Abstract
Glioma proliferation is a multistep process during which a sequence of genetic and epigenetic alterations randomly occur to affect the genes controlling cell proliferation, cell death and genetic stability. microRNAs are emerging as important epigenetic modulators of multiple target genes, leading to abnormal cellular signaling involving cellular proliferation in cancers.In the present study, we found that expression of miR-195 was markedly downregulated in glioma cell lines and human primary glioma tissues, compared to normal human astrocytes and matched non-tumor associated tissues. Upregulation of miR-195 dramatically reduced the proliferation of glioma cells. Flow cytometry analysis showed that ectopic expression of miR-195 significantly decreased the percentage of S phase cells and increased the percentage of G1/G0 phase cells. Overexpression of miR-195 dramatically reduced the anchorage-independent growth ability of glioma cells. Furthermore, overexpression of miR-195 downregulated the levels of phosphorylated retinoblastoma (pRb) and proliferating cell nuclear antigen (PCNA) in glioma cells. Conversely, inhibition of miR-195 promoted cell proliferation, increased the percentage of S phase cells, reduced the percentage of G1/G0 phase cells, enhanced anchorage-independent growth ability, upregulated the phosphorylation of pRb and PCNA in glioma cells. Moreover, we show that miR-195 inhibited glioma cell proliferation by downregulating expression of cyclin D1 and cyclin E1, via directly targeting the 3′-untranslated regions (3′-UTR) of cyclin D1 and cyclin E1 mRNA. Taken together, our results suggest that miR-195 plays an important role to inhibit the proliferation of glioma cells, and present a novel mechanism for direct miRNA-mediated suppression of cyclin D1 and cyclin E1 in glioma.
Highlights
The cyclins and their catalytic partners, the cyclin dependent kinases (CDKs), are cell cycle regulators
We demonstrated that miR-195 promotes glioma cell proliferation by directly targeting the 39-UTRs of cyclin D1 and cyclin E1, reducing phosphorylation of protein retinoblastoma (pRb) and downregulating the proliferative marker proliferating cell nuclear antigen (PCNA)
We demonstrated that upregulation of miR-195 in glioma cells led to the downregulation of phosphorylated pRb and proliferative marker PCNA through downregulation of cyclin D1 and cyclin E1 via directly targeting the 39-UTR of cyclin D1 and cyclin E1
Summary
The cyclins and their catalytic partners, the cyclin dependent kinases (CDKs), are cell cycle regulators. The first functions of cyclin D1 and cyclin E1 to be identified were related to control of G1-S phase cell cycle progression [2]. Cyclin D1 and cyclin E1 are thought to promote progression to the G1 phase of the cell cycle, on the basis of their cyclic pattern of mRNA expression, with maximal expression levels detected near the G1/S boundary [3,4,5]. During the G1 phase, the cyclin D1/CDK4 complex is phosphorylated by CDKactivating kinase (CAK). Phosphorylation of pRb leads to dissociation of the E2 promoter-binding protein dimerization partners (E2F) from the pRb/E2F complex, and dissociated E2F induces transcription of cyclin E1, which is required for entry to the S phase of the cell cycle [7]
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