The expression levels of miR-655-3p, miR127-5p, miR-369-3p, miR-544a in gastric cancer

Introduction

Gastric cancer is the fourth frequently seen among malignant tumors in the world, while the fifth in Turkey and one of the most common causes of cancer-related deaths worldwide [1]. Gastric cancer has a low survival rate because of the lack of early diagnosis techniques is usually noticed late [2]. Gastric cancer is a heterogeneous disease; genetic factors, Helicobacter pylori infection, heavy alcohol consumption, smoking, high salt consumption and various environmental factors contribute to carcinogenesis [3]. The anomalies of various oncogene, tumor suppressor genes, transcription factors and growth factors might have crucial roles in tumor pathogenesis [4], [5], [6]. miRNAs play crucial role in the regulation of numerous metabolic and cellular pathways, particularly in the control of apoptosis and cell proliferation [7]. MicroRNAs (miRNAs) regulate gene expression targeting mRNA by causing translation inhibition or mRNA degradation and consist of 19–25 nucleotides [8], [9]. It is predicted that more than 60% of all protein-coding genes is controlling by miRNAs [10]. Several miRNAs such as miR-655-3p, miR-127-5p, miR-369-3p, miR-544a encoded in the 14q32 locus was found associated with metastatic phenotype in cancer [11], [12]. We aimed to determine the expressions of these miRNAs in our study and to evaluate their potential for biomarker and treatment of gastric cancer.

Materials and methods

MicroRNA isolation

The 200 μL of the serum samples was spiked with 2 μL of 25 fmol synthetic ce-miR-39 (miRNA mimic 10 pmol) as the external reference. Total RNA enriched for small RNAs was isolated simultaneously from the serum with the miRNeasy serum/plasma Isolation Kit (Qiagen, Germany, Product Number: 217184). The purities and concentrations were measured at 260 and 280 nm with NanoDrop spectrophotometer (NanoDrop 2000, Wilmington, DE, USA).

Figure 1:

Relative expression normalized to U6 for miR-127-5p, miR-544a, miR-369-3p and miR-655-3p.

*p<0.05; ns, Non-statistical.

Reverse transcription and quantitative real-time polimerase chain reaction

The extracted microRNA was reverse transcribed to cDNA in 20 μL using miscript II RT cDNA Synthesis Kit (Qiagen, Germany, Product Number: 218161) and total volume was placed in thermal cycler with 60 min at 37°C followed by 5 min at 95°C. Reverse transcription components and volumes were given in Table 1 and run in duplicate. The polimerase chain (PCR) reaction was performed for amplification using miscript SYBR Green PCR kit (Qiagen, Germany, Product Number: 218073) and miScript Primer Assay kit (for each microRNA: Qiagen, Germany, Product Number: 218300) on ABI PRISM™ 7000 Sequence Detection System. U6 was used as housekeeping gene. Each quantitative PCR reaction solution contained diluted cDNA, 2×Quanti Tect (with SYBR), the microRNA-specific forward primer and a universal reverse primer to a total volume of 25 μL and the components were given in Table 2. The qPCR reaction parameters were predenaturation at 94°C for 15 min, 40 cycles of 94°C for 15 s, 55°C for 30 s and extension at 72°C for 30 s.

Results

Gastric cancer group consisted of 26 women and 40 men, while the number of women was 35 and the number of men was 31 in the control group. Five people reported that they consumed alcohol and 23 people said they were smoking in the gastric cancer group. The mean age was 57.53±19.87 in the gastric cancer group and 57.42±16.98 in the females and 57.6±21.76 in the males. The mean age was 39.2±9.74 in the control group, 36.58±8 in the females and 42.25±10.77 in the males. The age difference in the patient and control groups was statistically significant (p<0.001).

The expression levels of four microRNAs (miR-127-5p, miR-544a, miR-369-3p and miR-655-3p) of gastric cancer group compared to the control group with 2^−ΔΔCT method and relative fold change was determined. miR-655-3p was found 100-fold less expressed in the gastric cancer group compared to the control group (fold change: 0.01, p=0.026). The expression levels of miR-127-5p were found about 48-fold less expressed in the gastric cancer group than the control group (fold change: 0.021). This fold change in the groups was found statistically significantly (p<0.001). miR-369-3p was found approximately 1.6-fold less expressed in the gastric cancer group to the control group (fold change: 0.63, p=0.054). It was found that miR-544a was expressed about 15.5-fold more in gastric cancer group to the control group (fold change: 15.47, p<0.001) (Table 3) (Figure 1).

Metastasis was detected in 23 patients in gastric cancer group. When the correlation between the gene expression levels of microRNAs and metastasis is examined; correlation was determined with miR-127-5p and miR-369-5p (p<0.001). It was observed that miR-127-5p and miR-369-5p expression levels were lower in the presence of metastases (data not shown).

Discussion

A case control study was conducted in gastric cancer and we found miR-655-3p (fold change: 100, p=0.026), miR-127-5p (fold change: 48, p<0.001) and miR-369-3p (fold change: 1.6, p>0.05) downregulated in the gastric cancer. However, miR-544a was found 15.5-fold more expressed in the patient group than control group (fold change: 15.47, p<0.001).

The overexpression of miR-655 targeted pituitary tumor-transforming gene-1 (PTTG1) acting as a tumor suppressor [13]. Han et al. found that expression levels of miR-655-3p significantly decrease in cervical cancer [14]. Downregulated miR-655-3p levels were found correlated with advanced tumor stage and lymph node metastasis in hepatocellular carcinoma [15]. miR-655-3p also directly targets ADAM10 and inhibits β-catenin pathway. Wu et al. showed decreased expression levels of miR-655-3p in in hepatocellular carcinoma tissues and cell lines. Tumor stage was negatively related to low miR-655-3p expression [16]. However, tumor growth was suppressed using miR-655-3p with conventional cytotoxic agents such as oxaliplatin via tumor-specific nanoscale coordination polymers on colorectal liver metastases [17]. In our study, we found miR-655-3p was found 100-fold less expressed in the patient group (fold change: 0.01, p=0.026). Our result shows similarity with other cancer types and miR-655-3p may be downregulated via methylation. However, we did not evaluate methylation status.

The miR-127 is known to target the proto-oncogene B-cell CLL/lymphoma 6 (BCL6) [18]. It was shown that downregulation of miR-127 in rat liver cells and overexpression of miR-127 directly inhibits BCL6 expression and suppresses cell growth [19]. miR-127-5p was found downregulated in more than half of hepatocellular carcinoma samples. Moreover, researchers showed that miR-127-5p suppresses nuclear factor-κB activity by directly targeting biliverdin reductase B [20]. Downregulation of miR-127-5p was also found associated with colon cancer [21]. The expression levels of miR-127-5p were found about 48-fold less expressed in the gastric cancer in our study (fold change: 0.021, p<0.001). Pronina et al. found an association between hypermethylation of miR-127, breast cancer progression and metastasis [22]. We also found miR-127-5p expression levels were lower in the presence of metastases (p<0.001).

Low expressions of miR-369-3p were found associated with shorter overall survival in thyroid cancer patients and overexpression of miR-369-3p promoted apoptosis and suppressed proliferation in papillary thyroid cancer [23]. Thayanithy et al. reported that miR-369-3p in 14q32 locus may contributes to osteosarcoma pathogenesis [12]. The reprogramming of miR-369-3p or -5p inhibited cell proliferation and invasion in colon cancer cells [24]. Another study showed that glycolysis and tumor growth is suppressed by miR-369-3p through inhibition of lactate dehydrogenase A. The expression levels of miR-369-3p were found lower in primary colorectal cancer specimens [25]. We observed miR-369-3p approximately 1.6-fold less expressed in the gastric cancer group to the control group (fold change: 0.63, p=0.54). Moreover, the correlation was found between metastasis and miR-369-5p expression (p<0.001) and miR-369-5p expression levels were lower in the presence of metastases.

Liu et al. reported that upregulation of miR-544a in lung cancer tissues and cells and proliferation and invasion is facilitated by miR-544a [26]. Upregulated miR-544a expression was found in metastatic tumor samples and colorectal cancer cell lines. Furthermore, researchers suggested that metastatic and invasive properties of colorectal cancer are regulated by miR-544a via modulating Homeobox A10 [27]. The expression levels of cadherin 1 were found negatively correlate with miR-544a and miR-544a expression was higher in breast cancer MDA-MB-231 cells compared to the normal breast Hs578Bst cells [28]. It was demonstrated that the expression of e-cadherin is reduced via overexpression of miR-544a and resulting epithelial-mesenchymal transition (EMT) in gastric cancer [29]. It was found that miR-544a was expressed about 15.5-fold more in gastric cancer group (fold change: 15.47, p<0.001).

Conclusion

Our study demonstrated that miR-655-3p, miR-127-5p and miR-544a might be assessed as biomarker in gastric cell. However, these microRNAs expressions should be evaluated at methylation levels in both of cell lines and human tissues. Furthermore, miR-655-3p and miR-127-5p might be introduced to the gastric cancer cell lines to show their therapeutic effects because of the downregulated in gastric cancer. On the contrary, miR-544a can be silenced in gastric cancer cell lines to observe the effects on carcinogenic profile.