OS is one of the common malignant bone tumors. However, breakthroughs are failed to realize in the OS treatment. This disease is mainly treated with surgery and chemotherapy, but the prognosis of OS patients is still unsatisfactory. Previous studies have shown that many genes are associated with the development and progression of OS. However, the emphasis has been on protein-coding genes or miRNAs17. The molecular mechanisms of OS are still unknown, and recent studies show that some non-coding RNAs play an important role in the development of OS18,19. For example, lncRNA(TMPO-AS1) acts as a ceRNA that promotes OS tumorigenesis by regulating the miR-199a-5p to WNT7B axis 20. MiRNA-1236-3p is significantly overexpressed in OS, inhibiting cell proliferation and inducing apoptosis by targeting KLF8.21.
In contrast to other non-coding RNAs, lncRNAs, miRNAs, and circRNAs are highly conserved and stable in mammalian cells, making circRNAs potentially ideal biomarkers and therapeutic targets22. In recent years, increasing research reveals the important role of circRNAs in different biological processes, especially in tumorigenesis, development, and metastasis, such as the development of gastric cancer23, colorectal cancer24, lung cancer25, and cervical cancer26. A previous study showed that circTADA2A is differentially expressed between OS cell lines and corresponding noncancer cell lines27. However, the role of many circRNAs in the development of OS is still largely unknown. In this study, we focused on the significantly upregulated expression of hsa-circ-0064636 in OS cell lines and normal tissue cell lines and further predicted the regulatory network between its targeted regulatory miRNAs and corresponding target genes. MiR-326 and miR-503-5p were predicted to be the targeting miRNAs of hsa-circ-0064636 according to multiple databases of circRNA-miRNA interactions. To verify the expression of miR-326 and hsa-miR-503-5 in OS, they were also used in the GSE65071 dataset and the difference-in-differences analysis showed that they were under-expressed in the OS samples compared to normal samples. As mentioned in a previous study, hsa-circ-0064636 is significantly upregulated in OS, regulates down-regulation of miR-326 and promotes cervical cancer progression through up-regulation of ELK1. MiR-326 overexpression transfection experiments verified that it could inhibit proliferation and invasion of cervical cancer cells and induce their apoptosis and autophagy28. MiR-326 is also significantly under-expressed in prostate cancer29 and correlated with prognosis. However, miR-503-5p has been reported to inhibit tumorigenesis, angiogenesis, and lymphangiogenesis of colon cancer through directly downregulating VEGF-A30. Hepatocellular carcinoma studies showed miR-503-5p could regulate epithelial to mesenchymal transformation, metastasis, and prognosis of hepatocellular carcinoma cells by inhibiting WEE131. Thus, miR-326 and miR-503-5p play a key role in the suppression of many cancers, with low expressions in cancers. Therefore, hsa-circ-0064636 may lead to the development of OS by suppressing the expression of miR-326 and miR-503-5p in OS. However, the function of miR-326 and miR-503-5p in OS are rarely studied.
This study also used the miRNA prediction mRNA database to analyze the significantly differentially expressed genes in OS to screen the potential target genes of miR-326 and miR-503-5p and obtain a circRNA-miRNA-mRNA ceRNA Interaction Network. Based on that, we screened the mRNAs with prognostic impact to obtain a circRNA-miRNA-mRNA regulatory subnetwork. UBE4A was a potential direct target of miR-326, and VDAC1 was a potential target of miR-503-5p. We also found significant prognostic differences between UBE4A and VDAC1 in the survival analysis of OS patients. The results of survival analysis plots showed the prognosis of UBE4A and VDAC1 was significantly worse in the high expression group compared to the low expression group. UBE4A belongs to the U-box ubiquitin ligase class of enzymes. The encoded protein is involved in polyubiquitin chain assembly and plays a key role in chromosome condensation and polyubiquitination segregation via securin. Autoantibodies against the encoded protein may become a marker for scleroderma and Crohn’s disease32,33. UBE4A is significantly upregulated in the comparison between ovarian plasmatic cystic carcinoma and adjacent normal tissues. The VDAC1 voltage-dependent anion channel protein is a major component of the outer mitochondrial membrane34. VDAC1 is expressed in all compartments, including mitochondria, the plasma membrane and other cells. It regulates major metabolic and energetic functions of cells, including Ca2+ homeostasis, oxidative stress, and mitochondria-mediated apoptosis. The overexpression of VDAC1 triggers cell death, which may be related to the destruction of nerve cells 35. VDAC1 was identified as a tumor promoter in cervical cancer, and knock-down of VDAC1 in cell lines of cervical cancer increase the incidence of apoptosis, which could be partially reversed by overexpression of VDAC136.
The qRT-PCR experiments showed that hsa-circ-00063636, VDAC1 and UBE4A were highly expressed in OS cell lines, and miR-326 and miR-503-5p were lowly expressed in OS cell lines. These results were consistent with those of our bioinformatics analysis. However, the combination of the regulatory network needs to be verified in the subsequent experiments.