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. 2021 Oct;22(4):1157.
doi: 10.3892/etm.2021.10591. Epub 2021 Aug 10.

Upregulation of miR-144-3p expression attenuates glioma cell viability and invasion by targeting BCL6

Jingru Zhou  1 , Ruen Liu  2
Affiliations

Upregulation of miR-144-3p expression attenuates glioma cell viability and invasion by targeting BCL6

Jingru Zhou et al. Exp Ther Med. 2021 Oct.

Abstract

Glioma remains to be an aggressive type of cancer with poor prognosis irrespective of the type of standard treatment applied. Therefore, identification of accurate early diagnostic methods and therapeutic strategies for glioma is imperative for the treatment of this disease. The expression of a number of miRNAs in glioma have been reported to be associated with the regulation of tumorigenic progression, cancer cell proliferation, metastasis, invasion, angiogenesis and drug resistance. The aim of the present study was to assess the function of the microRNA (miR/miRNA)-144-3p/BCL6 axis in glioma. Reverse transcription-quantitative PCR was used to measure miR-144-3p and BCL6 expression. Western blotting was used for measuring BCL6 expression. Luciferase reporter assay was used to assess the association between miR-144-3p and BCL6 and a tumor xenograft model was established for assess tumor growth. The data demonstrated that miR-144-3p was decreased whereas BCL6 expression was increased in glioma tissues compared with those in healthy human brain tissues, where miR-144-3p suppressed BCL6 expression by targeting the 3'-UTR sequence of BCL6. miR-144-3p overexpression alleviated proliferation and invasion in U251 cells whereas transfection with the BCL6-overexpressing plasmid rescued the suppressive effects of miR-144-3p upregulation on the proliferation and invasion of U251 cells. In addition, miR-144-3p overexpression and BCL6 downregulation inhibited tumor progression in a mouse tumor xenograft model. The present findings suggest that miR-144-3p and BCL6 may serve to be indicator of proliferation and invasion for patients with glioma. Furthermore, BCL6 may serve an important role in the miR-144-3p-mediated regulation of proliferation and invasion of glioma cells, where the miR-144-3p/BCL6 axis can be used to target patients with glioma therapeutically.

Keywords: BCL6; glioma; invasion; microRNA-144-3p; viability.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
miR-144-3p and BCL6 expression in glioma tisseus. Reverse transcription-quantitative PCR was used for the measurement of the relative (A) miR-144-3p and (B) BCL6 expression in 25 glioma and 10 normal brain tissues. ***P<0.001 vs. Normal. miR, microRNA.
Figure 2
Figure 2
BCL6 is a target of miR-144-3p. (A) Targetscan software was used to predict that BCL6 is a target of miR-144-3p where the binding sequences of WT or MUT BCL6 3'-UTR for miR-144-3p are shown. (B) Assessment of luciferase activity following transfection with WT or MUT BCL6 3'UTR and miR-NC or miR-14-3p mimics using a Glomax luminometer and subsequent normalization. All data are presented as the mean ± SEM (n=4). ***P<0.001 vs. the control-WT group. WT, wild-type; MUT, mutant; 3'-UTR, 3' untranslated region; miR, microRNA.
Figure 3
Figure 3
miR-144-3p overexpression suppresses BCL6 expression. (A) Reverse transcription-quantitative PCR analysis was used to measure miR-144-3p expression in miR-144-3p mimic- or inhibitor-transfected U251 cells. (B) Western blot analysis was conducted to examine BCL6 protein expression following transfection. All data are presented as the mean ± SEM (n=4). ***P<0.001 vs. Control (untransfected). miR, microRNA; NC, negative control.
Figure 4
Figure 4
miR-144-3p overexpression decreases the malignancy of glioma cells. Following transfection with the miR-144-3p mimic or inhibitor, (A) Cell Counting Kit-8 assays and (B) Transwell assays were performed to measure cell viability and invasion of U251 cells, respectively. All data are presented as the mean ± SEM (n=4). *P<0.05, **P<0.01 and ***P<0.001 vs. Control. ###P<0.001 vs. miR-144-3p mimic. Scale bar, 20 µm. Control, non-transfected U251 cells; OD, optical density; miR, microRNA.
Figure 5
Figure 5
BCL6 plasmid co-transfection partly abolishes the inhibitory effects of miR-144-3p on U251 cell viability and invasion. U251 cells were co-transfected with the miR-144-3p mimic and BCL6-activation plasmid before (A) western blotting, (B) Cell Counting Kit-8 and (C) Transwell assays were performed to measure BCL6 expression, cell viability and cell invasion, respectively. *P<0.05, **P<0.01 and ***P<0.001 vs. miR-144-3p. Scale bar, 20 µm. miR, microRNA; OD, optical density.
Figure 6
Figure 6
miR-144-3p upregulation or BCL knockdown suppress glioma tumor growth in vivo. Stably-transfected U251 cells were subcutaneously injected into the flanks of nude mice. In total, four groups of nude mice are shown with the corresponding tumor sizes 4 weeks following inoculation. Representative images of tumors (A) after injection of cells transfected with miR-NC or miR-144-5p mimic and (B) after injection of cell transfected with NC-shRNA or BCL6-shRNA. Tumor volumes (C) after injection of cells transfected with miR-NC or miR-144-5p mimic and (D) after injection of cell transfected with NC-shRNA or BCL6-shRNA were measured every week. Red arrows to indicate the location of the tumors on the mice. Scale bar, 10 mm. All data are presented as the mean ± SEM, n=6 mice per group. *P<0.05, **P<0.01 and ***P<0.001 vs. the NC (miR-NC or NC-shRNA) group. miR, microRNA; sh, short hairpin; NC, negative control.
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