You are on page 1of 15

MiR-31 e miR-200 expression in gastric cancer cell lines from Brazilian patients.

AMP Cruz1, AP Barros1, CDP Lopes1, LDS Paradela1, RC Montenegro1, S Darnet1,*,RR Burbano1,, AK Ribeiro-dos-Santos1

Instituto de Cincias Biolgicas, Universidade Federal do Par, Belm, Brazil

*Corresponding Author: sylvain@ufpa.br

E-mails: Aline Maria Pereira Cruz : nurse.alinecruz@gmail.com Amanda Paiva de Barros : amandabarros@ufpa.br Camile de Barros Lopes: camilelopes@bol.com.br Luciana de Sousa Paradela : lucianasparadela@hotmail.com Raquel Carvalho Montenegro : rcm.montenegro@gmail.com Sylvain Darnet : sylvain@ufpa.br Rommel Rodriguez Burbano : rommel@ufpa.br ndrea Kely Ribeiro-dos-Santos : akely@ufpa.br

Key words: miRNA, Gastric cancer, cell lines

Abstract Gastric cancer remains the second most common cause of cancer-related deaths worldwide and few specific tumor markers with high sensitivity and specificity are available for the diagnosis. This study is about the expression of miR-31 and miR-200 by qRT-PCR in three gastric cancer cell lines from Brazilian patients. Our results demonstrated that the both microRNAs are down regulated in cell lines and should be listed as new putative biomarkers of gastric cancer tumors.

Background Gastric cancer (GC) remains the second most common cause of cancer-related deaths worldwide and it is likely to remain as one of the leading cause of all deaths in near future[1]. Few specific tumor markers with high sensitivity and specificity are available for the diagnosis of GC [2], however none of those could solve the problem of early onset of this type of cancer. An emerging class of small non-coding RNA species, known as microRNAs (miRNAs), has been associated with critical functions across several biological processes and tumor formation [2]. Lu et al. (2005) showed that the miRNA profiles were surprisingly informative in terms of developmental lineage, differentiation and state of the tumors, can they accurately classify samples poorly differentiated tumors [3]. These findings highlight the potential of miRNA profiling in cancer diagnosis. Recent studies show that miRNAs could be used as a novel noninvasive biomarker in gastric cancer (GC) detection [4-6], mainly because they are crucial regulators of a variety of physiologic and pathologic processes, histological differentiation, tumor development and metastatic progression [7-9]. Also, metastasis is responsible for the overwhelming majority of carcinoma-associated mortality [8]. In this way, miRNA-200 (miR-200) family has been reported as tumor suppressor in gastric cancer, since this family regulates cell proliferation, invasion, and migration by directly targeting ZEB1 and ZEB2, thus, indirectly increasing the level of E-cadherin that favors cancer invasion and metastasis. On the other hand, the function of miR-31 in gastric cancers is unclear, but clinical trials have indicated inverse correlations between miR-31 levels and malignant phenotypes [10, 11]. In addition, miR-31 was reported as a potent inhibitor of metastasis via the pleiotropic suppression of a cohort of prometastatic target genes that include integrin alpha (5) (ITGA5), radixin (RDX), and RhoA in metastatic breast cancer [8]. The miR-31 also acts as a master regulator of integrins as it targets multiple subunit partners (2, 5, and V) of 1 integrins and also 3 integrins [12]. Valastyan et al. (2009) observed that the ectopic expression of miR-31 inhibited several steps of the metastatic cascade, including local invasion, extravasation or initial survival at a distant site, and metastatic colonization in metastatic breast cancer. In addition, it

was characterized as an inducer of cell cycle arrest and apoptosis in a large number of cancer cell lines in which the p53 pathway was upregulated [8, 13]. Cell lines behave as a biological model to obtain information about the clonal evolution of this malignancy. In addition, they are widely used in studies of cytotoxicity and cell survival for new chemotherapeutic agents [10, 14]), justifying himself for being difficult to monitor and reproducibility. There are few cytogenetic studies in vitro lines derived from solid tumors, especially gastric cancer, because of technical limitations such as proper cultivation of clinical samples, bacterial contamination, low mitotic metaphases of poor quality and complex chromosomal changes suffered by these cells, which carries a modest number of gastric tumor cell lines available [15-18]. The Most strains available in the market comes from the Asian (Japan and China), justified by the epidemiology of this continent in the world ranking, whose investments were focused on the research of this disease. However, three gastric tumor cell lines were the firsts stabilized in northern Brazil of patients with gastric cancer ACP02 (diffuse type with staging T3N2M0 from cardia), ACP03 (intestinal type with staging T4N1M0 from antrum) and AGP01(intestinal type with staging T3N2M1, from ascites fluid of tumor in the body and antrum)[15]. The aim of the present study was to investigate the expression of miR-31, miR-200a and miR-200b in these three gastric cancer lines cells stabilized in northern Brazil.

Results
Identification of the expression reduced of miR-31 in gastric tumor cell lines We found that the expression levels of miR-31 in the three gastric cancer cells line were significantly lower than those in non-tumor tissues (Figure 1). The value of fold change of miR-31 expression was calculated relative to gastric cancer cell lines to non-tumor. We identified that miR-31 presented the 2-Ct values of 0.0012, 0.0003 and 0.0005 in ACP02, ACP03 and AGP01, respectively. Whereas, the expression of miR-31 was higher compared to the cardia with antrum non-tumor tissues (2-Ct 43.07 and 21.47 respectively) as shown the table 1. miR-200a had reduced expression in gastric tumor cell lines

qRT-PCR analysis also indicated that the expression levels of miR-200a were significantly lower in the three gastric cancer cells line than those in non-tumor tissues (Figure 2). From the analysis of the non-tumor tissues, we observed that 2-Ct values were 220.75 and 161.88 in cardia and antrum respectively (Table 2). Aberrant expression the miR-200b in gastric tumor cell lines The expression levels of miR-200b were aberrant due to deviant behavior within the three gastric tumor cell lines, which it presented with significantly reduced expression of ACP02 and high in others, especially AGP01 relative to non-tumor tissues (Figure 3).On the other hand, we observed that 2-Ct values were and 95.57 and 7.94 in crdia and antrum respectively (Table 3).

Discussion
Expression reduced of miR-31 in gastric tumor cell lines The miR-31 is reported as a potent inhibitor of metastasis, since it has as target genes pro-metastatic [19]. In addition, it was characterized as an inducer of cell cycle arrest and apoptosis in a large number of cancer cell lines in which the p53 pathway was upregulated [13, 20]. In our study, we identified the high expression of this miRNA in non-tumor gastric tissue, especially in the cardia (relative expression in the 2-Ct 43.07) according to Guo et al. and Ribeiro dos Santos et al. [10, 21]. Moreover, the MIR-31 expression was observed with significantly reduced in studies conducted in gastric tumors when paired with the primary non-tumor tissue and controls [22, 23], which was also convergent with the results found in ACP02, ACP03 and AGP01 cell lines (Table 1).This new information may help to clarify the molecular mechanisms involved in gastric carcinogenesis and to indicate that miR-31 may be a novel diagnostic biomarker of gastric cancer. Aberrant expression of two members of the family miR-200 in gastric tumor cell lines The miR-200 family is formed by five entities (among them the 200a miR-c and miR200b), characterized as a potent tumor suppressor by preventing epithelial-mesenquimal transition (EMT) indirectly, by inhibiting ZEB1 and ZEB2 [24-26]. These two genes suppress gene expression of E-cadherin [27, 28], resulting in the reduction of cell

adherence and increase in motility and metastasis [25, 26]. Thus, miR-200 hyperexpression was found in various tumors, including stomach [29], consistent with our results where we observed the reduced expression in the three gastric tumor cell lines (Table 3). Moreover, the MIR-200b showed aberrant expression to express in a manner reduced by ACP02 (relative expression of 2-ct 0.05) and larger in ACP03 (relative expression of 2-ct 5.93) and AGP01 (relative expression by 2-ct of 19.56), these findings were not correlated to specific anatomical region or gastric adenocarcinoma. However, in other tumors, miR-200b underexpressed found to have its Smad3-induced transcription or p53 [30].

Conclusions
In summary, we report here the comparison of the expression profile of seven miRNAs in three tumor cell lines and gastric tissue samples from five gastric cardia and antrum of patients undergoing endoscopy. Identified the increased expression of miR-31 and miR-200a evaluated in non-tumor tissue compared to tumor cell lines. The aberrant expression of miR-200b was higher in non-cardia tumor, then the line AGP01, which demonstrates variable expression profile and prevents it from being characterized as a biomarker. The study of the expression profile of miRNAs in gastric tumor cell lines stabilized patients in northern Brazil is unprecedented, characterizing it as a challenging opportunity can reveal phylogeographic particularities related to the group in a country as heterogeneous and mainly contribute as a model study of gastric carcinogenesis, since its results correlated with the provisions in the current literature. Therefore, further studies with higher sample are required to ensure this expression pattern diverging in order to characterize the expression of target genes involved in gastric cancer.

Material and Methods


MicroRNA expression levels were detected using quantitative reverse transcriptionpolymerase chain reaction (qRT-PCR) in three gastric cancer cells lines and five nontumor tissues obtained from antrum and cardia by endoscopic biopsy and compare them. The results were normalized with endogenous RNU24. Data are presented as mean SD from at least three independent experiments. Statistical analysis was

performed with Students t-test using GraphPad Prism software 5.0. Differences were considered statistically significant at p < 0.05.

References
1. 2. 3. Murray CJ, Lopez AD: Alternative projections of mortality and disability by cause 1990-2020: Global Burden of Disease Study. Lancet 1997, 349(9064):1498-1504. Zhang M, Zhu G, Zhang H, Gao H, Xue Y: Clinicopathologic features of gastric carcinoma with signet ring cell histology. J Gastrointest Surg 2010, 14(4):601-606. Lu J, Getz G, Miska EA, Alvarez-Saavedra E, Lamb J, Peck D, Sweet-Cordero A, Ebert BL, Mak RH, Ferrando AA et al: MicroRNA expression profiles classify human cancers. Nature 2005, 435(7043):834-838. Brenner B, Hoshen MB, Purim O, David MB, Ashkenazi K, Marshak G, Kundel Y, Brenner R, Morgenstern S, Halpern M et al: MicroRNAs as a potential prognostic factor in gastric cancer. World J Gastroenterol 2011, 17(35):3976-3985. Lu L, Li Y, Li S: Computational identification of potential microRNA network biomarkers for the progression stages of gastric cancer. Int J Data Min Bioinform 2011, 5(5):519-531. Lu XF, Wang LJ, Zhou WY, Si JM: [Small interfering RNA inhibits cell proliferation in gastric cancer cell lines highly expressing RegIalpha]. Zhejiang Da Xue Xue Bao Yi Xue Ban 2011, 40(1):57-63. Yasui W: Future perspectives of gastric cancer treatment: from bench to bedside. Pathobiology : journal of immunopathology, molecular and cellular biology 2011, 78(6):293-294. Valastyan S, Weinberg RA: Assaying microRNA loss-of-function phenotypes in mammalian cells: emerging tools and their potential therapeutic utility. RNA Biol 2009, 6(5):541-545. Ventura A, Jacks T: MicroRNAs and cancer: short RNAs go a long way. Cell 2009, 136(4):586-591. Ribeiro-dos-Santos A, Khayat AS, Silva A, Alencar DO, Lobato J, Luz L, Pinheiro DG, Varuzza L, Assumpcao M, Assumpcao P et al: Ultra-deep sequencing reveals the microRNA expression pattern of the human stomach. PLoS One 2010, 5(10):e13205. Zhang Z, Li M, Zhang G, Fang P, Yao H, Xiao Z, Chen Z: Identification of human gastric carcinoma biomarkers by differential protein expression analysis using 18O labeling and nanoLC-MS/MS coupled with laser capture microdissection. Med Oncol 2010, 27(2):296-303. Augoff K, Das M, Bialkowska K, McCue B, Plow EF, Sossey-Alaoui K: miR-31 is a broad regulator of beta1-integrin expression and function in cancer cells. Mol Cancer Res 2011, 9(11):1500-1508. Creighton CJ, Fountain MD, Yu Z, Nagaraja AK, Zhu H, Khan M, Olokpa E, Zariff A, Gunaratne PH, Matzuk MM et al: Molecular profiling uncovers a p53-associated role for microRNA-31 in inhibiting the proliferation of serous ovarian carcinomas and other cancers. Cancer Res 2010, 70(5):1906-1915. Lee HS, Park MH, Yang SJ, Jung HY, Byun SS, Lee DS, Yoo HS, Yeom YI, Seo SB: Gene expression analysis in human gastric cancer cell line treated with trichostatin A and S-adenosyl-L-homocysteine using cDNA microarray. Biological & pharmaceutical bulletin 2004, 27(10):1497-1503. Leal MF, Martins do Nascimento JL, da Silva CE, Vita Lamarao MF, Calcagno DQ, Khayat AS, Assumpcao PP, Cabral IR, de Arruda Cardoso Smith M, Burbano RR: Establishment and conventional cytogenetic characterization of three gastric cancer cell lines. Cancer Genet Cytogenet 2009, 195(1):85-91. Han X, Papadopoulos AJ, Jones TA, Sheer D, Raju KS: SR8--the establishment and characterisation of a new ovarian carcinoma cell line and xenograft model. Eur J Cancer 1996, 32A(1):160-167.

4.

5.

6.

7.

8.

9. 10.

11.

12.

13.

14.

15.

16.

17.

18.

19. 20.

21.

22.

23.

24. 25.

26. 27.

28.

29. 30.

Lima EM, Rissino JD, Harada ML, Assumpcao PP, Demachki S, Guimaraes AC, Casartelli C, Smith MA, Burbano RR: Conventional cytogenetic characterization of a new cell line, ACP01, established from a primary human gastric tumor. Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas / Sociedade Brasileira de Biofisica [et al] 2004, 37(12):1831-1838. Chun YH, Kil JI, Suh YS, Kim SH, Kim H, Park SH: Characterization of chromosomal aberrations in human gastric carcinoma cell lines using chromosome painting. Cancer Genet Cytogenet 2000, 119(1):18-25. Griffiths-Jones S, Saini HK, van Dongen S, Enright AJ: miRBase: tools for microRNA genomics. Nucleic Acids Res 2008, 36(Database issue):D154-158. Creighton CJ, Benham AL, Zhu H, Khan MF, Reid JG, Nagaraja AK, Fountain MD, Dziadek O, Han D, Ma L et al: Discovery of novel microRNAs in female reproductive tract using next generation sequencing. PLoS One 2010, 5(3):e9637. Guo W, Dong Z, Guo Y, Chen Z, Yang Z, Kuang G, Shan B: Polymorphisms of transforming growth factor-beta1 associated with increased risk of gastric cardia adenocarcinoma in north China. Int J Immunogenet 2011, 38(3):215-224. Li WB, Zuo XL, Zuo F, Gu XM, Yu T, Zhao YA, Zhang TG, Zhang JP, Li YQ: Characterization and identification of gastric hyperplastic polyps and adenomas by confocal laser endomicroscopy. Surg Endosc 2010, 24(3):517-524. Zhang Y, Guo J, Li D, Xiao B, Miao Y, Jiang Z, Zhuo H: Down-regulation of miR-31 expression in gastric cancer tissues and its clinical significance. Med Oncol 2010, 27(3):685-689. Brabletz S, Brabletz T: The ZEB/miR-200 feedback loop--a motor of cellular plasticity in development and cancer? EMBO Rep 2010, 11(9):670-677. 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 2008, 283(22):14910-14914. Korpal M, Kang Y: The emerging role of miR-200 family of microRNAs in epithelialmesenchymal transition and cancer metastasis. RNA Biol 2008, 5(3):115-119. Gregory PA, Bert AG, Paterson EL, Barry SC, Tsykin A, Farshid G, Vadas MA, KhewGoodall Y, Goodall GJ: The miR-200 family and miR-205 regulate epithelial to mesenchymal transition by targeting ZEB1 and SIP1. Nat Cell Biol 2008, 10(5):593-601. Park SM, Gaur AB, Lengyel E, Peter ME: The miR-200 family determines the epithelial phenotype of cancer cells by targeting the E-cadherin repressors ZEB1 and ZEB2. Genes Dev 2008, 22(7):894-907. Belair C, Darfeuille F, Staedel C: Helicobacter pylori and gastric cancer: possible role of microRNAs in this intimate relationship. Clin Microbiol Infect 2009, 15(9):806-812. Shinozaki S, Yamamoto H, Yano T, Sunada K, Miyata T, Hayashi Y, Arashiro M, Sugano K: Long-term outcome of patients with obscure gastrointestinal bleeding investigated by double-balloon endoscopy. Clin Gastroenterol Hepatol 2010, 8(2):151-158.

Figure 1 Relative expression of miR-31 in tumor cell lines ACP02, ACP03 and AGP01 (in red) and non-tumor tissue in the cardia and antrum (in green). Data were normalized and submitted RNU24 2-Ct Figure 2. Relative expression of miR-200a in tumor cell lines ACP02, ACP03 and AGP01 (in red) and non-tumor tissue in the cardia and antrum (in green). Data were normalized and submitted RNU24 2-Ct.

Figure 3. Relative expression of miR-200b in tumor cell lines ACP02, ACP03 and AGP01 (in red) and non-tumor tissue in the cardia and antrum (in green). Data were normalized and submitted RNU24 2-Ct Table 1. Relative expression of miR-31 in tumor cell lines ACP02, ACP03 and AGP01 and non-tumor tissue in the cardia and antrum, represented by 2-Ct, calculating their respective fold change. Table 2. Relative expression of miR-200a in tumor cell lines ACP02, ACP03 and AGP01 and non-tumor tissue in the cardia and antrum Table 3. Relative expression of miR-200b in tumor cell lines ACP02, ACP03 and AGP01 and non-tumor tissue in the cardia and antrum region.

Table 1
Mdia de expresso na crdia 2-CT Tumoral 2-CT No tumoral 43,07 43,07 43,07 Fold change Mdia de expresso no antro 2-CT Tumoral 2-CT No tumoral 21,47 21,47 21,47 Fold change

ACP02 ACP03 AGP01

1,2E-03 3,2E-04 5,1E-04

2,8E-05 7,6E-06 1,1E-05

1,2E-03 3,2E-04 5,1E-04

4,4E-05 1,1E-05 1,8E-05

Table 2
Mdia de expresso na crdia 2-CT Tumoral 2-CT No tumoral 220,75 220,75 220,75 Fold change Mdia de expresso no antro 2-CT Tumoral 2-CT No tumoral 161,88 161,88 161,88 Fold change

ACP02 ACP03 AGP01

1 1 1

4,4E-03 4,4E-03 4,4E-03

1 1 1

7,9E-02 7,9E-02 7,9E-02

Table 3
Mdia de expresso na crdia 2-CT Tumoral 2-CT No tumoral 95,57 95,57 95,57 Fold change Mdia de expresso no antro 2-CT Tumoral 2-CT No tumoral 7, 94 7, 94 7, 94 Fold change

ACP02 ACP03 AGP01

5,0-03 5, 93 19, 55

5,2E-05 6,2E-02 2,0E-01

5,0-03 5, 93 19, 55

6,2E-04 7,4E-01 2, 46

Figure 1

Figure 2

Figure 3

You might also like