Hsa-miR-323a-3p functions as a tumor suppressor and targets STAT3 in neuroblastoma cells

Abstract

Background: Studies conducted in the last decades have revealed a role for the non-coding microRNAs (miRNAs) in cancer development and progression. Several miRNAs within the chromosome region 14q32, a region commonly deleted in cancers, are associated with poor clinical outcome in the childhood cancer neuroblastoma. We have previously identified miR-323a-3p from this region to be downregulated in chemotherapy treated neuroblastoma cells compared to pre-treatment cells from the same patients. Furthermore, in neuroblastoma tumors, this miRNA is downregulated in advanced stage 4 disease compared to stage 1-2. In this study, we attempt to delineate the unknown functional roles of miR-323a-3p in neuroblastoma.

Methods: Synthetic miRNA mimics were used to overexpress miR-323a-3p in neuroblastoma cell lines. To investigate the functional roles of miR-323a-3p, cell viability assay, flow cytometry, reverse transcription-quantitative polymerase chain reaction, luciferase reporter assay and western blot were conducted on the neuroblastoma cell lines Kelly, SH-SY5Y and SK-N-BE(2)-C.

Results: Ectopic expression of miR-323a-3p resulted in marked reduction of cell viability in Kelly, SH-SY5Y and SK-N-BE(2)-C by causing G1-cell cycle arrest in Kelly and SH-SY5Y and apoptosis in all the cell lines tested. Furthermore, mRNA and protein levels of signal transducer and activator of transcription 3 (STAT3) were reduced upon miR-323a-3p overexpression. A direct binding of the miR-323a-3p to the 3'UTR of STAT3 was experimentally validated by luciferase reporter assay, where miR-323a-3p reduced luminescent signal from full length STAT3 3'UTR luciferase reporter, but not from a reporter with mutation in the predicted seed sequence.

Conclusions: miR-323a-3p inhibits growth of neuroblastoma cell lines through G1-cell cycle arrest and apoptosis, and the well-known oncogene STAT3 is a direct target of this miRNA.

Keywords: STAT3; chemotherapy; chromosome region 14q32; microRNAs; neuroblastoma; non-coding.

Figure 1

Low expression of miR-323a-3p is associated with poor overall survival in a primary neuroblastoma tumors dataset. Kaplan-Meier overall survival curve for patients with high (blue, n = 97) and low (red, n = 98) expression of miR-323a-3p.

Figure 2

miR-323a-3p overexpression suppressed the growth and survival of neuroblastoma cells. (A) The basic expression of miR-323a-3p with respect to stably expressed miR-4286 in Kelly, SH-SY5Y and BE(2)-C cell lines were detected by RT-qPCR analysis. Data is presented as mean ± SEM of at least three independent experiments, each repeated in triplicates. (B) Cell viability of the cell lines Kelly, SH-SY5Y and BE(2)-C at 24 h, 48 h, 72 h and 96 h post transfection with miR-323a-3p, measured with the alamarBlue cell viability assay. Data is presented as mean ± SD of at least three independent experiments, each repeated in triplicates. *P < 0.05, **P < 0.01 vs. the NC. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; NC, negative control; miR, microRNA; SD, standard deviation.

Figure 3

miR-323a-3p induces G1-cell cycle arrest and apoptosis in neuroblastoma cells. (A) Kelly and SH-SY5Y cell lines were transfected with miR-323a-3p or NC mimics and cell cycle distribution was measured by flow cytometry assay. Data is presented as mean ± SD of three independent experiments. (B) Total PARP and PARP-cleavage (represents apoptosis) was detected on western blot in Kelly, SH-SY5Y and BE(2)-C cell lines transfected with miR-323a-3p. Quantification of cleaved PARP protein levels on the western blots (n = 3). (C) (B) BCL2 was detected on western blot in Kelly, SH-SY5Y and BE(2)-C cell lines transfected with miR-323a-3p. Quantification of BCL2 protein levels on the western blots (n = 3). Data is presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the NC. SD, standard deviation; NC, negative control; miR, microRNA; c-PARP, cleaved PARP.

Figure 4

STAT3 is a direct target of miR-323a-3p in neuroblastoma. (A) The putative binding site of miR-323a-3p (nucleotides 1,097 to 1,103) in the 3′UTR of STAT3 was mutated as shown in the figure with bold and italics. (B) Dual-luciferase reporter assay demonstrating the luciferase activity of a construct with a wild-type or a mutated 3′UTR of STAT3 in SH-SY5Y transfected with miR-323a-3p or NC mimics. Data is presented as mean ± SD of three independent experiments, each repeated in triplicates. **P < 0.01 vs. the NC. SD, standard deviation; miR, microRNA; NC, negative control; STAT3, signal transducer and activator of transcription; ORF, open Reading frame; UTR, untranslated region; wt, wild-type; MUT, mutated.

Figure 5

miR-323a-3p reduces mRNA and protein levels of STAT3 in neuroblastoma cells. (A) The RT-qPCR analysis of STAT3 mRNA levels in Kelly, SH-SY5Y and BE(2)-C cell lines transfected with miR-323a-3p. Data is presented as mean ± SEM of three independent experiments, each repeated in triplicates. (B) Western blot assay demonstrating STAT3 protein levels in Kelly, SH-SY5Y and BE(2)-C cell lines transfected with miR-323a-3p. (C) Quantification of STAT3 protein expression on the western blots (n = 3). Data is presented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01 vs. the NC. RT-qPCR, reverse transcription-quantitative polymerase chain reaction; SD, standard deviation; miR, microRNA; NC, negative control; STAT3, signal transducer and activator of transcription.