MicroRNA-155 Inhibit The Degeneration Of Nucleus Pulposus Cells Through Targeting ERK1/2

Part 1 Construction of the immortalized nucleus pulposus cell by overexpression of p GFP/h TERT/NeoObjective: The nucleus pulposus(NP) cells are chondrocyte-like cells that are required for the resistance of compressive loads through the synthesis of collagen fibrils and proteoglycan aggrecans, and the generation of a hydrostatic swelling pressure, and thus play an important role in the intervertebral disc. The object was to construct the human telomerase reverse transcriptase(h TERT) recombinant green fluorescence expression vector and assess the feasibility of transfecting normal nucleus pulposus cells to procure an immortalized cell strain.Methods: The experimental procedures included target gene cloning, target gene sequencing in eukaryotic expression vector, construction of target gene eukaryotic expression plasmid, and detection of the h TERT expression. Bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide(XTT) was used to determine growth curves of normal nucleus pulposus cells and h TERT-transfected cells. RT-PCR was used to detect expression of two kinds of type II collagen, glycosaminoglycans, bone morphogenic protein-2, insulin-like growth factor-1, transforming growth factor-β, bone morphogenic protein-4, collagen I, platelet-derived growth factor, and fibroblast growth factor genes. ELISA was used to detect both type II collagen and glycosaminoglycan expression in extracellular fluid. The tumorigenicity of immortalized nucleus pulposus cells was determined in a nude mice transplantation assay.Results: Here, we report the production and characterization of an immortalized human NP cell line from normal NP cells using stable transfection of recombinanthuman telomerase reverse transcriptase(h TERT) gene. The h TERT-transfected NP cells exhibited morphological characteristics typical of native cells. When compared with the first generation of normal NP cells, the h TERT-transfected NP cells grew faster and had an increased level of IGF-1 and TGF-b gene expression. They were successfully passaged over 20 generations without significant change in the levels of type II collagen and proteoglycan aggrecan expression. In addition, they showed resistance to serum starvation-induced apoptosis, G1 cell cycle arrest, and gene expression of p53, CCNE1, Fas, and Caspase 3. Moreover, histology revealed that no tumorigenicity of NP cells over expressing h TERT was observed after they were implanted in nude mice.Conclusion: Taken together, an immortalized human NP cell line was established, which has an extended lifespan, retains phenotypic features similar to primary parent NP cells, and should provide a suitable model for studying the biology of NP cells.Part 2 Micro RNA-155 inhibit the degeneration of nucleus pulposus cells through targeting ERK1/2Objective: Emerging evidence indicates that micro RNAs(mi RNAs) play essential roles in regulating intervertebral disc degeneration. However, the role of mi RNA in nucleus pulposus cells remains to be defined. In this study, we aimed to investigate if mi RNAs regulate nucleus pulposus cells’ degeneration.Methods: We first investigated the difference of micro RNA expression between normal NP cells and degenerated NP cells using gene chip. Using bioinformatic analyses and subsequently confirmed by quantitative Real-Time PCR(q RT-PCR), using mi RDB, mi Randa, mi RBase, mi RWalk database Target Scan to detect the microRNA,using mimic-155 and inhibitor-155 to overexpress and decrease the micro RNA 155 expression, using RT-PCR and western blot to dectcet the relative gene and proteins expression(p MEK、MEK、p Raf、Raf 、Ras、p Ras).Results: we found that mi R-155 was identified as a novel mi RNA and was fully characterized. mi R-155 were downregulated during NP cells degeneration. The expression of micro RNA-155,which would decrease in degenerative nucleus pulposus cell have been proved by using gene chip and RT-PCR in this dissertation. Overexpression and decrease of micro RNA-155 with has-mi R-155 mimics and has-mi R-155 inhibitor were done. Expression of ERK1/2 decreases when overexpression of micro RNA-155 in normal nucleus pulposus cell. When micro RNA-155 is inhibited, expression of ERK1/2 increases. Overexpression or inhibition of micro RNA-155 has no effects on expression level of m RNA ERK1/2 in nucleus pulposus cell, which indicates micro RNA-155 influence expression of p ERK1/2 after transcription of ERK1/2 m RNA. It declares ERK1/2 is a new target protein regulated by mi R-155. In the degeneration of intervertebral disc, inhibited mi R-155 decrease the expressions of extracellular main matrix collagen II and glycosaminoglycan increase expression of ERK1/2.Conclusion: Taken together, our data suggest that mi R-155 is the identified mechano-sensitive mi RNA that regulates NP cells degenerated via directly targeting ERK1/2, and therapeutic overexpression of mi R-155 may be an efficient anabolic strategy for intervertebral disc degeneration.