MicroRNA-221/222 Inhibits the Radiation-Induced Invasiveness and Promotes the Radiosensitivity of Malignant Meningioma Cells

Abstract

The controversy of adjuvant radiotherapy of meningiomas is at least partially due to the insufficient understanding on meningioma cells' response to irradiation and the shortage of radiosensitivity-promotion methods. MicroRNA-221 and microRNA-222 were identified as critical regulators of radiosensitivity in several other tumors. However, their effect in meningiomas has yet to be confirmed. Therefore, the malignant meningioma IOMM-Lee cells were adopted, transfected with microRNA-221/222 mimics or inhibitors, and irradiated with different dosages. The effects of radiation and microRNA-221/222 were then assessed in vitro and in vivo. Radiation dose increases and microRNA-221/222 downregulation synergistically inhibited cell proliferation and colony formation, prevented xenograft tumor progression, and promoted apoptosis, but antagonistically regulated cell invasiveness. Pairwise comparisons revealed that only high-dose radiations (6 and 8 Gy) can significantly promote cell invasiveness in comparison with unirradiated counterparts. Further comparisons exhibited that downregulating the microRNA-221/222 expression can reverse this radiation-induced cell invasiveness to a level of untransfected and unirradiated cells only if cells were irradiated with no more than 6 Gy. In addition, this approach can promote IOMM-Lee's radiosensitivity. Meanwhile, we also detected that the dose rate of irradiation affects cell cycle distribution and cell apoptosis of IOMM-Lee. A high dose rate irradiation induces G0/G1 cell cycle arrest and apoptosis-promoting effect. Therefore, for malignant meningiomas, high-dose irradiation can facilitate cell invasiveness significantly. Downregulating the microRNA-221/222 level can reverse the radiation-induced cell invasiveness while enhancing the apoptosis-promoting and proliferation-inhibiting effects of radiation and promoting cell radiosensitivity.

Keywords: IOMM-Lee; dose rate; epithelial–mesenchymal transition-inducing transcription factors; invasiveness; microRNA-221/222; radiosensitivity.

Figure 1

The effects of the expression of miR-221/222 and radiation dose on IOMM-Lee cells in vitro. (A,B) Present the relative expressions of miR-221 and miR-222 in different groups after transfection, respectively. (C) Shows that increasing the radiation dose and downregulating miR-221/222 expression level can synergistically inhibit the proliferation of IOMM-Lee cells, while (D,E) show their synergistical promotion on cell apoptosis. (F,G) Exhibit that both increasing the radiation dose and downregulating miR-221/222 expression level can separately increase the sub-G0/G1 population and induce G0/G1 cell cycle arrest. (H,I) Revealed that increasing the radiation dose and downregulating miR-221/222 expression level can antagonistically regulate the cell invasiveness, while synergistically inhibit the colony formation, respectively. Furthermore, (J) exhibits that downregulating the miR-221/222 expression enhances the radiosensitivity of IOMM-Lee cells. The abovementioned experiments were performed at least in triplicate.

Figure 2

Phosphatase and tensin homolog (PTEN) is a target gene of miR-221 and miR-222 in IOMM-Lee cells. (A,B) Significantly decreased luciferase activities were revealed after the cotransfection of miR-221 mimics or miR-222 mimics and psi-check2-PTEN in dual luciferase reporter assay. (C,D) Western blot analysis exhibited that the expression of PTEN was positively correlated with radiation dose but negatively correlated with miR-221/222 expression.

Figure 3

Downregulating miR-221/222 expression along with radiation suppresses subcutaneous tumor growth in vivo. (A) Exhibits the xenograft tumors from different treatment subgroups. (B) Reveals that both inhibition of the miR-221/222 expression level and ionizing radiation significantly suppress tumor growth in nude mice.

Figure 4

Downregulation of miR-221/222 expression and ionizing radiation promote the expression of phosphatase and tensin homolog (PTEN) in vivo. (A,B) Show the relative expressions of miR-221 and miR-222 in subcutaneous tumors of different subgroups, respectively. (C) Exhibits immunohistochemical staining for PTEN (bottom, 400 ×) and their corresponding H&E staining (top, 400 ×) of tissue sections from each subgroup.