Upregulation of long non-coding RNA NNT-AS1 promotes osteosarcoma progression by inhibiting the tumor suppressive miR-320a

Abstract

Objective: To investigate the role and mechanism of action of nicotinamide nucleotide transhydrogenase antisense RNA 1 (NNT-AS1) in osteosarcoma (OS).

Methods: Bioinformatic analysis suggested miR-320a as potential target of NNT-AS1. Influence of NNT-AS1 overexpression or knockdown on OS cell proliferation, colony-formation, apoptosis, migration and invasion capacity was first investigated. Expression levels of NNT-AS1, miR-320a, beta-catenin, RUNX2, IGF-1R, c-Myc, Cyclin D1 and MMP13 were also evaluated by RT-qPCR and western blotting accordingly. Xenograft models using U2OS and OS-732 cells with different NNT-AS1 gene modifications were constructed for tumor formation assay as well as evaluation of miR-320a, beta-catenin and RUNX2 expression in primary lesion. NNT-AS1-overexpressing U2OS cells and NNT-AS1-knockdown OS-732 cells were subject to miR-320a mimic and inhibitor transfection, respectively, to investigate the miR-320a dependency of the osteosarcoma-promoting role of NNT-AS1.

Results: NNT-AS1 overexpression significantly increased proliferation, survival and mobility of U2OS cells in vitro as well as its tumor formation ability in vivo, while NNT-AS1 knockdown showed opposite effect on OS-732 cells. In both in vitro and in vivo model, NNT-AS1 expression level significantly correlated with that of beta-catenin, RUNX2, IGF-1R, c-Myc, Cyclin D1 and MMP13 as well as Akt phosphorylation level, and inversely correlated with miR-320a expression. Transfection of miR-320a mimic significantly inhibiter the promoting effect of NNT-AS1 on cell proliferation, survival and mobility of U2OS cells, while miR-320 inhibitor partially rescued that of OS-732 cells.

Conclusion: NNT-As1 functions as a cancer-promoting lncRNA by downregulating miR-320a, thus increasing the protein expression level of beta-catenin, RUNX2 and IGF-1R as well as activation of Akt in osteosarcoma.

Keywords: NNT-AS1; long noncoding RNA; miR-320a; osteosarcoma.

Figure 1.

NNT-AS1 is involved in osteosarcoma cell proliferation, survival and metastasis in vitro. Cells were assayed 24 hours after transfection for NNT-AS1 overexpression (O/E) or knockdown (KD). A, evaluation of NNT-AS1 in U2OS and OS-732 OS cells with NNT-AS1 overexpression or knockdown by transfection. Data of each group was normalized to that of wildtype (WT) U2OS cells transfected with control vector and presented as fold change. B, cell proliferation evaluated by CCK-8 method. Density of cells in each well on a 96-well plate correlated to absorbance at 450 nm (O.D 450 nm) after incubation with CCK-8 for 1 hour. C and D, colony formation assay evaluating clonogenicity of OS cells with or without NNT-AS1 overexpression or knockdown. C is a representative result of colony formation assay. E and F, flow cytometry evaluating Annexin V/PI surface staining of each group of cells after NNT-AS1 overexpression or knockdown. E is a representative result of flow cytometry. G and H, trans-well assay evaluating the invasiveness of OS cells with different NNT-AS1 expression. G is a representative result of trans-well assay. I and J, scratch assay evaluating cell mobility. I is a representative result of scratch assay. Vertical lines in each image indicate the width of the initial scratch at 0 hour. Data in panel D, H and J were normalized to WT group in the same panel and presented as fold change. Students’ t test was used for significance test in panel A, D, F, H and J, and two-way ANOVA was used for significance test in panel B. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 2.

NNT-AS1 increased protein expression level of beta-catenin, RUNX2 as well as their down-stream genes MYC, Cyclin D1 and MMP-13 in OS cells in vitro, while inhibiting miR-320a. Cells were assayed 24 hours after transfection. A, B and C, western blot evaluating protein expression level of beta-catenin (CTNNB1) and RUNX2 in different group of cells. GAPDH was used as housekeeping genes. C is a representative result of western blot. D, RT-qPCR evaluating miR-320a expression level in each group of cells. E ~ H, western blot evaluating protein expression level of c-Myc (MYC), cyclin D1 (CCND1) and MMP-13 (MMP13). GAPDH was used as housekeeping genes. F is a representative result of western blot. Data normalized to the WT group and presented as fold change. Students’ t test was used for significance test. *, p < 0.05; **, p < 0.01.

Figure 3.

Western blot showed that NNT-AS1 increased protein expression level of IGF-1R and Ser473 phosphorylation level of Akt in OS cells in vitro. Cells were treated and analyzed as described in Figure 2. Total protein expression level of Akt was intact under NNT-AS1 overexpression or knockdown conditions. E is a representative result of western blot. Data in panel A ~ D were normalized to the WT in the same panel group and presented as fold change. Students’ t test was used for significance test. *, p < 0.05.

Figure 4.

MiR-320a antagonized the OS-promoting role of NNT-AS1 in vitro. Cells were assayed 24 hours after transfection. A ~ D, western blot evaluating protein expression level of beta-catenin (CTNNB1), RUNX2 and IGF-1R in different group of cells. GAPDH was used as housekeeping genes. D is a representative result of western blot. E and F, cell proliferation assay by CCK-8 (as described in Figure 1A), G and H, evaluation of cell apoptosis by flow cytometry. Cell apoptosis was determined by Annexin V positive staining. G is a representative result of flow cytometry. I and J, tran-well assay evaluating the invasiveness of each group of cells. I is a representative result of trans-well assay. Sidak’s multiple comparisons test was used for significance test in panel E and F, while students’ t test was used for significance test panel A, B, C, H and J. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Figure 5.

NNT-AS1 facilitated OS development in xenograft model. A and B, tumor formation assay evaluating tumor volume in tumor bearing nude mice. OS cells of different groups were subcutaneously injected on the left back of mice. Assay began when initial tumor volume formed by WT cells reached 150 mm³. C and D, tumor weight at the end-point of tumor formation assay. E and F, RT-qPCR evaluating NNT-AS1 and miR-320a in dissected tumor tissue. G ~ J, western blot evaluating protein expression level of beta-catenin (CTNNB1), RUNX2 and IGF-1R in dissected tumor tissues using GAPDH as housekeeping gene. J is a representative result of western blot. Data in panel C, E ~ I were normalized to WT group in the same graph. Sidak’s multiple comparisons test was used for significance test in panel A and B, while students’ t test was used for significance test in panel C, E ~ I. *, p < 0.05; **, p < 0.01; ***, p < 0.001.