MicroRNA PC-3p-40996 Targeted And Regulated CALM1 And CAMK2D Expression In Antler Chondrocytes

MicroRNA PC-3p-40996(PC-40996)is a novel microRNA found in the anther of red deer(Cervus elaphus)at fast-growing period.Target gene prediction results showed that calmodulin(CALM1)and calcium/calmodulin dependent protein kinase 2D(CAMK2D),which play an vital role in the calcium signaling pathway,are potential targets of PC-40996.To explore the functions of PC-40996 in deer antler chondrocytes,we explored the regulation of PC-40996 on CALM1 and CAMK2D by luciferase assay and western blot assay.The function of PC-40996 on cancer cells was also explored in this study.The results showed that:(1)when 293 T were transfected with CALM1 3’UTR wild-type recombinant plasmid and PC-40996 mimics or control mimics,respectively,the luciferase activity in the experimental group was significantly decreased compared with that in the control group;When PC-40996 mimics or control mimics transfected 293 T with CALM13’UTR mutant recombinant plasmids respectively,there was no significant change in luciferase activity between two groups.Luciferase experiments confirmed that PC-40996 can bind to the3’UTR region of CALM1.(2)When PC-40996 mimics or control mimics were transfected into 293 T with CAMK2D3’UTR wild-type recombinant plasmid,the luciferase activity of the experimental group was significantly decreased compared with the control group;When PC-40996 or Control mimics transfected into 293 T with CAMK2D 3’UTR mutant recombinant plasmid,there was no significant change in luciferase activity between two groups.Luciferase experiments confirmed that PC-40996 can bind to the 3’UTR region of CAMK2D.(3)When PC-40996 was over-expressed in antler chondrocytes,western blot results showed that the expression of endogenous CALM1 and CAMK2D proteins were significantly decreased,which confirmed the targeted regulation of PC-40996 on CALM1 and CAMK2D.(4)When PC-40996 was over-expressed in human breast cancer cells(MCF7)and human liver cancer cells(Huh7),the results of cell migration and proliferation experiments showed that the invasion and proliferation of cancer cells were significantly enhanced.The results show that: PC-40996 has a biological function,which regulates the proliferation and migration of MCF7 cells and Huh7 cells;PC-40996 can specifically target the3’UTR of CALM1 and CAMK2D m RNA and negatively regulate the expression of endogenous CALM1 and CAMK2D.