miR-526b-3p serves as a prognostic factor and regulates the proliferation, invasion, and migration of glioma through targeting WEE1

Abstract

Background: MicroRNAs play important roles in cancer progression including glioma. In this study, we aimed to explore the expression pattern, prognostic potential, and functional role of miR-526b-3p in human glioma. Materials and methods: The expression of miR-526b-3p in glioma tissues and the adjacent non-tumor tissues was determined by quantitative RT-PCR. The chi-square test was performed to evaluate the statistical associations between miR-526b-3p level and patient characteristics. The prognostic value of miR-526b-3p was analyzed by Kaplan-Meier and Cox regression analyses. The function of miR-526b-3p was analyzed by MTT, colony formation assay, transwell assay, and flow cytometry analysis in vitro. The binding between miR-526b-3p and predicted target WEE1 was verified using dual luciferase assay and Western blot analysis. Results: We found that miR-526b-3p expression was significantly downregulated in both glioma tissues and cell lines. Downregulation of miR-526b-3p was significantly associated with advanced WHO grade, lower KPS score, and inferior patient outcomes. Functional investigation indicated that overexpression of miR-526b-3p suppressed cell proliferation, migration, and invasion, and promoted apoptosis in glioma cell lines. Mechanically, WEE1 was identified as direct targets of miR-526b-3p and overexpression of WEE1 significantly suppressed the levels of WEE1. Moreover, re-introduction of WEE1 abrogates the suppression of motility and invasiveness induced by miR-526b-3p in glioma cells. Conclusion: These findings indicate that miR-526b-3p may target WEE1 and inhibit glioma tumorigenesis and progression.

Keywords: WEE1; glioma; miR-526b-3p; prognosis; tumorigenesis.

Figure 1

Expression levels of miR-526b-3p in glioma and its clinical significance. (A) Measured by qRT-PCR, miR-526b-3p expression was significantly lower in glioma tissues than adjacent tissues. (B) The relative expression of miR-526b-3p was downregulated in human glioma cell lines (T98G, U251, LN18, LN229, and A172) compared with NHAs. (C and D) Kaplan-Meier survival analysis of the correlation between miR-526b-3p expression and overall survival and progression-free of the 158 glioma patients. The median expression level of miR-526b-3p was used as the cut-off. **P<0.01. Abbreviations: miR-526b-3p, microRNA-526b-3p; qRT-PCR, quantitative real-time polymerase chain reaction.

Figure 2

The effects of miR-526b-3p on the proliferation and apoptosis of U251 and LN299 cells. (A) Levels of miR-526b-3p in U251 and LN299 cells transfected with miR-526b-3p mimics were found to be higher than that in the cells transfected with NC mimics. (B and C) The cell viabilities of U251 and LN299 cells were determined by CCK-8 assays. (D and E) Transfection of miR-526b-3p mimics reduced the cell colony number of U251 and LN299 cells. (F and G) Flow cytometry analysis evaluated the apoptotic rates of U251 and LN299 cells transfected with NC mimic or miR-526b-3p mimic. **P<0.01. Abbreviations: miR-526b-3p, microRNA-526b-3p; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; CCK-8, cell counting kit.

Figure 3

Overexpression of miR-526b-3p inhibited the migration and invasion of U251 and LN299 cells. (A and B) Transwell migration assays were conducted with U251 and LN299 cells transfected with NC mimics or miR-526b-3p mimics, respectively. (C and D) Transwell invasion assays were performed using U251 and LN299 cells transfected with NC mimics or miR-526b-3p mimics, respectively. (E, F) Western blot was utilized to detect the protein levels of N-cadherin and vimentin. **P<0.01. Abbreviations: miR-526b-3p, microRNA-526b-3p; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control.

Figure 4

WEE1 was targeted by miR-526b-3p at its 3ʹ-UTR. (A) The predicted binding site through “starBase”. (B) The luciferase activities of U251 and LN299 cells were examined by dual-luciferase reporter assays. (C and D) Transfection of miR-526b-3p mimics reduced the mRNA or protein levels of WEE1 in U251 and LN299 cells which were assessed by qRT-PCR or Western blot, respectively. **P<0.01. Abbreviations: miR-526b-3p, microRNA-526b-3p; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; 3ʹ- UTR, 3ʹ-untranslated region.

Figure 5

Ectopic expression of WEE1 abrogated the tumor suppressive roles of miR-526b-3p. (A) Relative mRNA expression levels of WEE1 in U251 and LN299 cells transfected with NC mimic, miR-526b-3p mimic or co-transfected with miR-526b-3p mimic and pcDNA3.1-WEE1 vector. (B and C) CCK-8 assays evaluated the proliferation of U251 and LN299 cells in different groups. (D and E) Cell colony formation assays were conducted to determine the clonogenic abilities of U251 and LN299 cells in different groups. (F and G) Transwell invasion assays were carried out to assess the invasive cell number of U251 and LN299 cells in various experimental conditions. **P<0.01. Abbreviations: miR-526b-3p, microRNA-526b-3p; qRT-PCR, quantitative real-time polymerase chain reaction; NC, negative control; 3ʹ- UTR, 3ʹ-untranslated region; CCK-8, cell counting kit.