Effect of dexmedetomidine on miR-144-3p expression and epithelial mesenchymal transition in gastric cancer cells

Purpose: To investigate the effect of dexmedetomidine (DEX) on epithelial mesenchymal transition (EMT) in gastric cancer cells, and the role of microRNA-144-3p (miR-144-3p) in the process. Methods: The effect of DEX on miRNA expression profile was analyzed using GEO database (https://www.ncbi.nlm.nih.gov/gds/). Human gastric cancer cells were cultured in vitro, and one group of cells was treated with saline for 48 h (control group). Cells treated with DEX at doses of 0.01, 0.1 and 1.0 μmol/L for 48 h were marked as low-, mediumand high-DEX concentration groups. The mRNA expression levels of miR-144-3p, ZEB1, E-cadherin and vimentin were determined using real-time quantitative polymerase chain reaction (RT-PCR), while the protein expressions of ZEB1, E-cadherin and vimentin were assayed with Western blotting. Cell proliferation was determined with CCK-8 assay, while metastasis was measured using Transwell assay. Results: The GEO database demonstrated that the expression of miR-144-3p in rat cardiomyocytes was significantly decreased after DEX treatment (p < 0.05). The expression of miR-144-3p was decreased in all groups, when compared to the control group, but the expressions of ZEB1 and vimentin were increased, while that of E-cadherin was down-regulated (p < 0.05). Cell proliferation in the highDEX concentration group was decreased (p < 0.05). The degrees of cell invasion and migration were increased in the mediumand high-DEX concentration groups (p < 0.05). Conclusion: DEX promotes the metastasis of gastric cancer cells by regulation of epithelial mesenchymal transition (EMT) and the expression of miR-144-3p. This finding provides a new insight into the treatment of gastric cancer.


INTRODUCTION
Gastric cancer is second only to lung cancer as a leading cause of death in the world [1]. The onset of most gastric cancer cases is insidious, but they progress very rapidly. An understanding of the key factors that affect the malignant behavior of gastric cancer cells is very important guide in the clinical diagnosis and treatment of the disease. Dexmedetomidine (DEX), a frequently-used intravenous anesthetic, affects different biological processes in the body by regulating a variety of signal pathways [2,3]. The importance of DEX in malignant tumors has been progressively revealed. Many studies have indicated that DEX is involved in regulating the biological behavior of tumor cells [4,5]. However, the effect of DEX on gastric cancer cells, and its exact mechanism remain to be elucidated.
Micro-RNAs (miRNAs) are important posttranscriptional regulatory factors which participate in various biological processes, including the pathogenesis of malignant tumors [6,7]. In this study, bioinformatics methods and in vitro experiments were used to investigate the regulatory effect of DEX on gastric cancer cellrelated miRNA and malignant behavior, in order to provide a new insight into the use of DEX in malignant tumors. cell incubator at relative humidity > 95 %. Cells in logarithmic growth phase were divided into control group treated with normal saline, and three groups treated with DEX at doses of 0.01, 0.1 and 1 μmol/L. All treatments lasted for 48 h.

Quantitative real-time polymerase chain reaction (qRT-PCR)
Total RNA was extracted from the cells in each group with TRIzol reagent. Then, the total RNA was reverse-transcribed to cDNA using reverse transcription kit (Takara, Japan) according the manufacturer's instructions. The primers for miR-144-3p, ZEB1, E-cadherin, Vimentin and β-actin which were used for qPCR (Table 1) were synthesized by Sangon Biotech (Sangon, Shanghai, China). The qRT-PCR reaction system was constituted in line with the operating instructions in a 7500 real-time fluorescence quantitative PCR instrument (Applied Biosystems, American, CA). The reaction steps were as follows: 50 ℃ for 2 min, 95 ℃ for 10 min, 95 ℃ for 15 min x 40 cycles; 95 ℃ for 15 sec, 60 ℃ for 1 min, 95 ℃ for 30 min, and 60 ℃ for 15 sec. The mRNA expression levels of the genes were calculated using the 2 -ΔΔCt method.

Western blot assay
Total protein was extracted with IP lysing solution containing protease inhibitor, and the protein concentration of the lysate was determined using BCA kit. Equal amounts of protein (50-μg) were resolved on 10 % polyacrylamide gel electrophoresis, followed by transfer to PVDF membrane. The membrane was blocked with 10 % skimmed milk at room temperature for 2 h, prior to incubation with primary antibodies for ZEB1, E-cadherin and Vimentin (each at 1:1000 dilution) at 4 ℃ overnight. Thereafter, the membrane was incubated with secondary antibody at room temperature for 1 h. The blots were subjected to enhanced chemiluminescence (ECL) analysis for 1 h. Image analysis was done with ChemiDoc XRS Chemiluminescence Imager (Bio-Rad, USA).

Determination of cell proliferation
Cells in each group were inoculated in 96-well plate at 0, 12, 24, 48 and 72 h, with 5 replicates at each time point. After adherence, 10 μL CCK-8 solution was added to each well. The wells were cultured in the dark for 2 h, after which the optical density (OD) of each well was read at 450nm in a microplate reader.

Transwell assay
Matrigel glue (50 mg/L) was diluted in serum-free RPMI-1640 medium at a volume ratio of 8:

Statistical analysis
The SPSS 23.0 software was used for statistical analysis. Statistical differences in measurement data amongst the groups were analyzed using analysis of variance, while Student's t-test was used for pairwise comparison. Statistical significance was assumed at p < 0.05.

Effect of DEX on the proliferation of HGC-27 cells
The results of RT-qPCR and Western blot assays showed that, compared with the control group, the expressions of miR-144-3p in low, medium and high DEX concentration groups were decreased significantly, while the mRNA and protein expressions of ZEB1, vimentin were markedly up-regulated (p < 0.05. Moreover, the expression of E-cadherin was significantly decreased (p < 0.05). These data are shown in Figure 2.

Effect of DEX on the invasion and migration of HGC-27 cells
As shown in Figure 4, the invasion and migration potential of cells in the middle-and high-DEX concentration groups were significantly higher than those in the control group (p < 0.05).

DISCUSSION
Dexmedetomidine (DEX) is a frequently-used intravenous anesthetic in clinics. In recent years, it has been discovered that DEX regulates the malignant behaviors of tumor cells. A study revealed that DEX significantly upregulated the phosphorylation levels of the transmembrane protease serine 2 (TMPRSS2) and STAT3 Tyr705 in breast cancer cells i.e., MCF-7 and MDA-MB-231, and promoted their migration [4]. Moreover, it has been reported that DEX enhanced the invasion and metastasis of A549 lung cancer cells and HCT116 colorectal cancer cells, through a mechanism related to upregulation of matrix metalloproteinases MMP-2 and MMP-9, and hypoxia-induced factor-1α (HIF-1α) [8]. Dexmedetomidine (DEX) up-regulated the expression of miR-155 in ovarian cancer cells, thereby inhibiting the tumor-promoting effect mediated by HIF-1α [9]. It has also been demonstrated that DEX promoted the expression of miR-143-3p and inhibited the malignant behavior of esophageal cancer cells [10]. These results indicate that DEX exerts different effects on different malignant tumor cells. However, its effect on gastric cancer cells, and the related mechanisms are still unclear. In the present study, it was found that DEX significantly downregulated the expression level of miR-144-3p in rat myocardium in the GEO database chip (GSE126106). Thus, it can be reasonably speculated that miR-144-3p may serve as a potential target of DEX.
Low expression of miR-144-3p is often closely associated wth malignant tumors [11][12][13]. It has been reported that in gastric cancer, the expression level of miR-144-3p was significantly lower than those of adjacent tissues, and it was negatively correlated with lymph node metastasis and TNM stage, suggesting that miR-144-3p may be a protective factor for gastric cancer [14]. In vitro studies have shown that miR-144-3p promoted the radiosensitivity of gastric cancer cells by inhibiting the expression of zinc finger transcription factor ZEB1 [15]. Moreover, it has been reported that miR-144-3p regulated the target molecules PBX3 and CBX4, and inhibited epithelial-mesenchymal transformation, proliferation and metastasis of gastric cancer cells [16,17].
In the present study, human gastric cancer cells HCG-27 were used as an in vitro model. It was found that the expression of miR-144-3p was significantly and dose-dependently reduced after DEX treatment. This finding was consistent with the sequencing data of the GSE126106 chip. At the same time, the expression of EMT-related molecules downstream of miR-144-3p were significantly changed, and the EMT process was enhanced. Moreover, the levels of invasion and migration were significantly increased. These results indicate that DEX inhibited the expression of miR-144-3p and promoted the EMT process of gastric cancer cells. Interestingly, cell proliferation was inhibited on treatment with the high concentration of DEX, due to the cytotoxicity produced the drug through other mechanisms.

CONCLUSION
DEX promotes the metastasis of gastric cancer cells by regulation of miR-144-3p and EMT. This finding provides a new insight into the treatment of gastric cancer.