Fig 1: LNCAROD enhanced the stability of CCAR2 and facilitated the protein–protein interaction between CCAR2 and AROS. (A) RNA pull-down and Western blot analysis demonstrated the interaction between LNCAROD, CCAR2, and AROS proteins. (B) RIP-qPCR detection indicated the binding of CCAR2 to LNCAROD. (C) Subcellular fractionation and Western blot analysis showed localization of CCAR2 and AROS proteins in the nucleus of SK-MES-1 and NCI-H2170 cells. (D) Schematic representation depicting the full-length transcript of LNCAROD along with its deletion fragments. (E) The association between CCAR2, AROS proteins, and LNCAROD deletion fragments was confirmed through Western blot analysis. (F) Co-IP experiments indicated the coimmunoprecipitation of exogenous and endogenous AROS with CCAR2 in SK-MES-1 cells. (G) Co-IP experiments showed a weakened association between CCAR2 and AROS proteins in SK-MES-1 cells following treatment with RNase A. (H) Co-IP analysis indicated a correlation between CCAR2 and AROS proteins in SK-MES-1 cells transfected with LNCAROD-shRNA or LNCAROD-OE constructs. (I) Effects of silencing or overexpressing CCAR2 on the expression of LNCAROD. (J) Effects of silencing or overexpressing AROS on the expression of LNCAROD. (K) Effects of silencing or overexpressing LNCAROD on the protein expression of CCAR2. (L) Effect of silencing and overexpressing LNCAROD on AROS mRNA expression. (M) Effect of silencing and overexpressing LNCAROD on CCAR2 mRNA expression. (N) Cycloheximide chase analysis of CCAR2 protein levels in CHX-treated SK-MES-1 cells that silenced or overexpressed LNCAROD. (O) MG132 treatment prevented the decrease in CCAR2 protein levels in SK-MES-1 and NCI-H2170 after LNCAROD depletion. (P) Effect of AROS knockdown on CCAR2 protein expression. (Q) Effect of AROS knockdown on CCAR2 mRNA expression. **, P<0.01; ns, no significance. Co-IP, coimmunoprecipitation; NC, negative control; OE, overexpression; qPCR, quantitative polymerase chain reaction; RIP, RNA Binding Protein Immunoprecipitation Assay.
Fig 2: CCAR2 inhibited the activating effect of AROS on SIRT1 and suppressed the deacetylation of p53. (A) LNCAROD inhibited the deacetylation of p53 by CCAR2. (B) Cycloheximide chase analysis clarified the effect of LNCAROD and CCAR2 on the stability of p53 in SK-MES-1 cells. (C) Immunofluorescence staining confirmed the effect of LNCAROD and CCAR2 on the nuclear export of p53 in SK-MES-1 cells (200×). (D) Activation of SIRT1 or overexpression of AROS both facilitated p53 deacetylation. (E) Cycloheximide chase analysis demonstrated the effects of SIRT1 and AROS on p53 stability in SK-MES-1 cells. (F) Immunofluorescence staining confirmed the effect of SIRT1 and AROS on the nuclear export of p53 in SK-MES-1 cells (200×). NC, negative control; OE, overexpression.
Fig 3: Schematic of the mechanism. LNCAROD hinders the activation of SIRT1 by facilitating the interaction between CCAR2 and AROS in LUSC cells, thereby suppressing p53 deacetylation. This leads to the acetylation and mitochondrial translocation of p53, thereby activating mitochondrial apoptosis and inducing tumor cell death. Thus, LNCAROD plays a role in suppressing cancer hallmarks in LUSC. LUSC, lung squamous cell carcinoma.
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