Experimental Study Of MiR-340-5p Regulating Intervertebral Disc Degeneration In Rats Through FMOD

Objective: Lumbar disc herniation is a syndrome caused by the rupture of the fibrous ring and the stimulation or compression of the nerve root and cauda equina nerve by the protrusion of the nucleus pulposus tissue.Currently,approximately 200 million patients in China suffer from the disease.The intervertebral disc is composed of the nucleus pulposus,annulus fibrosus,and cartilage endplate.Its secretion of various factors not only regulates NP cells,but also plays a very important role in regulating the function or activity of AF cells.This study aims to explore the interaction between degenerative NP and AF through cellular and animal experiments,and clarify the mechanism of miR-340-5p exacerbating intervertebral disc degeneration by inhibiting FMOD expression,providing new ideas for future treatment.Method: 1.Fifteen male SD rats were randomly divided into three groups.MRI examinations were performed at weeks 0,4,and 12 in each group.After the animals were euthanized,intervertebral disc tissue and cells were obtained.The histological morphology,gene expression,and protein expression of the intervertebral discs in each group were observed by HE,qRT PCR staining,and Western blot.2.Thirty six male SD rats were injected with miR-340-5p Lentivirus overexpression vector via tail vein to construct miR-340-5p overexpression animal model in vivo.They were randomly divided into three groups: control group,empty transfection group and transfection group.The tail intervertebral disc tissue was taken to observe the morphological changes of the intervertebral disc tissue using HE staining,real-time fluorescent PCR,immunoblotting test and other techniques,Simultaneously detect the expression of extracellular matrix related components(miR-340-5p,FMOD,Col Ⅰ,Col Ⅱ,Aggrescan).3.The rat intervertebral disc degeneration model was selected and randomly divided into three groups: the control group,the empty transfection group and the transfection group.The miR-340-5p knockdown Lentivirus was used to construct the miR-340-5p low expression animal model.The HE staining,real-time fluorescent PCR,immunoblotting test and other techniques were used to observe the morphological changes of the intervertebral disc tissue,Simultaneously detect the expression of extracellular matrix related components(miR-340-5p,FMOD,Col Ⅰ,Col Ⅱ,Aggrescan).Results: 1.The double Luciferase report experiment reported that there was a miR-340-5p binding site in the 3’UTR of FMOD mRNA.To further verify the relationship between FMOD and miR-340-5p,we found that miR-340-5p can significantly inhibit the fluorescence activity of cells transfected with the report vector with its binding sequence through the double Luciferase reporting experiment.FMOD,as the target gene of miR-340-5p,regulates the degeneration of rat intervertebral discs.To further validate,a rat model of compressive degeneration was established.HE staining: visible under a microscope,compared to the control group;The gap and arrangement of the fibrous ring tissue structure in the tail intervertebral disc of rats in the model 4-week group increased,and the degeneration was more severe in the model 12-week group compared to the model4-week group.The qRT-PCR experiment results showed that compared with the control group,the expression levels of FMOD in the tail intervertebral disc tissue of the model 4week group and model 12 week group rats were significantly reduced,while the expression levels of miR-340-5p were significantly increased.Among them,the model 12 week group was the most significant,and the differences between the groups were statistically significant compared to the control group(P<0.05).The Western Blot experiment results showed that compared with the control group,the protein expression levels of FMOD,Col Ⅱ,and Aggrescan in the tail intervertebral disc tissue of the model 4week and model 12 week groups were significantly reduced,while the protein expression levels of Col Ⅰ were significantly increased.Among them,the model 12 week group was the most significant,and the differences between the groups were statistically significant compared to the control group(P<0.05).2.The miR-340-5p Lentivirus overexpression vector was transfected in vivo to construct the rat miR-340-5p overexpression animal model;QRT PCR was used to detect the expression of miR-340-5p in the tail intervertebral disc of rats,verifying the construction of a rat overexpression model.Western blot analysis was performed on the protein expression levels of FMOD,Col Ⅰ,Col Ⅱ,and Aggrescan in the tail intervertebral disc tissue of rats after 1 week,2 weeks,and 3weeks of virus injection.The results showed that compared with the control group,the protein expression levels of Col Ⅰ in the tail intervertebral disc tissue of the transfected group were significantly increased,while the protein expression levels of FMOD,Col Ⅱ,and Aggrescan were significantly reduced.The most significant difference was observed after 3 weeks of injection,with statistical differences between the groups compared to the control group(P<0.05).3.The rat model of miR-340-5p knockdown was constructed by transfection of miR-340-5p knockdown Lentivirus in vivo.The results of qRT PCR and Western Blot experiments showed that compared with the control group,the gene and protein expression levels of Col Ⅰ in the tail intervertebral disc tissue of the transfected group rats were significantly reduced,while the gene and protein expression levels of FMOD,Col Ⅱ,and Aggrescan were significantly increased.The most significant difference was observed after 3 weeks of injection,and the differences between the groups were statistically significant compared to the control group(P<0.05).Conclusion: The abnormal expression of miR-340-5p in IDD inhibits the synthesis of FMOD,accelerates intervertebral disc degeneration,and regulates miR-340-5P to have a significant effect on delaying the process of intervertebral disc degeneration.