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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

1 Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, Brazil

*Corresponding Author: [email protected]

E-mails:

Aline Maria Pereira Cruz : [email protected]

Amanda Paiva de Barros : [email protected]

Camile de Barros Lopes: [email protected]

Luciana de Sousa Paradela : [email protected]

Raquel Carvalho Montenegro : [email protected]

Sylvain Darnet : [email protected]

Rommel Rodriguez Burbano : [email protected]

Ândrea Kely Ribeiro-dos-Santos : [email protected]

Key words: miRNA, Gastric cancer, cell lines

mailto:[email protected]

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 α subunit partners (α2, α5, and αV) of β1 integrins and

also β3 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 cárdia

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-ΔCt

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 miR-

200b), 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-Δct

0.05) and larger in ACP03

(relative expression of 2-Δct

5.93) and AGP01 (relative expression by 2-Δct

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 transcription-

polymerase chain reaction (qRT-PCR) in three gastric cancer cells lines and five non-

tumor 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 Student’s t-test using GraphPad Prism software 5.0. Differences were

considered statistically significant at p < 0.05.

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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-ΔCt

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-ΔCt

.

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-ΔCt

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-ΔCt

, 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

Média de expressão na cárdia Média de expressão no antro

2-∆CT

Tumoral 2-∆CT

Não tumoral

Fold change

2-∆CT

Tumoral 2-∆CT

Não tumoral

Fold change

ACP02 1,2E-03 43,07 2,8E-05

1,2E-03 21,47 4,4E-05

ACP03 3,2E-04 43,07 7,6E-06

3,2E-04 21,47 1,1E-05

AGP01 5,1E-04 43,07 1,1E-05

5,1E-04 21,47 1,8E-05

Table 2


2-∆CT

Tumoral 2-∆CT

Não tumoral

Fold change

2-∆CT

Tumoral 2-∆CT

Não tumoral

Fold change

ACP02 1 220,75 4,4E-03 1 161,88 7,9E-02 ACP03 1 220,75 4,4E-03 1 161,88 7,9E-02 AGP01 1 220,75 4,4E-03 1 161,88 7,9E-02

Table 3


2-∆CT

Tumoral 2-∆CT

Não tumoral

Fold change

2-∆CT

Tumoral 2-∆CT

Não tumoral

Fold change

ACP02 5,0-03 95,57 5,2E-05 5,0-03 7, 94 6,2E-04 ACP03 5, 93 95,57 6,2E-02 5, 93 7, 94 7,4E-01 AGP01 19, 55 95,57 2,0E-01 19, 55 7, 94 2, 46

Figure 1

Figure 2

Figure 3

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