Dysregulation of LINC00470 and METTL3 promotes chemoresistance and suppresses autophagy of chronic myelocytic leukaemia cells

Abstract

Cytoplasmic lncRNAs have been found to directly interact with target mRNAs and regulate their stability. In this study, we aimed to study the molecular mechanism underlying the function of m⁶ A as a central regulator in chemoresistance and CML proliferation. In this study, we established three mice groups (control group, ADR-R group and ADR-R + shLINC00470 group). We detected PTEN mRNA expression in the presence of LINC00470 in the mice models, as well as in the KCL22 and K562 cells. LINC00470 was significantly enriched for PTEN mRNA to exhibit a negative regulatory relationship between LINC00470 and PTEN mRNA. However, the alteration of LINC00470 had no effect on the luciferase activity of PTEN promoter, while the half-life of PTEN mRNA was affected. It was further validated that LINC00470 down-regulated PTEN expression by positively regulating the m6A modification of PTEN mRNA via RNA methyltransferase METTL3. Moreover, the relative expression of LC3II, Beclin-1, ATG7 and ATG5 was all decreased in cells treated with LINC00470, and down-regulated PTEN expression was observed in chemo-resistant cells, while the expression of PTEN was rescued by the transfection of shMETTL3 into chemo-resistant cells. Moreover, the knockdown of METTL3 also restored the normal level of PTEN m⁶ A modification and LINC00470 expression in chemo-resistant cells. In conclusion, our results demonstrated the molecular mechanism underlying the effect of LINC00470 on CML by reducing the PTEN stability via RNA methyltransferase METTL3, thus leading to the inhibition of cell autophagy while promoting chemoresistance in CML.

Keywords: AKT; Chronic myelocytic leukaemia; LINC00470; METTL3; PTEN; autophagy; chemoresistance.

FIGURE 1

LINC00470 down‐regulated PTEN expression and promoted the degradation of PTEN mRNA. A, LINC00470 in KCL22 and K562 cells was enriched for PTEN mRNA in comparison with the empty vector or IgG (*P value < .05 vs IgG group); B, RNA pull‐down assay of PTEN mRNA expression in KCL22 cells (*P value < .05 vs Beads group); C, Western blot analysis indicated that the knockdown of LINC00470 up‐regulated PTEN expression (*P value < .05 vs shNC group); D, Western blot analysis indicated that the overexpression of LINC00470 down‐regulated PTEN expression (*P value < .05 vs NC group); E, Luciferase reporter assay of PTEN promoter showed that the knockdown of LINC00470 in KCL22 cells had no effect on the luciferase activity of PTEN promoter; F, Luciferase reporter assay of PTEN promoter showed that the overexpression of LINC00470 in KCL22 cells had no effect on the luciferase activity of PTEN promoter; G, The knockdown of LINC00470 extended the half‐life of PTEN mRNA in KCL22 cells; H, The overexpression of LINC00470 shortened the half‐life of PTEN mRNA in K562 cells

FIGURE 2

LINC00470 down‐regulated PTEN expression by positively regulating the m6A modification of PTEN mRNA via RNA methyltransferase METTL3. A, The MeRIP assay showed that PTEN mRNA was significantly enriched by m⁶A antibody compared to the negative control IgG in KCL22 and K562 cells (*P value < .05 vs IgG group); B, The relative enrichment of m⁶A was inhibited by the knockdown of LINC00470 in KCL22 cells (*P value < .05 vs. shNC group); C, The relative enrichment of m⁶A was promoted by the overexpression of LINC00470 in K562 cells (*P value < .05 vs shNC group); D, An RIP assay using METTL3, METTL14 and WTAP antibodies showed that METTL3 antibody enriched the transcripts of LINC00470 compared with METTL14 and WTAP antibodies (*P value < .05 vs IgG group); E, RNA pull‐down assay exhibited an obvious interaction between LINC00470 and METTL3; F, The binding of METTL3 to PTEN mRNA was suppressed in LINC00470‐depleted KCL22 cells (*P value < .05 vs shNC group); G, The binding of METTL3 to PTEN mRNA was augmented in LINC00470‐overexpressing K562 cells (*P value < .05 vs NC group); H, The knockdown of METTL3 in K562 cells rescued the down‐regulation and degradation of PTEN mRNA induced by LINC00470 (*P value < .05 vs NC group; **P value < .05 vs LINC00470 group); I, The knockdown of METTL3 in K562 cells rescued the down‐regulation and degradation of PTEN protein induced by LINC00470; J, The knockdown of METTL3 in K562 cells recovered the normal level of PTEN m⁶A modification increased by LINC00470 (*P value < .05 vs NC group; **P value < .05 vs LINC00470 group); K, The knockdown of LINC00470 increased the luciferase activity of wild‐type PTEN in KCL22 cells (*P value < .05 vs shNC group); L, The overexpression of LINC00470 decreased the luciferase activity of wild‐type PTEN in K562 cells (*P value < .05 vs NC group)

FIGURE 3

LINC00470 down‐regulated the expression of LC3II, Beclin‐1, ATG7 and ATG5 via RNA methyltransferase METTL3 (*P value < 0.05 vs NC group; **P value < .05 vs LINC00470 group). A, The expression of LC3II, Beclin‐1, ATG7 and ATG5 proteins in different K562 cell groups was measured with Western blot; B, The relative expression of LC3II was decreased in the presence of LINC00470; C, The relative expression of Beclin‐1 was decreased in the presence of LINC00470; D, The relative expression of ATG7 was decreased in the presence of LINC00470; E, The relative expression of ATG5 was decreased in the presence of LINC00470

FIGURE 4

Knockdown of LINC00470 recovered the expression of PTEN inhibited in chemo‐resistant cells (*P value < .05 vs Parental group; **P value < .05 vs Resistant group). A, Western blot analysis showed down‐regulated PTEN protein expression in the chemo‐resistant cell group, and the transfection of shLINC00470 rescued PTEN protein expression; B, PCR showed down‐regulated PTEN mRNA expression in the chemo‐resistant cell group, and the transfection of shLINC00470 rescued PTEN mRNA expression; C, The knockdown of LINC00470 recovered the normal level of PTEN m⁶A modification boosted in the chemo‐resistant cell group; D, The knockdown of LINC00470 recovered the normal level of METTL3 mRNA expression boosted in the chemo‐resistant cell group

FIGURE 5

Knockdown of LINC00470 reversed the inhibited expression of LC3II, Beclin‐1, ATG7 and ATG5 proteins in chemo‐resistant cells (*P value < .05 vs Parental group; **P value < .05 vs Resistant group). A, Western blot analysis of LC3II, Beclin‐1, ATG7 and ATG5 protein expression in different cell groups; B, LC3II protein expression was inhibited in chemo‐resistant cells, which were inhibited by the knockdown of LINC00470; C, Beclin‐1 protein expression was inhibited in chemo‐resistant cells, which were inhibited by the knockdown of LINC00470; D, ATG7 protein expression was inhibited in chemo‐resistant cells, which were inhibited by the knockdown of LINC00470; E, ATG5 protein expression was inhibited in chemo‐resistant cells, which were inhibited by the knockdown of LINC00470

FIGURE 6

Knockdown of LINC00470 recovered the inhibited PTEN expression in ADR mice (*P value < .05 vs Control group; **P value < .05 vs ADR‐R group). A, Tumour volume was evidently increased in ADR‐R mice, and the presence of shLINC00470 suppressed the tumour growth in ADR mice; B, PTEN mRNA expression was evidently reduced in ADR‐R mice and recovered in ADR‐R + shLINC00470 mice; C, MeRIP assay showed that the knockdown of LINC00470 in vivo could reduce the up‐regulated PTEN m⁶A modification in ADR‐R mice; D, MeRIP assay showed that the knockdown of LINC00470 in vivo could reduce the up‐regulated LINC00470 expression in ADR‐R mice; E, Upon the ADR treatment, tumour volume was evidently increased in ADR‐R mice, and the presence of shLINC00470 suppressed the tumour growth in ADR mice; F, Upon the ADR treatment, PTEN mRNA expression was evidently reduced in ADR‐R mice and recovered in ADR‐R + shLINC00470 mice; G, Upon the ADR treatment, the knockdown of LINC00470 in vivo could reduce the up‐regulated PTEN m⁶A modification in ADR‐R mice; H, Upon the ADR treatment, the knockdown of LINC00470 in vivo could reduce the up‐regulated LINC00470 expression in ADR‐R mice

FIGURE 7

LINC00470 down‐regulated the expression of LC3II, Beclin‐1, ATG7 and ATG5 via RNA methyltransferase METTL3 (*P value < .05 vs Control group; **P value < .05 vs ADR‐R group). A, The expression of LC3II, Beclin‐1, ATG7, and ATG5 proteins in different mouse groups was measured with Western blot; B, The relative expression of LC3II was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of LC3II induced by ADR; C, The relative expression of Beclin‐1 was decreased in ADR = R mice, while the knockdown of LINC00470 attenuated the relative expression of Beclin‐1 induced by ADR; D, The relative expression of ATG7 was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of ATG7 induced by ADR; E, The relative expression of ATG5 was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of ATG5 induced by ADR; F, The expression of LC3II, Beclin‐1, ATG7 and ATG5 proteins in different mouse groups with ADR treatment was measured with Western blot; G, Upon the ADR treatment, the relative expression of LC3II was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of LC3II induced by ADR; H, Upon the ADR treatment, the relative expression of Beclin‐1 was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of Beclin‐1 induced by ADR; I, Upon the ADR treatment, the relative expression of ATG7 was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of ATG7 induced by ADR; J, Upon the ADR treatment, the relative expression of ATG5 was decreased in ADR‐R mice, while the knockdown of LINC00470 attenuated the relative expression of ATG5 induced by ADR

FIGURE 8

Flow chart of molecular mechanism underlying the effect of LINC00470 on CML