Quantitative Detection of miRNA-21 Expression in Tumor Cells and Tissues Based on Molecular Beacon

As a new tumor marker, the microRNA-21 (miRNA21) level can provide important information for early diagnosis, drug treatment, and prognosis of gastric cancer. With the tool of molecular beacons which can hybridize specifically with target miRNA-21 and generate fluorescence signal change, this paper develops a direct, simple, and rapid method for miRNA-21 detection with detection limit of 0.5 nM. Under the optimal conditions, the method was used to detect the expression of miRNA-21 in tumor cells and tissues. The results showed significant differences of miRNA-21 levels in tumor cells which have different origins and different degree of malignancy. In 8 cases of gastric cancer tissues and adjacent tissues, the level of miRNA-21 in 6 cases was higher than that in adjacent tissues, 1 case had lower expression level than that in adjacent tissues, and 1 case had no significant difference. Furthermore, qRT-PCR method was used to verify the detection results based on the fluorescent probe detection method. The consistent results show that the molecular beacon assay has a good prospect in direct and rapid detection of miRNA-21 expression and will be widely used in the functional research and clinical diagnosis of microRNA.


Introduction
Since Lee et al. [1] first reported on microRNA (miRNA) in 1993, research on miRNA has quickly become a hot research topic in various fields. MiRNAs are a class of small RNA with about 18-25 nucleotides of endogenous noncoding whose expression level changes directly affect cell proliferation, differentiation, development, and apoptosis in many biological processes and regulate more than a third of the gene expression of body [2]. Clinical study results indicate that, due to specific expression in tissues and cells, miRNA not only is directly involved in the development and progression of tumors, but also can be served as noninvasive markers in the early diagnosis and treatment of a tumor [3][4][5][6][7][8][9][10][11]. Located in the 17q23.2, the coding gene miRNA-21 belongs to the coding region of the vacuole membrane protein gene (VMP1), and is the tenth intron of the VMP1 gene. A large number of studies show that miRNA-21 in tumor tissues and cells is abnormally high [12,13] and the expression level directly reflects the degree of tumor malignance [14,15]. Therefore, the detection of miRNA-21 has significant theoretical and practical value in the early diagnosis, treatment, and prognosis evaluation of clinical tumor. At present, methods that can be used for miRNA detection are microarray [16], northern blotting [17], qRT-PCR [18], etc. However, in varying degrees, these methods have disadvantages such as high cost, being time-consuming, having large demand of sample size, and fussy process; in addition, miRNA is very short and its characteristics of high sequence similarity between members of the same family make it difficult to achieve fast, simple, and good reproducibility of miRNA detection. Since the molecular beacon probe has the advantages of strong specificity, short hybridization time, and easy operation [19][20][21][22], it is expected to be applied in the miRNA rapid detection and analysis.
2 International Journal of Analytical Chemistry

Materials and Instruments.
Instruments were Hitachi F-2500 fluorescence spectrophotometer (Japan), Thermo Newslab water bath (USA), Bio-Rad fluorescence quantitative PCR (USA), and Thermo trace UV spectrophotometer (USA). All sequences of RNA (miRNA-21 simply "miR21" for short; miRNA-21 with 3 mutation bases simply "miR21m3" for short) and probes (miRNA-21 molecular beacon simply "miR21-MB" for short) were synthesized by Takara and shown in Table 1. Other reagents such as HRP (Promega), TRIzol (Life Technology), miRNA qPCR kit, and DNaseI (Promega) were used. 8 specimens of gastric cancer (cancerous tissues and adjacent tissues) were collected from the Department of Pathology, Chenzhou First People's Hospital, Hunan Province, including 6 males and 2 females, aged between 44 and 74 years old, with an average age of 58 ± 10 years. All pathological specimens were confirmed by pathological diagnosis, including 1 case of high differentiation, 2 cases of middle differentiation, and 5 cases of low differentiation. The tissue samples were stored at −80 ∘ C after liquid nitrogen treatment.

Fluorescence Measurement.
A final concentration of 100 nM of MiR21-MB was added to total volume of 100 L standard buffer solution; then the fluorescence intensity was measured using the scanning time and wavelength scanning at the indicated temperature (37 ∘ C, ex = 521 nm, em = 578 nm). Both of excitation and emission slit widths were set at 10 nm. The background intensity of solution was monitored until it kept stable at the test temperature. Total RNA was then added and the subsequent change of fluorescence intensity was recorded. The emission spectra were measured by exciting samples at 521 nm and scanning the emission between 550 and 650 nm. Fluorescence emission peaks were measured at 578 nm.

Construction of Detection Standard
Curve. The solutions containing 100 nM MiR21-MB and different concentrations of miR21 were incubated in PCR amplifier for 3 h at 37 ∘ C. Fluorescence emission spectra were recorded as in Section 2.2. Only the fluorescence data at 578 nm (the maximum emission of TAMRA) were used for analysis. Each sample was incubated at 37 ∘ C for 10 min before detection to obtain a steady status of fluorescence and detected three times.

Results and Discussion
3.1. Effects of Temperature and Mg 2+ on the Stability of Molecular Beacon. Since temperature and Mg 2+ can affect the stability of molecular beacon [23][24][25], in order to select the best conditions to maintain the stability of molecular beacon, we investigated the effect of these two factors. As shown in Figure 1(a), at temperatures below 45 ∘ C, fluorescence intensity does not change with increasing temperature, which indicates that molecular beacon can maintain a stable stemloop structure. When temperature is higher than 45 ∘ C, the fluorescence intensity increases slowly with increasing temperature, which indicates that the stem-loop structure of molecular beacon is affected to a certain extent. Figure 1(b) shows that Mg 2+ plays an important role in maintaining the stability of the molecular beacon. When the concentration of Mg 2+ is about 10 mM, the fluorescence background signal is the lowest. In order to ensure the stability and repeatability of the experimental results, the reaction temperature was 45 ∘ C. And the Mg 2+ will affect the hybridization between miR21-MB and miR21, so next the effects will be investigated.

The Effect of pH and Mg 2+ on the Hybridization between miR21-MB and miR21.
We further investigated the influence of Mg 2+ and pH on the hybridization between miR21-MB and miR21. Figure 2(a) shows that Mg 2+ also affects the hybridization process of molecular beacon and miR21. The fluorescence intensity of hybridization process is concentration dependence, and it is pink when Mg 2+ concentration is 10 mM. The results indicate that more Mg 2+ may maintain a more stable hairpin structure to decrease the fluorescence signal. Therefore, it is concluded that 10 mM is optimal concentration of Mg 2+ . In addition, Figure 2(b) shows that, at a certain range (5.5-8.0), the fluorescence intensity increases with the increase of pH value, and then the fluorescence signal starts to decrease when the pH value continues to increase. As a result, it is concluded that pH value of 8 is the best condition of pH.

Specificity of Molecular
Beacon. The specificity of the hybridization between miR21-MB and target molecules was further investigated by using 3 kinds of miR21 chains including those in random sequence (miR21-r), those with 3 mismatched bases (miR21-m3), and those with complete matching bases (miR21). The fluorescence wavelength scanning results (Figure 3(a)) showed that the fluorescence intensity of miR21-m3 and miR21-r were similar to that of the background, indicating that miR21-MB could not be hybridized with random RNA sequences or mismatched RNA sequences, and the stem cannot be opened to produce fluorescence. But miR21-MB can be opened by the sequence of the complete match of the miR21 hybrid and then produced a significant fluorescence signal. This result indicates that the molecular beacon can distinguish the RNA molecule with different base composition, showing good specificity. We also analyzed the results of miR21-MB/miR21 hybridization using polypropylene gel electrophoresis to verify the results of fluorescence experiments. As shown in Figure 3 Fluorescence emission spectra of miR21-MB with miR21 are shown in Figure 4(a). A standard curve of miR21 was drawn by displaying relative fluorescence intensities (F-F0) at 578 nm and their correlation was analyzed with linear regression. The relative fluorescence intensities (F-F0) were linearly proportional to the concentration of miR21 in the range from 1 to 50 nM (Figure 4(b)). The regression equation is Y = 0.8057X + 0.2516. The regression coefficient is 0.9915. The limit of detection was lower to 0.5 nM (estimated from three times the standard deviation in the blank solution). The detection sensitivity was similar to the reported method [26,27], but more simple with less reagents and steps.

Detection of miRNA-21 in Liver Cancer Cell by Molecular
Beacon. To demonstrate that molecular beacons have the sensitivity and specificity to detect mature miRNA in a heterogeneous RNA sample, we mixed 100 nM MiR21-MB with 3 L total RNA that had been isolated from cancer cells (Huh7, HCC1102, and HCC20986). Then the mixtures were incubated at 37 ∘ C and fluorescence measurements of all samples were carried out on FL-2500 fluorometer. According to the fluorescence signal of 578 nm, the concentration of miRNA-21 was calculated by the standard curve, and the relative expression level was calculated. The expression of miRNA-21 in HCC1102 cells was higher, and the expression of HCC20986 was the lowest in Huh7 cells ( Figure 5(a)). qRT-PCR is the standard technique to assess miRNA expression. The results of the two methods are similar ( Figure 5(b)) indicating that the MB method will be used in bioassay.

Detection of miRNA-21 in Gastric Cancer Tissue by Molecular Beacon.
We mixed 100 nM miR21-MB with 3 g total RNA that had been isolated from gastric carcinoma. Then the mixtures were incubated at 37 ∘ C and fluorescence measurements of all samples were carried out on FL-2500 fluorometer. According to the fluorescence signal of 578 nm, the concentration of miRNA-21 was calculated by the standard curve ( Figure 6(a)). The expression level of miRNA-21 in 6    International Journal of Analytical Chemistry cases of cancer tissues was higher than that in adjacent tissues and in 1 case was lower than the adjacent tissue, and the expression level of the 1 case was similar to that in the adjacent tissues. qRT-PCR was compared with the molecular beacon, and the results of the two methods are similar (Figure 6(b)). The results showed that the microRNA expression level of tumor samples could be detected quickly and accurately by using MB method. And the results indicated that the miR21 was related to the cancer development; the expression level would be the marker to diagnosis.

Conclusions
In this paper, a fast, safe, and specific miRNA-21 quantitative method was established, with its linear range of detection in the range of 1 ∼ 50 nm and the detection limit of 0.5 nm. It is used to detect the changes of miRNA-21 expression in different tumor cells and gastric cancer tissues and get similar results with qRT-PCR. So, this method will provide a reliable experimental basis for the further elucidation of the role of miRNA-21 in tumorigenesis.

Data Availability
The data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.