LncRNA Hotairm1 Promotes Osteosarcoma Progression By Sponging MiR-106a-5p And Regulating XIAP Expression

Objective: Osteosarcoma is the most common primary malignant bone tumor.Its characteristics including rapid progression,high invasiveness,early metastasis,and drug resistance.All above bring great difficulties to the treatment of osteosarcoma.With the continuous progress of neoadjuvant chemotherapy,targeted therapy and immunotherapy,the five-year survival rate of patients with osteosarcoma has been improved.In recent years,the therapeutic effect of osteosarcoma has reached a plateau.Therefore,it is of great significance to explore the occurrence and development mechanism of osteosarcoma,find more effective targets for diagnosis and treatment,and further improve the treatment plan of osteosarcoma.In recent years,people have found lnc RNAs have the potential to serve as the therapeutic targets of tumors.Increased studies have identified lnc RNAs hotairm1 played important roles in a variety of tumors,but its role in osteosarcoma is not clear.In this paper,the specific high expression of Lnc RNA hotairm1 in osteosarcoma and its correlations with prognosis and other clinical indicators of osteosarcoma patients were discussed,as well as its regulation and specific mechanism on proliferation,migration,invasion,apoptosis and tumorigenesis of osteosarcoma cells.We hope our study could provide a new potential biological target for the diagnosis and treatment of osteosarcoma and a theoretical basis for the clinical treatment of osteosarcoma by targeting Lnc RNA hotairm1.Methods: Firstly,we detected the expression of Lnc RNA hotairm1 in osteosarcoma and adjacent normal tissues by q PCR,and analyzed the relationship between Lnc RNA hotairm1 and prognosis of osteosarcoma.Secondly,we confirmed the localization of Lnc RNA hotairm1 in osteosarcoma cells by nucleo-cytoplasmic separation and FISH assay.By overexpression and silencing of Lnc RNA hotairm1 in osteosarcoma cell lines,the effects of Lnc RNA hotairm1 on proliferation,migration,invasion,apoptosis and tumorigenesis in nude mice of osteosarcoma were investigated based on CCK8,colony formation,wound healing test,transwell test,flow cytometry and tumor bearing in nude mice.Thirdly,we predicted the targeted mi RNA of Lnc RNA hotairm1 based on bioinformatics,and verified the targeted binding relationship based on double luciferase reporter gene assay and q PCR.Moreover,through salvage experiments,we verified that Lnc RNA hotairm1 regulated osteosarcoma cells by regulating mi R-106a-5p.Fourthly,we predicted and verified that XIAP was the targeted gene of mi R-106a-5p.Based on q PCR and western blot assay,we identified that Lnc RNA hotairm1 could regulate XIAP by targeting mi R-106a-5P and affect apoptosis-related pathways in osteosarcoma cells.Results: Firstly,through q PCR analysis,we found that the expression level of Lnc RNA hotairm1 in osteosarcoma tissues was significantly higher than that in normal tissues.According to immunohistochemistry assay,the expression level of Ki67 protein in the group of high Lnc RNA hotairm1 expression was significantly higher than the group of low Lnc RNA hotairm1 expression.In correlation analysis of clinical variables,the results suggested that the group of high Lnc RNA hotairm1 expression had a higher risk of metastasis and recurrence.Furthermore,kaplan-Meier survival analysis also showed that patients with high expression of Lnc RNA hotairm1 had a worse prognosis.Secondly,q PCR results indicated that Lnc RNA hotairm1 expression in osteosarcoma cell lines was significantly higher than that in osteoblast cell lines.According to nucleo-cytoplasmic analysis and FISH assay,we found that Lnc RNA hotairm1 was distributed in both the nucleus and cytoplasm of osteosarcoma cell,and its expression was higher in the cytoplasm.CCK8,clonogenesis,striation assay,Transwell assay,flow cytometry and tumor-bearing nude mice assay showed that pc DNA-hotairm1 group had stronger proliferation,migration,invasion,tumorigenesis abilities and lower apoptosis rate than the control group.Furthermore,sh-hotairm1 group had weaker proliferation,migration,invasion,tumorigenesis abilities and higher apoptosis rate.Thirdly,through bioinformatics analysis,double lucifase reporter gene and q PCR assay,we found that Lnc RNA hotairm1 could target mi R-106a-5p,and they were negatively correlated in osteosarcoma.In addition,the q PCR results of tissue and cell suggested that mi R-106a-5p was significantly lowly expressed in osteosarcoma tissues and cell lines.sh-hotairm1+mi R-106a-5p inhibitor group had stronger proliferation,migration and invasion abilities and lower apoptosis rate than sh-hotairm1 inhibitor group.Fourthly,bioinformatics analysis,double lucifase reporter gene assay and q PCR results suggested that mi R-106a-5p could target XIAP,and XIAP was negatively correlated with mi R-106a-5p in osteosarcoma tissues,and positively correlated with Lnc RNA hotairm1.In addition,XIAP expression was significantly increased in osteosarcoma tissues and cell lines.The m RNA and protein expression of XIAP in sh-hotairm1 mice group were significantly lower than those in control group.In osteosarcoma cells,the protein expression of XIAP in sh-hotairm1+mi R-106a-5p inhibitor group was higher than that in sh-hotairm1 inhibitor group while the protein expression of Caspase7 and Caspase9 protein were lower.Conclusions: Lnc RNA hotairm1 is higher expressed in osteosarcoma tissues and cells comparing to the normal.The prognosis of patients with higher expression of Lnc RNA hotairm1 were worse while they also had the higher risk of metastasis and recurrence.Lnc RNA hotairm1 can regulate XIAP by combining with mi R-106a-5p through competitive endogenous RNA(ce RNA)mechanism,thereby promote proliferation,migration and invasion of osteosarcoma while inhibit osteosarcoma apoptosis and apoptosis-related pathways.Thus,Lnc RNA hotairm1 might serve as a potential target for the diagnosis and treatment of osteosarcoma.