miRNA-765 mediates multidrug resistance via targeting BATF2 in gastric cancer cells

Abstract

Elucidation of the mechanisms underlying multidrug resistance (MDR) is required to ensure the efficacy of chemotherapy against gastric cancer (GC). To investigate this issue, here we identified that microRNA-765 (miRNA-765) is up-regulated both in MDR GC cell lines and in specimens from patients who are not responding to chemotherapy. In addition, down-regulation of miRNA-765 increased the sensitivity of GC cells to anticancer drugs, whereas its overexpression had the opposite effect. Moreover, miRNA-765 suppressed drug-induced apoptosis and positively regulated the expression of MDR-related genes. Finally, we showed that the basic leucine zipper ATF-like transcription factor 2, a tumor suppressor gene, is the functional target of miRNA-765. In summary, these results suggest that miRNA-765 may promote MDR via basic leucine zipper ATF-like transcription factor 2 in GC cells.

Keywords: basic leucine zipper ATF-like transcription factor 2; gastric cancer; miRNA-765; multidrug resistance.

Fig. 1

The expression of miRNA‐765 in GC tissues and drug‐resistant GC cells. (A) The expression of miRNA‐765 in tumor tissues obtained from responding patients (n = 16) was significantly lower than that observed in tumor tissues from nonresponding patients (n = 28). (B) The expression of miRNA‐765 in SGC7901/VCR and SGC7901 cells was detected using qRT‐PCR. The error bars indicate standard deviation. t‐Test was used for the analysis of differences. The experiments were carried out independently three times. ***P < 0.005.

Fig. 2

Suppression of miRNA‐765 enhances the sensitivity of GC cells to anticancer drugs. (A) qRT‐PCR analyzed the expression of miRNA‐765 after transfection of anti‐miRNA‐765 into SGC7901/VCR cells. (B) IC₅₀ values for VCR after transfection of anti‐miRNA‐765 into SGC7901/VCR cells. (C) Representative colony‐forming assay images of SGC7901/VCR cells after transfection of anti‐miRNA‐765 and quantifications of colony numbers after crystal violet staining. (D) Flow cytometry was performed to assess the cell cycle distribution of SGC7901/VCR cells stained with Propidium Iodide (PI) after transfection with anti‐miRNA‐765. A representative result is shown. Red: cells at G0/G1 or G2/M phase; cyan: cell debris; dashed lines: cells at S phase. (E) Flow cytometry was used to assess the percentage of apoptotic SGC7901/VCR cells after transfection with anti‐miRNA‐765. Representative scatterplots are shown. The error bars indicate standard deviation. t‐Test was used for the analysis of differences. The experiments were carried out independently three times. ***P < 0.005; **P < 0.01; *P < 0.05. 7‐AAD, 7‐aminoactinomycin D.

Fig. 3

Overexpression of miRNA‐765 decreases the sensitivity of GC cells to anticancer drugs. (A) qRT‐PCR analyzed the expression of miRNA‐765 after transfection of miRNA‐765 into SGC7901 cells. (B) IC₅₀ values for VCR after transfection of miRNA‐765 into SGC7901 cells. (C) Representative colony‐forming assay images of SGC7901 cells after transfection of miRNA‐765 and quantifications of colony numbers after crystal violet staining. (D) Flow cytometry was performed to assess the cell cycle distribution of SGC7901 cells stained with PI after transfection with miRNA‐765. A representative result is shown. Red: cells at G0/G1 or G2/M phase; cyan: cell debris; dashed lines: cells at S phase. (E) Flow cytometry was used to assess the percentage of apoptotic SGC7901 cells after transfection with miRNA‐765. Representative scatterplots are shown. The error bars indicate standard deviation. t‐Test was used for the analysis of differences. The experiments were carried out independently three times. ***P < 0.005; **P < 0.01.

Fig. 4

miRNA‐765 targets BATF2. (A) The miRNA‐765 target site predicted in the 3′ UTR of BATF2 mRNA and its mutant are shown. (B) WT BATF2 3′ UTR or the Mut luciferase constructed and miRNA‐765 or the control mimic were cotransfected into 293T cells for 48 h. Subsequently, luciferase activity assay was performed. (C) Anti‐miRNA‐765 was transfected into SGC7901/VCR cells. The mRNA and protein expression levels of BATF2 were measured 48 h after transfection. (D) The miRNA‐765 mimic was transfected into SGC7901 cells. The mRNA and protein expression levels of BATF2 were detected 48 h after transfection. The error bars indicate standard deviation. t‐Test was used for the analysis of differences. The experiments were carried out independently three times. **P < 0.01; *P < 0.05.

Fig. 5

miRNA‐765 functions by targeting MDR‐related genes. (A) qRT‐PCR analysis of the expression of MDR1, LRP and MRP1 48 h after transfection of anti‐miRNA‐765 in SGC7901/VCR cells. (B) The protein levels of P‐gp, LRP and MRP were detected 48 h after transfection of anti‐miRNA‐765 into SGC7901/VCR cells. The error bars indicate standard deviation. t‐Test was used for the analysis of differences. The experiments were carried out independently three times. *P < 0.05.