All animal experiments were approved by the Ethics Committee of Shanghai Tenth People’s Hospital, Shanghai, China. Surgical procedures were performed under anesthesia and every effort was made to minimize suffering of animals. All mice were anesthetized via intraperitoneal injection of sodium pentobarbital (30 mg/kg).
Cell lines and cultures
Both SKOV3 and HEK293T cell lines were obtained from the American Type Culture Collection (Manassas, VA, USA). SKOV3 cells were cultured in RPMI 1640 (Invitrogen, California, USA), and HEK293T in Dulbecco’s Modified Eagle Medium (Invitrogen, California, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen, California, USA) at 37 °C in 5% CO2.
Transfection of cells with miR-219-5p mimic or HMGA2 overexpression vector
The miR-219-5p mimic vector was synthesized by GenePharma (Shanghai, China). Full-length HMGA2 from the human cDNA library was cloned into pCDNA3.1 vector. Cells were transfected using the Lipofectamine 2000 reagent (Thermo Scientific, Massachusetts, USA) according to manufacturer’s instructions.
Qualitative PCR analysis
Total RNA was extracted from tissues and cells using TRIzol reagent (Invitrogen, California, USA) in keeping with the reagent kit protocol and cDNA amplified using the TaqMan miRNA reverse transcription kit (Invitrogen, California, USA). HMGA2, miR-219-5p and U6 mRNA levels were determined via qPCR using the TaqMan human miRNA assay kit. Relative fold difference was measured using the 2−∆∆CT method. The following primers were employed: HMGA2 (F) 5′-CTCAAAAGAAAGCAGAAGCCACTG-3′ and (R) 5′-TGAGCAGGCTTCTTCTGAACAACT-3′; miR-219-5p (F) 5′-ACACTCCAGCTGGGTGATTGTCCAAACGCAAT-3′ and (R) 5′-CTCAACTGGTGTCGTGGA-3′; U6 (F) 5′-CTCGCTTCGGCAGCACA-3′ and (R) 5′-AACGCTTCACGAATTTGCGT-3′.
Cell proliferation was detected with the CCK-8 assay according to the manufacturer’s protocol (Invitrogen, California, USA). SKOV3 cells from different groups were cultured in 96-well plates under the same conditions. After 0, 24, 48 and 72 h, a mixture of 90 μl fresh culture medium and 10 μl CCK-8 solution was added to wells. Following further incubation of SKOV3 cells at 37 °C for 2 h, cells were examined using a microplate reader at a wavelength of 450 nm.
Luciferase reporter assay
HMGA2 with wild-type or mutant 3′-UTR was generated and cloned into the firefly luciferase-expressing vector, psiCHECK-2. For the luciferase assay, cells were seeded in triplicate in 12-well plates the day before transfection and subsequently transfected with WT or Mut 3′-UTR reporter vector, either in combination with or without the miR-219-5p mimic. Next, cells were harvested and lysed for luciferase activity analysis using the Dual-Luciferase Reporter System (Promega, WI, USA). Three independent experiments were performed.
Western blot assay
Cells or tissues were lysed, protease inhibitors added to the lysates and centrifuged at 12,000 rpm at 4 °C. Protein concentrations were examined with the BCA kit (Pierce, USA). Next, proteins were separated via 10% SDS-PAGE and transferred to PVDF membranes. The following antibodies were employed for detection of protein expression: HMGA2 (1:500, Santa Cruz, California, USA) or anti-GAPDH (1:2000, Santa Cruz, California, USA) primary antibodies and horseradish peroxidase-conjugated secondary antibody (1:1000, Santa Cruz, California, USA). The ECL chemiluminescent kit (Millipore, MA, USA) was applied to visualize protein bands.
Cells were transfected with miR-219-5p mimic or HMGA2 overexpression vector or pretreated with miR-219-5p inhibitor. After 48 h, cells were cultured in serum deprivation medium for 12 h and digested with trypsin before seeding on the top chambers of 24-well transwell culture inserts (Promega). Medium supplemented with 20% serum was used as a chemoattractant in the lower chambers. After 24 h, cells were fixed for 10 min with 4% formalin before staining with 0.005% crystal violet and counted under a phase contrast microscope.
Invasion assays were performed using the BD Bio-Coat Matrigel invasion assay system (BD Biosciences, NJ, USA) according to the manufacturer’s instructions. The non-motile or noninvasive cells were removed while the lower side of the filter was stained with 0.005% crystal violet and counted.
Tumor growth in vivo
After transfection, 2 × 106 SKOV3 cells were transplanted into nude mice (25–30 g, 6 weeks old, n = 6). Gross tumor volumes were measured with vernier calipers every 5 days from days 5 to 25. Mice were subsequently killed for western blot analysis.
Continuous variables were expressed as mean ± standard deviation (SD). One-way ANOVA was performed for multiple comparisons using GraphPad Prism software, version 5.0 (GraphPad, La Jolla, CA, USA). P-values ≤ 0.05 indicated statistically significant differences.