The miR-106b-25 polycistron, activated by genomic amplification, functions as an oncogene by suppressing p21 and Bim

Abstract

Background & aims: Barrett's esophagus (BE) is a highly premalignant disease that predisposes to the development of esophageal adenocarcinoma (EAC); however, the involvement of microRNAs (miRs) in BE-EAC carcinogenic progression is not known.

Methods: Esophageal cultured cells (HEEpiC, QhTRT, ChTRT, GihTRT, and OE-33) and esophageal tissues (22 normal epithelia, 24 BE, and 22 EAC) were studied. MiR microarrays and quantitative reverse-transcription polymerase chain reaction (RT-PCR) were employed to explore and verify differentially expressed miRs. Quantitative genomic PCR was performed to study copy number variation at the miR-106b-25 polycistron and MCM7 gene locus on chromosome 7q22.1. In vitro cell proliferation, cell cycle, and apoptosis assays and in vivo tumorigenesis experiments were performed to elucidate biologic effects of the miR-106b-25 polycistron. Western blotting and luciferase assays were performed to confirm direct messenger RNA (mRNA) targeting by the miR-106b-25 polycistron.

Results: The miR-106b-25 polycistron exerted potential proliferative, antiapoptotic, cell cycle-promoting effects in vitro and tumorigenic activity in vivo. MiRs-93 and -106b targeted and inhibited p21, whereas miR-25 targeted and inhibited Bim. This polycistron was upregulated progressively at successive stages of neoplasia, in association with genomic amplification and overexpression of MCM7. In addition, miRs-93 and -106b decreased p21 mRNA, whereas miR-25 did not alter Bim mRNA, suggesting the following discrete miR effector mechanisms: (1) for p21, mRNA degradation; (2) for Bim, translational inhibition.

Conclusions: The miR-106b-25 polycistron is activated by genomic amplification and is potentially involved in esophageal neoplastic progression and proliferation via suppression of 2 target genes: p21 and Bim.

Figure 1. Quantitative RT-PCR of miR-106b-25 polycistron in esophageal cultured cells and tissues

(A–C) Microarray and qRT-PCR results for miRs -25, -93 and -106b are shown in esophageal cells. The values of HEEpiC are set at 1 for both experiments. As shown, results of qRT-PCR correlated closely with those of microarrays. Both methods revealed that miR-106b-25 polycistron expression was gradually upregulated progressively at successive stages of neoplasia from NE to BE to EAC cells. (D–F) 22 NE (normal esophageal epithelial tissue), 24 BE, and 22 EAC tissues were studied. The average value of NE is set at 1. Horizontal bars indicate average levels. MiRs -25, -93 and -106b were significantly upregulated in EAC relative to NE and BE.

Figure 2. MCM7 mRNA expression and CNV in esophageal cultured cells and tissues

(A) qRT-PCR and gene expression microarray results of MCM7 are shown in cultured esophageal cells. The value of HEEpiC is set at 1. Both methods revealed that MCM7 expression was gradually upregulated during neoplastic progression from NE to BE to EAC cells. MCM7 exhibited an expression pattern similar to that of the miR-106b-25 polycistron (Figures 1A–1C). (B) 22 NE, 24 BE, and 22 EAC tissues were studied. The average value of NE is set at 1. Horizontal bars indicate average levels. MCM7 was significantly upregulated in EAC relative to NE and BE. (C) qPCR of MCM7 locus is shown in cultured esophageal cells. The value of HEEpiC is set at 1. The CNV pattern of the MCM7 locus in the esophageal cultured cells correlated with those of both miR-106b-25 polycistron expression (Figures 1A–1C) and MCM7 mRNA expression (Figure 2A). (D) 24 NE, 22 BE, and 24 EAC tissues were enrolled. The average value of NE is set at 1. Horizontal bars indicate average levels. CNV of the MCM7 locus in tissues showed a gradual and significant upregulation during neoplastic progression from NE to BE to EAC. CNV ratio in EAC averaged 1.37-fold across all specimens, but 29.2% (7/24) of EACs exhibited significant genomic copy number increase (≥1.5-fold vs. NE). (E) CNV of the MCM7 locus in 11 paired tissue specimens. The MCM7 locus was statistically significantly amplified in EAC tissues relative to paired NE tissues. Paired average fold change (EAC/NE) equalled 1.67 (p = 0.0037, paired t test).

Figure 3

Expression levels of miRs -25, -93 and -106b correlated significantly with CNV levels in primary esophageal tissues (Pearson’s correlation coefficient test, p = 0.0014, 0.011 and 0.019, respectively). The average value of NE was set at 1 for both miR expression and CNV determinations. 11 NE, 5 BE, and 9 EAC specimens were analyzed.

Figure 4. Cell Proliferation Assessed by WST-1 and in vivo Tumorigenesis Assays

OE-33 cells were transfected with either a nonspecific control inhibitor (NSC-INH), miR-25-INH, miR-93-INH or miR-106b-INH. WI-38 and ChTRT cells were transfected with either NSC mimic (NSC-MM), miR-25-MM, miR-93-MM or miR-106b-MM. WI-38 cells were also transfected with a miR cocktail (20nM each of miRs -25, -93 and -106b). Each experiment was performed in 6 replicates. (A) MiR-25, -93 and -106b inhibitors significantly inhibited proliferation in OE-33 cells (*p= 1.3×10⁻⁵, **p= 6.6×10⁻⁹, ***p= 3.6×10⁻⁷, at Day 5, respectively). (B) MiR-25, -93 and -106b mimics significantly increased the number of ChTRT cells (^†p= 2.3×10⁻⁹, ^††p= 7.1×10⁻⁶, ^†††p= 1.6×10⁻⁷, at Day 5, respectively). (C) MiR-93, -106b, and miR cocktail mimics significantly increased the number of WI-38 cells (^$p= 8.2×10⁻⁷, ^$$p= 7.5×10⁻⁷, ^$$$p= 3.3×10⁻⁷, at Day 5, respectively), while miR-25 significantly increased cell numbers at Day 3 (^$$$$p= 1.0×10⁻⁶), but not at Day 5. (D) 2 × 10⁶ OE-33 cells transfected (Day -1) with either miR-25, -93, -106b or NSC-INH were implanted (Day 0) subcutaneously into the flanks of nude mice (N=8 each). MiR-25, -93 and -106b inhibitors significantly inhibited tumorigenesis of OE-33 cells relative to NSC-INH in nude mice.

Figure 4. Cell Proliferation Assessed by WST-1 and in vivo Tumorigenesis Assays

OE-33 cells were transfected with either a nonspecific control inhibitor (NSC-INH), miR-25-INH, miR-93-INH or miR-106b-INH. WI-38 and ChTRT cells were transfected with either NSC mimic (NSC-MM), miR-25-MM, miR-93-MM or miR-106b-MM. WI-38 cells were also transfected with a miR cocktail (20nM each of miRs -25, -93 and -106b). Each experiment was performed in 6 replicates. (A) MiR-25, -93 and -106b inhibitors significantly inhibited proliferation in OE-33 cells (*p= 1.3×10⁻⁵, **p= 6.6×10⁻⁹, ***p= 3.6×10⁻⁷, at Day 5, respectively). (B) MiR-25, -93 and -106b mimics significantly increased the number of ChTRT cells (^†p= 2.3×10⁻⁹, ^††p= 7.1×10⁻⁶, ^†††p= 1.6×10⁻⁷, at Day 5, respectively). (C) MiR-93, -106b, and miR cocktail mimics significantly increased the number of WI-38 cells (^$p= 8.2×10⁻⁷, ^$$p= 7.5×10⁻⁷, ^$$$p= 3.3×10⁻⁷, at Day 5, respectively), while miR-25 significantly increased cell numbers at Day 3 (^$$$$p= 1.0×10⁻⁶), but not at Day 5. (D) 2 × 10⁶ OE-33 cells transfected (Day -1) with either miR-25, -93, -106b or NSC-INH were implanted (Day 0) subcutaneously into the flanks of nude mice (N=8 each). MiR-25, -93 and -106b inhibitors significantly inhibited tumorigenesis of OE-33 cells relative to NSC-INH in nude mice.

Figure 5. p21 Western Blotting and Luciferase Assays

(A and B) Western blotting for p21. WI-38 cells were transfected with miR-93, -106b or NSC-MM, while OE-33 cells were transfected with miR-93, -106b or NSC inhibitors-INH. (C and D) Luciferase assays for p21 3′UTR. ChTRT and GihTRT cells were performed. Each procedure was performed in triplicate. The average values of NSC-MM were set at 1. p21 protein expression was reduced by miR-93 and -106b-MM (A), and increased by miR-93 and -106b-INH (B). MiR-93-MM and miR-106b-MM significantly decreased luciferase activity in both ChTRT (C) and GihTRT (D) cells.

Figure 6. Bim Western Blotting and Luciferase Assays

(A and B) Western blotting for Bim. WI-38 cells were transfected with miR-25 or NSC-MM, while OE-33 cells were transfected with miR-25 or NSC-INH. (C and D) Luciferase assay for Bim 3′UTR. GihTRT cells and OE-33 cells were used. Procedure was the same as Figure 5. Bim protein expression was reduced by miR-25-MM (A) and increased by miR-25-INH (B). (C) MiR-25-MM significantly decreased luciferase activity in GihTRT. (D) MiR-25-INH significantly increased luciferase activity in OE-33 cells.

Figure 7. p21 and Bim mRNA Expression Following miR Mimic Transfection

WI-38 cells were transfected with NSC, miR-25, -93 or -106b-MM as well as an siRNA against p21 mRNA (p21-siRNA) for comparison. p21 and Bim mRNA expression levels were assayed by qRT-PCR. Data was normalized to beta-actin. The value of NSC-MM was set at 1. (A) p21 mRNA was significantly (approximately 40%) decreased by miR-106b-MM, while p21-siRNA decreased p21 mRNA by approximately 94%. (B) Bim mRNA was not significantly changed by miR-25-MM.