Downregulation of lncRNA CASC2 facilitates osteosarcoma growth and invasion through miR-181a

Abstract

Objectives: Long non-coding RNA cancer susceptibility candidate 2 (CASC2) is a novel lncRNA and has been indicated as playing tumour suppressor gene in several tumours. However, the role of CASC2 in osteosarcoma is still uncovered.

Materials and methods: The CASC2 and miR-181a expressions were measured via qRT-PCR. CCK-8 assay and colony formation assay were performed to determine the cell growth, and transwell assay was performed to assess the cell invasion.

Results: We showed that CASC2 expression was downregulated in osteosarcoma samples and cell lines. Moreover, we showed that downregulated expression of CASC2 was correlated with advanced TNM stage. Furthermore, overexpression of CASC2 inhibited osteosarcoma cell proliferation, colony formation, and invasion. In addition, we indicated that ectopic expression of CASC2 suppressed miR-181a expression and enhanced the expression of Ras association domain family member 6 (RASSF6), PTEN and ATM in osteosarcoma cell, which were the direct target gene of miR-181a. Moreover, we indicated that RASSF6 expression was downregulated in osteosarcoma samples and cell lines and downregulated expression of RASSF6 was correlated with advanced TNM stage. We found that the expression of RASSF6 was positively correlated with the expression of CASC2 in osteosarcoma tissues. Ectopic expression of CASC2 suppressed the osteosarcoma cell proliferation, colony formation and invasion through regulating RASSF6 expression.

Conclusions: Our data illuminated that CASC2 acted as a tumour suppressor in osteosarcoma progression.

Figure 1

Cancer susceptibility candidate 2 (CASC2) expression was downregulated in the osteosarcoma tissues. A, The expression of CASC2 in the osteosarcoma samples and non‐tumour tissues was determined by qRT‐PCR. U6 was used as the internal control. B, The CASC2 expression level in the osteosarcoma samples was lower than in the non‐tumorous samples. C, The lower expression of CASC2 was associated with the advanced TNM stage. *P < .05

Figure 2

Cancer susceptibility candidate 2 (CASC2) suppressed the osteosarcoma cell proliferation. A, The expression of CASC2 in the osteosarcoma cell lines (MG‐63, U2OS, SOSP‐9607 and Saos‐2) and human osteoblasts cell line (hFOB) was measured by qRT‐PCR. U6 was used as the internal control. B, The expression level of CASC2 in the MG‐63 cell with transfecting pcDNA‐CASC2 vector was determined using qRT‐PCR. C, The expression level of CASC2 in the U2OS cell was measured by qRT‐PCR. D, Ectopic expression of CASC2 decreased the MG‐63 proliferation. The cell proliferation was determined by CCK‐8 assay. E, CCK‐8 assay was performed to measure the U2OS cell proliferation. *P < .05, **P < .01 and ***P < .001

Figure 3

Cancer susceptibility candidate 2 (CASC2) inhibited the osteosarcoma cell colony formation and invasion. A, Elevated expression of CASC2 decreased the MG‐63 and U2OS cell invasion. Relative ratio of invasive cells per field is shown. B, Ectopic expression of CASC2 suppressed the MG‐63 and U2OS cell colony formation. The relative colony numbers were shown. ***P < .001

Figure 4

Cancer susceptibility candidate 2 (CASC2) suppressed the miR‐181a expression in the osteosarcoma cell. A, Ectopic expression of CASC2 suppressed the expression of miR‐181a in the MG‐63 cell. B, Overexpression of CASC2 promoted the Ras association domain family member 6 (RASSF6) expression in the MG‐63 cell. C, Ectopic CASC2 expression elevated the PTEN expression in the MG‐63 cell. D, CASC2 overexpression enhanced the ATM expression in the MG‐63 cell

Figure 5

Ras association domain family member 6 (RASSF6) expression was downregulated in the osteosarcoma tissues. A, The RASSF6 expression in the osteosarcoma samples and non‐tumour tissues using qRT‐PCR. GAPDH was used as the control. B, The RASSF6 expression was significantly downregulated in the osteosarcoma tissues compared with non‐tumorous samples. C, The lower expression of RASSF6 was associated with the advanced TNM stage. D, The expression of RASSF6 was positively correlated with the expression of CASC2 in the osteosarcoma tissues. E, The expression level of RASSF6 in the osteosarcoma cell lines (MG‐63, U2OS, SOSP‐9607 and Saos‐2) and human osteoblasts cell line (hFOB) was determined by qRT‐PCR. *P < .05 and **P < .01

Figure 6

Cancer susceptibility candidate 2 (CASC2) suppressed the osteosarcoma cell proliferation, colony formation and invasion through regulating Ras association domain family member 6 (RASSF6) expression. A, The expression of RASSF6 in the MG‐63 cell transfected with si‐RASSF6 was determined by qRT‐PCR. B, The protein expression of RASSF6 was measured by Western blot. C, Knockdown of RASSF6 promoted the CASC2 overexpressing MG‐63 cell proliferation. D, Downregulation of RASSF6 enhanced the CASC2‐overexpressing MG‐63 cell colony formation. The relative colony numbers were shown. E, Downregulation of RASSF6 enhanced the CASC2‐overexpressing MG‐63 cell invasion. Relative ratio of invasive cells per field is shown. *P < .05, **P < .01 and ***P < .001