Two OSA cell lines, SaOS2 and U2OS (American Type Culture Collection, Manassas, VA, USA) were investigated in the present study. Cells were cultured in Dulbecco's modified Eagle medium (DMEM; Invitrogen; Thermo Fisher Scientific, Inc., Paisley, Scotland) containing 10% fetal calf serum, 10,000 U/ml penicillin and 10,000 mg/ml streptomycin, at 37°C in a humidified atmosphere containing 5% CO₂ in air.

Tissue samples were collected from 37 patients diagnosed with OSA at the Department of Orthopedic Surgery, Shanghai Sixth People's Hospital (Shanghai, China). All specimens were acquired from patients who underwent surgical resection and provided informed consent. Among the 37 patients, 25 were male and 12 were female. The ages of the patients ranged from 14–45 years. Specimens of tumor and adjacent normal tissue were collected from each patient, and the diagnosis of OSA was validated by pathological examination. Specimens were frozen at −80°C for DNA/RNA extraction. The Ethics Committee of Shanghai Sixth People's Hospital provided ethical approval.

Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc., Cergy Pontoise, France). Next, 3 mg total RNA was denatured for 10 min at 70°C and then reversed transcribed into cDNA at 37°C for 90 min using 300 U Moloney murine leukemia virus reverse transcriptase, 15 mg oligo dT primers (both Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1 mM deoxynucleoside triphosphate (Bioline, London, UK) in a total volume of 30 ml. qPCR was then performed using a SYBR Green PCR Master Mix kit (ABgene; Thermo Fisher Scientific, Inc., Courtaboeuf Cedex, France) supplemented with 0.5 mM primers. The PCR mixture contained 7.5 µl SYBR Green, 4.5 µl water, 1 µl forward and reverse primers, respectively, and 2 µl DNA template. The primers were: Human TMEM35 forward, 5′-TGGGGACTATCAAGCTGACC-3′, and reverse, 5′-CAATGCTTTTTCGGAGGAGA-3′; β-actin forward, 5′-AATCGTGCGTGACATTAAGGAG-3′, and reverse, 5′-ACTGTGTTGGCGTACAGGTCTT-3′. The thermal cycling conditions used were as follows: 95°C for 15 min, then 40 cycles at 95°C for 20 sec, 58°C for 15 sec, and 72°C for 15 sec. Signals with a threshold cycle (Cq) value of >39 were considered to indicate no transcription of the target gene. The relative expression of mRNA was calculated using the 2^−ΔΔCq method (25).

siRNAs specifically targeting the TMEM35 gene (TMEM35-si-1 and TMEM35-si-2), and negative control (si-LUC) were transfected into SaOS2 and U2OS cells. The siRNA sequences were TMEM35-si-1: CCAGAACCGUAACUAUUGU and TMEM35-si-2: CAACCCUCCUUAUAUGAGA. The siRNAs and Lipofectamine 2000 (both Invitrogen) were combined in DMEM at room temperature. The mixture was added to the cells dropwise and incubated for 4–6 h. The medium was then changed to fresh medium containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 1% antibiotic, and cells were cultured for 3 days.

A wound-healing assay was performed using SaOS2 cells according to the manufacturer's protocol (ibidi GmbH, Martinsried, Germany). Cell migration assays were also performed using SaOS2 and U2OS cell lines, as described previously (26). Briefly, in the wound-healing assay, cells were seeded into 3-mm cell culture dishes and when cell confluence was ~90%, a scratch was made at the bottom of the dish. The width of the wound was measured to assess wound healing. The cell migration assay was a Boyden chamber assay in which cells were seeded in an insert with a porous membrane (50,000 cells/insert). DMEM without FBS was added to the upper chamber and DMEM with 10% FBS was added to the bottom of the plate. The insert was put into the well of a 12 well plate containing medium for 24 h. The number of cells that transferred to the bottom of the membrane from the upper side was determined. To do this, non-migrated cells were removed from the upper surface using cotton-tipped swabs. Cells that migrated to the underside were fixed with 3.7% paraformaldehyde in phosphate-buffered saline at 4°C and stained with crystal violet. Membranes were then cut from the insert and observed under a microscope. Five fields were randomly selected and a count for each assay was performed in duplicate.

A total of ~20,000 SaOS2 or U2OS cells were seeded into dishes and cultured at 37°C for 2 weeks in DMEM containing 10% fetal bovine serum and 1% antibiotic. Soft agar colony formation assays were conducted, in which the transfected cells were seeded in soft agar and incubated for approximately 3–4 weeks. At the end of the incubation, the cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet (Hangzhou DayangChem Co., Ltd.; Hangzhou, China) or 1% Coomassie Brilliant Blue. The colonies were then counted under a dissecting microscope (AmScope SE306-AZ-E; AmScope, Irvine, CA, USA).

Flow cytometry with propidium iodide staining was used to analyze cell cycle progression. The cells were fixed in 70% ethanol and rehydrated in phosphate-buffered saline and adjusted to a concentration of 1–5×10⁶ cells/ml. Cells were treated with 10 mg/ml RNase A for 30 min and 10 µg/ml PI (both Sigma-Aldrich, St. Louis, MO, USA) for 15 min at room temperature. The cells were kept in the dark on ice or at 4°C in a fridge until analysis. Results were analyzed using a BD Accuri C6 Plus flow cytometer with Flowjo software (BD Biosciences, San Jose, CA, USA).

In order to analyze the structural characteristics and secondary structure of TMEM35, SPLIT software version 4.0 (split.pmfst.hr/split/4/) was used (27). For investigation of the poorly understood TMEM35 function and the associated molecular mechanism, the STRING database (http://string-db.org) (27) was used to predict the proteins interacting with TMEM35. The database contains known and predicted protein interactions, including directly (physical) and indirectly (functional) relevant interactions. The data are derived from four sources: Genomic context, high-throughput experiments, coexpression and previous knowledge.

Two-factor analysis of variance was performed to compare the data. Student's t-test was used to determine the statistical significance. The results are presented as mean ± standard deviation. SPSS statistical analysis software, version 21.0 (IBM SPSS, Armonk, NY, USA) was used to analyze the results. P<0.05 was considered to indicate a statistically significant difference.

SPLIT software was used to analyze the structural characteristics and secondary structure of TMEM35 (27). On the basis of the results presented in Fig. 1, TMEM35 is a low molecular weight, cell membrane surface protein with four highly hydrophobic domains. TMEM35 has a structure typical of a tetraspanin, the functions of which have been reviewed previously (28), and include extensive involvement in cell proliferation, adhesion and migration. Therefore, we hypothesize that cell signal transduction pathways are directly or indirectly influenced by TMEM35, due to an effect on tumor cell adhesion, differentiation, migration and invasion.

In order to investigate the TMEM35 expression in OSA, RT-qPCR assays were performed in human OSA samples and non-tumor tissues. As shown in Fig. 2, TMEM35 was found to be upregulated in 24/37 (64.86%) OSA samples, among which 22 samples presented a 2-fold upregulation of TMEM35 (two tailed t-test, P<0.05). These results indicated that TMEM35 overexpression may play an important role in OSA.

To further investigate the effects of TMEM35 expression on OSA cell growth, TMEM35 knockdown was performed by transfection with siRNA, and the effect of TMEM35 knockdown on soft agar colony formation was evaluated. siRNAs specifically targeting the TMEM35 gene (TMEM35-si-1 and TMEM35-si-2) were transfected into SaOS2 and U2OS cells, and RT-qPCR was performed to evaluate the efficiency of gene knockdown. The results indicated that TMEM35 expression decreased significantly following transfection, indicating that the siRNAs silenced TMEM35 expression in the SaOS2 and U2OS cells (Fig. 3A). Subsequently, the effect of TMEM35 knockdown on OSA cell colony formation was examined.

A soft agar colony formation assay of SaOS2 and U2OS cells was performed to evaluate the contact inhibition effect on these tumor cells, and the results are shown in Fig. 3B. Results indicated that the number and size of the cell colonies decreased significantly compared with those in the negative control (si-LUC). Subsequently, the number of SaOS2 and U2OS cell colonies were investigated with crystal violet staining (Fig. 3C). Results demonstrated that TMEM35 knockdown significantly inhibited SaOS2 and U2OS cell growth in the soft agar (two tailed t-test, P<0.05), implying that TMEM35 may play an role in the adherence-independent growth of OSA cells.

In order to explain how the cytological metabolism of TMEM35 knockdown inhibits cell growth in OSA cells, flow cytometric analysis was performed to analyze changes in the cell cycle progression following TMEM35 silencing in SaOS2 cells. Cells transfected with either si-LUC or TMEM35-si-1 were collected and stained with PI. As shown in Fig. 4, the proportion of cells in the G0/G1 phase increased (si-LUC, 61.07±3.21%; TMEM35-si-1, 73.35±4.52%), and the proportion of cells in the S phase decreased (si-LUC, 28.16±1.23%; TMEM35-si-1, 14.55±2.31%) when compared with the cell population in the negative control (si-LUC) following TMEM35 silencing in SaOS2 cells. These collective data indicate that TMEM35 knockdown reduced the S phase cell population and resulted in G1 arrest in SaOS2 cells, which suggests that TMEM35 participated in cell cycle progression.

The migratory potential of transduced OSA cell lines was determined, since metastasis is the main deleterious characteristic of OSA cells. The results indicated that cell migration was reduced subsequent to TMEM35 silencing when compared with that in the parental cells (Fig. 5A). A Boyden chamber assay confirmed the strong association between TMEM35 expression and migratory potential (Fig. 5B and C). These data indicated that TMEM35 expression regulates the migratory potential, in vitro cell migration and invasive abilities of human OSA cells. In addition, the results demonstrated that a reduced TMEM35 expression attenuates the OSA cell aggressiveness.

For investigation of the poorly understood TMEM35 function and the associated molecular mechanism, the STRING database (27) was used to predict the proteins interacting with TMEM35. The database contains known and predicted protein interactions, including directly (physical) and indirectly (functional) relevant interactions. As demonstrated in Fig. 6, TMEM35 may be associated with neuropeptide Y (NPY), nerve growth factor (NGF), NGF receptor (NGFR), brain-derived neurotropic factor (BDNF), vasoactive intestinal peptide (VIP) and calcitonin-related polypeptide β (CALCB). These predicted functional partner proteins indicate that TMEM35 may play an important role in cell differentiation and survival, further revealing the important function of TMEM35.