A total of 36 surgically resected OS tissue specimens were obtained (prior to the administration of neoadjuvant chemotherapy) from the Department of Orthopedic Surgery at the First Affiliated Hospital of Harbin Medical University (Harbin, China). Written informed consent was obtained from all patients, and the present study was approved by the Ethics Committee of Harbin Medical University (Harbin, China).

Genomic DNA was extracted using a DNA extraction kit (Promega, Fitchburg, WI, USA). DNA quality and quantity was assessed using the Nanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA). The T>G variant at the single nucleotide polymorphism (SNP) rs61764370 was verified by the purification of the PCR product amplified with forward primer, 5′-TTTTGGGGTGGTGGTGTGCCAA-3′ and reverse primer, 5′-AGTCACCACACAAGGCACTG-3′ and sequenced using the forward primer. Sequencing and mutational analysis was performed.

Two OS cell cultures, which were genotyped as rs61764370 TT (named as OSTT) and TG (named as OSTG), were derived from the biopsies obtained from the Department of Orthopedic Surgery at the First Affiliated Hospital of Harbin Medical University (Harbin, China) in accordance with the protocol approved by the Ethics Committee of Harbin Medical University. The MG-63 cell line was purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA) and maintained in Dulbecco's modified Eagle's medium (DMEM)/F12 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA). We obtained scramble RNA, let-7a mimics (5′-AGUGGGGAACCCUUCCAUGAGG-3′) and KRAS siRNA (5′-AGTACTGGCGATTCTCTGA GGC-3′) from GenePharma (Shanghai, China). The transfection of cells with blank control (no RNA was transfected), scramble control, let-7a mimics and KRAS siRNA was performed using Lipofectamine 2000 (Invitrogen Life Technologies, Carlsbad, CA, USA).

A 6-well plate was used to culture OSTT and OSGG cells. After 24 h, when the cells reached 80–90% confluence, we removed the attached cells with a sterile plastic pipette tip to create a single wound in the center of the well. Serum-free medium was used to remove the debris. An inverted microscope (IX83; Olympus, Tokyo, Japan) was employed to visualize and capture images of the cells which had migrated into the wounded area after 24 h of culturing. Each experiment was performed independently at least three times.

The full length of the KRAS 3′-UTR was subcloned into the pmirGLO vector (Promega, Madison, WI, USA) to generate the luciferase reporter vector. The MG-63 cells were seeded in 48-well plates at a density of 1×10⁴ cells/well. Let-7a mimics and luciferase reporter vectors were co-transfected using Lipofectamine 2000 (Invitrogen Life Technologies). After 48 h, the cells were harvested and assayed for luciferase activity using the Dual-Luciferase Reporter Assay system (Promega) according to the manufacturer's instructions. The firefly luciferase activities were normalized to Renilla luciferase activity. Each treatment was performed in triplicate in three independent experiments.

The viability of the cells was measured using an MTT assay according to the manufacturer's instructions (Sigma, St. Louis, MO, USA). Briefly, the cells were seeded at a density of 2,000 cells/well in 96-well plates containing 100 µl culture medium and incubated overnight. To each well, 20 µl of 5 mg/ml MTT was added and the plates were incubated for an additional 4 h at 37°C. After removing the medium, the formazan complex was solubilized with 100 µl dimethyl sulfoxide (DMSO; Sigma). Optical density (OD) was determined by measuring the absorbance at a test wavelength of 490 nm and a reference wavelength of 630 nm. Wells without cells (DMSO alone) were used as blanks. Each group contained six wells; experiments were repeated three times independently and the results are expressed as the means ± standard deviation.

Forty-eight hours after transfection, the total RNA (including miRs) was extracted using an miRNANeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration of RNA was measured using a Beckman DU-640 spectrophotometer (Beckman Coulter, Inc., Fullerton, CA, USA). The quality of RNA was determined by 1% formaldehyde-agarose gel electrophoresis. PCR amplification for the quantification of let-7a and KRAS mRNA was performed using a TaqMan^® miRNA reverse transcription kit and TaqMan miRNA assay kits (both from Applied Biosystems, Foster City, CA, USA). The following PCR protocol was used: 40 cycles at 95°C for 15 sec, 60°C for 15 sec and 72°C for 30 sec on a real-time PCR system (Applied Biosystems). RNU48 (Applied Biosystems) was used as the endogenous control. The relative gene expression levels were then normalized to RNU48 and calculated using the 2^−ΔΔCT method.

Proteins were extracted from the transfected cells or the tissue samples using radioimmunoprecipitation assay (RIPA) lysis buffer (Upstate Biotechnology, Inc., Lake Placid, NY, USA). The protein level was determined using protein assay reagents (Bio-Rad Laboratories, Hercules, CA, USA) according to standard protocols. Briefly, 25 µg of total protein was loaded onto a 12% PAGE gel (NuPAGE; Invitrogen Life Technologies) and subjected to electrophoresis. After blotting onto pure nitrocellulose membranes (Bio-Rad Laboratories) the proteins were blocked with Odyssey^® Blocking Buffer (LI-COR Biosciences, Lincoln, NE, USA) for 2 h. Subsequently, the membranes were incubated in primary antibodies buffer (primary antibodies, Odyssey Blocking Buffer, 0.1% Tween^®-20) overnight at 4°C. The primary antibodies, KRAS and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). The following day, the membranes were washed four times for 5 min in Tris-buffered saline and Tween-20 (TBST). Finally, the membranes were incubated with secondary antibodies, goat anti-rabbit lgG (Cell Signaling Technology, Inc.). The blots were then treated with an ECL kit and the relative density of the target bands was calculated using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

After transfection, the OSTT or OSGG cells (5×10³) were suspended in serum-free medium and seeded onto the upper part of the chamber for the cell migration assay. Complete medium with 20% serum was added to the lower chamber as a chemoattractant. After 48 h, the Transwell membrane was fixed with methanol, and the cells that had migrated to the lower surface of the filters were stained with 0.1% crystal violet stain solution (Sigma). Migration was determined by counting the cell number under a microscope (Olympus). The average number of migrating cells in five random fields was taken as the cell migration number of the group.

All statistical analyses were performed using SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Comparison of datasets for each cell group was performed using the independent t-test or one-way ANOVA, applying Bonferonni's multiple comparison test. A P-value <0.05 was considered to indicate a statistically significant difference. The values reported correspond to the means ± standard deviation.

It has been previously shown that KRAS was a target of let-7a in human cells (15–17). Based on computational analysis, the KRAS 3′-UTR rs61764370 polymorphism was found to be located in the flanking sequence close to the predicted 'binding site' for let-7a (Fig. 1). To determine whether let-7a targets KRAS 3′-UTR in OS cells, we constructed reporter vectors carrying wild-type or mutant KRAS 3′-UTR, as described in Fig. 1. Subsequently, we used them for transient transfection of the OS cells with let-7a mimics or scrambled controls. As shown in Fig. 2, only the luciferase activity from the cells cotransfected with wild-type KRAS 3′-UTR and let-7a mimics decreased by 50% compared with the control, and all other groups were comparable. The results confirmed that KRAS is a validated target of let-7a in OS cells, and the rs61764370 polymorphism compromised the interaction between let-7a and KRAS 3′-UTR, and the nucleotide substitution completely abrogated the miRNA/mRNA interaction in OS cells.

The OS tissues of three different genotypes (TT, n=22, GT, n=10, GG, n=4) were used to further explore the impact of the polymorphism on the interaction between let-7a and KRAS 3′-UTR. Using RT-qPCR, we found that let-7a expression was comparable among the TT, GT and GG groups (Fig. 3). Additionally, to characterize the effect of KRAS 3′-UTR rs61764370 polymorphism on the miRNA/mRNA interaction in OS cells, which plays a central role in the development of OS, we quantified the expression of KRAS using RT-qPCR as well as western blot analysis. As shown in Fig. 3, the expression of let-7a was similarly distributed among each genotype group, whereas in Fig. 4 the mRNA and protein expression of KRAS in the TT genotype group was significantly lower than that in the GT and GG groups. We then analyzed the correlation between the mRNA expression of let-7a and KRAS among the OS tissues (n=36), and found a negative correlation between them (Fig. 5).

To further validate the hypothesis of the negative regulatory relationship between let-7a and KRAS and to explore the effect of the polymorphism in such a relationship, we examined the mRNA/protein expression of KRAS in two primary OS cell cultures which were genotyped as TT (OSTT) and TG (OSTG). We transfected the two OS cell groups with either scrambled control, let-7a mimics or KRAS siRNA. As shown in Fig. 6A and C, the mRNA and protein expression of KRAS in the OSTT cells treated with let-7a mimics and KRAS siRNA was lower than that in the scrambled control. The OSTG cells treated with KRAS siRNA showed substantial downregulation of the mRNA and protein expression of KRAS whereas let-7a mimics exerted minimal effects on the expression of KRAS compared with the control (Fig. 6B).

We also examined the relative viability of OS cells following transfection with either scrambled control, let-7a mimics or KRAS siRNA. The OSTT cells transfected with let-7a mimics and KRAS siRNA showed comparably lower viability compared with the scrambled control group. The viability of the OSTG cells was comparable between the scrambled control and the let-7a mimic group, and the viability of both groups was notably higher than that of KRAS siRNA group (Fig. 7A and B).

We also examined the relative invasion (Fig. 8) and migration (Fig. 9) of the two primary OS cell cultures following transfection with either scrambled control, let-7a mimics or KRAS siRNA. The invasiveness (Fig. 8A) and migratory ability (Fig. 9A–C) of the OSTT cells transfected with KRAS siRNA and let-7a mimics was notably downregulated when compared with the scrambled controls. The OSTG cells transfected with let-7a mimics and scramble controls showed comparable invasiveness and migratory abilities which were markedly higher compared with the OSTG cells treated with KRAS siRNA (Fig. 8B and Fig. 9D–F).