LINC01006 inhibits proliferation and invasion and promotes apoptosis of nephroblastoma cells by down-regulating miR-148a-3p to block MEOX2/PI3K/Akt pathway

Abstract

Objective To investigate the role of long noncoding RNA LINC01006 in regulating the proliferation, invasion and apoptosis of nephroblastoma cells and explore the molecular mechanism. Methods We detected the expression of LINC01006 in nephroblastoma tissues and cell lines using real-time quantitative PCR (RT-qPCR).We subsequently tested the effects of transfection with an adenovirus vector for LINC01006 overexpression or a siRNA targeting LINC01006 on cell viability, proliferation, invasion and apoptosis in human nephroblastoma cells using cell counting kit-8 (CCK-8), 5-bromo-2-deoxyuridine (BrdU) cell proliferation assay kit, Transwell assay or flow cytometry as appropriate.Bioinformatics analysis was conducted to identify the target gene of LINC01006, and the results were verified by luciferase reporter gene analysis.The expression of miR-148a-3p and its target gene mesoderm homeobox2 (MEOX2) was detected in human nephroblastoma cells with overexpression and knockdown of LINC01006.The tumor cell lines transfected with an empty adenovirus vector or a LINC01006-overexpressing vector were injected subcutaneously into nude mice to establish mouse models bearing transplanted nephroblastoma, in which the tumor growth dynamics were observed.The expression levels of LINC01006, miR-148a-3p, MEOX2, phosphorylated-phosphatidylinositol- 3 kinase (p-PI3K), and phosphorylated-protein kinase B (p-PKB /p-Akt) were examined in the tumor tissues. Results The nephroblastoma tissues and cell lines expressed significantly lower level of LINC01006 than normal tissues and control cell lines (P < 0.01).LINC01006 over-expression significantly inhibited the viability (P < 0.01), proliferation (P < 0.01) and invasion (166.00±13.75 vs 103.00±9.24, P < 0.05) and promoted apoptosis (11.30 ± 0.24 vs 37.25 ± 1.57, P < 0.01)of nephroblastoma cells in vitro.Luciferase reporter gene analysis confirmed that LINC01006 bound directly to miR-148a-3p, which targets MEOX2 gene.Overexpression of LINC01006 obviously inhibited the expression of miR-148a-3p (1.07±0.02 vs 0.32±0.03, P < 0.01), while inhibiting miR-148a-3p promoted the expression of MEOX2 (P < 0.01)and inhibited the activation of the PI3K /Akt signaling pathway (P < 0.05).Compared with the cells transfected with pcDNA- LINC01006, the cells co-transfected with LINC01006 and miR-148a-3p exhibited a lowered expression of MEOX2 (P < 0.01) with enhanced activity of PI3K /Akt signaling pathway (P < 0.01), significantly increased cell viability, proliferation and invasion (P < 0.01), and decreased cell apoptosis (P < 0.01).Overexpression of LINC01006 in the tumor cells significantly inhibited the growth of the transplanted tumors in nude mice (P < 0.01). Conclusion LINC01006 can directly interact with miR-148a-3p to down-regulate the expression of the latter, thus blocking the activity of MEOX2 /PI3K /Akt pathway to inhibit the proliferation and invasion and promote apoptosis of nephroblastoma cells.

Keywords

• nephroblastoma
• linc01006
• mir-148a-3p
• meox2/pi3k/akt pathway
• proliferation and
• nvasion
• apoptosis