Effect Of TRPV6 Gene Overexpression On The Expression Of Genes Related To Calcium Transport And Apoptosis In Mouse Osteoclasts

Bone remodelling consists of bone resorption and formation throughout an organism’s lifespan.This process is necessary for skeletal growth and maintains normal bone structure.Osteoclasts,the only cells capable of resorbing bone,are multinuclear cells derived from haematopoietic precursors of the monocyte and macrophage lineage under the stimulation of two essential factors:monocyte/macrophage colony stimulating factor(M-CSF)and receptor activation of NF-kB ligand(RANKL).Transient receptor potential vanilloid 6(TRPV6),a member of the TRP family,is a highly selective Ca2+channel and is considered the rate-limiting step for Ca2+ transportation in osteoclasts in vitro.Intracellular Ca2+ is an important second messenger in osteoclasts,and it has been shown to regulate numerous functions,including signal transduction and apoptosis.Cell apoptosis is both a normal process of tissue homeostasis and a necessary physiological cellular response to many noxious stimuli that is executed by a cascade of molecular events involving a number of membrane receptors or cytoplasmic proteins.In this study,we examined the interaction between TRPV6 overexpression and calcium transport and the influence on apoptosis in mouse osteoclasts in vitro using TRPV6 overexpression lentivirus vector-transfected osteoclasts.The lentivirus vector transfection is an effective and common tool used in cell tansfection.We constructed the transient receptor potential vanilloid 6 overexpression lentivirus vectors that provided an optimal path for mouse osteoclasts transfection.The full-length TRPV6 gene was obtained from GenBank and synthesized by PCR amplification.The PCR product was cloned into a linearized vector,which was cut by restriction enzymes.The recombinant vector was transformed into E.coli DH5a competent cells,and PCR was used to analyse the colonies after growth on a LB agar plate containing ampicillin at 37℃ for 12-36 h.The identified positive clones were transferred into LB liquid medium and cultured for 12-16 h,and the recombinant plasmid was extracted for gene sequencing.The recombinant plasmid,the pHelper1.0 vector,the pHelper2.0 vector,and transfection reagent were incubated with 239T cells at 37℃ in a humidified atmosphere with 5%CO2.The plasmid expression was confirmed,and TRPV6 overexpression was assessed by western blotting.The femoral and tibial bone marrow cavities of 8-10-week-old BALB/cJ mice were flushed,and the monocyte/macrophage lineage cells were collected after centrifugation with a Ficoll-Hypaque gradient.The cells were cultured in complete α-MEM containing 10%FBS,2 mM/L-glutamine,100 units/ml penicillin,100 μg/ml streptomycin,25 ng/ml M-CSF and 50 ng/ml RANKL for three days at 37℃ in a humidified atmosphere of 5%CO2,and the complete α-MEM was replaced every 2 days.After cultivation for 5 days,the TRPV6 overexpression lentivirus were added to different groups of cells with the completeα-MEM was replaced after co-culturing for 10-12 h.Then,the osteoclasts were cultured for 3-4 d,and confirm the most appropriate condition which the vast majority of the osteoclasts showed fluorescence under a fluorescence inverted microscope.Detecting the relatative mRNA and protein expression of calcium transportation genes and cell apoptosis genes by Realtime-PCR and Western-Blot,respectively.Detecting cell apoptosis ratio by Annexin V-APC/PI dyeing with flow cytometry.The result of qRealtime-PCR shows:compared with the blank control,the relative mRNA expression of TRPV6 increased significantly after transfection with the TRPV6 overexpression lentivirus vector(P<0.01).The relative mRNA expression of calbindin-D28K and NCX1 was significantly increased with TRPV6 overexpression(P<0.05),but PMCA-1B showed no significant change(Fig 3B).Along with the changes in calcium transport-related genes,intracellular calcium concentration was also altered.Thus,the transfection may affect cell apoptosis,and we therefore detected the relative mRNA expression of apoptosis-related genes.The results showed that the apoptosis-related genes decreased significantly(P<0.05),except Fas and FasL.We also noted that the negative control group showed no obvious changes.The result of Western-blot shows:compared with the blank control,the relative protein expression of TRPV6 increased significantly,and CaBP-D28K and NCX1 showed a similar tendency(P<0.01);in contrast,the relative protein expression of apoptosis-related genes decreased significantly(P<0.01).We also noted that the negative control group showed no obvious changes.The apoptotic rate of LV-TRPV6 decreased to 6.33%±0.235%compared with the blank group(12.623%±0.469%)and the negative control group(13.163%±0.221%).We found that the apoptosis rate of the LV-TRPV6 group decreased significantly compared with the blank control group(P<0.01).We also noted that the negative control group had no obvious changesConclusion:The results show that we structured the TRPV6 overexpressing lentivirus vectors successfully.TRPV6 overexpression on mouse osteoclasts leads to an increasion expression of calcium transportation genes and a descend ratio of cell apoptosis.