Long noncoding RNA DNAJC3-AS1 promotes osteosarcoma progression via its sense-cognate gene DNAJC3

Abstract

Long noncoding RNAs have been proved to play essential roles in tumor development and progression. In this study, we focused on DNAJC3-AS1 and investigated its biological function and clinical significance in osteosarcoma. We detected the expression of DNAJC3-AS1 in 30 pairs of matched osteosarcoma and adjacent nontumorous specimens and osteosarcoma cell lines and analyzed association between DNAJC3-AS1 levels and clinicopathological factors. We found that DNAJC3-AS1 expression was up-regulated in osteosarcoma. High level of DNAJC3-AS1 was correlated with high differentiated degree (P = 0.018) and advanced Enneking stage (P = 0.016). Mechanistically, DNAJC3-AS1 enhanced cell proliferation, migration, and invasion in vitro and in vivo and reduced sensitivity of osteosarcoma to cisplatin. These effects of DNAJC3-AS1 were reversed by down-regulation of its sense-cognate gene DNAJC3. Thus, DNAJC3-AS1 promotes osteosarcoma development and progression by regulating DNAJC3 and might be a biomarker and therapeutic target for osteosarcoma.

Keywords: DNAJC3-AS1; cisplatin; eIF2α; metastasis; osteosarcoma; proliferation.

Figure 1

Osteosarcoma specimens and cell lines exhibit higher DNAJC3‐AS1 expression level. A, The expression of DNAJC3‐AS1 in OS specimens (n = 30) was compared with the pair‐matched noncancerous specimens (n = 30). B, The expression level of DNAJC3‐AS1 in HOS and SAOS‐2 were compared with hFOB1.19. C, The subcellular location of DNAJC3‐AS1 was identified using qRT‐PCR in HOS cells and SAOS‐2 cells. D, Fold change of DNAJC3‐AS1 in stable transfected HOS cells detecting by qRT‐PCR analysis. E, Fold change of DNAJC3 in stable transfected HOS cells detecting by qRT‐PCR analysis. We define up‐regulated LncRNA DNAJC3‐AS1 as up‐Lnc, down‐regulated LncRNA DNAJC3‐AS1 as sh‐RNA1 or 2 and their respective control group as up‐ctrl and sh‐ctrl. Data were expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 2

DNAJC3‐AS1 promotes cells proliferation, migration, and invasion capacity of HOS cells (A) CCK‐8 assay, (B (left) and D) Clone formation and (B(right) and E) Soft agar clone formation showed that down‐regulated DNAJC3‐AS1 suppressed proliferation of HOS cells, and up‐regulated DNAJC3‐AS1 did the opposite. (F and C (left)) wound healing assay and (G and C (middle)) migration assay showed that up‐regulated DNAJC3‐AS1 improved migration ability of HOS cells, and down‐regulated DNAJC3‐AS1 did the opposite. (H and C (right)) Invasion assay showed that DNAJC3‐AS1 improved invasion capacity of HOS cells. All the photographs were randomly selected and taken at ×100 field. Scale bar, 200 μm. Data were expressed as the mean ± SD. The results were reproducible in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 3

DNAJC3‐AS1 not only promotes HOS cells proliferation, but also inhibits HOS cells apoptosis and increases drug resistance to cisplatin of HOS cells. A and B, Flow cytometer analysis indicated that up‐regulated DNAJC3‐AS1 increased the percentage of S phase cells and decreased the percentage of G0/G1 phase cells, and down‐regulated DNAJC3‐AS1 did the opposite. C and D, Cell apoptosis assay showed that down‐regulated DNAJC3‐AS1 promoted HOS cells apoptosis rate. And up‐regulated DNAJC3‐AS1 reduced HOS cells apoptosis rate. E, CCK8 assay showed that up‐regulated DNAJC3‐AS1 reduces sensitivity of HOS cells to cisplatin, and down‐regulated DNAJC3‐AS1 does the opposite. F, The IC50 of HOS cells with up‐regulated or down‐regulated DNAJC3‐AS1 and their respective control groups. Data were expressed as the mean ± SD. The results were reproducible in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 4

DNAJC3‐AS1 promotes OS growth and inhibits cells apoptosis and accelerates distant metastasis of OS cells in vivo. A, Representative photographs taking at 28th day after injecting transfected HOS cells subcutaneously into the scruff of male BALB/C‐nu mice (arrows show the tumor nodules), and the statistic showed that DNAJC3‐AS1 increased the growth speed of tumor nodules in vivo significantly more than in the control group, and down‐regulated DNAJC3‐AS1 did the opposite. B, Representative images and the average weight of tumor nodules collected from each group at 28th day after injection. C, Immunohistochemical staining of Ki‐67 (x400; scale bar: 100 μm) and (D left) statistic analysis of Ki‐67 positive staining cells rate indicated that DNAJC3‐AS1 promotes OS cells proliferation in vivo. E, Emblematic figures of TUNEL assay (400 × , scale bar: 100 μm) and (D middle) statistic analysis of TUNEL‐positive staining cells rate indicated that DNAJC3‐AS1 promotes OS cells proliferation in vivo. F, Emblematic photographs of nude mice lung taking at 28th day after injected transfected HOS cells via tail vein. And (D right) the statistics of tumor nodules number which appeared on the surface of lung (arrows show the tumor nodules). G, The microscopic sections of lung tissues with HE stain, the metastatic tumor was shown as indicated by the arrows. Data were expressed as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 5

DNAJC3‐AS1 associates positively with its sense‐cognate gene DNAJC3 in HOS cells. A, Schematic diagram of the structure of DNAJC3‐AS1 and DNAJC3. Arrows indicate the transcription direction of gene, and blocks are representative of exons. B, The expression of DNAJC3 in OS specimens (n = 30) was compared with the pair‐matched noncancerous specimens (n = 30). C, The expression level of DNAJC3 in HOS and SAOS‐2 were compared with hFOB1.19. D, Correlation analysis revealed apparent positive relationship between DNAJC3‐AS1 and DNAJC3 in OS specimens (r = 0.75, P < 0.001). E, Western blot analysis of DNAJC3 in HOS cells with down or up‐regulating DNAJC3‐AS1. Data were expressed as the mean ± SD. The results were reproducible in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 6

DNAJC3‐AS1 accelerates osteosarcoma progression via DNAJC3. A, HOS cells with stably down‐regulated (Sh‐RNA1 and Sh‐RNA2) or up‐regulated DNAJC3‐AS1 (up‐DNAJC3‐AS1) were over‐expressed with DNAJC3 (up‐DNAJC3) or interfered with DNAJC3 siRNA (si‐RNA1 and si‐RNA2). DNAJC3 expression in these cell sets was detected by RT‐PCR. B, Proliferation of the cell sets depicted in (A) was determined by CCK‐8 assay. C, Migration capacity of the cell sets was determined by wound healing assay. D, Western blot analysis of eIF2α and eIF2α‐pSer 51 in HOS cells with down‐regulated or up‐regulated DNAJC3‐AS1. Data were expressed as the mean ± SD, all the images are 400×, scale bar, 100 μm. The results were reproducible in three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001