Down-regulation of miR-27a might inhibit proliferation and drug resistance of gastric cancer cells

Human gastric cancer cell line, MKN45, was routinely maintained in DMEM medium (GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum at 37°C in humidified air containing 5% carbon dioxide air atmosphere.

Cells were plated in plates and cultured for 16 h, and then transfected with the antagomirs of miR-27a or control RNA (Lafayette, CO) as described previously [3].

Total RNAs from cells were extracted and cDNA synthesis and amplification were performed as described previously [4]. Primers were designed as: MDR1, forward: 5'-CCCATCATTGCAATAGCAGG-3', reverse: 5'-TGTTCAAACTTCTGCTCCTGA-3'; cyclinD1, forward: 5'-GGAGCTGCTCCTGGTGAACA-3', reverse: 5'-TGTTGGGGCTCCTCAG GTTCA-3'; P21, forward: 5'-CCCGTGAGCGATGGAACT-3', reverse: 5'-CGAGGCACAAGGG TACAAGA-3'; P27, forward: 5'-CAAGTACGAGTGGCAAGAGG-3', reverse: 5'-GTAGAA GAATCGTCGGTTGC-3'. Comparative real-time PCR was performed in triplicate, including no-template controls. Relative expression was calculated using the comparative Ct method.

Cells were seeded on a 96-well plate at 3 × 10⁴ cells/well. Each sample has four replicates. Viable cells were counted by the MTT assay after 2, 4, 6, and 8 days.

Soft agar assay was performed as described previously [5]. Each assay was performed in triplicate.

Female athymic nu/nu mice, 5-6 weeks of age, were used in the experiment. The cells were resuspended in D'Hanks solution, and 5 × 10⁶ cells in 0.2 ml were injected subcutaneously into the right flank of 4-week-age mice. Experimental and control groups had at least 6 mice each. Tumors were measured twice weekly, and the tumor volume was calculated.

Vincristine (VCR), adriamycin (ADR), cisplatin (CDDP) and 5-fludrouracil (5-flu) were prepared before experiment. Drug sensitivity was evaluated using MTT assay as described previously [3].

Fluorescence intensity of intracellular ADR was detected by FCM as described previously [3].

Cellular proteins were separated on SDS-PAGE gels, and western blot was performed as described previously [3].

The pGL3-cyclin D1 vector and the control vector were prepared as described previously [3]. Briefly, 0.4 μg of reporter gene constructs was transfected into MKN45 cells using LipofectAMINE (Invitrogen) reagent according to the manufacturer's protocol. This transfection was done concurrently with the transfection of the antagomirs of miR-27a. Cells co-transfected with scrambled antago-miR-NC served as controls.

As Figure 1A showed, MKN45 cells were transfected with either the antagomirs of miR-27a or control RNA. The antagomirs of miR-27a could significantly inhibit the expression of miR-27a by almost 67% as compared with that of control. Cell growth was assayed, and down-regulation of miR-27a significantly inhibited proliferation of MKN45 cells as compared with control (P < 0.05) (Figure 1B). MKN45 cells and their transfectants were seeded in soft agar and colon formation was assessed. As shown in Figure 1C, down-regulation of miR-27a significantly inhibited the number of colonies formed by gastric cancer cells. Tumorigenesis was found profoundly decreased in miR-27a-downregulating cells as compared with control groups (Figure 1D), suggesting that down-regulation of miR-27a might inhibit the growth of MKN45 cells in vitro and in vivo.

As shown in Table 1, the IC50 values of miR-27a antagomir cells for VCR, ADR and 5-flu were significantly decreased as compared with control cells. The ADR intracellular accumulation and releasing were explored using FCM assay. As shown in Figure 2A, B, increased accumulation and decreased releasing index of ADR of miR-27a antagomir cells was observed as compared with control cells (p < 0.05).

The expression of P-glycoprotein, cyclin D1, p21 and p27 was detected in the gastric cancer cells using real-time PCR (Figure 3) and western blot (Figure 4). Down-regulation of miR-27a could significantly decrease the expression of P-glycoprotein and cyclin D1, and up-regulate the expression of p21. To evaluate whether cyclin D1 was a genuine target of miR-27a, luciferase reporter assay was performed. As shown in Figure 5, co-transfection of increasing amounts of antagomirs of miR-27a with cyclin D1 reporter gene led to significantly decrease in cyclin D1 promoter activity, suggesting that miR-27a might target cyclin D1.