MicroRNA-210 and endoplasmic reticulum chaperones in the regulation of chemoresistance in glioma

Abstract

Gliomas are the commonest type of primary malignant brain tumours of the central nervous system (CNS). The highly aggressive and infiltrative characteristics of gliomas render them one of the most lethal cancers. Amongst all, the most malignant form of glioma is glioblastoma multiforme (GBM), a World Health Organization (WHO) grade IV astrocytoma. Despite well-developed multimodal treatment including surgery, radiotherapy, and chemotherapy, the prognosis of GBM patients remains poor with median survival of just over one year. This high mortality rate is commonly the result of relentless tumour recurrence secondary to the tumour’s intrinsic resistance towards its standard chemotherapeutic agent temozolomide (TMZ).

Prolyl 4-hydroxylase, beta subunit (P4HB) is an endoplasmic reticulum stress response (ERSR) chaperone protein that was previously found to be overexpressed in the chemoresistant glioma cell lines D54-MG and U87-MG. Differential expressions of numerous microRNAs (miRNAs) were also found between chemosensitive and chemoresistant glioma cell lines. As such, we surmised that the dysregulation of a P4HB-regulating miRNA may contribute to P4HB upregulation and therefore chemoresistance in glioma. MiR-210, a commonly dysregulated miRNA in various cancers, is one of the most highly downregulated miRNAs in chemoresistant glioma cells (compared to chemosensitive glioma cells), and, based on bioinformatics findings, may also regulate P4HB expression. MiR-210 was therefore selected for further investigations regarding its potential roles in glioma chemoresistance.

The regulatory relationship between P4HB and miR-210 was subjected for verifications. With the use of quantitative real-time polymerase chain reaction (qPCR) and western blotting, the intrinsic expressions of P4HB and miR-210 were studied. The upregulation of P4HB in D54 and U87 chemoresistant glioma (compared to the parental) cell lines were found to correlate reciprocally with the downregulation of miR-210 in the same chemoresistant glioma cells.

To delineate the potential regulatory role of miR-210, a gain of function approach was adopted. Transfection of a miR-210 mimic was performed into the D54 and U87 parental chemosensitive (D54-S and U87-S) and chemoresistant (D54-R and U87-R) cells, along with a negative control. The transfection efficiency of miR-210 as well as the subsequent P4HB expressions was verified. It was found that P4HB expression was downregulated as a result of miR-210 upregulation both at the mRNA and protein levels in glioma cells. Furthermore, the effects of miR-210 overexpression on chemoresistance in the glioma cells were tested by performing cell proliferation assay. Decrease in the half maximal inhibitory concentration (IC50) of TMZ were found in all cell lines overexpressing miR-210, suggesting that miR-210 upregulation may lead to P4HB inhibition, which would at least partially mediate an alleviation of glioma cells’ resistance towards its chemotherapeutic agent TMZ.

In summary, miR-210 is downregulated in chemoresistant glioma cells in vitro. It plays a potential role in regulating P4HB expression, hence chemoresistance in GBM cells. Future investigations may focus on its mechanism of action and potentiality for therapeutic intervention.