MUC21 induces the viability and migration of glioblastoma via the STAT3/AKT pathway

Abstract

Glioblastoma (GBM) is a malignant tumor with one of the fastest increasing morbidity and mortality rates. As such, more therapeutic targets need to be developed to combat this disease. Mucin 21 (MUC21) is a human counterpart of mouse epiglycanin and mediates multiple cellular functions. However, its possible effects on GBM and its possible mechanism remain unclear. The current study aimed to clarify the role or MUC21 in the progression of GBM by performing a series of in vitro assays, including Cell Counting Kit-8, colony formation, wound closure, transwell, and in vivo assays. In the present study, the aberrantly high expression of MUC21 in human GBM tissues and cell lines was observed and it was revealed that it was associated with the clinicopathological feature, tumor recurrence, in patients with GBM. MUC21 promoted the viability and motility of GBM cells in vitro and stimulated tumor growth in vivo. It was further confirmed that MUC21 promoted the progression of GBM via the STAT3/AKT pathway and it was considered that MUC21 could serve as a promising target for the treatment of GBM.

Keywords: STAT3/AKT pathway; glioblastoma; mucin 21; prognosis; vaibility.

Figure 1

MUC21 is highly expressed in human GBM tissues. (A) A reverse transcription-quantitative PCR assay were conducted to determine the mRNA levels of MUC21 in GBM and corresponding adjacent non-cancerous tissues. A paired Student's t-test was used for the analysis of tumor and adjacent non-tumor samples. (B) Western blotting was performed to determine MUC21 expression in three GBM cell lines. (C) Immunohistochemistry was performed to evaluate the protein levels of MUC21 in GBM tumor and corresponding adjacent non-cancerous tissues. Magnification, x100 and x200 (scale bar, 5 mm). ^*P<0.05 as indicated. GBM glioblastoma; MUC21, mucin 21.

Figure 2

MUC21 expression is downregulated in U251 and U87 cells after MUC21 shRNA transfection. (A) Reverse transcription-quantitative PCR assays were performed to measure MUC21 mRNA levels in U251 and U87 cells following control or MUC21 shRNA transfection. (B) Western blot analysis was performed to determine the protein expression of MUC21 in U251 and U87 cells following control or MUC21 shRNA transfection. ^*P<0.05 vs. shRNA. MUC21, mucin 21; NC, negative control; shRNA, short hairpin RNA.

Figure 3

MUC21 promotes GBM cell viability and migration in vitro. (A) Cell viability was assessed after MUC21 depletion in U87 and U251 through colony formation assays. (B) MTT assays were performed to detect the viability of cells transfected with the indicated shRNA. (C) Wound closure assay of GBM cells transfected with the indicated shRNA were performed for the quantification of migrated cells after 24 h. Magnification, x200. Western blotting assays were performed to analyze the expression of (D) Ki-67 and PCNA, and (E) MMP2 and MMP9 in GBM cells transfected with the indicated shRNA. Relative expression was analyzed. ^*P<0.05 and ^**P<0.01 vs. the shNC group. GBM glioblastoma; MUC21, mucin 21; PCNA, proliferating cell nuclear antigen; shRNA, short hairpin RNA; NC, negative control.

Figure 4

MUC21 contributes to GBM progression via the STAT3 and AKT signaling pathway. (A) The protein levels of p-STAT3, STAT3, p-AKT and AKT in shMUC21-transfected or control U251 cells were detected by immunoblot assays. (B) The protein levels of MUC21, p-STAT3, STAT3, p-AKT and AKT in MUC21-overexpressing or control U251 cells were detected by immunoblot assays. The relative expression was analyzed. ^*P<0.05 and ^**P<0.01 vs. shNC or Con. Con, control; MUC21, mucin 21; NC, negative control; p-, phosphorylated; shRNA, short hairpin RNA.

Figure 5

MUC21 stimulates glioblastoma progression through the STAT3 and AKT pathway in vivo. (A) U251 cells stably transfected with control or MUC21 shRNA vectors were subcutaneously implanted into nude mice. Tumor volume was monitored every week. Isolated tumors were displayed in (A). Tumor growth curves were compared between control and MUC21 depletion groups. Murine weight was also measured. (B) An immunohistochemistry assay was conducted to assess MUC21 protein levels in control or MUC21-depleted groups (scale bar, 200 µm). (C) Western blotting assays were performed to assess the expression levels of the indicated proteins in control or MUC21-depleted tumor tissues isolated from nude mice. ^*P<0.05 and ^**P<0.01 vs. shNC. MUC21, mucin 21; NC, negative control; PCNA, proliferating cell nuclear antigen; p-, phosphorylated; shRNA, short hairpin RNA.