Study On The Function And Regulation Mechanism Of Long Non-coding RNA LINC00511 In Osteosarcoma

Osteosarcoma is the most common primary malignant tumor in skeletal system.Its annual incidence is about 1-3/100 million.It can happen at any age.However,high grade osteosarcoma occurs mainly in 10-20 year old adolescents and children.Osteosarcoma involves the distal end of the long bone,especially the fast growing bones,such as the distal femur,the proximal tibia,the proximal humerus and so on.Thanks to the development of neoadjuvant chemotherapy and the progress of prosthesis and material technology,the limb salvage rate and survival rate of osteosarcoma have been greatly improved.However,the prognosis of patients with recurrent and distant metastasis is still poor,and the development of new drugs is also delayed,and the treatment of osteosarcoma has entered the bottleneck.Further clarifying the molecular mechanism of osteosarcoma development and developing new drugs are the future direction for the treatment of osteosarcoma.Long non-coding RNA(LncRNA)is an important class of non-coding RNA(ncRNA).A large number of studies have reported that the abnormal expression or mutation of LncRNA is closely related to the mechanisms of proliferation,invasion,metastasis and drug resistance of malignant tumors.In osteosarcoma,the related research of LncRNA is still in its infancy.Whether we can use the down regulated or up regulated LncRNA expression in the treatment of osteosarcoma or reverse the drug resistance of osteosarcoma still need further exploration.In addition,whether LncRNA can be used as an important reference for clinical diagnosis of osteosarcoma and a molecular marker to predict chemosensitivity and prognosis need clinical practice.LINC00511 is a relatively new LncRNA.In non-small cell lung cancer,pancreatic ductal adenocarcinoma and squamous cell carcinoma of the tongue,it has been confirmed that LINC00511 showed high expression of oncogene and as in which play a role in promoting the malignant tumor proliferation,migration,and poor prognosis is closely related.We found that LINC00511 showed low expression in osteosarcoma tissues compared to normal tissues adjacent to the cancer.The purpose of this study is to further explore the expression of LINC00511 in osteosarcoma,and to analyze the correlation between the expression and the clinical features,and prognosis of the patients.In addition,we will try to study the function of LINC00511 separately in vitro and in vivo,and preliminarily reveal the regulatory mechanism of LINC00511 in osteosarcoma.Part 1: Expression and significance of LINC00511 in osteosarcoma tissue and osteosarcoma cell lineIn the first part,we through quantitative PCR detection of LINC00511 expression in normal tissue specimens in osteosarcoma tissues and para carcinoma,combined with clinical data and correlation analysis between clinical features of LINC00511 expression level with age,sex,tumor staging in patients with osteosarcoma,chemotherapy and prognosis.In addition,quantitative PCR was used to detect the difference in expression of LINC00511 in different osteosarcoma cell lines and normal osteoblast cells.The results showed that LINC00511 showed low expression in osteosarcoma tissues and cell lines;expression of patients with tumor necrosis rate of LINC00511 was significantly related to high expression patients showing sensitivity to chemotherapy is better;the expression of LINC00511 was not associated with gender,age,tumor location,tumor stage,tumor volume,surgical method;LINC00511 significantly correlated the expression level and prognosis of patients with osteosarcoma,high expression in patients with 3 year survival rate is higher,the prognosis is better.These results suggest that LINC00511 may be an important tumor suppressor gene in the development and progression of osteosarcoma.Its elevated expression indicates better chemotherapy sensitivity and prognosis,and is expected to become an important molecular marker for diagnosis and prognosis of osteosarcoma.Part 2: The effect of LINC00511 on the biological behavior of osteosarcoma cells and its effectIn the second part,we constructed an over expression stable strain of osteosarcoma cell line MG-63 and Saos-2,and compared the biological behavior changes of tumor with that of LINC00511.At the cellular level,the expression of LINC00511 was upregulated,and the effects of LINC00511 on proliferation,apoptosis and migration of osteosarcoma cells were observed by CCK-8 cell proliferation assay,colony formation assay,cell apoptosis assay,cell scratch test and cell Transwell assay.At the body level,the role of LINC00511 in the development of osteosarcoma was further verified through the tumorigenesis experiment in nude mice.The results showed that MG-63 and Saos-2 overexpression of LINC00511 can inhibit the proliferation and migration of osteosarcoma cell line,but does not affect cell apoptosis;athymic mouse LINC00511 expression in experimental tumor growth in the same period of time slower,smaller tumor volume,tumor lighter.These results suggest that LINC00511 can inhibit the proliferation and migration of tumor cells in osteosarcoma,and it is an important tumor suppressor gene in the pathogenesis of the tumor.Part 3: Study on the regulation mechanism of LINC00511 on the progression of osteosarcomaIn the third part,we have conducted a preliminary study on the molecular mechanism of LINC00511 regulation of osteosarcoma.We observe two aspects including the epithelial mesenchymal transition of osteosarcoma and the regulation of the PI3K/Akt signaling pathway.In each group of Saos-2 cells,the expression of Western Blot were used to detect EMT related protein E-cadherin,Vimentin and?-SMA,and PI3 K and p-Akt in the PI3K/Akt signaling pathway protein expression,thus preliminary studying the molecular mechanism of LINC00511 to inhibit the proliferation and migration of osteosarcoma.The results showed that the expression of E-cadherin protein in osteosarcoma increased significantly after overexpression of LINC00511 in Saos-2,while the expression of Vimentin and ?-SMA protein decreased significantly,suggesting that EMT in osteosarcoma was significantly inhibited.In addition,the expression of PI3 K and p-Akt(308)protein was significantly decreased,while the expression of p-Akt(473)protein was not significantly changed,suggesting that the PI3K/Akt signaling pathway was significantly inhibited.These results suggest that LINC00511 may regulate the proliferation and migration of osteosarcoma cells by inhibiting the EMT and PI3K/Akt signaling pathways of osteosarcoma,and the phosphorylation site of PI3K/Akt signaling pathway is threonine 308(Thr308).