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Introduction
Osteosarcoma is a tumor characterized by the production of osteoid by malignant cells. This tumor commonly afflicts patients of 20 to 30 years of age, and the number has increased markedly in recent years. Tumor is a disease characterized by abnormal cell cycle phases. The first gap phase, G1, is unique among cell cycle phases because it is the point at which cells are responsive to extracellular cues. Cyclin D1 plays a key regulatory role during the G1 phase and its gene is amplified and overexpressed in many cancers, such as non-small cell lung cancer (1), stomach cancer, and mantle cell lymphoma (2). However, the mechanism that would explain CCND1 deregulation in these cancer cells is yet to be discovered. MicroRNAs are a new class of small non-coding RNAs found in both animals and plants. They bind to the complementary sequences in the 3′UTR of the protein coding genes and induce mRNA degradation or translational repression (3). Growing evidence has indicated that microRNAs control basic cell functions, including development, differentiation, apoptosis, and proliferation (4,5).
miR-15a and miR-16-1 are highly conserved RNAs that form a cluster at the chromosomal region 13q14.3 (6), which is frequently deleted in cancer. A number of studies have reported that miR-15a and miR-16-1 are missed or deleted in chronic lymphocytic leukemia (CLL), non-small cell lung carcinoma, liver cancer, breast cancer, ovarian cancer, prostatic cancer, stomach cancer, pituitary adenoma, multiple myeloma, and osteosarcoma (7-13). Numerous studies have reported that miR-15a and miR-16-1 induce apoptosis and inhibit cell proliferation by targeting multiple genes, but their specific mechanisms remain unclear.
In the current study, experimental evidence which shows that miR-15a and miR-16-1 induce apoptosis and cell cycle arrest in osteosarcoma was provided. In addition, a post-transcriptional regulatory mechanism of CCND1 expression mediated by miR-15a and miR-16-1 through direct interaction with the CCND1 mRNA at the 3′-UTR was discovered. CCND1 protein level expression is suppressed by miR-15a and miR-16-1, thus providing a potential strategy to prevent osteosarcoma proliferation by targeting the CCND1 oncogene.
Materials and methods
Cell lines
The human osteosarcoma cell lines SOSP-9607 and MG63 were grown in the RPMI-1640 medium and MEM medium with 10% heat-inactivated fetal bovine serum, 2 g/l sodium bicarbonate, and 4 g/l HEPES (Sigma) at 37°C in an...