| Objective: In the field of treatment of severe joint diseases,artificial joint replacement is of great significance,which can provide powerful support for the recovery of joint function of patients.However,this method is also prone to the problem of late prosthesis loosening after treatment.The relevant statistical studies show that after 15 years of prosthesis replacement,about 15% of the patients will suffer from prosthesis failure and 50% of the patients will suffer from knee joint revision.In the case of prosthesis loosening,revision surgery of prosthesis has poor therapeutic effect,obvious trauma and poor economy,so it is necessary to study non-surgical methods to prevent the above problems,which is also the current focus of joint surgery.Prosthesis failure is associated with a number of factors,the most important of which is aseptic loosening,followed by infection.The former factor appears relatively late,while infection occurs mainly in the early postoperative period.Osteolysis around the prosthesis is the main cause of aseptic loosening.Osteolysis around the prosthesis is a bone resorption after joint replacement.Studies have found that this complication is not easy to occur in the early postoperative period.At the same time,patients have no obvious symptoms after the occurrence,and they can only be sensed under the condition of severe bone defect.Under this condition,the difficulty and cost of revision are significantly increased,and more complications are likely to occur.According to clinical experience,the efficacy of late revision osteolysis is also poor.Previous studies have shown that the hip joint prosthesis is stable in the early stage mainly based on the press-fit between the bone and the joint prosthesis,while in the late stage it is stable through bone ingrowth.When the activity of osteoblasts near the prosthesis is low,new bone cannot be produced,while the proliferation of osteoclasts is relatively enhanced,which causes osteolysis around the hip prosthesis,resulting in aseptic loosening of the joint prosthesis.In view of this,it is very important to study the pathophysiological mechanism of osteolysis.It is important to identify the regulatory mechanism of osteoblast activity during aseptic loosening of joint prosthesis,which can provide support and basis for the prevention and treatment of this complication.At present,circular RNA(circ RNA)has attracted attention in the field of pathological research.Due to its stable closed-loop structure and wide expression range,circ CRNA plays a regulatory role in many pathological activities of the body.Studies have found that exon-derived circular RNA can be adsorbed by specific binding sites on it,and have different effects on the expression of the target gene.The experimental studies have revealed that they are involved in the pathological processes of many orthopedic diseases,such as osteoporosis and arthritis.Through high-throughput sequencing,we found that significantly differentially expressed circ RNA and m RNA were present in the peri-prosthetic tissues of patients with aseptic loosening of hip prostheses,so we conducted further relevant functional and mechanism studies.Methods: Part I:differential genes were screened out by high-throughput sequencing analysis from six collected clinical specimens,and the target circ RNA was selected as the research object.The expression in tissues was verified by q RT-PCR,and the stability of the selected circ＿0004496 was verified by RNase R digestion experiment and actinomycin D experiment.Titanium granule were cocultured in osteoblast cell line and a human osteoblast cell line overexpressing hsa＿circ＿000496 was constructed using lentivirus,and a knock-down hsa＿circ＿000496 human osteoblast cell line was construct simultaneously.The expression of hsa＿circ＿0004496 in osteoblasts and the transfection efficiency were detected by q RT-PCR.Western Blot was used to detect the expression of LC3II/LC3 I and P62 proteins in human osteoblasts through the observation of osteoblast autophagosomes based on electronic lenses.Western Blot was also used to detect the expression changes of osteoblast marker proteins ALP,osteocalcin and RUNX2 caused by the overexpression and knockdown of hsa＿circ＿0004496.The overexpression of hsa＿circ＿0004496 and the change in ALP activity in human osteoblasts of the knockout group were detected by an ALP activity assay in response to the effect on the activity of human osteoblasts.The CCK-8 assay and cell clone formation assay were used to determine the effect of hsa＿circ＿0004496on the proliferation of human osteoblasts.Effect of flow cytometry on apoptosis.Part II: First of all,the regulatory network of hsa＿circ＿000496/let-7b-5p/ATG7 was established by bioinformatics method.Fish experiment verified the localization of hsa＿circ＿000496 and let-7b-5p in cells,and the mutual binding between hsa＿circ＿000496 and let-7b-5p,as well as between let-7b-5p and ATG7 was verified by double luciferase gene reporting experiment.RIP experiment was used to further verify the direct binding of hsa＿circ＿000496 and let-7b-5p,and the expression of let-7b-5p and ATG7 in the loose tissue of the prosthesis was verified by tissue q RT-PCR.Cellular experiments were conducted to verify the regulatory relationship between hsa＿circ＿000496 and let-7b-5p,and between let-7b-5p and ATG7.Western Blot was then used to detect the expressions of autophagy proteins and osteoblast marker proteins.The CCK-8 experiment,cell clone formation experiment,ALP activity experiment,and flow cytometry apoptosis experiment were also used to verify that let-7b-5 and ATG7 affected autophagy,proliferation,differentiation,and apoptosis in osteoblasts.Part III: Study on the regulation of ATG7-activated osteoblastic autophagy mediated by endogenous competitive binding of let-7b-5p via ce RNA mechanism of CIRC0004496 in osteoblasts to intervene in the occurrence of periprosthetic osteolysis:A cell line co-transfected with overexpressing hsa＿circ＿000496+up-regulating let-7b-5p was constructed.Besides,a knock-down hsa＿circ＿0004496+ down-regulated let-7b-5p co-transfected cell line was constructed,as well as an over-expressed hsa＿circ＿0004496+knock-down ATG7 co-transfected cell line and a knock-down hsa＿circ＿0004496+over-expressed ATG7 co-transfected cell line.Western Blot was used to detect the expression changes of ATG7,ALP,osteocalcin and RUNX2 as well as the expression of autophagic proteins LC3II/LC3 I and P62 by each transfection group.The effects on the activity of human osteoblasts,cell proliferation and apoptosis were examined by ALP activity test,CCK-8,cell clone formation test,and flow cytometry.Finally,the mouse tibial osteolysis model was established,and the homology was converted from human circ RNA to mouse circ RNA: mmu-circ＿0004496.Then,the constructed transfected mouse osteoblasts expressing mmu-circ＿0004496 were injected into the joint cavity of the mouse tibial osteolysis model.After five weeks,the changes of the mouse bone mass were viewed by micro-CT,and the changes of bone mineral density was detected.Finally,the osteolytic tissue around the Ti bar of the tibia in the mouse osteolysis model was taken for Western Blot experiment to detect the changes of ATG7 and osteoblast marker proteins.Results: Part I:we selected hsa＿circ＿0004496 as the target circ RNA in our study from the differentially expressed circ RNAs screened in aseptic loosening tissues by high-throughput sequencing,and the results were verified to be consistent with the sequencing results by tissue q RT-PCR.The cyclic stability of hsa＿circ＿0004496 was confirmed by Actinomycin D experimental verification and RNase R digestion experiment.The results of q RT-PCR of human osteoblast cell lines co-cultured with Ti particles showed a downward trend of circ＿0004496,as well as the down-regulation of osteoblast marker proteins ALP,osteocalcin and RUNX2 detected by Western Blot.The results of transmission electron microscopy showed that the number of autophagosomes in the hsa＿circ＿0004496 overexpression group was significantly higher than that in the control group.The results of Western Blot showed that the ratio of LC3 II/ LC3 I in the hsa＿circ＿0004496 overexpression group was significantly higher than that in the control group,and the expression of P62 protein was significantly lower than that in the control group.The hsa＿circ＿0004496knockout group showed a significant decrease in the LC3 II/LC3 I ratio as compared with the control group,and the expression of P62 protein was also significantly increased as compared with the control group.When hsa＿circ＿0004496 was overexpressed,the expressions of osteoblast marker proteins ALP,osteocalcin and RUNX2 were correspondingly increased;when hsa＿circ＿0004496 was overexpressed,the expressions of proteins ALP,osteocalcin and RUNX2 were correspondingly decreased.In the ALP activity assay,alkaline phosphatase activity was significantly increased in the hsa＿circ＿0004496 overexpression group as compared with the control group,while alkaline phosphatase activity was significantly decreased in the hsa＿circ＿0004496 knockout group as compared with the control group.The CCK-8test and cell clone formation test showed that the overexpression group of hsa＿circ＿0004496 promoted the proliferation of osteoblasts as compared with the control group.However,after the knock-down of hsa＿circ＿0004496,the proliferation of human osteoblasts was weakened.The apoptotic rate was detected by flow cytometry.The apoptotic rate of human osteoblasts in the hsa＿circ＿0004496overexpression group was significantly lower than that in the control group,while the apoptotic rate in the hsa＿circ＿0004496 knockout group was significantly higher than that in the control group.All the experimental results above have significant statistical differences.Part II: Establishment of the hsa＿circ＿0004496/let-7b-5p/ATG7 Regulatory Network by Bioinformatics Technology.FISH experiments demonstrated that hsa＿circ＿0004496 was co-located in the cytoplasm with let-7b-5p.The results of double luciferase gene reporting experiments showed that both hsa＿circ＿0004496 and ATG7 could directly bind to let-7b-5p.RIP experiments have shown that hsa＿circ＿0004496 can bind directly to let-7b-5p.It could be seen from the q RT-PCR assay that let-7b-5p was highly expressed in the loose tissue of the prosthesis,while ATG7 was highly expressed.The q RT-PCR assay in human osteoblast lines revealed that in the hsa＿circ＿0004496 overexpression group,compared with the control group,the expression of let-7b-5p was decreased,and the expression of ATG7 was increased.Compared with the control group,the expression of let-7b-5p was increased and the expression of ATG7 was decreased in the hsa＿circ＿0004496 knockout group.q RT-PCR in human osteoblast lines revealed reduced expression of ATG7 in the let-7b-5p overexpression group as compared with the control group;ATG7 expression was increased in the let-7b-5p knockdown group compared with the control group.All the experimental results above were statistically significant.Western Blot analysis showed that in the let-7b-5p knockdown group,the ratio of LC3 II to LC3 I was significantly higher than that of the control group,and the expression of P62 protein was significantly lower than that of the control group.However,the let-7b-5p overexpression group showed a significant decrease in the LC3 II/LC3 I ratio compared with the control group,and the expression of P62 protein was significantly increased compared with the control group.Compared with the NC control group,the expressions of ATG7,ALP,osteocalcin and RUNX2 in the let-7b-5p down-regulated group were increased by Western Blot.In contrast,up-regulation of let-7b-5p resulted in decreased expression of ATG7,ALP,osteocalcin,and RUNX2.In the ALP activity assay,alkaline phosphatase activity was significantly higher in the let-7b-5p knockout group than in the control group,while alkaline phosphatase activity was significantly lower in the let-7b-5p overexpression group than in the control group.Detection of CCK-8 assay and cell clonal formation assay Promoting human osteoblast proliferation in the let-7b-5p knockdown group;Overexpression of let-7b-5p inhibits the proliferation of human osteoblasts.The experimental results of apoptosis showed that the down-regulated group of let-7b-5p was able to inhibit the apoptosis rate,and the up-regulated group of let-7b-5p was able to increase the apoptosis rate.All the experimental results above were statistically significant.The autophagy-related proteins were detected by Western Blot.In the ATG7 overexpression group,the ratio of LC3II/LC3 I was significantly higher than that of the control group,and the expression level of P62 protein was significantly lower than that of the control group.However,the LC3II/LC3 I ratio was significantly reduced and P62 protein expression was significantly higher in the ATG7 knockout group than in the control group.In the same western blot analysis,compared with the control group,the expressions of ATG7,ALP,osteocalcin and RUNX2 in the ATG7 overexpression group were increased.In contrast,the expression levels of ATG7,ALP,osteocalcin,and RUNX2 were decreased in the ATG7 group with low knockdown.In the ALP activity assay,alkaline phosphatase activity was significantly increased in the ATG7 overexpression group and significantly decreased in the ATG7 knockout group as compared with the control group.The CCK-8 assay and cell cloning assay showed that the ATG7 overexpression group promoted the proliferation of human osteoblasts,while the knock-down of ATG7 inhibited the proliferation of human osteoblasts.The experimental results of apoptosis showed that overexpression of ATG7 could inhibit the apoptosis rate,and knocking down the ATG7 group could increase the apoptosis rate.All the experimental results above were statistically significant.Part III:(1)Up-regulation of let-7b-5p reverses the up-regulation of ATG7 by overexpression of hsa＿circ＿0004496 in both let-7b-5p co-transfected cells.In contrast,in the down-regulated let-7b-5p coinfected cell lines with hsa＿circ＿0004496knockdown,the results of the q RT-PCR assay showed that the down-regulated let-7b-5p reversed the down-regulation of ATG7 by knocking down hsa＿circ＿0004496.In Western Blot experiments,the up-regulation of let-7b-5p reversed the up-regulation of the target protein ATG7 by overexpression of hsa＿circ＿0004496 in cell lines overexpressing hsa＿circ＿0004496 and up-regulating let-7b-5p.In contrast,in cell lines co-transfected with the down-regulated let-7b-5p and the down-regulated hsa＿circ＿0004496,Western Blot experiments showed that the down-regulated let-7b-5p could reverse the down-regulation of the target protein ATG7 due to the down-regulation of hsa＿circ＿0004496.Up-regulation of let-7b-5p reversed the increased conversion of the autophagy-related protein LC3 I to LC3 II by overexpression of hsa＿circ＿0004496 and the decreased expression of P62 protein.Conversely,down-regulation of let-7b-5p reversed the decrease in the conversion of the autophagy-related protein LC3I-LC3 II due to the knock-down of hsa＿circ＿0004496,and reversed the increase in P62 protein expression.Up-regulation of let-7b-5p reversed the up-regulation of osteoblast marker proteins ALP,osteocalcin,and RUNX2 by overexpression of hsa＿circ＿0004496.Conversely,down-regulation of let-7b-5p reversed the down-regulation of the osteoblast marker ALP,osteocalcin,and RUNX2 by knocking down hsa＿circ＿0004496.In the CCK-8 experiment and cell clone formation experiment,the up-regulation of let-7b-5p was able to reverse the promotion of osteoblast proliferation by overexpression of hsa＿circ＿0004496.In contrast,the down-regulation of let-7b-5p reversed the inhibition of osteoblast proliferation by knocking down hsa＿circ＿0004496 in both let-7b-5p co-transfected cell lines.The apoptotic rate was detected by flow cytometry,and up-regulation of let-7b-5p could reverse the inhibition of apoptotic rate by overexpression of hsa＿circ＿0004496.Conversely,down-regulation of let-7b-5p reversed the rate-of-apoptosis-boosting effect achieved by knocking down hsa＿circ＿0004496.All the experimental results above were statistically significant.Western Blot experiments showed that in the co-transfected cell lines overexpressing hsa＿circ＿0004496 and knockout ATG7,the knockout ATG7 could reverse the increase in the conversion of autophagy-related proteins LC3 I to LC3 II due to overexpression of hsa＿circ＿0004496 and the decrease in the expression of P62 protein.Conversely,overexpression of ATG7 reversed the decrease in the conversion of the autophagy-related protein LC3I-LC3 II due to the knockdown of hsa＿circ＿0004496,and reversed the increase in P62 protein expression.Knocking down ATG7 could reverse the up-regulation of osteoblast marker proteins ALP,osteocalcin,and RUNX2 due to overexpression of hsa＿circ＿0004496.In contrast,overexpression of ATG7 reversed the down-regulation of the osteoblast marker proteins ALP,osteocalcin,and RUNX2 by knocking down hsa＿circ＿0004496.In the CCK-8 assay and cell clonal formation assay,the knock-down of ATG7 could reverse the proliferation-promoting effect of osteoblast induced by over-expression of hsa＿circ＿0004496.In contrast,overexpression of ATG7 reversed the osteoblast proliferation inhibition by knockdown of hsa＿circ＿0004496 in both hsa＿circ＿0004496and ATG7 co-transfected cells.The apoptotic rate was detected by flow cytometry,and knocking down ATG7 could reverse the inhibitory effect of overexpression of hsa＿circ＿0004496 on apoptosis.Conversely,overexpression of ATG7 reverses the pro-apoptotic effects of knock-down hsa＿circ＿0004496.All the experimental results above were statistically significant.(2)Animal experiments: micro-CT showed that the cortex around the original Ti rod channel in the control group was thinner and more obvious,and the loss of trabecular bone around the implant was increased in comparison with the group overexpressing mmu-circ＿0004496.And the group overexpressing mmu-circ＿0004496 had higher bone mineral density than the control group.Finally,Western Blot analysis of the osteolytic tissue in the proximal tibia of the mouse near the periphery of the Ti rod showed that the expression levels of proteins ATG7,ALP,osteocalcin,and RUNX2 in the group overexpressing mmu-circ＿0004496 were significantly higher than those in the control group.The above experimental results were statistically significant.Conclusion:The let-7b-5p/ATG7 pathway mediated by circ RNA hsa＿circ＿0004496 is involved in positively regulating osteoblast autophagy,inhibiting apoptosis,promoting osteoblast activity and proliferation,and negatively interfering with osteolysis around artificial prosthesis. |
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