The human osteosarcoma cell lines MG63 and U2OS cells were bought from American Type Culture Collection (ATCC) Company. The MG63 and U2OS cells were maintained in DMEM medium with 1% penicillin/streptomycin and 5% fetal bovine serum at 37°C incubator under 5% CO₂ atmosphere.

The human osteosarcoma MG63 and U2OS cells were seeded in 6-well plates for overnight. Then, osteosarcoma cells were transfected with negative control or miR-26a mimics, miR-26a inhibitors, ZEB2 cDNA, shR-ZEB2, shR-SCAMP1, or SCAMP1 cDNA by Lipofectamine 2000 according to the instruction’s approach as described previously (25). After different time transfection, further analysis was performed in transfected osteosarcoma cells for their viability, migration and invasion.

Total RNA was extracted from the transfected osteosarcoma cells using 1ml TRLzol Reagent. Then, 1microgram RNA was used for reverse transcription to generate first-strand cDNA. PCR was conducted using SYBR Green Kit following the manufacturer’s instructions as described before (26).

The transfected osteosarcoma cells were seeded in 96-well plates for overnight. After 24, 48 and 72 hours, CCK8 cell viability assay was used to measure the viability of osteosarcoma cells. Briefly, after different time treatments, 10 μL CCK8 reagent was put into each well and maintained for 3 hours at 37 °C in a humidified incubator. The OD values were measured by the microplate reader at 450 nm wavelength.

The human osteosarcoma MG63 and U2OS cells were seeded in 6-well plates and cultured with full DMEM medium for 10-14 days at 37°C incubator under 5% CO₂ atmosphere. Then, the cells were washed by PBS three times after discarding the medium. 4% paraformaldehyde was used to fix the osteosarcoma cells for 30-40 minutes. The colonies were stained by 0.1% crystal violet for 15 minutes. The images were taken by microscope and the colony numbers were calculated.

The transfected osteosarcoma cells were seeded in top Transwell plates with serum-free DMEM on 24-well plates. The bottom Transwell plates were filled with full DMEM medium, which make the cells transfer from the top plates to the bottom plates via the transwell membranes with Matrigel. After 20 hours, the culture medium in the top plates were removed and the plates were washed three times by PBS. Then, the plates were fixed by 4% paraformaldehyde for half hour and stained by 0.1% crystal violet. The invasive cells were imaged by an inverted microscope.

The transfected osteosarcoma cells were seeded on 6-well plates. Until the cell fluence reached high than 90%, the pipette tip was used to scratch a wound. The cells were washed three times to remove the floatage cells. After 20 hours, the wound location was taken photographs as described before (27).

To confirm the interaction between lncRNA SCAMP1 and miR-26a-5p, we performed luciferase report assay. The mutant binding sequences of miR-26a-5p in lncRNA SCAMP1 were cloned into pmirGLO dual-luciferase vector. The osteosarcoma cells were transfected with various plasmids. The lncRNA SCAMP1 mutant had the mutant binding sequences of miR-26a-5p. The luciferase activity was measured following manufacturer’s instructions.

In this study, all data were analyzed by GraphPad Prism 5.0. Student t test was performed to measure significance between two groups. ANOVE was used to compare three or more groups. The results were descripted as means ± SD. P < 0.05 was considered as statistically significant.

To check the function of lncRNA SCAMP1 in osteosarcoma cells, we transfected the SCAMP1 cDNA into MG63 and U2OS cells using Lipofectamine 2000. Our RT-PCR data indicated that lncRNA SCAMP1 was highly elevated in MG63 and U2OS cells after SCAMP1 cDNA transfection (Figure 1A). Moreover, shR-SCAMP1 was transfected into MG63 and U2OS cells by Lipofectamine 2000. We found that lncRNA SCAMP1 expression was remarkable reduced in MG63 and U2OS cells after shR-SCAMP1 transfection (Figure 1A). To examine whether the cell viability was governed by lncRNA SCAMP1, CCK8 assay was utilized in MG63 and U2OS cells after lncRNA SCAMP1 modulation. As expected, shR-SCAMP1 transfected MG63 cells exhibited the less viability at 48 hours and 72 hours (Figure 1B). Consistently, SCAMP1 cDNA transfection led to high viability at 48 hours and 72 hours in U2OS cells (Figure 1C). To validate this role of lncRNA SCAMP1 in osteosarcoma cells, colony formation assay was conducted in MG63 and U2OS cells after lncRNA SCAMP1 knockdown and overexpression, respectively. The data showed that knockdown of lncRNA SCAMP1 reduced the number of colony formation in MG63 cells (Figure 2A). In line with this result, lncRNA SCAMP1 overexpression increased the number of colony formation in U2OS cells (Figure 2B). Therefore, lncRNA SCAMP1 could govern the viability of osteosarcoma cells.

It is known that Transwell invasion assay is a good approach for detection of cell invasive ability in cancer. We used Transwell invasion assay to test the function of lncRNA SCAMP1 in osteosarcoma cells. As shown in Figures 3A, B, SCAMP1 cDNA transfection resulted in promotion of invaded cell numbers in both MG63 and U2OS cells. The invasiveness ability in shR-SCAMP1 transfected osteosarcoma cells was also examined using Transwell invasion assay. Expectedly, shR-SCAMP1 transfected cells had a reduction in invaded cell numbers in U2OS and MG63 cells (Figures 3A, B). Wound healing assay is often used for measuring the migrative ability in cancer cells. Therefore, we used the wound healing assay in our study to check the role of lncRNA SCAMP1 in regulation of migration in osteosarcoma cells. The wound healing assay data showed that shR-SCAMP1 transfected cells had a slower rate to close the wound area in U2OS and MG63 cells (Figures 3C, D). On the contrary, both osteosarcoma cell lines with SCAMP1 cDNA transfection had a faster rate to close the wound area (Figures 3C, D). Hence, lncRNA SCAMP1 could govern the invasive and migrative abilities in osteosarcoma cells.

It has been known that lncRNAs often sponge with miRNAs to suppress their expressions. Therefore, we aimed to explore the lncRNAs that could bind to lncRNA SCAMP1 in osteosarcoma cells. Based on the TargetScan database from website, we saw the binding sites between lncRNA SCAMP1 and miR-26a-5p (Figure 4A). The luciferase reporter gene assay is a regular approach to confirm the interaction between lncRNAs and miRNAs. The data from the dual luciferase assay showed that miR-26a-5p mimic transfection led to a reduction in the luciferase activity in the lncRNA SCAMP1 wild-type group (Figure 4B). On the contrary, this phenotype was not observed in the lncRNA SCAMP1 mutant group (Figure 4B). Moreover, miR-26a-5p inhibitor increased the luciferase activity in lncRNA SCAMP1 wild-type group, but not in lncRNA SCAMP1 mutant group (Figure 4B). We also measured the expression of miR-26a-5p in U2OS and MG63 cells after miR-26a-5p mimics transfection and miR-26a-5p inhibitor treatment, respectively. Our data showed that miR-26a inhibitors decreased the expression of miR-26a-5p in MG63 cells, while miR-26a mimic transfection increased the miR-26a-5p expression levels in U2OS cells (Figure 4C). Notably, upregulation of wild-type lncRNA SCAMP1 suppressed the expression of miR-26a-5p in MG63 cells, but this phenotype did not exhibit in mutant lncRNA SCAMP1 group (Figure 4D). Strikingly, shR-SCAMP1 group showed the high expression of miR-26a-5p compared with shR-NC group in U2OS cells (Figure 4D). Hence, lncRNA SCAMP1 can interact with and regulate miR-26a-5p inU2OS and MG63 cells.

To check whether miR-26a-5p participates in lncRNA SCAMP1-mediated promotion of cell viability in osteosarcoma cells, the MG63 cells were treated with miR-26a-5p mimics in combination with lncRNA SCAMP1 cDNA transfection. Our CCK8 results indicated that miR-26a-5p mimic transfection suppressed viability of MG63 cells at 48 hours and 72 hours (Figure 5A). Overexpression of lncRNA SCAMP1 abolished miR-26a-5p mimics-mediated inhibition of cell viability in MG63 cells (Figure 5A). Moreover, inhibition of miR-26a-5p enhanced viability of U2OS cells, which was abrogated by shR-SCAMP1 transfection (Figure 5B). In line with these results, colony formation data revealed that miR-26a-5p mimics reduced the colony numbers in MG63 cells, while miR-26a-5p inhibitors increased the colony numbers in U2OS cells (Figure 5C and 65D). In SCAMP1-overexpressing MG63 cells, miR-26a-5p mimics-mediated suppression of colony formation was abolished in MG63 cells (Figure 5C). In U2OS cells with shR-SCAMP1 transfection, miR-26a-5p-inhibitor-induced colony formation was blocked in U2OS cells (Figure 5D). Taken together, lncRNA SCAMP1 overexpression promotes cell viability via miR-26a-5p in osteosarcoma cells.

It is known that miRNAs performed their functions in tumorigenesis via inhibiting the expression of their downstream genes. According to the TargetScan database from website, we observed the binding sites between miR-26a-5p and ZEB2, indicating that miR-26a-5p could interact with ZEB2 and suppress its expression (Figure 6A). In MG63 cells, we found that miR-26a-5p mimics attenuated the mRNA levels of ZEB2 (Figure 7B). Similarly, in U2OS cells, we found that miR-26a-5p inhibitors elevated the mRNA levels of ZEB2 (Figure 6B). Moreover, we tested whether lncRNA SCAMP1 regulated the expression of ZEB2 in osteosarcoma cells. We found that SCAMP1-overexpressing cells had the high expression of ZEB2 in MG63 cells (Figure 6B). In shR-SCAMP1 transfected U2OS cells, ZEB2 mRNA levels were remarkable downregulated (Figure 6B). Altogether, miR-26a-5p could target ZEB2 in osteosarcoma cells.

To determine whether lncRNA SCAMP1 increased viability of osteosarcoma cells via targeting miR-26a/ZEB2 axis, MG63 cells were co-transfected with miR-26a-5p mimics and ZEB2 cDNA, or SCAMP1 and sh-ZEB2. Our CCK8 assay data demonstrated that pZEB2 cDNA transfection increased cell viability in MG63 cells (Figure 7A). Upregulation of ZEB2 abrogated miR-26a-5p-induced inhibition of cell viability in MG63 cells (Figure 7A). Moreover, SCAMP1 overexpression elevated cell viability, which was blocked by downregulation of ZEB2 in MG63 cells (Figure 7A). Similarly, ZEB2 cDNA transfection increased colony formation of MG63 cells, which was abrogated by miR-26a mimics transfection (Figure 7B). LncRNA SCAMP1 upregulation elevated colony formation of MG63 cells, which was blocked by sh-ZEB2 transfection (Figure 7B).

To examine whether lncRNA SCAMP1 promoted invasion and migration of osteosarcoma cells via targeting miR-26a/ZEB2 axis, MG63 cells were co-transfected with miR-26a-5p mimics plus ZEB2 cDNA, or SCAMP1 plus shZEB2. Our Transwell invasion assay data dissected that pZEB2 cDNA transfection increased cell invasion ability in MG63 cells (Figure 7C). Upregulation of ZEB2 abrogated miR-26a-5p-induced suppression of cell invasion in MG63 cells (Figure 7C). Moreover, SCAMP1 overexpression elevated cell invasion, which was reduced by downregulation of ZEB2 in MG63 cells (Figure 7C). Similarly, ZEB2 cDNA transfection increased migrative ability of MG63 cells, which was abrogated by miR-26a mimics transfection (Figure 7D). LncRNA SCAMP1 upregulation elevated migrative ability of MG63 cells, which was blocked by sh-ZEB2 transfection (Figure 7D). In a word, lncRNA SCAMP1 regulated cell invasiveness and migrative ability through targeting miR-26a and ZEB2 pathways in osteosarcoma.