Breast cancer cells were purchased from the Cell Bank Type Culture Collection of Chinese Academy of Sciences (CBTCCCAS; Shanghai, China) and cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) with fetal bovine serum (10%) (Gibco; Thermo Fisher Scientific, Inc.) at 37°C, in a humidified atmosphere containing CO₂ (5%). The culture medium was added 100 U/ml of penicillin sodium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 100 mg/ml of streptomycin sulfate (Invitrogen; Thermo Fisher Scientific, Inc.). Cells were exposed to 2% sevoflurane for 6 h as previously described (5,20). 2% sevoflurane is clinically relevant and regulates the proliferation of human hepatocellular carcinoma cells (21). CO₂, O₂ and sevoflurane concentration was monitored by A Drager Vamos gas analyzer (Drager, Lübeck, Germany). The cells used for each experiments were culture in the 6 wells dish and seeded at the concentration of 1×10⁵/well.

Pre-miR-203, miRNA-203 inhibitor and the control miRNA were synthetic nucleic acids (Biotend, Shanghai, China). Pre-miRNAs can be processed by enzymes to become mature miRNAs. miRNA inhibitor has reverse complementary sequence of the miRNA. Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was used for transfection of the pre-miRNA or inhibitor into cells which grown to approximately confluence (80%).

Proliferation was detected by CellTiter 96^®. A Queous One Solution Cell Proliferation Assay kit (Promega Corporation, Madison, WI, USA) was used to perform the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sul-fophenyl)-2H-tetrazolium (MTS) assay by following the instruction. The record absorbance at 490 nm was detected by microplate reader. We performed the detection every 24 h during 3 days.

Cells were seeded in 96-wells (2×10³ cells/well) in the culture medium with BrdU incubation. After 2 h, phosphate-buffered saline (PBS) solution with paraformaldehyde (PFA) (4%) was used to fix cells for 20 min. Cells were washed by PBS and treated by DNase for 20 min at room temperature. Then, BrdU primary antibody (Abcam, Cambridge, MA, USA) was added and incubated at 4°C for 12 h. After washing by PBS, cells were incubated with secondary antibody at room temperature for 60 min. Cell nucleus were dyed by 4′,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich; Merck KGaA). BrdU-positive cells were counted by microscope.

RNAiso (Takara Bio, Inc., Otsu, Japan) was used for isolating total RNA. For reverse-transcription of miRNA, miRNA specific stem-loop reverse-transcription primer (Ribobio, China) was used. The amount of gene expression (2-ΔΔCt) was normalized tothe endogenous nuclear RNA U6 using miRNA-specific primers (RiboBio Co., Ltd., Guangzhou, China). For mRNA RT-qPCR, cDNA was reverse-transcribed from mRNA by M-MLV Reverse Transcriptase (Takara) using random primers and oligo (dT) primers. The quantification of level of target gene expression (2^−ΔΔCt) was normalized to the endogenous GAPDH mRNA (7,22). The reaction conditions of PCR were according to the SYBR-Green qPCR Mix instruction (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primers sequences are as follows: Cyclin E1, PF, 5′-ACTCAACGTGCAAGCCTCG-3′, PR, 5′-GCTCAAGAAAGTGCTGATCCC-3′; Cyclin D1, PF, 5′-CAATGACCCCGCACGATTTC-3′, PR, 5′-CATGGAGGGCGGATTGGAA-3′; P21, PF, 5′-TGTCCGTCAGAACCCATGC-3′, PR, 5′-AAAGTCGAAGTTCCATCGCTC-3′; P27, PF, 5′-TAATTGGGGCTCCGGCTAACT-3′, PR, 5′-TGCAGGTCGCTTCCTTATTCC-3′; RB1, PF, 5′-TTGGATCACAGCGATACAAACTT-3′, PR, 5′-AGCGCACGCCAATAAAGACAT-3′. The primers of miRNA RT-qPCR were purchased from the RiboBio Co., Ltd. The thermocycling condition for mRNA is as follows: For mRNA: 95°C for 30 sec followed by 40 cycles of 95°C for 10 sec, 60°C for 30 sec, 70°C for 30 sec. For miRNA: 95°C for 30 sec followed by 40 cycles of 95°C for 5 sec, 60°C for 20 sec, 70°C for 10 sec.

Proteins from MDA-MB-231 cells were lysed and then transfered onto PVDF membranes (What man, England). Then incubated the membranes with the primary antibodies diluted by TBST (Beyotime, China) at room temperature for 2 h. The primary antibodies are as follows: Anti-GAPDH (ab9485, Abcam, USA), anti-cyclin E (ab135380, Abcam), anti-cyclin D (ab134175, Abcam), anti-P21 (ab109520, Abcam), anti-P27 (ab32034, Abcam), anti-Rb1 (ab181616, Abcam). Then incubated the membranes with secondary antibodies diluted by TBST (Beyotime) according to the primary antibodies at room temperature for 1 h. Protein signals were detected using enhanced chemiluminescence (ECL).

For statistical analyses, student's t-test or one way ANOVA with Boferroni's correction was used. The values were showed as the mean ± SD. P<0.05 was defined as statistically significant.

In order to determine the function of sevoflurane on breast cancer cells proliferation, MDA-MB-231 or MCF-7 cells were treated with sevoflurane (2%) for 6 h and cultured for another 24, 48 and 72 h without sevoflurane. The proliferation capacity was detected by MTS assay. The results showed that sevoflurane decreased proliferation of MDA-MB-231 or MCF-7 cells (Fig. 1A). The BrdU incorporation assay in MDA-MB-231 cells was performed and cells treated with sevoflurane (2%) for 6 h and then cultured for another 24 h without sevoflurane were found showing decreased proliferation capacity (Fig. 1B). We further detected the cycle-related genes in MDA-MB-231 cells and found that the level of cyclin D, cyclin E, which have been reported to be the critical regulatory genes of G1 phase, were significantly downregulated. In contrary, the expression of Rb1, P21, P27, the cell cycle-inhibiting genes, were upregulated compared with control group on both mRNA and protein levels (Fig. 1C). Flow cytometry assay showed that proportion of cells at G1 phase was increased and cells at the S, G2/M phase was decreased after treating by sevoflurane in MDA-MB-231 cells (Fig. 1D).

Using RT-qPCR, we detected that miR-203 could be upregulated in MDA-MB-231 cells treated with sevoflurane (Fig. 2A). To determine the effect of miR-203, we transfected the pre-miR-203 into MDA-MB-231 cells and found that miR-203 significantly repressed the proliferation in both MDA-MB-231 and MCF-7 cells (Fig. 2B and C). In contrast, transfection of the miR-203 inhibitor whose efficiency was determined by detecting the target gene SNAI2 level (23) promoted the proliferation of MDA-MB-231 and MCF-7 cells detected by MTS assay (Fig. 2B). BrdU incorporation assay in MDA-MB-231 cells also indicated the similar results (Fig. 2C). Expression level of cyclin D, cyclin E was downregulated in cells transfected with pre-miR-203 (Fig. 2D) and upregulated in the MDA-MB-231 cells transfected with miR-203 inhibitor (Fig. 2E). The expression of P21, P27, Rb1 was upregulated in cells transfected with pre-miR-203, while downregulated in cells transfected with miR-203 inhibitor (Fig. 2D and E). Flow cytometry assay showed that proportion of cells was increased at G1 phase and decreased at the S and G2/M phase in MDA-MB-231 cells transfected with pre-miR-203 (Fig. 2F). Whereas, the proportion of cells was decreased at G1 phase and increased at the S and G2/M phase in MDA-MB-231 cells transfected with miR-203 inhibitor (Fig. 2F).

In order to determine whether miR-203 actually mediated the function of sevoflurane, we performed the rescue experiment (Fig. 3). Results indicated that miR-203 inhibitor significantly attenuated the proliferation repression caused by sevoflurane treatment in both MDA-MB-231 (Fig. 3A and C) and MCF-7 cells (Fig. 3B). miR-203 inhibitor also resisted the effect of sevoflurane on the expression of cell cycle-related genes in MDA-MB-231 cells (Fig. 3D). Flow cytometry assay showed that, compared with MDA-MB-231 cells treated with sevoflurane, the increase of cells at G1 phase and the decrease of cells at the S and G2/M phase can be reversed by miR-203 inhibitor in MDA-MB-231 cells (Fig. 3E).