MicroRNA-221 Promoted Cell Proliferation By Enhancing N-myc Expression In Neuroblastoma And Its Mechanism

ObjectiveN-myc amplification is closely associated with malignant phenotype and progression in neuroblastoma(NB). However, the mechanism of N-myc amplication is not fully clear. It has been reported that microRNA-221(mi R-221) was upregulated in the neuroblastoma with amplified N-myc. The aims of the study are: 1) to examine the patterns of miR-221 level in human NB tumors and cells with or without N-Myc overexpression, and the correlation between miR-221 expression and various clinical features of these NB patients; 2) to explore the impact of mi R-221 on the cell proliferation and N-myc expression in vitro and in vivo in NB and its potential mechanism. Methods1. To explore the association between miR-221 and N-Myc expression in human NB tumors and cells, and the correlation between miR-221 expression and various clinical features of NB patients: A total of 31 human confirmed NB by pathology were enrolled. The clinical data of 31 NB patients were collected from the medical records and the formalin-fixed, paraffin-embedded(FFPE) NB tumor tissues were obtained. The expression of mi R-221 in NB tissues was detected by LNA-ISH(locked nucleic acid-in situ hybridization) and qRT-PCR(real-time quantitative PCR). The expression of N-myc in NB tissues was determined by immunohistochemistry assay. The relation of miR-221 and N-myc expression in NB tissues was analyzed. The correlation between miR-221 expression and clinical features in NB patients were assessed. The expression of mi R-221 and N-myc mRNA and protein level in NB cells(SH-SY5 Y, SK-N-SH, SK-N-DZ and IMR-32) were tested by qRT-PCR and Western blot, respectively.2. To examine the effect of miR-221 on cell proliferation in NB: Lentivirus-based vectors for overexpression of miR-221 and control were constructed. SH-SY5 Y cells were transfected with above lentivirus or control, then sorted by flow cytometry, named as miR-221-up and control cells. The expression of miR-221 was detected by qRT-PCR. The cell proliferation was measured by CCK8 and colony-forming assay. The distribution of cell cycle was tested by flow cytometry. SH-SY5 Y, control, and miR-221-up cells were injected subcutaneously in the backs of nude mice, respectively. The growth of transplanted tumors was dynamically observed. After injection 30 days, the mice were sacrificed; the xenografts were excised and weighed. The cell proliferation in tumor xenografts was assessed by staining Ki67.3. To discuss the potential mechanism of N-myc expression enhanced by miR-221: The expression of NLK, LEF1, p-LEF1, N-myc mRNA and protein level in cells and xenografts were tested by qRT-PCR and Western blot, respectively. The expression of NLK, p-LEF1 and N-myc protein in cells and xenografts were analyzed by immunofluorescence and immunohistochemistry assay. Result1. The miR-221 level both by q RT-PCR and by LNA-ISH were indicated to be significantly related to high level of N-myc in NB tissues(p<0.05). The Kaplan-Meier survival curve showed that there was a trend for poor survival for patients with high level of miR-221(p=0.045). However, there was no significant correlation of miR-221 expression with clinical features, including age, gender, tumor size, tumor differentiation, metastasis potential and tumor stage. The expression of mi R-221 and N-myc were higher in IMR-32 and SK-N-DZ cells(p<0.05), compared with SH-SY5 Y cells, but there was no significant alteration in SK-N-SH cells(p>0.05).2. The recombinant vectors were verified by DNA Sanger sequencing. The expression of miR-221 in mi R-221-up cells, stable overexpression of miR-221, was as seven times as that in SH-SY5 Y cells(p<0.05), with no difference between control and SH-SY5 Y cells(p>0.05). The data of CCK8 and colony-forming assay showed that overexpression of miR-221 promoted cell proliferation(p < 0.05). The results of flow cytometry demonstrated that miR-221 significantly decreased the G1-S checkpoint arrest(p<0.05). A significant increase in tumor size and weight were observed in the xenografts of miR-221-up cells-injected group(p<0.05), compared to those of SH-SY5 Y cells-injected group. No significant change in tumor size and weight were found in control cells-injected group(p>0.05). The Ki-67 proliferative index was significantly higher in the xenografts of SH-SY5Y-up cells-injected mice than that in SH-SY5 Y cells-injected group(p<0.05).3. NLK and p-LEF1 protein were significantly decreased(p<0.05), while N-myc was markedly increased in miR-221-up cells(p < 0.05), compared with those in SH-SY5 Y cells. However, there was no significant differences in the levels of LEF1 protein after overexpression of miR-221(p>0.05). N-myc mRNA level was higher in miR-221-up cells than in SH-SY5 Y cells(p<0.05), but no significant change of NLK and LEF1 mRNA were observed at the presence of stable overexpression of miR-221(p>0.05). The same results were observed in vivo. Conclusion1. miR-221 is upregulated in human NB tissues and cells with N-myc overexpression and high miR-221 expression predicts NB poor prognosis.2. miR-221 promotes to the cell proliferation by rescue of G1-S checkpoint arrest in neuroblastomas.3. Overexpression of miR-221 suppresses NLK expression and phosphorylation of LEF1, thus to induce an enhancement of N-myc expression.