Circ-MTDH Regulates Autophagy Through ER Stress Preliminary Experimental Studies In Osteosarcoma Cell Proliferation

Objective: Osteosarcoma(OS)is the most common primary bone malignancy affecting children and adolescents.The study found that circular RNA(circ-RNA)is closely related to the physiological activity of osteosarcoma cells,while isopolyucin(MTDH)is associated with the drug resistance of osteosarcoma.Further study confirmed that circ-MTDH can affect the proliferation and invasion of tumor cells.In this experiment,in vitro cell experiments were conducted to verify whether circ-MTDH could regulate autophagy through ER stress to enhance osteosarcoma cell proliferation,and explore the correlation between circ-MTDH and ER stress and its role in osteosarcoma cell proliferation,providing new ideas for the treatment of osteosarcoma.Methods: This study was mainly performed by in vitro cell experiments,which were divided into two parts.The first part is the expression level of circ-MTDH in the osteosarcoma cell line,human normal bone cells(hFOB 1.19)by quantitative fluorescence PCR.In the second part,after osteosarcoma cells were cultured and passaged,the cells were transfection with circ-MTDH-NC and circ-MTDH inhibitor and divided into control group(control group),circ-MTDH-NC(scrambled control group)group and si-circ-MTDH group(interference group).After meeting the requirements of MTDH expression level of the three groups of cells by quantitative PCR for the subsequent experiments,the proliferation of osteosarcoma cells was detected and compared using CCK 8;the levels of P62 and LC3 / I by WB experiment to compare the autophagy level of each group of cells after transfection;the levels of PERK,XBP 1,ATF 4,and CHOP protein in each group of cells by Western bloting experiment to explore the relationship with the endoplasmic reticulum stress.Finally,statistical methods were used to analyze the differences in autophagy levels and proliferation levels between each group,and whether the differences between the proteins responding to ER stress were statistically significant.Results:1 Fluorescence quantitative PCR showed that the expression levels of circ-MTDH and MTDH in osteosarcoma cells was significantly increased compared with human normal bone cells.2.The results of quantitative PCR showed that the expression level of MTDH in the interfering group was significantly downregulated compared with the other two groups.3.WB experiment showed that the LC3 / I level of cells in the interference group decreased significantly,and the expression level of P62 increased.Combined with the MDC staining experiment,it was known that after the knockdown of circ-MTDH,the autophagy ability of cells in the interference group decreased significantly compared with the other two groups.4CCK 8 experiments confirmed that the proliferation level of cells in the scrambled group after knockdown of circ-MTDH was significantly inhibited compared with the scrambled control and control groups.5 WB experiments confirmed that the protein expression levels of ER stress-related factors PERK,XBP 1 and ATF 4 were significantly reduced in the interference group,while Chop protein levels were increased in the cells of the interference group.Accordingly,it can be preliminarily speculated that knockdown of circ-MTDH can downregulate the proliferation level of osteosarcoma cells and is associated with ER stress as well as autophagy level of osteosarcoma cells.Conclusion:The expression level of circ-MTDH is higher in osteosarcoma cells than in human normal bone cells.Knockdown of circ-MTDH effectively inhibited the proliferation capacity of osteosarcoma cells,but also downregulated the autophagy level in osteosarcoma cells,and knockdown of circ-MTDH also inhibited the ER stress level in osteosarcoma cells.