Effect and Mechanism of Dexmedetomidine on Inhibiting Proliferation, Migration and Invasion of Ovarian Cancer Cell Strain SKOV3

*Corresponding Author:

Qin Tang
Department of Gynaecology and Obstetrics, Zhejiang Xinda Hospital, Huzhou, Zhejiang 313000, China
E-mail: 75052798@qq.com

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Abstract

To investigate the effect and mechanism of dexmedetomidine on SKOV3 proliferation, migration and invasion in ovarian cancer cell strains and the effect on high mobility group protein A2 expression. Western blot was used to detect the protein expression of high mobility group protein A2 in SKOV3 ovarian cancer cells; control group (without any treatment), (dexmedetomidine group 100 ng/ml was treated for 24 h), si-control group (transfection with si-control), si-high mobility group protein A2 group (transfection with si-high mobility group protein A2), dexmedetomidine+plasmid cloning deoxyribonucleic acid group (after transfection with plasmid cloning deoxyribonucleic acid group followed by dexmedetomidine 100 ng/ml treated for 24 h), dexmedetomidine+plasmid cloning deoxyribonucleic acid group-high mobility group protein A2 group (after transfection with plasmid cloning deoxyribonucleic acid-high mobility group protein A2 followed by dexmedetomidine 100 ng/ml treated for 24 h), were all transfected into SKOV3 cells using Lipofectamine; the proliferation of cells was detected by methyl thiazolyl tetrazolium assay; cell migration and invasion were detected by transwell assay; high mobility group protein A2 expression was measured by quantitative reverse transcription-polymerase chain reaction. The proliferation, migration and invasion of SKOV3 cells treated with dexmedetomidine were significantly down-regulated and high mobility group protein A2 expression was significantly down-regulated compared with the control group (p<0.05); knockdown of high mobility group protein A2 significantly inhibited SKOV3 cell proliferation, migration and invasion and overexpression of high mobility group protein A2 reversed the inhibitory effects of dexmedetomidine on ovarian cancer cell proliferation, migration and invasion. Dexmedetomidine can inhibit the proliferation, migration and invasion of ovarian cancer cells SKOV3 and the mechanism may be related to the direct down-regulation of high mobility group protein A2, which will provide a theoretical basis for dexmedetomidine treatment of ovarian cancer.

Keywords

Dexmedetomidine, high mobility group protein A2, ovarian cancer, proliferation, migration

Ovarian cancer is a common gynecological malignancy, the incidence of which is second only to cervical cancer and the mortality rate ranks first among female neoplasms, seriously threatening women’s physical and mental health[1]. Ovarian cancer is highly susceptible to drug resistance during the later course of treatment and although new drugs are continuously influx to the market, there are still patients who are insensitive to the therapeutic agents[2]. Dexmedetomidine (DEX) is novel highly selective alpha (α) 2 adrenergic receptor agonists with sedative, analgesic, anxiolytic, and oxygen consumption lowering effects in the myocardium[3]. Recently, DEX was found to effectively inhibit the stress response in the perioperative period and relieve the suppression of postoperative cellular immune function in breast cancer patients[4], but the role and mechanism in ovarian cancer are still unclear. Members of the High Mobility Group Protein A (HMGA) family are expressed abundantly in the early embryo and include HMGA1, HMGA2[5]. The HMGA1 gene is mainly expressed in proliferating epithelial cells and areas of parenchymal organ tissue, whereas the HMGA2 gene is highly expressed in all mesenchymal cell condensations and mesenchymal derivatives[6]. HMGA2 may be completely silenced in adult tissues, but its expression is observed in many human malignancies, including ovarian cancer[7]. The aim of this study was to investigate the mechanisms underlying the effects of DEX on the proliferation, migration and invasion of ovarian cancer cells.

Materials and Methods

Materials:

The human ovarian cancer cell strain SKOV3 was purchased from American Type Culture Collection (ATCC); DEX (batch No.: 113775-47-6) was purchased from the Yangtze River Pharmaceutical Group; Dulbecco's Modified Eagle Medium (DMEM) and Methyl Thiazolyl Tetrazolium (MTT) were purchased from GIBCO, USA; Lipofectamine™ 2000 was purchased from Shanghai Sunshine Biotechnology Co., Ltd; Polyvinylidene Fluoride (PVDF) membranes were purchased from Roche Diagnostics GmbH, Germany; Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) kit and Electrochemiluminescence (ECL) luminescent solution were purchased from Beyotime Biotechnology; HMGA2 monoclonal antibody was purchased from Shanghai Xinyu Biotechnology Co., Ltd; horseradish peroxidase labeled secondary antibodies were purchased from Beijing Bersee Science and Technology Co., Ltd. All plasmids and primers were designed and synthesized by Shanghai Gene Pharm Co., Ltd.

Methods:

Cell culture: SKOV3 cells were cultured with DMEM (with 10 % fetal bovine serum) and routinely grown in an incubator at 37°, 5 % Carbon dioxide (CO₂).

Cell transfection: SKOV3 cells were treated with DEX at concentrations of 0 and 100 ng/ml for 24 h, which were labeled as Control (Con) group and DEX group respectively. si-con, si-HMGA2, plasmid cloning Deoxyribonucleic Acid (pcDNA) group and pcDNA-HMGA2 were transfected into SKOV3 cells following the Lipofectamine™ 2000 protocol, labeled as si-con group, si-HMGA2 group, pcDNA group and pcDNA-HMGA2 group; after transfection of pcDNA and pcDNA-HMGA2 into SKOV3 cells and treatment with 100 ng/ml DEX for 24 h, the cells were labeled as DEX+pcDNA group, DEX+pcDNA-HMGA2 group and the efficiency of low expression or overexpression was verified by quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR) and SKOV3 cells were subsequently collected for subsequent assays.

qRT-PCR experiments: Grouping SKOV3 cells were collected, Ribonucleic Acid (RNA) was extracted following the instructions of RNA extraction kit and complementary DNA (cDNA) preparation and amplification reactions were performed by reverse transcription kit, qRT-PCR kit protocol from Takara Co., Ltd. Dalian, China, respectively. HMGA2 expression was finally calculated as 2^-ΔΔCt. HMGA2 upstream primer 5′-CTCAAAAGAAAGCAGAAGCCACTG-3′, reverse primer 5′-TGAGCAGGCTTCTTCTGAACAACT-3′; the internal reference glyceraldehyde 3-phosphate dehydrogenase was used as the universal primer. All primers were synthesized by Shanghai GenePharma Co., Ltd.

Western blot experiments: Grouping SKOV3 cells in cell transfection were collected, total protein was extracted in the lysate, proteins were quantified by Bicinchoninic Acid (BCA) and bath denatured in boiling water. Next, protein electrophoresis and transmembrane were performed; after blocking with 5 % nonfat dry milk for 2 h at room temperature, the membranes were washed with the addition of primary antibody (HMGA2 monoclonal antibody, 1:1000), overnight incubation at 4°, added secondary antibody (horseradish peroxidase labeled immunoglobulin G Heavy and Light (H&L) chains, 1:2000) and incubation at 37° for 2 h, washed the membranes. Add luminescent solution and expose.

MTT experiments: SKOV3 cells were treated with 0 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/ml and 20 μl, 5 g/l MTT solution was added at 24 h, 48 h, 72 h and was reacted for 4 h and 150 μl Dimethyl Sulfoxide (DMSO) was added and the SKOV3 cell absorbance (Optical Density (OD)) after crystal dissolution was detected at a wavelength of 490 nm. Grouping SKOV3 cells in cell transfection also detected SKOV3 cell absorbance (OD).

Transwell chamber assay for cell migration and invasion: In the upper chamber of a transwell chamber coated with (to detect invasion) or without (to detect migration) Matrigel, 100 μl cell transfection grouped SKOV3 cells (10⁵/ml serum-free medium) were added and 600 μl of serum containing medium was added in the lower chamber and incubated for 24 h. SKOV3 cells on the lower surface of the chamber were fixed with methanol for 30 min, stained with 0.1 % crystal violet and after 20 min, three fields were randomly selected, photographed under a microscope and the invaded or migrated cells were counted and the average value was taken as the number of invaded or migrated cells.

Statistical treatment:

All data in the experiments were analyzed using Statistical Package for the Social Sciences (SPSS) 21.0 software. The measurement data were expressed as the mean±standard deviation (x̄±s) and data comparisons among multiple groups, pairwise comparisons were performed using one-way Analysis of Variance (ANOVA) and t-test and a value of p<0.05 was considered statistically significant.

Results and Discussion

Results are as shown in Table 1, SKOV3 ovarian cancer cells in the 50 ng/ml, 100 ng/ml and 200 ng/ml groups exhibited significantly decreased cell proliferation compared with the 0 ng/ml group at 48 h and 72 h (p<0.05). A concentration of 100 ng/ml was chosen for subsequent experiments.

Table 1: Proliferation Effects of DEX on Ovarian Cancer Cells (x̄±s, n=3)

Results are as shown in fig. 1 and Table 2, the number of migration and invasion of SKOV3 cells were significantly decreased in the DEX group compared with the Con group (p<0.05).