The reagents, kits and antibodies included: Adult SaOS-2 osteosarcoma cells (Kunming Cell Bank, Chinese Academy of Sciences, Kunming, China), RNA extraction kit (Takara), SYBR Premix Ex Taq kit and Prime Script RT reagent Kit (Takara), Dulbecco's modified Eagle's medium (DMEM) (Gibco, Rockville, MD, USA), fetal bovine serum (FBS) (Gibco; Thermo Fisher Scientific, Inc.), TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.), Radioimmunoprecipitation assay (RIPA) lysis buffer (Wuhan Guge Biotech), methyl thiazolyl tetrazolium (MTT) kit (Wuhan Boster Biological Technology Co., Ltd.), apoptosis assay kit (R&D Systems, Inc.), reactive oxygen species (ROS) assay kit (Beyotime Institute of Biotechnology), and mouse anti-human caspase-3 (cat. no. ab13847; Abcam), cleaved caspase-3 (cat. no. ab32042; Abcam), B-cell lymphoma-2 (Bcl-2) (cat. no. ab32124; Abcam), Bcl-2 associated X protein (Bax) (cat. no. ab32503; Abcam), phosphorylated (p-)JAK2 (cat. no. ab32101; Abcam), p-STAT3 (cat. no. ab76315; Abcam), JAK2 (cat. no. ab108596; Abcam), STAT3 (cat. no. ab68153; Abcam) and myeloid cell leukemia-1 (Mcl-1) (cat. no. ab32087; Abcam) antibodies. Other reagents of unspecified sources are listed in the manuscript.

The expression of miR-19a in the SaOS-2 osteosarcoma cell line was knocked down by GenePharma using lentiviral transfection. At 48 h after transfection, the successfully transfected cells were selected to extract the total RNA. The expression level of miR-19a in cells was verified via polymerase chain reaction (PCR), and the transfection efficiency was determined. The cell lines with low expression of miR-19a were selected for stable subculture as the miR-19a-inhibitor group. The blank plasmid group (NC-inhibitor group) and blank control group (Control group) were also set up.

The cells in the logarithmic growth phase in each group were collected and centrifuged at 10,500 × g for 10 min, the supernatant was discarded, and appropriate amount of TRIzol was added to extract the total RNA. The total RNA concentration in each group was determined using the nucleic acid-protein quantometer (NanoDrop 2000), and the optical density (OD) value was 1.8-2.0. The reverse transcription system was prepared, and the reaction conditions are as follows: 37°C for 15 min and 85°C for 5 sec. The total RNA was reversely transcribed into complementary deoxyribonucleic acid (cDNA), and stored for later use. Then the qPCR system was prepared in strict accordance with the instructions of kit, and the reaction conditions were as follows: 95°C for 30 sec, 95°C for 5 sec, 60°C for 35 sec for a total of 35 cycles. With U6 as an internal reference, the relative expression level of miR-19a in each group was calculated by 2^−ΔΔCq (9). The primers were synthesized by Invitrogen; Thermo Fisher Scientific, Inc., and the primer sequences are shown in Table I.

The cells in the logarithmic growth phase in each group were collected, inoculated into a 96-well plate (1×10⁴ cells/well), and cultured using complete medium with 5% CO₂ under a constant temperature of 37°C. Then 20 µl of MTT was added into each well after 12, 24, 36, 48, 60 and 72 h respectively, followed by culture in an incubator for another 4 h. After the supernatant was discarded, 150 µl of dimethyl sulfoxide (DMSO) (Wuxi Yangshan Biochemical Co., Ltd.) was added into each well and shaken gently. Finally, the optical density (OD) value of the cells in each group was measured at a wavelength of 450 nm using a microplate reader, based on which the cell proliferation level was calculated.

The cells in the logarithmic growth phase in each group were collected, inoculated into a 6-well plate (1×10⁵ cells/well), and cultured using complete medium with 5% CO₂ under a constant temperature of 37°C. After 24 h, the supernatant was discarded, and the cells were washed twice with pre-cooled phosphate-buffered saline (PBS) and centrifuged using a centrifuge at 950 × g for 10 min to prepare the cell suspension. Then the cells were resuspended, centrifuged, added with fluorescence solution and incubated in the dark at room temperature for 15 min, followed by detection of the apoptosis level via flow cytometry strictly according to the instructions of the apoptosis kit.

The ROS level in each group was determined using DCFH-DA. The cells in the logarithmic growth phase in each group were collected, inoculated into the 6-well plate (1×10⁵ cells/well), and cultured using complete medium with 5% CO₂ under a constant temperature of 37°C. After 24 h, the reactive oxygen species (ROS) level in each group was detected strictly according to the instructions of the ROS assay kit: An appropriate amount of DCFH-DA (10 µM) was added into the 6-well plate, and incubated in the incubator in the dark at 37°C for 20 min. After the medium was discarded, the cells were washed with pre-cooled PBS and washing solution, and observed under a confocal microscope (excitation wavelength of 488 nm, and emission wavelength of 525 nm). The higher the ROS level, the higher the brightness.

The cells in the logarithmic growth phase in each group were collected, inoculated into the 6-well plate (1×10⁵ cells/well), and cultured using complete medium with 5% CO₂ under a constant temperature of 37°C. After 24 h, the supernatant was discarded, and the cells in each group were collected for later use. The total protein was extracted from cells in each group, and the total protein concentration was determined using the bicinchoninic acid (BCA) protein quantification kit (Wuhan Boster Biological Technology Co., Ltd.). After the sodium dodecyl sulphate (SDS) 10% gel was prepared, the protein was subjected to electrophoresis, transferred (40 mg) onto polyvinylidene fluoride (PVDF) membranes (Millipore), and sealed with freshly-prepared 5% skim milk powder for 1 h. Then the corresponding target bands were cut, incubated with caspase-3 (dilution: 1/2,000), Bcl-2 (dilution: 1/2,000), Bax (dilution: 1/2,000), p-JAK2 (dilution: 1/2,000), p-STAT3 (dilution: 1/2,000), JAK2 (dilution: 1/1,000), STAT3 (dilution: 1/2,000), Mcl-1 (dilution: 1/1,000) and GAPDH (dilution: 1/500) antibodies at 4°C overnight, washed with Tris-buffered saline with Tween^®20 (TBST) for 3 times, incubated again with horseradish peroxidase-conjugated secondary antibodies (dilution: 1/2,000; cat. no. ab6721; Abcam) at room temperature for 1 h, and washed again with TBST for 3 times. Finally, bands were exposed by enhanced chemiluminescence (ECL) detection kit (Amersham Biosciences, and analyzed by ImageJ Software (version 1.38; National Institutes of Health), and the protein bands were scanned to calculate the protein expression levels in each group.

The data are expressed as mean ± standard deviation and were analyzed using Statistical Product and Service Solutions (SPSS) 22.0 software (IBM, Corp.). One-way analysis of variance was adopted for the data in each group. The homogeneity test of variance was performed. Bonferroni's method was adopted for the pairwise comparison in the case of homogeneity of variance, while Welch's method was used in the case of heterogeneity of variance. P<0.05 suggested that the difference was statistically significant.

The expression of miR-19a in SaOS-2 osteosarcoma cell lines was downregulated using lentiviral transfection. After successful transfection, the expression level of miR-19a in each group was detected via qPCR. As shown in Fig. 1, the expression level of miR-19a in the miR-19a-inhibitor group was significantly lower than that in the control group and NC-inhibitor group (P<0.01), indicating that the transfection was successful and subsequent experiments could be performed.

The effect of miR-19a downregulation on the proliferation of SaOS-2 cells was detected using MTT assay. As shown in Fig. 2, the cell proliferation ability in the miR-19a-inhibitor group was significantly weaker than that in the control group and NC-inhibitor group (P<0.01), and the proliferation ability in the miR-19a-inhibitor group became weaker with time.

The apoptosis level in each group was detected through flow cytometry. The results showed that the miR-19a-inhibitor group had an obviously higher apoptosis level (38.76%) than the control group (4.16%) and NC-inhibitor group (13.92%) (P<0.01) (Fig. 3).

According to the detection results of ROS level in each group using DCFH-DA, the content of ROS (60%) in the miR-19a-inhibitor group was evidently higher than that in the control group (42%) and NC-inhibitor group (40%) (P<0.01) (Fig. 4).

Western blotting was performed to detect the expression levels of apoptosis-related proteins in each group. The results revealed that compared with those in the control group and NC-inhibitor group, the ratio of expression of Bcl-2/Bax was significantly decreased (P<0.01), while that of cleaved caspase-3/caspase-3 was significantly increased in the miR-19a-inhibitor group (P<0.01) (Fig. 5).

Western blotting was also performed to detect the expression levels of JAK2/STAT3 signaling pathway-related proteins in each group. It was found that the miR-19a-inhibitor group had significantly lower protein expression ratio of p-JAK2/JAK, p-STAT3/STAT3 and Mcl-1/GAPDH than the control group and NC-inhibitor group (P<0.01) (Fig. 6).