Methylation of miR-129-5p CpG island modulates multi-drug resistance in gastric cancer by targeting ABC transporters

Abstract

Recent studies have reported that hyper-methylation in the promoter region of miRNAs could silence the expression of tumor suppressive miRNAs and might play significant roles in the process of tumor development. However, the potential mechanisms regarding how methylation of miRNA CpG Island could regulate cancer cell chemo-resistance have not yet been studied. Using microarray and BSP (Bisulfate Sequencing PCR) assays, we found that compared with the parent SGC7901/VCR cells, expression of miR-129-5p was restored in SGC7901/VCR gastric cancer multi-drug resistant cell line treated by de-methylation reagent (5-AZA-dC). Using gain or loss of function assays, we found the over-expressed miR-129-5p reduced the chemo-resistance of SGC7901/VCR and SGC7901/ADR cells, while down-regulation of miR-129-5p had an opposite effect. Furthermore, three members of multi-drug resistance (MDR) related ABC transporters (ABCB1, ABCC5 and ABCG1) were found to be direct targets of miR-129-5p using bioinformatics analysis and report gene assays. The present study indicated that hyper-methylation of miR-129-5p CpG island might play important roles in the development of gastric cancer chemo-resistance by targeting MDR related ABC transporters and might be used as a potential therapeutic target in preventing the chemo-resistance of gastric cancer.

Figure 1. MiR-129-5p was hypo-methylated in gastric cancer MDR cell lines after 5-AZA-dC treatment

A. IC50 values of cells to 5-FU, VCR and DDP calculated from MTT assays showing the effects of 5-AZA-dC on MDR in SGC7901/VCR cells compared with the parent SGC7901 cells or SGC7901/VCR cells. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01). B. ADR accumulation and retention values were tested using Flow Cytometer analysis and the luciferase intensities of accumulation and retention on 5-AZA-dC treated or untreated SGC7901/VCR cells were indicated. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01, *p<0.05). C. Fore ground minus back ground signals from microarray analysis. Signals of six miRNAs were found to be significantly increased in 5-AZA-dC treated SGC7901/VCR cells. Error bars correspond to the mean ± SD. (**p<0.01).

Figure 2. MiR-129-5p was hyper-methylated and was down-regulated in MDR gastric cancer cell lines

A. The methylation status of miR-129-5p in SGC7901/VCR, SGC7901/ADR and SGC7901 cell lines was determined by BSP assay. Shown was the methylation percent of in tested cells. Error bars correspond to the mean ± SD. (**p<0.01, *p<0.05). B. Real-time PCR was used to test the miR-129-5p expression in SGC7901/VCR, SGC7901/ADR and SGC7901 cell lines and the relative expression in these cells was indicated. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01). C. The methylation status of miR-129-5p in SGC7901/VCR, SGC7901/ADR cell lines after the treatment of 5-AZA-dC (2μM and 4μM) was determined by BSP assay and the methylation percent was analyzed. Error bars correspond to the mean ± SD. (**p<0.01, *p<0.05). D. MiR-129-5p expression in SGC7901/VCR and SGC7901/ADR cell lines after treatment of 2μM and 4μM 5-AZA-dC was determined by real-time PCR and the relative miR-129-5p expression value was indicated. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01).

Figure 3. MiR-129-5p modulate multi-drug resistance in gastric cancer cell lines

Transient transfection efficiency was determined by real-time PCR. The relative expression of miR-129-5p in antagomirs transfected SGC7901 cells, pre-miRs transfected SGC7901/VCR and SGC7901/ADR cells compared with the parent cell lines and the negative controls (pre-NC or anti-NC) transfected cells was indicated. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01). B. C and D. IC50 values of cells to 5-FU, VCR and DDP calculated from MTT assays showing the effects of miR-129-5p antagomirs on MDR in SGC7901 cells (B), miR-129-5p pre-miRs on MDR in SGC7901/VCR cells (C), and miR-129-5p pre-miRs on MDR in SGC7901/ADR cells (D) compared with the parent cells or the negative control (NC) transfected cells. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01, *p<0.05).

Figure 4. Members of the ABC transporter family were direct targets of miR-129-5p

A. Bioinformatics analysis showing the conserved putativebinding sites for miR-129-5p in ABCB1, ABCC5 and ABCG1. B. C and D. Luciferase assays were performed with Luc-B1(B), Luc-C5(C),Luc-G1(D) and the mutant constructLuc-B1M(B), Luc-C5M(C), Luc-G1M(D). Bars indicate the ratio of firefly luciferase (normalized to Renilla luciferase) activity measured following transfection with miR-129-5p or miR-150 pre-miRNA compared with the activity measured following transfection with the pre-miR-control (pre-NC) for the same construct. Each experiment was independently repeated at least 3 times. Error bars correspond to the mean ± SD. (**p<0.01, *p<0.05). E and F. Western blot (E) or Real-time PCR (F) showing the changes in the protein levels (E) or RNA levels (F) of ABCB1, ABCC5 and ABCG1 after transient transfection of pre-miR-129-5p compared with the negative controls (Control) respectively. Error bars correspond to the mean ± SD. (**p<0.01).

Figure 5. MiR-129-5p antagomirs modulated MDR in tumor-bearing nude mice

A and B. Representative images showing the luciferase signals of tumors treated or untreated with miR-129-5p on the back of nude mice on the day 25, 32 and 39 under the treatment of 5-FU, VCR and DDP (A). Comparison of luciferase signals on the day 39 was analyzed and indicated (B). Error bars correspond to the mean ± SD. (**p<0.01, *p<0.05). C. Representative images show H&E staining of the miR-129-5p treated or untreated tumors (×400) generated in nude mice and immunohistochemistry staining using ABCB1, ABCC5 and ABCG1 antibodies on these tumors (×400). D. Relative miR-129-5p expression in tumors generated from nude mice was determined by real-time PCR. Expression of miR-129-5p was significantly down regulated in antagomir injected tumors compared with anti-NC injected ones. Error bars correspond to the mean ± SD. (**p<0.01).