Abstract
ABSTRACT The long noncoding RNA X-inactive specific transcript (XIST) plays vital roles in tumor progression. However, the underlying mechanisms remain unclear. This study investigated the effects and mechanisms of targeting XIST on osteosarcoma (OS) cells in vitro and in vivo. We used shRNA to knockdown XIST to evaluate cell growth and apoptosis in U2OS cells in vitro and xenograft formation in vivo. An observed relationship between XIST and the p53 upregulated modulator of apoptosis (PUMA) and nuclear factor-kappa B (NF-kB) pathway was further explored by using small interfering RNA (siRNA). Our results showed that suppression of XIST by short hairpin RNA (shRNA) impeded U2OS cell growth, induced apoptosis and lessened OS xenograft tumor growth. Targeting XIST increased NF-kB-dependent PUMA upregulation in U2OS cells. Upregulation of PUMA is correlated with suppression of XIST-induced apoptosis in U2OS cells. Therefore, inhibition of XIST could promote U2OS cell death via activation of NF-kB/PUMA pathways.
Highlights
Osteosarcoma (OS) is the most common primary tumor of bone in children and adolescents
We demonstrated that p53 upregulated modulator of apoptosis (PUMA) is activated by NF-κB in response to short hairpin RNA (shRNA) X-inactive specific transcript (XIST), and that PUMA is a critical mediator of shRNA XIST induced apoptosis in OS cells in vitro and in vivo
Lv-XIST shRNA or Lv-scramble were transfected into U2OS cells for 72 h. p65 nuclear translocation, PUMA, bax,cleaved-caspase-3 protein expression and the binding activity of NF-Kb was gradually increased in U2OS cells by western blot (Figure 2A) and Electrophoresis Mobility Shift Assay (EMSA) (Figure 2B)
Summary
Osteosarcoma (OS) is the most common primary tumor of bone in children and adolescents. It has a poor prognosis due to local recurrence, metastasis, and chemotherapy resistance [1]. Patients without clinical signs of systematic spread show. 5-year survival rates of 60–80%, whereas, the 5-year survival for patients with metastatic disease is 20% [2]. Treatment relies on the use of chemotherapy and surgery, but treatment paradigms and survival rates have not improved for the past three decades [3].
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