MEX3A contributes to development and progression of glioma through regulating cell proliferation and cell migration and targeting CCL2

Abstract

Glioma is one of the most commonly diagnosed intracranial malignant tumors with extremely high morbidity and mortality, whose treatment was seriously limited because of the unclear molecular mechanism. In this study, in order to identify a novel therapeutic target for glioma treatment, we explored the functions and mechanism of MEX3A in regulating glioma. The immunohistochemical staining of MEX3A in glioma and normal tissues revealed the upregulation of MEX3A and further indicated the relationship between high MEX3A expression and higher malignancy as well as poorer prognosis of glioma. In vitro loss-of-function and gain-of-function experiments comprehensively demonstrated that MEX3A may promote glioma development through regulating cell proliferation, cell apoptosis, cell cycle, and cell migration. In vivo experiments also suggested the inhibition of glioma growth by MEX3A knockdown. Moreover, our mechanistic study identifies CCL2 as a potential downstream target of MEX3A, which possesses similar regulatory effects on glioma development with MEX3A and could attenuate the promotion of glioma induced by MEX3A overexpression. Overall, MEX3A was identified as a potential tumor promoter in glioma development and therapeutic target in glioma treatment.

Fig. 1. MEX3A was upregulated in glioma tissues and expressed in glioma cells.

A The expression level of MEX3A was detected by IHC analysis in glioma and normal tissues. B The mining of RNA-seq data of TCGA showed the upregulated mRNA expression of MEX3A in tumor tissues of glioma patients compared with that in normal tissues. C The association between MEX3A expression and prognosis of glioma patients was evaluated with Kaplan–Meier survival analysis. D The mRNA expression of MEX3A in U87, U251, U373, and SHG-44 cell lines was detected by qPCR. The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 2. MEX3A knockdown inhibited glioma development in vitro.

A, B Cell models with or without MEX3A knockdown were constructed by transfecting shMEX3A or shCtrl. The knockdown efficiency of MEX3A in BGC-823 and U251 cells was assessed by western blotting (A). B MTT assay was employed to show the effects of MEX3A on cell proliferation of U87 and U251 cells. C The number of colonies formed by U87 and U251 was counted to show the effects of MEX3A on their colony formation ability. D Flow cytometry was performed to detect cell apoptosis of U87 and U251 cells with or without MEX3A knockdown. E Human apoptosis antibody array was utilized to analyze the regulatory ability of MEX3A on the expression of apoptosis-related proteins in U251 cells. F The effects of MEX3A on the cell migration ability of U87 and U251 cells were evaluated by Transwell assay. G The expression of EMT-related proteins, including N-cadherin, Vimentin, and Snail, was detected by western blotting in U87 and U251 cells of shMEX3A and shCtrl groups. The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. MEX3A knockdown inhibited glioma development in vivo.

A Seven days post injection of U251 cells with or without MEX3A knockdown, the volume of tumors formed in mice was measured and calculated at the indicated time intervals. Inset: tumors were removed 26 days post injection for collecting photos. B Mice were sacrificed at day 26 post injection, and the tumors were removed for weighing. C In vivo imaging was performed to evaluate the tumor burden in mice of shMEX3A and shCtrl groups at day 26 post tumor inoculation. The bioluminescence intensity was scanned and used as a representation of tumor burden in mice of shMEX3A and shCtrl groups. D The expression of Ki-67 was detected by IHC to show the proliferative activity of tumors. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. The exploration and verification of downstream-underlying MEX3A-induced regulation of glioma.

A A PrimeView human gene expression array was performed to identify the differentially expressed genes (DEGs) between shMEX3A and shCtrl groups of U251 cells. B qPCR and C western blotting were used to detect the expression of several selected DEGs in U251 cells with or without MEX3A. D A MEX3A-associated interaction network constructed by IPA analysis revealed the potential linkage between MEX3A and CCL2. E The expression of CCL2 in glioma and normal tissues was evaluated by IHC analysis. F The mRNA expression of MEX3A in U87, U251, U373, and SHG-44 cell lines was detected by qPCR. G A co-IP assay was performed to show the direct interaction between MEX3A and CCL2. The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 5. CCL2 knockdown inhibited glioma development, MEX3A overexpression promoted glioma development in vitro.

A, B Cell models with or without CCL2 knockdown were constructed by transfecting shCCL2 or shCtrl. The knockdown efficiency of CCL2 in U251 cells was assessed by qPCR (A) and western blotting (B). C Celigo cell counting assay was employed to show the effects of CCL2 on cell proliferation of U251 cells. Flow cytometry was performed to detect cell apoptosis (D) and cell cycle distribution (E) of U251 cells with or without CCL2 knockdown. F, G The effects of CCL2 on the cell migration ability of U251 cells were evaluated by wound-healing assay (F) and Transwell assay (G). H, I Cell models with or without MEX3A overexpression were constructed by transfecting control or MEX3A-overexpression plasmids. The overexpression efficiency of MEX3A in U251 cells was assessed by qPCR (H) and western blotting (I). J Celigo cell counting assay was employed to show the effects of MEX3A on cell proliferation of U251 cells. Flow cytometry was performed to detect cell apoptosis (K) and cell cycle distribution (L) of U251 cells with or without MEX3A overexpression. M, N The effects of MEX3A on the cell migration ability of U251 cells were evaluated by wound-healing assay (M) and Transwell assay (N). The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 6. Knockdown of CCL2 attenuated the effects of glioma cells by MEX3A overexpression.

U251 cells transfected with NC(OE + KD), MEX3A-overexpression plasmids, and simultaneous MEX3A-overexpression plasmids, and shCCL2 was subjected to the detection of expression (A, B) cell proliferation by Celigo cell counting assay (C), cell apoptosis by flow cytometry (D), cell migration by wound-healing assay (E), and cell migration by Transwell assay (F). The representative images were selected from at least three independent experiments. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.