Arsenic trioxide-mediated suppression of miR-182-5p is associated with potent anti-oxidant effects through up-regulation of SESN2

Abstract

Arsenic trioxide (ATO) is a traditional Chinese medicine that can induce oxidative stress for treatment of cancer cells. However, ATO may generate anti-oxidative responses to compromise the cytotoxic effect, but the underlying mechanisms remain unclear. Here we found that ATO could inhibit miR-182-5p expression in patient-derived primary S1 glioblastoma (GBM) cells accompanied by up-regulation of Sestrin-2 (SESN2) mRNA, a known anti-oxidant molecule. This phenomenon was also detected in a U87MG glioma cell line, human lung adenocarcinoma H1299 cell line and A549 cell line. Pretreatment with a free radical scavenger N-acetylcysteine (NAC) reduced the oxidative stress induced by ATO. Concomitantly, ATO mediated suppression of miR-182-5p and enhancement of SESN2 expression were also compromised. The MTT assay further showed that ATO induced cytotoxicity was enhanced by transfection of miR-182-5p mimics. Overexpression of miR-182-5p mimics significantly suppressed the expression of SENS2 and a firefly luciferase reporter gene fused to 3'- untranslated region (UTR) of SESN2 mRNA. Use of ribonucleoprotein immunoprecipitation (RNP-IP), ATO mediated suppression of miR-182-5p led to the stabilization of SESN2 mRNA as a result of Argonaute-2 (AGO2) dependent gene silencing. Furthermore, high expression of miR-182-5p and low expression of SESN2 mRNA tend to be associated with longer survival of glioma or lung cancer patients using public available gene expression datasets and online tools for prediction of clinical outcomes. Taken together, current data suggest that the miR-182-5p/SENS2 pathway is involved in ATO induced anti-oxidant responses, which may be important for the design of novel strategy for cancer treatment.

Keywords: anti-oxidant effect; arsenic trioxide; miR-182; oxidative stress; sestrin 2.

Figure 1. Effect of ATO on miR-182-5p expression in S1 GBM cells

(A) Dose-dependent suppression of miR-182-5p expression by ATO (n=4). (B) The melting curves of miR-182-5p and U6 snRNA during the qRT-PCR. (C) The Venn diagram involved four databases that predict putative mRNA targeted by miR-182-5p. The number of each colored circle was the amount of predicted mRNAs. There were 327 predicted mRNAs repeatedly presented in these four databases. (D) Flow cytometric analysis of ATO-induced oxidative stress. (E) NAC rescued ATO-mediated suppression of miR-182 expression. (F) Reporter gene assay results showed that ATO suppressed miR-182 targeting of the 3’UTR of the SESN2 gene. (G) Suppression of ATO-mediated induction of SESN2 by NAC. (H) The results of M TT assay showed that transduction with a miR-182 mimic enhanced the cytotoxic effects of ATO. ^*: p<0.05.

Figure 2. MiR-182-5p suppressed the expression of SESN2 and TP53INP1

(A) Sequence complementary between miR-182-5p and 3’-UTR of SESN2 and TP53INP1. (B) Confirmation of miR-182-5p mimic transduction into GBM cells by qRT-PCR. (C) SESN2 mRNA and TP53INP1 mRNA expression was inhibited following transduction of the miR-182-5p mimic. (D) SESN2 and TP53INP1 protein expression were inhibited following transduction of the miR-182-5p mimic. (E) and (F) Reporter gene assay results demonstrating that the miR-182-5p mimic targets the 3’UTRs of SESN2 mRNA and TP53INP1 mRNA. (G) Tetracycline-induced miR-182-5p expression in H1299 cells transduced with a tet-on-miRNA system. (H) Suppression of SENS2 protein expression following tet-on induction of miR-182-5p expression. (I) Suppression of ATO induced SENS2 expression, but not HO-1 expression, following tet-on induction of miR-182-5p expression. ^*: p<0.05.

Figure 3. Suppression of miR-182-5p mediated binding of AGO2 to SESN2 mRNA following treatment with ATO

(A) RNP-IP was used to evaluate the binding ability of AGO2 to SENS2 mRNA and TP53INP1 mRNA before and after ATO treatment (see text) (n=3). (B) The influence of manipulating miR-182-5p expression on AGO2 binding to SESN2 mRNA and TP53INP1 mRNA. (C) Suppression of miR-183/96/182 gene cluster expression by ATO. (D) ATO-mediated suppression of miR-182-5p expression was rescued by TGF-β. (E) A putative model shows ATO inhibiting the binding of AGO2 to mRNA via suppression of miR-182-5p expression. ^*: p<0.05.

Figure 4. Association of miR-182-5p and SESN2 expression with overall survival using publicly available datasets

(A) Comparison of high and low miR-182-5p expressive levels on overall survivals of glioma (TCGA Brain Lower Grade Glioma data) and lung cancer patient (TCGA Lung Squamous Cell Carcinoma data); (B) Comparison of high and low SESN2 mRNA expressive levels on overall survivals of glioma (accession No. GSE16011) and lung cancer patient (accession No. GSE 11969). A p-value below 0.05 represented a significant difference.