Mechanosensitive ion channel PIEZO1 promotes glioma invasion and aggression by regulating Yes-associated protein / 中华神经医学杂志

ABSTRACT

Objective:To investigate the molecular mechanism of PIEZO1 in promoting the invasion and aggression of glioma via Yes-associated protein (YAP) delivering mechanical signals.Methods:(1) Specimen detection: specimens from 94 patients accepted glioma resection in our hospital from February 2015 to March 2017 were chosen; immunohistochemical staining was used to detect the PIEZO1 expression in these glioma tissues of different grades. (2) Cell experiment: human glioma cell lines U87 and U251 were cultured under different matrix; the expressions of PIEZO1 and YAP were detected by immunofluorescent staining, and the PIEZO1 protein expression was detected by Western blotting. (3) Cell experiment after lentivirus transfection: the U87 and U251 cells were divided into negative control group and sh-PIEZO1 group according to the presence or lack of shRNA lentivirus vector; the role of PIEZO1 in glioma proliferation and invasion was detected by clone formation assay, proliferation and invasion assay, and Western blotting; after PIEZO1 silencing, Western blotting was used to detect the expressions of mechanical signal pathway proteins, YAP, FAK, and β1-integrin; real time-PCR was used to detect the mRNA expressions of CTGF and CYR61, which were downstream target genes of YAP; immunofluorescent staining was used to detect the YAP expression after PIEZO1 silencing. Results:(1) The PIEZO1 expression in glioma specimens increased with glioma grading, and the PIEZO1 expression in IDH wild-type patients was higher than that in IDH mutant patients. (2) The PIEZO1 and YAP expressions increased with the increase of matrix stiffness; as compared with that in the 0.2 kPa group, the PIEZO1 protein expression in stromal cultured cells of 16 and 64 kPa groups was significantly increased ( P<0.05). (3) After PIEZO1 silencing, U87 cells became large and tentacles increased, while U251 cells were mostly with long fusiform; as compared with those in the negative control group, the number of cell clones, and the proliferation rate and invasive cell count at each time point in the sh-PIEZO1 group were significantly smaller/decreased ( P<0.05); as compared with those in the negative control group, the epithelial marker E-cadherin expression was significantly increased, while the expressions of stromal markers Vimentin, Snailin and Slug were significantly decreased in the sh-PIEZO1 group ( P<0.05). Finally, Western blotting and immunofluorescent staining showed that PIEZO1 silence significantly increased the phosphorylated-YAP expression, significantly decreased the YAP nuclear expression, significantly down-regulated the CYR61 and CTGF expressions, and significantly reduced the levels of integrin β1 and phosphorylated-FAK; there were significant differences on these indexes between the negative control group and sh-PIEZO1 group ( P<0.05). Conclusion:PIEZO1 regulates the response of glioma cells to mechanical signal and promotes proliferation and invasion of glioma cells through YAP, which is an attractive therapeutic target for glioma treatment.