Study On Influences Of RAMP1-siRNA On CGRP-induced Proliferation And Differentiation Effects Of MG-63 Cells

Over 50 years ago,calcitonin as a polypeptide hormone secreted from parathyroid glands was discovered.Calcitonin is a peptide consisting of 32 amino acids and it is linked by disulfide bond at the terminals of cysteine residue and carboxyl acetamide at 1 and 7 loci.Calcitonin family contains many polypeptide hormones which are mostly similar to calcitonin in structure,where CGRP,amylin and adrenomedullin（MSH）are representative ones.Among them,CGRP as the crossed molecular between nervous system and immune system is the most extensively-distributed sensory neuropeptide in bone tissue,and thus its role in bone formation and remodeling is also receiving more and more attention.Many in vivo and in vitro experiments have proved that CGRP is equipped with fine biological activity,for it can promote the proliferation and differentiation of osteoblasts and play its biological effect through CRLR,RAMP1 and RCP on the osteoblast surface.During the early experiments,the research group discovers that RAMP1 expression can strengthen CGRP role in promoting osteoblast differentiation,and increase the expression of ALP mRNAon osteoblast cells.In order to further understand the relationship between CGRP receptor functions and receptors,this experiment firstly utilizes RNA interference technology to explore the influences of RAMP1 expression on the biological activities of CGRP receptor and MG-63 cells based on the previous experiment work,hoping to illustrate the receptor functions of RAMP1 and the repair mechanism of neuropeptide in bone trauma more deeply.CGRP is a polypeptide with 37 amino acids which is from the same single copy gene as calcitonin（CT）and produced by different splices after transcription.CGRP performs its functions through G-protein-coupled receptors.The receptor subunits confirmed currently include CRLR,RAMP 1 and RCP,of which the functions and mating types of CRLR strictly depend on the RAMP 1-3.The union of CRLR and RAMP forms CGRP.Though the research difficulty still lies in revealing the molecular structure features of RAMP1 through molecular model strategies,the current molecular structure and functions of RAMP 1 are still unknoown.To further define the RAMP1 functions,this experiment observes the influences of interfering the expression of RAMP Ion the expression and film distribution of CRLR and RCP and explores the influences of interfering RAMP1 on CGRP’s role in the biological activity of MG-63 through constructing RAMP 1 interference fragments and transfecting MG-63 cells.Methods1.Routine culture and passage of MG63 cells:from the Shanghai cell bank of Chinese Academy of Sciences,the MG63 cells are inoculated into the routine medium（high glucose DMEM with 10%FBS）of 100ml culture flask in 5%C02 incubator for passage.2.Design and synthesis of siRNA:according to the Ambion siRNA software,3 pairs of siRNA sequences aiming at RAMP1 mRNA target positions of human can be synthesized through transcription.The specificity of the 3 pairs of siRNA is determined through Blast.It is determined that RAMP 1-homo-185 sense:5c-CAUCACCUCUUCAUGACCATT-3c and antisense:5c-UGGUCAUGAAGAGGUGAUGTT-3c are use sequences and synthesized by Shanghai Invitrogen.3.Grouping of experiment:in vitro transcription synthesis method is applied to synthesize and screen siRNA,and the MG-63 cells transfected to subculture can be divided into:RAMP1 interference group,non-carrier group and blank control group.4.The regulatory effect of RAMP1 interference to CRLR and RCP of MG-63 cell membranes:4.1 Influences of transfected RAMP 1-siRNA on the expression of CRLR and RCP:after successful transfection of RAMP1-siRNA,fluorescent quantitative PCR will test the relative expression of CRLR and RCP in RAMP1 interference group,non-carrier group and blank control group.4.2 Influences to the distribution and expression of CRLR and RCP in MG63 cell membranes:confocal laser immunofluorescence assay is adopted to test the distribution and expression of CRLR and RCP in MG63 cell membranes of RAMP1 interference group,non-carrier group and blank control group.5.Influences of RAMP1 interference on CGRP in promoting MG63 cell proliferation:CCK-8 method is used to test the cell proliferation of RAMP1 interference group,non-carrier group and blank control group at 0,24,48,72 and 96 hours.6.Influences of RAMP1 interference on CGRP in promoting MG63 cell differentiation:Elasa approach is utilized to test the ALP activity expression in RAMP1 interference group,non-carrier group and blank control group.Fluorogenic quantitative PCR is used to test the expression of relevant osteoblast marker gene demRNA in RAMP1 interference group,non-carrier group and blank control group.7.Statistical analysis:SPSS17.0 statistical software is applied;all data are expressed with x±s;and P<0.05 difference is of statistical significance.Results:1.Design and screen of siRNA sequences:Design and synthesis of siRNA:according to the Ambion siRNA software,3 pairs of siRNA sequences aiming at RAMP1 mRNA target positions of human can be synthesized through transcription.The specificity of the 3 pairs of siRNA is determined through Blast.Real-time fluorogenic quantitative PCR is adopted to screen the sequence with the optimal interfering effect as the use sequence.2.Influences of transfected RAMP1-siRNA on the expression of CRLR and RCP:After successful transfection of RAMP1-siRNA,the relative expression of CRLR and RCP in RAMP1 interference group,non-carrier group and blank control group is tested by fluorescent quantitative PCR and the results are 1.00±0.06;2.05±0.36;and 2.14±0.18 respectively.Compared with non-carrier group and blank control group,the CRLR mRNA expression of RAMP1 interference group significantly declines（P<0.05）.Meanwhile,the mRNA expression of RCP significantly increased（P<0.05）.The results of immunofluorescence also shows that compared with non-carrier group,the green fluorescence of CRLR in RAMP1 interference group obviously weakens while the red fluorescence of RCP strengthens.3.Influences of RAMP1 interference on CGRP in promoting MG63 cell proliferation and differentiation:3.1 According to the cell proliferative activity results tested by CCK-8 method,after 18h of stable transfection,the cell proliferation efficiency of RAMP1 interference group is significantly lower than the other two groups（p<0.05）.The testing results of 24h,36h and 48h are consistent with the above result.This proves that if the expression of RAMP1 is restrained,the proliferation efficiency of MG63 promoted by CGRP will also be restrained obviously.3.2 After RAMP1 interference and 10^-8 mol/L CGRP is applied to treat and induce MG63 cells for 3d,the expression of osteoblast-related markers changes.The results of fluorogenic quantitative PCR show that the relative expression amounts of ALP,Colla 1,Runx2 and OCNmRNA in RAMP1 interference group are1.0±0.12;1.11±0.36;and 0.91±0.17;1.11±0.29 respectively,and the relative expression amounts of ALP,Colla 1,Runx2 and OCNmRNA in non-carrier group1.74±0.153;1.40±0.10;1.95±0.25;1.27±0.12are respectively.The expression of ALP,Colla 1,Runx2 and OCN in RAMP1 interference group is significantly lower than that of non-carrier group（P<0.05）,where only the relative expression of mRNA of BMP2 in RAMP1 interference group is significantly lower than that of non-carrier group（P>0.05）.3.3 After RAMP1 interference and 10^-8 mol/L CGRP is applied to treat and induce MG63 cells for 3d,the ALP activity expression changes are tested by Elasa method.As the results reveal,the ALP activity of RAMP1 interference group is 36.66±10.59,and the non-carrier group is 84.33±15.53.The ALP activity of RAMP1 interference group is also significantly lower than the non-carrier group（P<0.05）.Conclusions:1.RAMP1 interference can reduce the expression of CRLR protein in MG63 cell membranes and increase RCP distribution in membranes in the meantime.2.RAMP 1 interference will restrain proliferation promoting effect of CGRP to MG63 cells.3.RAMP 1 interference will restrain the expression of osteoblast-related markers stimulated by CGRP,but the expression of BMP2 is not obviously restrained.Meanwhile,the RAMP1 interference will also restrain the activity expression of ALP in MG63 cells stimulated by CGRP.