Effects Of Human Core Clock Gene Overexpression On Circadian Rhythm And Phenotype Of U2OS Cells

Some core clock genes are found to be down-regulated in many tumor cells,such as colorectal cancer cell lines and osteosarcoma cell lines.Overexpression of these genes is helpful for disease treatment and prognosis.However,the changes of cell rhythm and phenotype after the overexpression of circadian clock genes are still unclear.The research contents of this paper are as follows:(1)The plasmid expression vectors of the core clock genes bmal1,clock,cry1 and cry2 were constructed using seamless cloning technology.(2)Overexpression of bmal1 and clock,cry1 and cry2 respectively by transient transfection in osteosarcoma cells U2 OS.After both m RNA and protein expression levels were up-regulated,effects on the expression of other core clock genes were examined.The m RNA levels of cry2,per1 and per2 genes were significantly increased after transfection with bmal1 and clock,while the protein expression level of PER2 was decreased.The m RNA levels of clock,per1,per2,and npas2 genes and the protein levels of BMAL1 and CLOCK were significantly increased after transfection of cry1 and cry2.(3)BAML1 and CLOCK,CRY1 and CRY2 were overexpressed in osteosarcoma cells U2 OS by transient transfection,and the cells were treated with DEX to synchronize their rhythms,and the rhythm changes of other core clock genes were detected.After overexpression of BMAL1 and CLOCK,the m RNA expression levels of cry1,cry2,per1 and per2 were significantly increased in cycle 1;the cycles of bmal1 and clock genes were prolonged,the phase shifted to the left,and the amplitude was significantly increased;among other core clock genes,only cry2 cycle was prolonged;The phase of cry2 shifted to the left,and the phase of npas2 shifted to the right;the period,phase and amplitude of other genes did not change significantly.After overexpression of CRY1 and CRY2,only the m RNA levels of bmal1 and clock were significantly increased in cycle 2;the phase of cry1 and cry2 was shifted to the right,and the amplitude was significantly increased,the period of cry2 and npas2 was shortened,the period of clock was prolonged,and the phase was shifted to the left;the phase of per2 was shifted to the left There were no significant changes in other core clock genes.(4)The changes of cell phenotype were detected after overexpression of BMAl1 and CLOCK,CRY1 and CRY2,respectively,in osteosarcoma cells U2 OS.It was found that after overexpression of BMAl1 and CLOCK,there was no significant change in cell proliferation ability,cell migration rate decreased,and cell invasion number decreased.After overexpression of CRY1 and CRY2,the cell proliferation ability was inhibited,the cell migration rate was significantly reduced,and the number of cell invasion was significantly reduced.This paper shows that overexpression of BMAl1 and CLOCK,CRY1 and CRY2 in osteosarcoma cells can promote the expression of other core circadian clock genes,and the cell rhythms of other core clock genes are also changed.Therefore,overexpression of core clock genes is a treatment for circadian rhythm disorders.research provides new research methods.In addition,both BMAl1 and CLOCK or CRY1 and CRY2 gene overexpression have beneficial effects on cell phenotype,providing strategies for the treatment of many diseases.