Abstract
BackgroundThere is evidence that the extent of the G2/M arrest following irradiation is correlated with tumour cell survival and hence therapeutic success. We studied the regulation of cellular response to radiation treatment by miR-21-mediated modulation of cell cycle progression in breast cancer cells and analysed miR-21 expression in breast cancer tissue samples with long-term follow up.MethodsThe miR-21 expression levels were quantified (qRT-PCR) in a panel of 86 cases of invasive breast carcinomas in relation to metastasis free survival. The cellular radiosensitivity of human breast cancer cells after irradiation was determined comparing two cell lines (T47D and MDA-MB-361) by cell proliferation and colony forming assays. The influence of miR-21 overexpression or downregulation on cell cycle progression and G2/M checkpoint arrest after irradiation was assessed by flow cytometric analysis.ResultsThe expression of miR-21 was transiently increased 8 hours after irradiation in the radioresistant T47D cells and significantly changed with lower extent in radiosensitive MDA-MB-361 cells. Anti-miR-21 treated breast cancer cells failed to exhibit the DNA damage-G2 checkpoint increase after irradiation. Apoptotic activity was significantly enhanced from 7% to 27% in T47D cells and from 18% to 30% in MDA-MB-361 cells 24 hours after 5 Gy irradiation. Additionally, we characterized expression of miR-21 in invasive breast carcinomas. In comparison to non-cancerous adjacent breast tissue, tumours samples had increased miR-21 expression that inversely correlated with the distant metastases-free survival of patients (p = 0.029).ConclusionsOur data indicate that miR-21 expression in breast cancer cells contributes to radiation resistance by compromising cell cycle progression. These data point to the potential of combining radiotherapy with an anti-miR-21 as a potent G2/M check point inhibitor in overcoming radiation resistance of tumours.
Highlights
There is evidence that the extent of the G2/M arrest following irradiation is correlated with tumour cell survival and therapeutic success
Growth and maintenance of cell lines The breast cancer cell line MDA–MB–361 was cultured in DMEM (Dulbecco Modified Eagles medium) with 20% FCS, (Invitrogen, Carlsbad, CA) and T47D was maintained in RPMI 1640 (Roswell Park Memorial Institute medium) supplemented with 10% FCS and human insulin (10 μg/ml)
Functional analysis of miR-21 overexpression and inhibition in breast cancer cellular response to radiation Since miR-21 is well expressed in non irradiated T47D cells (Figure 3A) and is dramatically elevated by radiation we proposed that inhibition of miR-21 would decrease the degree of radiation resistance
Summary
There is evidence that the extent of the G2/M arrest following irradiation is correlated with tumour cell survival and therapeutic success. We studied the regulation of cellular response to radiation treatment by miR-21-mediated modulation of cell cycle progression in breast cancer cells and analysed miR-21 expression in breast cancer tissue samples with long-term follow up. A more detailed knowledge about radiation influences on miRNA expression in tumour cells is important for improving the effectiveness and reducing the side effects of radiotherapy. A possible explanation was provided by a genome wide search for miR-21 targets. This suggested a functional link between miR-21 and the p53 tumour suppressor pathway [17,19], where p53-induced proteins provoke apoptosis in response to DNA damage after irradiation in cancer. If the G2 checkpoint is selectively disrupted the cancer cells with impaired G1 checkpoint would become more sensitive to the DNA-damaging treatment compared with normal cells because normal cells still retain G1 checkpoint intact [21]
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