Expression Of LncRNA ANRIL In Osteosarcoma And The Effects On Proliferation, Apoptosis, Invasion And Migration Of Osteosarcoma Cells

Research background and purpose Osteosarcoma is a common malignant tumor of bone surgery,which originated in bone marrow mesenchymal stem cells and often occurs in young children.Surgery and radiotherapy and chemotherapy combined with the current treatment of the disease is the basic means,with the improvement of surgical methods and the application of new chemotherapy drugs make the prognosis of this disease has been significantly improved,but still about 40% of patients after treatment of tumor metastasis,the five-year survival rate has not improved significantly,suggesting that new treatments are still needed to improve the prognosis of patients and that the effect of new genes on osteosarcoma appears to be an effective means.Long-non-coding RNA(lncRNA)is a non-coding RNA with a length greater than 200 nucleotides.Studies have shown that lncRNA plays an important role in many life activities such as dose compensation,epigenetic regulation,cell cycle regulation and cell differentiation regulation,and becomes a hotspot in genetic research.lncRNA expression or dysfunction is closely related to the occurrence of human diseases,including cancer,degenerative neurological diseases,including a variety of serious harm to human health,a major disease,specifically for long-chain non-coding RNA in the sequence and spatial structure abnormalities of expression levels,abnormalities associated with binding proteins,and the like.lncRNA ANRIL is an antisense non-coding RNA in the INK4 Locus,which is associated with the apparent genetic silencing of INK4 a,which is a cell cycle kinase inhibitor 4b(INK4b)INK4b-ARF-INK4 a plays an important role in regulating cell cycle,cell senescence and apoptosis.However,the effect of lncRNA ANRIL on the biological behavior of osteosarcoma cells has not been reported.Therefore,to explore the effect of lncRNA ANRIL on the biological behavior of human osteosarcoma cells,so as to provide new ideas and strategies for the clinical treatment of osteosarcoma.In this study,we collected the specimens of osteosarcoma and adjacent tissues,and compared the expression of lncRNA ANRIL in cancer tissues and adjacent tissues.To investigate the effect of lncRNA on the proliferation,apoptosis and invasion and migration of osteosarcoma cells,Prevention and treatment to provide experimental basis.Method 1.In the orthopedic operating room of the First Affiliated Hospital of Zhengzhou University,25 cases of cancerous tissues and adjacent tissues of paired osteosarcoma were collected and stored in the-80 ° C refrigerator.The expression of lncRNA ANRIL gene was detected by real-time quantitative PCR(RT-q PCR)in 25 cases of paired osteosarcoma and adjacent tissues.2.Reagent company Synthetic lncRNA ANRIL inhibitor: siRNA-ANRIL.The experiment was divided into three groups: the experimental group(siRNA-ANRIL): siRNA-ANRIL transfected into osteosarcoma cell MG63,negative control group(Nagetive control,NC): transfection lncRNA ANRIL Nagetive control,blank group(Blank): only transfected liposomes.After transfection,the cells were incubated in a carbon dioxide(CO2)incubator for subsequent experiments.3.After transfection for 48 hours,the expression of ANRIL gene in lncRNA was measured by RT-q PCR technique,and the inhibitory effect of siRNA-ANRIL on the expression of lncRNA ANRIL was observed.4.Using CCK-8 cell counting Kit(Cell Counting,Kit-8,CCK-8)in 24 hours,48 hours,72 hours,96 hours after the three groups were measured to observe cell proliferation,inhibition of lncRNA ANRIL expression on the proliferation of osteosarcoma cells.5.The apoptosis of three groups of cells was measured by flow cytometry and caspase-3 activity assay kit.The effect of inhibition of lncRNA ANRIL on the apoptosis of osteosarcoma cells was observed.6.Transwell invasion assay was performed to observe the effect of inhibition of lncRNA ANRIL expression on the invasive ability of osteosarcoma cells after 12 hours of treatment.7.Scratch test,three groups of osteosarcoma cells scratch treatment,after treatment for 12 hours to observe the inhibition of lncRNA ANRIL expression on the migration of osteosarcoma cells.8.The expression of tumor metastasis-associated gene 1(MTA1),E-cadherin(E-cadherin)in epithelial cells and B lymphocyte neoplasia-2 protein(bcl-2)were detected by Western blotting.Result 1.Compared with the adjacent tissues,the expression level of lncRNA gene in osteosarcoma was significantly higher than that of ANRIL,and the difference was statistically significant(P<0.05).2.RT-q PCR results showed that the expression level of siRNA-ANRIL group lncRNA ANRIL gene after transfection was significantly lower than the negative control group and blank control group,the difference was statistically significant(P<0.05),no significant difference between negative control group and blank group the lncRNA expression of ANRIL(P>0.05).3.CCK-8 results showed that the absorbance of osteosarcoma cells in siRNA-ANRIL group at OD450 was significantly decreased,and the longer the time,the absorbance value and the negative control group and blank group compared the difference was more obvious,the difference was statistically significant(P<0.05),negative control group and blank group the absorbance values were similar,and did not change with time obviously,the difference was not statistically significant(P>0.05).4.Flow cytometry and caspase-3 activity assay kit showed that the apoptosis rate and Caspase-3 activity of osteosarcoma cells in siRNA-ANRIL group were significantly higher than those in negative control group and blank group(P<0.05).There was no significant difference in the apoptotic rate and Caspase-3 activity between the negative control group and the blank group(P>0.05).5.Transwell invasion assay showed that the number of siRNA-ANRIL group of osteosarcoma cells through the basement membrane was significantly lower than the negative control group and blank control group,the difference was statistically significant(P<0.05),while there was no significant difference in the number of negative control group and blank group of osteosarcoma cells through the basement membrane(P>0.05).6.The three group cells were scratching after 12 h results suggest that the migration ability and negative group siRNA-ANRIL osteosarcoma cells control group decreased significantly compared with the blank group,no significant difference between the migration ability of the negative control group and blank group of osteosarcoma cells.7.Western blot result showed that in siRNA-ANRIL group the expression of MTA1 was decreased,the expression of E-cad was increased and the bcl-2 protein was decreased,compared with the negative control group and the blank group,the difference was statistically significant(P< 0.05).The expression of MTA1,E-cad and bcl-2 protein in negative control group and blank group was similar,the difference was not statistically significant(P> 0.05).Conclusion 1.The expression of lncRNA ANRIL in osteosarcoma was higher than that in adjacent tissues,and siRNA-ANRIL could inhibit the expression of lncRNA ANRIL in osteosarcoma cells.2.Inhibition of the expression of lncRNA ANRIL gene can promote the apoptosis of osteosarcoma cells,and can reduce the proliferation,invasion and migration of osteosarcoma cells.