Knockingdown Of KLF8 Suppresses Proliferation And Invasion In Human Osteosarcoma Cells

Osteosarcoma(OS) is the most common primary malignancy bone tumor in children and paegiatric age group. Patients with matastasis OS who have been treated with resection surgery usually had poor prognosis. The 5-year survival rates for these patients are lower than 20%. Without early diagnosis and timely treatment, in most case, OS metastasizes to lung through hematogenous metastasis within 6-12 months. The mechanisms about the metastatic process in OS are still remained to be studied, but the metastasis of OS has already become the main reason for the poor prognosis.KLF8(Krüppel-like factor 8) is in the group I of KLF transcription factor family. KLF family members have three highly conserved zinc finger domains in the N-terminal of their proteins. KLFs bind to the GC-rich promoter region and regulate various genes in human. KLFs play key roles in human growth and development and also work in human disease including tumor progress. KLF8 has elevated expression in a lot of tumors. KLF8 play important roles in oncogenic transformation and epithelial to mesenchymal transition. Although there are many of studies working on the function of KFL8 in human tumors, the function of KLF8 in OS has not been reported yet. The aim of this research is to investigate the regulatory function of KLF8 in OS especially in cell metastasis.The real-time PCR and western blotting found that KLF8 had higher expression in both m RNA and protein level in OS cells compared with the osteoblast. KLF8 had the highest expression in the most malignant cells. The western blotting in OS tissues found that the expression levels of KLF8 in primary tumor and metastasis tumor in lung were higher than that in normal adjacent tissue. The KLF8 expression in metastasis was even higher than in primary tumor tissue. According the KLF8 m RNA sequence, we designed the lentivirus-sh RNA packing system for knocking down KLF8 in OS cell line Saos-2 cells. The random si RNA was used as the negative control. The KLF8 was efficiently knocked down in both m RNA and protein level.We analysed the effect of KLF8 for the cell proliferation in KLF8 stablely knocked down cells. The cell numbers were tested during the five days after virous infection using MTT assay. Compared with negative control cells, the cell number of KLF8 knocking down cells decreased significantly. Colony formation assay and soft agar assay showed that the decrease of the KLF8 protein affected the colony formation. The results demonstrated that KLF8 is important in the regulation of cell proliferation. The cell cycle analysis found that the KLF8 knockdown in OS cell induced the cell cycle arrest at G0/G1 phase. The results also showed that KLF8 knockdown damaged the DNA replication. Cell apoptosis assay showed that the decreased expression of KLF8 in OS cells induced the early stage cell spoptosis. The decreased cell number of knocking down cells is due to the cell cycle arrest and cell apoptosis.Transwell@ invasion assay found that knockdown of KLF8 significantly inhibited the invasion of OS cells. Compared with negative control cells, the numbers of migrated cells observed in knocking down group are significantly decreased.These data suggest that KLF8 may exhibit an important role in osteosarcoma tumorigenesis and that KLF8 may be a potential therapeutic target for the treatment of osteosarcoma.