LINC00963 facilitates acute myeloid leukemia development by modulating miR-608/MMP-15

Abstract

Despite continuous improvements of AML therapy, the prognosis of AML patients remains unsatisfactory. Recently, lncRNAs have been reported to participate in the development of AML. Our data demonstrated that MMP15 and LINC00963 were upregulated and miR-608 was decreased in AML cells (THP-1, HL-60, HEL and MOLM-13) compared to HS-5 cells. RT-qPCR results showed that LINC00963 levels were higher in the serum and bone marrow of AML cases than in controls. Moreover, overexpression of LINC00963 promoted AML cell growth and EMT progression in both THP-1 and HL-60 cells. Furthermore, miR-608 levels were downregulated in the serum and bone marrow of AML cases compared with controls, and Pearson's correlation analysis indicated that LINC00963 was negatively correlated with miR-608 in the serum and bone marrow of AML samples. In addition, we demonstrated that LINC00963 sponged miR-608 expression and that MMP-15 was a target of miR-608 in AML cells. Finally, rescue experiments indicated that ectopic expression of LINC00963 accelerated cell growth and EMT development by modulating MMP-15. These data demonstrated that LINC00963 acted as an oncogene and may be a potential target for AML treatment.

Keywords: LINC00963; MMP-15; acute myeloid leukemia; miR-608.

Figure 1

MMP15, LINC00963 and miR-608 levels in AML cells. (A) MMP15 was overexpressed in AML cells (THP-1, HL-60, HEL and MOLM-13) compared with HS-5 cells. GAPDH was used as the internal control. (B) The expression of LINC00963 was determined by RT-qPCR analysis. GAPDH was used as the internal control. (C) miR-608 was decreased in AML cells (THP-1, HL-60, HEL and MOLM-13) compared to HS-5 cells. U6 was used as the internal control for miR-608.

Figure 2

LINC00963 level in AML specimens. (A) LINC00963 levels were higher in the bone marrow of AML cases than in controls. (B) The expression of LINC00963 in the serum of AML cases and controls was measured by RT-qPCR. GAPDH was used as the internal control.

Figure 3

miR-608 levels in AML specimens. (A) The expression of miR-608 in the bone marrow of AML cases and controls was measured by RT-qPCR. (B) Pearson’s correlation analysis indicated that LINC00963 was negatively correlated with miR-608 in the bone marrow of AML samples. (C) miR-608 levels were downregulated in the serum of AML cases compared with controls. (D) Pearson’s correlation analysis sowed that LINC00963 was negatively correlated with miR-608 in the serum of AML samples. U6 was used as the internal control.

Figure 4

Overexpression of LINC00963 promoted AML cell growth and EMT progression. (A) The expression of LINC00963 was measured by RT-qPCR in both THP-1 and HL-60 AML cells. (B) CCK-8 assay showed that overexpression of LINC00963 accelerated cell growth in both THP-1 and HL-60 cells. (C) Elevated expression of LINC00963 facilitated ki-67 expression in both THP-1 and HL-60 cells. (D) RT-qPCR assay indicated that elevated expression of LINC00963 accelerated PCNA levels in both THP-1 and HL-60 cells. (E) Ectopic expression of LINC00963 enhanced Vimentin, N-cadherin and ZEB1 expression and decreased E-cadherin expression in both THP-1 and HL-60 cells. *p<0.05, **p<0.01 and ***p<0.001. GAPDH was used as the internal control.

Figure 5

LINC00963 inhibited miR-608 expression in AML cells. (A) An online database (starBase) predicted that miR-608 was a potential target of LINC00963. (B) The level of miR-608 was determined by RT-qPCR. (C) Luciferase reporter assays verified that elevated expression of miR-608 decreased the luciferase value in the LINC00963-wt group but did not change the luciferase value in the LINC00963-mut group. (D) Overexpression of LINC00963 suppressed miR-608 levels in THP-1 cells. **p<0.01. U6 was used as the internal control.

Figure 6

MMP-15 was a target of miR-608. (A) An online database (TargetScan) predicted that MMP-15 was a potential target of miR-608. (B) Luciferase reporter assays verified that elevated expression of miR-608 decreased the luciferase value in the MMP-15-wt group but did not change the luciferase value in the MMP-15-mut group. (C) Overexpression of miR-608 suppressed MMP-15 levels in THP-1 cells. (D) Ectopic expression of LINC00963 upregulated MMP-15 levels in THP-1 cells. *p<0.05. GAPDH was used as the internal control.

Figure 7

Ectopic expression of LINC00963 increased cell growth and EMT development by modulating MMP-15. (A) The level of MMP-15 was determined by RT-qPCR. (B) CCK-8 assay was performed to detect cell proliferation. (C) The level of ki-67 was measured by RT-qPCR. (D) The expression of PCNA was measured by RT-qPCR. (E) Downregulation of MMP-15 suppressed Vimentin, N-cadherin and ZEB1 levels and upregulated E-cadherin expression in LINC00963-overexpressing THP-1 cells. *p<0.05 and**p<0.01. GAPDH was used as the internal control.