Cell Culture
The human HLE-B3 LC line was from Shanghai Genechem (Shanghai, China). Cells were grown in DMEM (Gibco, MA, USA) containing 15% FBS (Zhejiang Tianhang Biotechnology, Hangzhou, China), along with penicillin and streptomycin in a 37°C 5% CO2 incubator.
Cell Transduction
HLE-B3 cells were transduced with control or miR–19-encoding lentiviral particles (Shanghai Genechem) in a 96-well plate, with 5x104 cells/mL in 100 uL per well. Lentiviral particles were added to cells at a range of multiplicities of infection (MOIs), and then after 72 h the GFP expression in transduces cells was assessed via fluorescent microscope (Leica DMI6000B).
RT-qPCR
The RNAiso for Small RNA kit (TaKaRa, Dalian, China) was used to extract total RNA from WT or lentivirally-transduced cells, with a BioSpectrometer (Eppendorf, Germany) used to gauge RNA quality. A total of 0.25–8 μg RNA in a 3.75 μl volume was then used with the Mir-X miRNA First-Strand Synthesis Kit (TaKaRa) for reverse transcription based on provided directions, and the Step One Plus Real-time PCR System (Applied Biosystems) was used to measure relative miR–19a expression. TB Green Premix Ex Taq II (Tli RNaseH Plus; TaKaRa) was used for all reactions, which consisted of a total of 25 µl made up of 12.5 µl TB Green Advanced premix, 0.5 µl ROX, 0.5 µl of the appropriate miR–19a primer, 0.5 µl of the mRQ 3′ primer, 2 µl cDNA, and 9 µl dd H2O. Thermocycler settings were: 95°C for 30 sec; 40 cycles of 95°C for 5 sec, 60°C for 30 sec. PCR products were confirmed via melting curve analyses. Relative miR–19a levels were normalized to levels of the U6 RNA, with the 2-△△Ct method used for quantification. miR–19a primer sequences were as follows: Forward - 5′-GGAACGATACAGAGAAGATTAGC–3′; Reverse - 5′-TGGAACGCTTCACGAATTTGCG –3′ (TaKaRa).
Cell proliferation assay
The MTT assay approach was used to quantify the proliferation of HLE-B3 cells, which were plated into 96-well plates (2×104 cells/ml, 100uL/well). After 24 h post-plating, cells were lentivirally transfected. After an additional 24, 48, 72, or 96 h, 20 μl MTT (5 mg/ml; Biofroxx, Germany) was added per well, and cells were incubated at 37°C for 4 h. Media was then exchanged for 150 µl DMSO (Beijing Solarbio, Beijing, China). A microplate reader (SepectraMax M3, Molecular Devices, Shanghai, China) was then used to assess absorbance values at 490 nm.
Wound healing assay
At 72 h post-lentiviral transduction, HLE-B3 cells were replated into 12-well plates at 3.5×105 cells/well. After an additional 24 h, a wound was generated in the monolayer within each well using a sterile 20µl pipette tip, and PBS was used to wash away non-adherent cells. After washing, fresh media was added and cells were imaged for 24 h, with the size of the wound being imaged at 0 and 24 h via an inverted microscope (Leica DMI6000B), with Image J used to quantify wound size changes over time.
Cell Migration Assay
At 72 h post-lentiviral transduction, HLE-B3 cells (5x104) were resuspended into 200 µl of serum-free DMEM, and were then added to the upper portion of a Transwell insert (Corning, High Wycombe, UK). This insert was then inserted into a well, with media supplemented with 20% FBS added to the lower chamber. Cells were incubated for 24 h, after which 4% paraformaldehyde was used to fix membranes prior to crystal violet (Beyotime) staining and counting of cells via Axio vert A1 inverted fluorescence microscope.
Western blotting
After a 30 minute lysis step on ice, HLE-B3 cell lysates were spun at 12,000 rpm for 10 minutes at 4°C, with supernatants being collected and protein levels therein being quantified via a BCA assay. After boiling for 5 min in sample buffer, proteins from each sample were separated via SDS-PAGE, transferred to PVDF membranes, blocked for 4 h using 5% non-fat dried milk at room temperature, and probed overnight with primary antibodies specific for AKT, pAKT, and PTEN (1:1000, Wanlei Biotechnology, China) at 4°C. Secondary HRP-conjugated antibodies were then used for antigen detection for 1 h at room temperature. Blots were then washed using TBST, and an ECL detection kit (Beyotime) was used to quantify protein levels. Actin served as a loading control
Statistical analysis
Data are means ± SD. Values were compared via one-way ANOVAs with Bonferroni post-hoc testing for multiplecomparisons using SPSS. P < 0.05 was the significance threshold.