Hsa-miR-181a-5p Expression and Effects on Cell Proliferation in Gastric Cancer

Gastric cancer (GC) is one of the most common cancers and ranks as the second leading cause of cancer death worldwide, probably accounting for about 10% of newly diagnosed cancers (Murray et al., 1997). MicroRNAs (miRNAs) are small endogenous, noncoding, single-stranded RNAs. Mature miRNAs target the 3’untranslated region (3’UTR) of a specific mRNA by base pairing, leading to translational repression or mRNA degradation (Bartel, 2004; He et al., 2004). Bioinformatics prediction indicates that 30% of all the genes are regulated by miRNAs (Yu et al., 2007). miRNAs have been shown to play crucial roles in diverse biological processes, such as development, differentiation, apoptosis and proliferation (Chen et al., 2004; Harfe et al., 2005; Hwang et al, 2006). Accumulating evidence has strongly suggested that altered miRNA expression is a common and important feature of human malignancies (A. Esquela-Kerscher et al., 2006; S. Sassen et al., 2008) Emerging researches found that aberrant miRNAs could be important in tumorigenesis as oncogenes and tumor suppressor genes in GC (Du et al., 2009; DingL et al., 2010; Guo et al., 2010; Tie et al., 2010; Wang et al., 2010; Guo et al., 2011). In this study, we choose miR-181a-5p as a candidate miRNA according to the miRNA microarray and then validate its expression in GC tissues and cell lines. Further research to assess


Introduction
Gastric cancer (GC) is one of the most common cancers and ranks as the second leading cause of cancer death worldwide, probably accounting for about 10% of newly diagnosed cancers (Murray et al., 1997).
MicroRNAs (miRNAs) are small endogenous, noncoding, single-stranded RNAs. Mature miRNAs target the 3'untranslated region (3'UTR) of a specific mRNA by base pairing, leading to translational repression or mRNA degradation He et al., 2004). Bioinformatics prediction indicates that 30% of all the genes are regulated by miRNAs (Yu et al., 2007). miRNAs have been shown to play crucial roles in diverse biological processes, such as development, differentiation, apoptosis and proliferation (Chen et al., 2004;Harfe et al., 2005;Hwang et al, 2006). Accumulating evidence has strongly suggested that altered miRNA expression is a common and important feature of human malignancies (A. Esquela-Kerscher et al., 2006;S. Sassen et al., 2008) Emerging researches found that aberrant miRNAs could be important in tumorigenesis as oncogenes and tumor suppressor genes in GC (Du et al., 2009;DingL et al., 2010;Guo et al., 2010;Tie et al., 2010;Wang et al., 2010;Guo et al., 2011). In this study, we choose miR-181a-5p as a candidate miRNA according to the miRNA microarray and then validate its expression in GC tissues and cell lines. Further research to assess

Hsa-miR-181a-5p Expression and Effects on Cell Proliferation in Gastric Cancer
Gang Chen 1& , Zhi-Li Shen 1& , Ling Wang 2& , Chun-Ye Lv 1 , Xin-En Huang 3 *, Rong-Ping Zhou 1 * the effects of miR-181a-5p on GC cell growth. Finally, bioinformatics was used to predict the target genes of miR-181a-5p accounting for its function in GC.

Cell culture and tissue samples preparation
Human gastric adenoma cell lines (GES-1, SGC-7901, MGC-803, BGC-823 and AGS) were purchased from the Cell Bank of Shang Hai. Cells were routinely cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere with 5% CO 2 .
Forty pairs of histopathologically confirmed GC and adjacent non-cancer tissue samples were obtained from patients in Nanjing Jiangning Hospital, China. Informed consent was taken from all patients and the procedure was approved by the Medical Ethics Committee of Nanjing Jiangning Hospital.

miRNA microarray analysis.
Prior to experimentation, five pairs of GC and adjacent non-cancer tissue samples were analyzed by miRNA microarray. Total RNA was harvested using TRIzol (Invitrogen) and an RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. After RNA quantification using a Nanodrop spectrophotometer, the samples were labeled using the miRCURYHy3/Hy5 Power Labeling Kit and hybridized to the miRCURYLNAArray (v. 11.0). The samples were hybridized using a hybridization station and the arrays were scanned with the Axon GenePix 4000B Microarray Scanner. The raw intensity of the image was read using GenePix Pro V6.0. The intensity of the green signal was calculated after background subtraction, and four replicated spots for each probe on the same slide were averaged. The Median Normalization Method was used to obtain ʻNormalized Data' [Normalized Data = (foreground-background)/median]. The median was defined as the 50% quantile of microRNA intensity that was > 50 in all samples after background correction. The statistical significance of the differentially expressed miRNA was analyzed using the Student's t-test.

Real-time reverse transcriptase quantitative PCR
Total RNA was extracted from cells and tissues samples with Trizol reagent (Invitrogen). The quality and quantity of the RNA samples were assessed by standard electrophoeresis and spectrophotometric methods. Realtime reverse transcriptase quantitative PCR (qRT-PCR) analysis were performed with locked nucleic acids (LNAs) linear primers (EXIQON) and SYBR Green I, and U6 small nuclear RNA was used as normalized PCR primers. All reagents for stem-loop RT and Q-PCR were obtained from TAKARA. The primers used for stem-loop RT-PCR and Q-PCR were synthesized and purified by RiboBio. The PCR conditions were 95℃ for 30 s, followed by 40 cycles of 95℃ for 30 s, 60℃ for 30 s,72℃ for 30 s. The reactions were monitored using a preheated real-time instrument (ABI step one).
The relative expression ratio of miR-181a-5p in gastric cancer tissues and cells was quantified by the 2 −rrCT method.
One day before transfection, 5.0 x 103 BGC cells in 100ul growth medium were plated in each well of a 96-well plate. The cells were then transfected with 50 nM of various synthetic miRNAs mimics and 100 nM inhibitor using Lipofectamine2000 (Invitrogen) according to the manufacturer's instruction. Cell proliferation was assessed at different time points (0 h, 24 h, 48 h, and 72 h), using Cell Counting Kit 8 (Dojindo, Tokyo, Japan) according to manufacturer's protocol. The absorbance at a wave length of 450 nm, which shows positive relation to the capacity of cellular proliferation, was measured by a spectrophotometer.

Statistical analysis
Data were presented as mean ± SD, the significance was analyzed with the Student's t-Test, the statistical significance of correlation was calculated by chi-square test and Spearman's rank correlation. Statistical analysis was performed using SPSS 16.0 software and differences were considered statistically significant when p< 0.05.

miR-181a-5p expression pattern and expression level was analyzed in forty matched GC tissues by qPCR
Q-PCR was used to validate miR-181a-5p expression in 40 pairs GC tissues. Compared to the normal tissues, hsa-miR-181a-5p was upregulated in GC tissues, accounting for 40% ( Figure 1B).

Correlation between miR-181a-5p expression and clinical features
To investigate miR-181a-5p role which is involved GC tumorigenesis and progression, we analysed the correlation between miR-181a-5p expression and clinical features. The results reveals that -miR-181a-5p expression is correlation with Lymph node invasion, nerve invasion, vascular invasion (Table 2).

Discussion
Recently, accumulating evidence indicated that miRNA is aberrant expressed in various human malignancies and play a role as oncogenes or tumor suppressor genes. Previous researches using microarray analysis suggested that a number of differentiated expression miRNAs were associated with GC, and could be potential markers for diagnosing and monitoring GC (Guo et al., 2012;Poliseno et al., 2012). It is noted that miR-181a is downregulated expression in breast cancer, oral, hepatocellular, ovarian and involve many biological process , e.g. cell proliferation, cell apoptosis, etc (Guo et al., 2012;Poliseno et al., 2012;Shin et al., 2012;Zhou et al., 2012). However, few studies focus on the expression and function of miR-181a in GC, and no consistent result regarding miR-181a expression and its function in GC.
In this study, depending on miRNA microarray, we showed miRNAs were differentially expressed between normal gastric and GC tissue, and miR-181a-5p was significantly upregulated. Therefore, we choose miR-181a-5p as a candidate miRNA. We validated hsa-miR-181a-5p expression level in forty matched GC tissues and GC cell lines. Our results showed that expression of miR-181a-5p was upregulated in GC tissues (40%), but the difference was not statistically significant. While, the expression of miR-181a-5p in SGC-7901,MGC-803, BGC-823 cell lines is upregulated compared to immortalize GES-1, and to some extent, was consistent with the results of microarray analysis. Meanwhile, we analyzed the correlation between hsa-miR-181a-5p expression and clinical features. These results demonstrate that hsa-miR-181a-5p expression is correlated with lymph node, nerve, and vascular invasion (p<0.05), suggesting that hsa-miR-181a-5p could be associated with GC tumorigenesis and progression. We found that hsa-miR-181a-5p overexpression promotes cell proliferation, by assessing the effects of miR-181a-5p on GC cell growth. Yang reported that miR-181a-5p was overexpressed and enhanced lymph-node metastasis through regulating migration in OSCC (Yang et al., 2011); and Bhattacharya proved that the expression of miR-181a-5p was upregulated in HCC (Bhattacharya et al., 2010).
In summary, our findings suggest that hsa-miR-181a-5p is correlated with GC tumorigenesis and progression. According to the relationship between the expression of miR-181a and the invasion of gastric cells, we suggest that hsa-miR-181a-5p could be a potential biomarker of GC and play a role in determining the prognosis of GC patients.