miR-23b-3p regulates the chemoresistance of gastric cancer cells by targeting ATG12 and HMGB2

Abstract

Chemotherapy is an important treatment modality for gastric cancer (GC); however, it usually fails because of drug resistance, especially multidrug resistance (MDR). Previously, we found a novel subset of MDR-associated microRNAs (miRNAs) through high-throughput functional screening. In this report, we investigated the exact roles and mechanisms of miR-23b-3p in the MDR of GC. Using gain or loss-of-function in in vitro and in vivo experiments, we found that overexpression of miR-23b-3p reversed cancer cell resistance to multiple chemotherapeutics in vitro and sensitize tumors to chemotherapy in vivo. Reporter gene assay and western blot analysis showed that ATG12 and HMGB2 were the direct targets of miR-23b-3p. Meanwhile, ATG12 and HMGB2 were positively associated with the occurrence of autophagy. Reducing the expression of these target genes by siRNA or inhibition of autophagy both sensitized GC cells to chemotherapy. These findings suggest that a miR-23b-3p/ATG12/HMGB2/autophagy-regulatory loop has a critical role in MDR in GC. In addition, miR-23b-3p could be used as a prognostic factor for overall survival in GC. In conclusion, our data demonstrated that miR-23b-3p inhibited autophagy mediated by ATG12 and HMGB2 and sensitized GC cells to chemotherapy, and suggested the potential application of miR-23b-3p in drug resistance prediction and treatment.

Figure 1

Expression and function of miR-23b-3p in GC cells. (a) Downregulation of miR-23b-3p was verified by qRT-PCR in SGC7901/VCR cells. (b and c) Expression of miR-23b-3p was studied after transfection of GC cells with inhibitors, mimics or NC. (d and e) miR-23b-3p negatively regulated the drug sensitivity of GC cells to chemotherapeutic agents in vitro. *P<0.05, **P<0.01

Figure 2

ATG12 and HMGB2 are direct target genes of miR-23b-3p. (A) Bioinformatic analysis predicted the target genes of miR-23b-3p. (B–D) ATG12 and HMGB2 were evaluated using qRT-PCR, western blotting and CLSM (blue fluorescence indicates nuclei stained with DAPI, green indicates cytoplasmic membrane stained with DIO and red indicates the proteins expressed by the target genes) in SGC7901/VCR cells and SGC7901 cells. (E and F) GC cells were transfected with miR-23b-3p mimics, inhibitor or NC, and the expression of ATG12 and HMGB2 was determined using qRT-PCR and western blot analysis. (G) Dual luciferase assays were performed in SGC7901/VCR cells after cotransfection with wild-type or mutant ATG12 and HMGB2 3'-UTR plasmids and NC or miR-23b-3p mimics. *P<0.05, **P<0.01

Figure 3

Reduced expression of ATG12 and HMGB2 increases the sensitivity of SGC7901/VCR cells to chemotherapeutic agents. (a–c) Expression levels of ATG12 and HMGB2 in SGC7901/VCR cells were significantly reduced by siRNAs. (d) Silencing of ATG12 or HMGB2 sensitized SGC7901/VCR cells to chemotherapeutic agents and decreased IC50 values. (e) ATG12 levels were decreased when HMGB2 was downregulated by siRNA. *P<0.05, **P<0.01

Figure 4

Chemoresistant cells exhibit increased autophagy and inhibition of autophagy restores the sensitivity of chemoresistant cells to chemotherapy. (A) Autophagy was evaluated in SGC7901 and SGC7901/VCR cells using TEM. (B) Western blot analysis was used to evaluate the expression level of LC3 and p62. (C) GC cells that stably express the mRFP-GFP-LC3 fusion protein were established and observed under CLSM. (D and E) Western blotting and CLSM were used to evaluate the effect of siRNA against ATG12 or HMGB2 on autophagy in SGC7901/VCR cells. (F) SGC7901/VCR and SGC7901 cells were not treated or treated with 20 μmol/l CQ for 24 h before being subjected to western blot analysis for LC3 expression. (G) CQ greatly enhanced the sensitivity of SGC7901/VCR cells to chemotherapeutic agents; however, this effect was not noticeable in SGC7901 cells *P<0.05, **P<0.01. The arrows indicate autophagosomes (A, C and E)

Figure 5

miR-23b-3p inhibits autophagy in chemoresistant cells by negatively regulating ATG12 and HMGB2. (a) GC cells stably expressing mRFP-GFP-LC3 were transfected with inhibitors, mimics or NC and then examined using CLSM. The white arrows indicate autophagosomes. (b) SGC7901/VCR and SGC7901 cells were transfected with mimics, inhibitors or NC. After 48 h, LC3-I/II proteins were detected using western blot analysis. (c– e) qRT-PCR and western blot analyses indicated that at 24 h after treating with low concentration of 5-Fu, miR-23b-3p expression in AGS and BGC823 cells was decreased, whereas the expressions of ATG12, HMGB2 and LC3-II were upregulated. (f) SGC7901 cells were cotransfected with inhibitors and siRNAs. After 48 h, ATG12 and HMGB2 were detected using western blot analysis. (g) Silencing of ATG12 or HMGB2 reverses the effect of miR-23b-3p knockdown on drug resistance. *P<0.05, **P<0.01

Figure 6

The restoration of miR-23b-3p reversed the drug resistance in vivo. (a) Expression of miR-23b-3p in SGC7901/VCR cells stably transfected with lenti-miR-NC or lenti-miR-23b-3p. (b) SGC7901/VCR-lenti-miR-NC- and SGC7901/VCR-lenti-miR-23b-3p-transfected cells were transplanted in the left and right side of the mice, respectively. (c and d) The tumor volumes were calculated as length × width² and measured at the indicated time points. *P<0.05, **P<0.01

Figure 7

miR-23b-3p expression is inversely correlated with the expression of its target genes and correlates with overall survival in patients with gastric cancer. (A and B) Compared with chemosensitive patients (S), ATG12 and HMGB2 were markedly upregulated in chemoresistant patients (R), whereas the expression of miR-23b-3p was significantly decreased (P<0.01). (C and D) In situ hybridization and Kaplan–Meier curves were used to analyze the correlation between miR-23b-3p expression and overall survival