nodal status (P = 0.287), TNM stage (P = 0.166), local invasion (P = 0.185), and distant metastasis (P = 0.151) [Table 2].
|Table 2: MiR - 25 expression and clinicopathological features in patients with gastric adenocarcinoma|
Click here to view
Kaplan-Meier analysis indicated that high expression of miR-25 (P < 0.001), stage of disease (P < 0.001), tumor status (P = 0.008), node status (P < 0.001), tumor size (P = 0.001), and distant metastasis (P = 0.005) were related to significantly shorter OS in patients with GC. Age, gender, and location did not correlate with OS (P > 0.05) [Table 3].
|Table 3: Univariate analysis of survival in patients with gastric cancer|
Click here to view
High expression of miR-25 (n = 112) remained a significant predictor of shorter mean OS compared with low/unchanged group (n = 68) (43.1 vs. 66.7 months, P < 0.001) [Figure 2]. After TNM stratification, high expression of miR-25 remained a significant predictor of poor OS in stage II (47.0 vs. 61.5 months, P = 0.0015, n = 56) and stage III GC (36.3 vs. 67.2 months, P < 0.001, n = 92) [Figure 2].
|Figure 2. Survival curves in patients with gastric carcinoma according to miR-25 levels. (a) Overall survival curves in 180 patients with gastric carcinoma according to miR-25 levels (P < 0.001). (b) Survival curves of patients with stage II gastric carcinoma according to miR-25 levels (P = 0.015). (c) Survival curves of patients with stage III gastric carcinoma according to miR-25 levels (P < 0.001)|
Click here to view
Univariate Cox regression analysis showed predictors of poor prognosis as following: tumor status (hazard ratio [HR]: 3.10; 95% confidence interval [CI]: 1.51-6.37; P = 0.002), stage (HR: 2.83; 95% CI: 1.88-4.27; P < 0.001), lymph node metastasis (HR: 2.82; 95% CI: 1.70-4.68; P < 0.001), high miR-25 expression (HR: 2.53; 95% CI: 1.64-3.90; P < 0.001), and tumor size (HR: 2.30; 95% CI: 1.42-3.71; P = 0.001). Gender, age, and tumor site were not predictors of poor prognosis [Table 4].
|Table 4: Multivariate Cox regression analysis of potential survival prognostic factors in 180 patients with gastric cancer|
Click here to view
Multivariate Cox regression analysis identified predictors of poor prognosis as following: tumor stage (HR: 2.26; 95% CI: 1.30-3.94; P = 0.004), miR-25 (HR: 2.39; 95% CI: 1.53-3.72; P < 0.001), age (HR: 1.21; 95% CI: 0.80-1.82; P = 0.372), and tumor site (HR: 1.82; 95% CI: 1.30-3.94; P = 0.03). Gender, tumor status, lymph node metastasis, tumor site, and tumor size were not predictors of poor prognosis [Table 4].
| Discussion|| |
To date, p21,  ERBB2,  FBXW7, , RhoGDI1,  SOCS4,  RGS3,  NEFL,  MOAP1,  CDKN1C,  KLF4, C/EBPα,  αv- and α6-integrins  have been reported as potential targets for miR-25 in various solid cancers. MiR-25 may act as an oncogenic miRNA to promote proliferation, migration, and invasion in many kinds of cancers, such as GC, ,,, colorectal cancer, , lung cancer, ,,,, ovarian cancer,  and esophageal squamous cell carcinoma.  However, miR-25 polymorphism can inhibit oncogenic function in GC. 
Circulating miR-25 in the plasma/serum could be used as a noninvasive biomarker for the early diagnosis of GC,  hepatocellular carcinoma,  and lung cancer.  According to recent publications, circulating miR-25 has diagnostic and prognostic value in both ovarian and hepatocellular cancer patients. ,
In our previous work, we found that circulating miR-25 was part of a five-miRNA signature for early diagnosis of GC.  Some groups have shown that high expression of miR-25 can be used as a predictive prognostic marker in hepatocellular carcinoma.  We compared miR-25 expression levels in GC and surrounding morphologically normal tissues and found that miR-25 expression was significantly increased in GC tissues overall. However, its expression level was decreased in 10.0% (18/180) of samples and unchanged in 27.8% (50/180), suggesting that heterogeneous miR-25 expression may have different prognostic values in different individuals.
To the best of our knowledge, this study is the first to systematically study the relationship of miR-25 to clinicopathological parameters and prognosis in patients with GC. In our previous work, we identified increased circulating miR-25 expression in the early stages of GC.  In addition, Li et al. found that high plasma concentrations of miR-25 in GC patients predicted poor prognosis.  This implies that higher levels of circulating and tissue miR-25 in GC may predict poor prognosis.
In this study, GC patients with higher miR-25 had a worse prognosis than those with lower or unchanged expression. Moreover, using multivariate Cox regression analysis, we identified high levels of miR-25 indicating poor prognosis (P < 0.001). In a univariate Cox regression analysis, high miR-25 expression was also independently associated with a worse prognosis (P < 0.001). Li et al. also found that circulating miR-25 in GC patients correlated with poor prognosis. As expected, the elevated level of miR-25 in plasma and GC tissues was consistent. Therefore, increased circulating miR-25 in the plasma may originate primarily from GC cells. In this study, we found that miR-25 was sometimes overexpressed in normal surrounding gastric tissues and its fundamental function is unknown. Thus, detection of miR-25 in the serum or tissues may provide new information to help early diagnosis and prognostic evaluation of GC. Although the molecular function of miR-25 in GC is largely unknown, miR-25 may act as an oncogenic driver in GC. ,,,
Although intervention including surgical resection may be different for each patient, higher expression of miR-25 remained a poor prognosticator in dependent of other clinical parameters. Other clinical and genetic factors may influence OS. For example, groups with a high expression of miR-25 trended toward more advanced TNM stage at presentation though this was not statistically significant [Table 2]. There are other factors that may influence tumor biology, such as p53 polymorphism, that could offset the efficacy of chemotherapy and affect the OS. , The molecular mechanism of increased miR-25 and its relationship to diagnosis and prognosis requires further investigation and validation.
| Conclusion|| |
Our study indicates that in patients with GC, increased expression of miR-25 predicts a relatively poor prognosis while low/unchanged miR-25 expression indicates a better prognosis. Although further research is needed to confirm our findings, this study suggests that miR-25 may represent a potential biomarker and target for the treatment of GC.
Financial support and sponsorship
This study was supported by the National Nature Science Foundation of China (Grant Number: 81573220), the Jiangsu provincial Six Talent Peaks (Grant Number: WSN107), the Foundation of China Postdoctoral Studies (Grant Number: M2013541699), the Foundation of Jiangsu Province Postdoctoral Studies (Grant Number: 1302149C), and the Foundation for Yong Scholar in Yangzhou (Grant Number: YZ2014046).
Conflicts of interest
There are no conflicts of interest.
| References|| |
McLean MH, El-Omar EM. Genetics of gastric cancer. Nat Rev Gastroenterol Hepatol
Ushijima T, Sasako M. Focus on gastric cancer. Cancer Cell
Lewis BP, Burge CB, Bartel DP. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell
Alvarez-Garcia I, Miska EA. MicroRNA functions in animal development and human disease. Development
Bartel DP. MicroRNAs: Genomics, biogenesis, mechanism, and function. Cell
Rupaimoole R, Calin GA, Lopez-Berestein G, Sood AK. miRNA deregulation in cancer cells and the tumor microenvironment. Cancer Discov
Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, Khew-Goodall Y, Goodall GJ. The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol
Korpal M, Lee ES, Hu G, Kang Y. The miR-200 family inhibits epithelial-mesenchymal transition and cancer cell migration by direct targeting of E-cadherin transcriptional repressors ZEB1 and ZEB2. J Biol Chem
Ueda T, Volinia S, Okumura H, Shimizu M, Taccioli C, Rossi S, Alder H, Liu CG, Oue N, Yasui W, Yoshida K, Sasaki H, Nomura S, Seto Y, Kaminishi M, Calin GA, Croce CM. Relation between microRNA expression and progression and prognosis of gastric cancer: A microRNA expression analysis. Lancet Oncol
Zhu C, Ren C, Han J, Ding Y, Du J, Dai N, Dai J, Ma H, Hu Z, Shen H, Xu Y, Jin G. A five-microRNA panel in plasma was identified as potential biomarker for early detection of gastric cancer. Br J Cancer
Zhou X, Zhu W, Li H, Wen W, Cheng W, Wang F, Wu Y, Qi L, Fan Y, Chen Y, Ding Y, Xu J, Qian J, Huang Z, Wang T, Zhu D, Shu Y, Liu P. Diagnostic value of a plasma microRNA signature in gastric cancer: A microRNA expression analysis. Sci Rep
Zhang X, Cui L, Ye G, Zheng T, Song H, Xia T, Yu X, Xiao B, Le Y, Guo J. Gastric juice microRNA-421 is a new biomarker for screening gastric cancer. Tumour Biol
Yu X, Luo L, Wu Y, Yu X, Liu Y, Yu X, Zhao X, Zhang X, Cui L, Ye G, Le Y, Guo J. Gastric juice miR-129 as a potential biomarker for screening gastric cancer. Med Oncol
Cui L, Zhang X, Ye G, Zheng T, Song H, Deng H, Xiao B, Xia T, Yu X, Le Y, Guo J. Gastric juice MicroRNAs as potential biomarkers for the screening of gastric cancer. Cancer
Kogo R, Mimori K, Tanaka F, Komune S, Mori M. Clinical significance of miR-146a in gastric cancer cases. Clin Cancer Res
Rotkrua P, Shimada S, Mogushi K, Akiyama Y, Tanaka H, Yuasa Y. Circulating microRNAs as biomarkers for early detection of diffuse-type gastric cancer using a mouse model. Br J Cancer
Liu X, Kwong A, Sihoe A, Chu KM. Plasma miR-940 may serve as a novel biomarker for gastric cancer. Tumour Biol
Ren C, Wang W, Han C, Chen H, Fu D, Luo Y, Yao H, Wang D, Ma L, Zhou L, Han D, Shen M. Expression and prognostic value of miR-92a in patients with gastric cancer. Tumour Biol
Zhou J, Zhou J, Wang W, Li W, Wu L, Li G, Shi J, Zhou S. The polymorphism in miR-25 attenuated the oncogenic function in gastric cancer. Tumour Biol
Gong J, Cui Z, Li L, Ma Q, Wang Q, Gao Y, Sun H. MicroRNA-25 promotes gastric cancer proliferation, invasion, and migration by directly targeting F-box and WD-40 Domain Protein 7, FBXW7. Tumour Biol
Li BS, Zuo QF, Zhao YL, Xiao B, Zhuang Y, Mao XH, Wu C, Yang SM, Zeng H, Zou QM, Guo G. MicroRNA-25 promotes gastric cancer migration, invasion and proliferation by directly targeting transducer of ERBB2, 1 and correlates with poor survival. Oncogene
He C, Jiang H, Geng S, Sheng H, Shen X, Zhang X, Zhu S, Chen X, Yang C, Gao H. Expression and prognostic value of c-Myc and Fas (CD95/APO1) in patients with pancreatic cancer. Int J Clin Exp Pathol
Chen H, Ren C, Han C, Wang D, Chen Y, Fu D. Expression and prognostic value of miR-486-5p in patients with gastric adenocarcinoma. PLoS One
Su X, Wang H, Ge W, Yang M, Hou J, Chen T, Li N, Cao X. An in vivo
method to identify microRNA targets not predicted by computation algorithms: p21 Targeting by miR-92a in cancer. Cancer Res
Xiang J, Hang JB, Che JM, Li HC. MiR-25 is up-regulated in non-small cell lung cancer and promotes cell proliferation and motility by targeting FBXW7. Int J Clin Exp Pathol
Wang C, Wang X, Su Z, Fei H, Liu X, Pan Q. MiR-25 promotes hepatocellular carcinoma cell growth, migration and invasion by inhibiting RhoGDI1. Oncotarget
Mei Z, Chen S, Chen C, Xiao B, Li F, Wang Y, Tao Z. Interleukin-23 facilitates thyroid cancer cell migration and invasion by inhibiting SOCS4 expression via MicroRNA-25. PLoS One
Chen Z, Wu Y, Meng Q, Xia Z. Elevated microRNA-25 inhibits cell apoptosis in lung cancer by targeting RGS3. In Vitro Cell Dev Biol Anim
Wu T, Chen W, Kong D, Li X, Lu H, Liu S, Wang J, Du L, Kong Q, Huang X, Lu Z. miR-25 targets the modulator of apoptosis 1 gene in lung cancer. Carcinogenesis
Zhang J, Gong X, Tian K, Chen D, Sun J, Wang G, Guo M. miR-25 promotes glioma cell proliferation by targeting CDKN1C. Biomed Pharmacother
Zoni E, van der Horst G, van de Merbel AF, Chen L, Rane JK, Pelger RC, Collins AT, Visakorpi T, Snaar-Jagalska BE, Maitland NJ, van der Pluijm G. miR-25 modulates invasiveness and dissemination of human prostate cancer cells via regulation of s and dissemination of human Pr. Cancer Res
Zhang M, Wang X, Li W, Cui Y. miR-107 and miR-25 simultaneously target LATS2 and regulate proliferation and invasion of gastric adenocarcinoma (GAC) cells. Biochem Biophys Res Commun
Zhao H, Wang Y, Yang L, Jiang R, Li W. MiR-25 promotes gastric cancer cells growth and motility by targeting RECK. Mol Cell Biochem
Zhou J, Wang J, Wu S, Zhu S, Wang S, Zhou H, Tian X, Tang N, Nie S. Angiopoietin-like protein 2 negatively regulated by microRNA-25 contributes to the malignant progression of colorectal cancer. Int J Mol Med
Li Q, Zou C, Zou C, Han Z, Xiao H, Wei H, Wang W, Zhang L, Zhang X, Tang Q, Zhang C, Tao J, Wang X, Gao X. MicroRNA-25 functions as a potential tumor suppressor in colon cancer by targeting Smad7. Cancer Lett
Zhao Z, Liu J, Wang C, Wang Y, Jiang Y, Guo M. MicroRNA-25 regulates small cell lung cancer cell development and cell cycle through cyclin E2. Int J Clin Exp Pathol
Yang T, Chen T, Li Y, Gao L, Zhang S, Wang T, Chen M. Downregulation of miR-25 modulates non-small cell lung cancer cells by targeting CDC42. Tumour Biol
Feng S, Pan W, Jin Y, Zheng J. MiR-25 promotes ovarian cancer proliferation and motility by targeting LATS2. Tumour Biol
Xu X, Chen Z, Zhao X, Wang J, Ding D, Wang Z, Tan F, Tan X, Zhou F, Sun J, Sun N, Gao Y, Shao K, Li N, Qiu B, He J. MicroRNA-25 promotes cell migration and invasion in esophageal squamous cell carcinoma. Biochem Biophys Res Commun
Mirzaei HR, Sahebkar A, Yazdi F, Salehi H, Jafari MH, Namdar A, Khabazian E, Jaafri MR, Mirzaei H. Circulating microRNAs in hepatocellular carcinoma: potential diagnostic and prognostic biomarkers. Curr Pharm Des
2016; [Epub ahead of print].
Wang P, Yang D, Zhang H, Wei X, Ma T, Cheng Z, Hong Q, Hu J, Zhuo H, Song Y, Jia C, Jing F, Jin Q, Bai C, Mao H, Zhao J. Early detection of lung cancer in serum by a panel of MicroRNA biomarkers. Clin Lung Cancer
Meng X, Joosse SA, Müller V, Trillsch F, Milde-Langosch K, Mahner S, Geffken M, Pantel K, Schwarzenbach H. Diagnostic and prognostic potential of serum miR-7, miR-16, miR-25, miR-93, miR-182, miR-376a and miR-429 in ovarian cancer patients. Br J Cancer 2015;113 (9):1358-66.
Sadeghian Y, Kamyabi-Moghaddam Z, Nodushan SM, Khoshbakht S, Pedram B, Yahaghi E, Mokarizadeh A, Mohebbi M. Profiles of tissue microRNAs; miR-148b and miR-25 serve as potential prognostic biomarkers for hepatocellular carcinoma. Tumour Biol
Ozen A, Kocak Z, Sipahi T, Oz-Puyan F, Cakina S, Saynak M, Ibis C, Karagol H. The prognostic significance of p21 and Her-2 gene expression and mutation/polymorphism in patients with gastric adenocarcinoma. Med Oncol
Yang C, Ma X, Liu D, Wang Y, Tang R, Zhu Y, Xu Z, Yang L. Promoter polymorphisms of miR-34b/c are associated with risk of gastric cancer in a Chinese population. Tumour Biol
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4]