This study revealed the role of one lncRNA STK4-AS1 in the proliferation of osteosarcoma. From clinical samples, we found an association between the expression of STK4-AS1 and osteosarcoma: the cancer bone tissue expressed a higher level of STK4-AS1 over noncancer bone tissues. Thus, STK4-AS1 can be developed as a potential clinical prognostic biomarker for osteosarcoma. We also found that the expression of STK4-AS1 in osteosarcoma is correlated with p53 and p21 expression in tissue samples. As p53 and p21 are critical regulators for cancers, this study revealed that STK4-AS1 can be a potential pharmacological target for osteosarcoma treatment. In addition, we suggested that STK4-AS1 might have different expression profiles in a certain fraction of cells within one sample. For example, cancer stem cells  might express a different level of STK4-AS1 from the other cancer cells.
Among the three osteosarcoma cell lines tested in this study, MG63 expressed most STK4-AS1. However, MG63 had no p53 expression, thus, it is a good negative control cell line for us to explore the role of p53 in STK4-AS1 regulation. We found that the actions of STK4-AS1 are different in U2OS and MG63. In U2OS, STK4-AS1 regulated the cell cycle while in MG63 STK4-AS1 showed not regulatory effects on cell viability or cell cycle. We suggested that the difference od p53 expression in U2OS and MG63 accounts for these differences. We also discovered that the viability of osteoblast cells fHOB only altered by overexpression of STK4-AS1 but not the knockdown. We suggested that this is because STK4-AS1 expression in fHOB is too low to regulate cell viability.
STK4-AS1 was found to be the “booster” in breast cancer , lung cancer, and colon cancer. In this study, we demonstrated that it promoted the progression of osteosarcoma by inhibiting the p53/p21 pathway. MTT assay with transfected cells revealed that the expression of STK4-AS1 level was associated with cell viability. Many pharmacological targets were discovered because they can regulate cell viability [59, 60]. We analyzed the effect of STK4-AS1 on the cell cycle of the p53 expressing osteosarcoma cell line U2OS and found that STK4-AS1 affected cell viability by regulating the cell cycle. Although some studies showed that the arresting of the cell cycle at the S or G2 phase can also lead to suppression of proliferation [61, 62], based on our viability results, we suggested that the increased cells in S and G2 phase resulted in more proliferation of U2OS cells.
In western blotting experiments, we observed the effect of STK4-AS1 on cyclin A, a regulator of the S phase regulation , cyclin B, a regulator of the G2 phase , and cyclin D/cyclin E, regulators of the G1 phase . Furthermore, we also determined p53/p21 pathway. P53 has long been reported to regulate the cell cycle in osteosarcomas . Here we tested it as a potential target underlying the effect of STK4-AS1 on osteosarcomas cells. The p53/p21 pathway is responsible for the negative regulation of cyclin B and cdk1, regulating activities of the G2 phase in cancer cells. The accumulation of p53 can increase the expression of its transcriptional target gene p21 which can potentially inactivate the cyclin B/cdk1 complex . Our results showed that the knockdown or overexpression of STK4-AS1 affects the expression of p53, and the p53 subsequently affected p21 and negatively regulates activities in the G2 phase. P53 rescue experiment showed that the abnormal cell viability, p21 expression, cyclin A expression, and cyclin B expression were largely recovered. Besides, p21 can also potentially binds and inactivate the cyclin E/CDK2 complex. Interestingly, although cyclin D and cyclin E have been found regulated by the p53/p21 pathway [65, 66], in this study the cyclin D and cyclin E levels were not affected even with p53/p21 level altered. We suggested that STK4-AS1 might have other impacts on cyclin D and cyclin E besides the p53/p21 pathway that subset the effect of the p53/p21 pathway (Fig. 10). We supposed that this unidentified regulation might involve other cancer-related mechanisms such as ion channel regulations [67, 68]. On the other hand, in the p53 negative control, MG63 cells, STK-AS1 failed to affect cyclin A and cyclin B, which was consistent with our hypothesis.
As STK4 has known roles in regulating cancer progression, it might be helpful to distinguish the impact of STK4-AS1 from the potential effects of STK4 upregulation. Although STK4-AS1 is transcribed from the non-coding region of the STK4 gene, we determined the levels of STK4 protein in tumor tissues and cell lines, as well as in cell lines involving knockdown and overexpression of STK4-AS1. Results showed that there is no correlation between STK4-AS1 and STK4, and the expression of STK4 was not affected by the alteration of STK4-AS1 expression in the knockdown and overexpression experiments (SFig.1). In addition, we suggested that the study might greatly benefit from the data on patient prognosis. Thus, we analyzed the available survival data in this study (SFig.2). We were also interested in the effect of STK4-AS1 on therapy response. However, the case number in this study was low and patients were treated with different therapy, including traditional medicine that was complex in their components, hence, we did not have enough cases to conclude the association of STK4-AS1 levels and patient survival or therapy response.
This study demonstrated that lncRNA STK4-AS1 is overexpressed in osteosarcoma and plays a role in the proliferation of p53 expressing osteosarcoma cells by affecting the cell cycle, which is mediated by the p53/p21 pathway. Given the potential value of STK4-AS1 for clinical osteosarcoma therapy and diagnosis, our study is conducive to the development of STK4-AS1 as a novel treatment target and diagnostic biomarker for osteosarcoma. In future clinical settings, STK4-AS1 can be determined using PCR or tested as one of the screening items in microarray probe assays. Potential gene therapy to reduce STK4-AS1 is also promising.