Long Non-coding RNA-UFC1 Promotes Proliferation Of Osteoarthritis Chondrocyte By Acting MiR34a

BackgroundOsteoarthritis(osteoarthritis,OA)is one type of degeneration of joint cartilage and the underlying bone.It causes pain and stiffness,especially in the hip,knee,and thumb joints.Severe cases can lead to joint deformity and loss of functionality.This disease is one of the most common joint diseases among people from middle age onward.The epidemiological statistical result shows that OA affects over 30 percentage of Chinese people who are over 40 years old.Among people who are over 65 years old,this rate can go up as high as 60 to 70 percentage.As China is facing increasing aging population,the percentage of people who are affected by OA is also rising,which eventually becomes a serious social problem.Currently,there is no cure but clinical treatments are available to manage symptoms,such as pain,stiffness and swelling..The clinical treatments are aimed to control pain,correct informality,help joint function recovery and ultimately improve quality of life.The conservative pharmacologic treatments should begin with acetaminophen and step up to nonsteroidal anti-inflammatory drugs.If conservative treatments do not show improvement,surgery may be recommended.The feasible surgery methods are arthroscopy cleaning,joint replacement and cartilage repair.New epidemiological evidence suggests that the occurrence and development of the OA is directly related to age but also affected by other factors,which are preventable and treatable.Given its pathological process is relatively complex and the mechanism also has diversified characteristics,according to different pathological process of OA and its related mechanism,some new treatment options,such as cartilage transplantation,matrix induce autologous chondrocyte transplantation and bone marrow mesenchymal stem cell transplantation,can be applied into the clinical practice.Those new treatments options had achieved some success but not sufficient to reach the ideal results.Currently further in-depth study of pathogenesis of OA and discovery of more efficient prevention and control methods are still needed.Long noncoding RNA(Lnc RNA)is a class of RNA coding,the length of which is more than 200 nt.Lnc RNA is highly abundant and functionally important RNAs and the expression is different based on different cells,tissues and its different development phase.Long noncoding RNA(Lnc RNA)is closely related with the occurrence and development of the various diseases.Some scholars noted that Lnc RNA plays important part in the mechanism of OA pathogenesis.Through the gene chip and high-throughput DNA sequencing technology,there are 2,166 Lnc RNA has been found up-regulated and 1,472 Lnc RNA was down-regulated in the process of cartilage differentiation.This provides new hope for the clinical treatment of OA.At present,many researches have been focus on the role of Lnc RNA but there are still many unknown pathological aspects to be explored.We have found that patients had OA chondrocytes LncRNA-UFC1 expression level below the normal cartilage cells in the prophase of this research.In tumor diseases,UFC1 can combine with miR-34a specifically to inhibit its activity and remove the inhibition of cell proliferation which is caused by miR-34a.The miR-34a is also an important cause of cartilage cell apoptosis and prohibits regeneration barrier in patients with OA.However,it is still unknown that if OA can combine with miR-34a and promote cartilage cell proliferation.This study is divided into three parts:First,I established an observation regarding the effect of Lnc-RNA-UFC1 on the proliferation and inflammation of apoptosis spongy cartilage cell,I analyzed whether Lnc-RNA-UFC1 plays an important role in OA pathological mechanism to establish its research value.Second,I analyzed the functionality of Lnc-RNA-UFC1 from the perspective of mutual regulation between Lnc-RNA-UFC1 with miR-34a I determined whether OA pathological mechanism mediated by Lnc-RNA-UFC1 is accomplished through miR-34a.Last,to provide evidence for exploring new therapeutic targets,I selected the related downstream signaling molecules which miR-34a regulated OA and made a preliminary discussion on the mechanism that Lnc-RNA-UFC1 promoted cartilage cell proliferation through miR-34a and eventually improved OA pathological mechanism.Objectives1.To observe the influence of UFC1 to the proliferation of OA chondrocyte,so as to analyze whether UFC1 play important role in OA pathomechanism and establish its research value.2.To analyze its UFC1's function through the mutual manipulation of UFC1 and miR-34a,so as to establish whether miR-34a is the bridge for UFC1 to regulate OA path mechanism.3.To explore the pathomechanism of UFC1 promoting OA chondrocyte proliferation through miR-34a by analyzing the downstream signal molecules of miR-34a,so as to provide evidence for completing OA pathological mechanism and exploring new therapeutic targetsMethods1.OA chondrocytes' culture and sample selection30 OA patients prepared for total knee arthroplasty were selected as pathology objects,and 20 femoral neck fracture patients prepared for hip orthroplasty were selected as the controls,which cartilaginous tissues were extracted in the surgery.The chondrocytes were separated by enzyme digestion and cultured.The second,third and fourth generations of cells were cultured for 14 days and the growth curve was prepared.The collagen ? expression was detected by immunohistochemistry,after,the cells grew well and with high collagen ? expression were selected.2.UFC1 expression and behavioristics of OA and healthy chondrocytesThe chondrocytes selected in the previous experiments were cultured with 96-well plates,37?,5%C02 for 48h.UFC1 mRNA expression was detected by real-time PCR assay,the cell proliferation activity was detected by CCK-8 assay,and the cell apoptotic rate were detected by flow cytometry test.3.The influence of UFC1 over expression and silence to cell behavioristicsUFC1 over expression and UFC1 disturb lentivirus vector(shRNA1 and shRNA2)were built and transfect to OA chondrocytes used siRNA technology,and cultured in 96-well plates.The control group(Controls 1),empty vector plasmid transfect group(Controls 2),UFC1 over expression and UFC1 silencing group(shRNA1 and shRNA2)were prepared.Controls 1 only had OA chondrocytes,Controls 2 both had OA chondrocytes and empty vector plasmid,UFC1 over expression,shRNA1 and shRNA2 both had OA chondrocytes and corresponding UFC1-siRNA-lipidosome recombination,all of the cells in 96-well plates were cultured for 48h in 37? 5%CO2.After,the cell proliferation activity and cell apoptosis rate were detected.Then,the cells were cultured for 14d to draw growth curve.4.Correlation study of UFC1 ? miR-34a expression in OA chondrocytesThe chondrocytes selected in the previous experiments were cultured with 96-well plates,37?,5%C02 for 48h.After,miR-34a mRNA expression level and UFC1 mRNA expression level were detected by real-time PCR experiments,and the correlation between UFC1? miR-34a was analyzed by pearson correlation analyze.5.Interaction of UFC1 and miR-34a in OA chondrocytesThe pcDNA3.1-MS2 and pcDNA3.1-MS2-UFC1 or pcDNA3.1-MS2-UFC1-mut and pMS2-GFP and OA chondrocytes were cotransfect to cells used lipidosome 2000,and cultured with 96-well plates,37?,5%C02 for 48h.Then,RIP cell analyse was completed by EZ-again Magna RIP RNA binding protein precipitation kits.Then,the original and variant UFC1 were cloned into pmirGLO plasmid receptors,and all of the original generation and variant carrier and miR-34a were transfect into cells,and cultured for 48h in 37?,5%CO2.The fluorescein activity was analyzed through the dual luciferase receptor measurement system.6.UFC1 regulation the behavioristics of OA chondrocytes through miR-34aOA chondrocytes were cultured with 96-well plates in a targeted intervention experiment,in which the control group,UFC1 silence group,miR-34a inhibitor group,UFC1 over expression group and miR-34a group were all prepared.The cells of the control group were cultured as normal.UFC1 silence group and miR-34a inhibitor group were both built UFC1 disturb plasmid through siRNA experiment,and miR-34a inhibitor group was given miR-34a inhibitor at the same time.UFC1 over expression group and miR-34a group were both built UFC1 over expression plasmid,and miR-34a group was given miR-34a at the same time.Then,all the cells were cultured in 37?,5%C02 for 48h.The cell proliferation activity and cell apoptosis rate were detected.Then,the cells were cultured for 14d to draw growth curve.7.Mechanism research of UFC1 to regulate behavioristics of OA chondrocytes through miR-34aOA chondrocytes were cultured with 96-well plates in a targeted intervention experiment,in which the control group(Controls 1),empty plasmid group(Controls 2),UFC1 silence group(shRNA),and miR-34a inhibitor group,UFC1 over expression group and miR-34a group were all prepared.Controls 1 only had OA chondrocytes and cultured as nomal.Controls 2 both had OA chondrocytes and empty vector plasmid.UFC1 silence group and miR-34a inhibitor group were both built UFC1 disturb plasmid through siRNA experiment,and miR-34a inhibitor group was given miR-34a inhibitor at the same time.Then,all the cells were cultured in 37?,5%C02 for 48h.The mRNA and protein expression of ADAMTS-4,ADAMTS-5,MMP-3,MMP-9 and MMP-13 were detected by real-time PCR and WB experiment.Results1.Culture in vitro and sample selection of OA chondrocytesOA chondrocytes grew in the third day of culture,and the growth velocity went to peak in the 7th to 10th day,then,went to deceleration.The cell growth status of the second and third generation was better than the 4th generation.The immunohistochemistry results of collagen ? showed that,the second generation colored obviously,the third generation colored a little,but there was clear positive cells,the 4th generation colored little,and showed serious cytometaplasia.So,we selected the second and third generation as the main sample.2 UFC1 expression and cell behavioristics of OA and healthy chondrocytesThe UFC1 mRNA expression level of OA chondrocytes was lower than the healthy chondrocytes,and the cell proliferation activity of OA chondrocytes was lower than the healthy chondrocytes too,while the apoptotic rate of OA chondrocytes was higher than the healthy chondrocytes,all the difference had statistical significance(P<0.01).3.Influence of UFC1 over expression and silence to behavioristics of OA chondrocytesThe UFC1 mRNA expression level of UFC1 over expression was higher than Controls 1,and its cell growth rate and proliferation activity increased,apoptotic rate decreased too,the difference compared to Controls 1 had statistical significance(P<0.01).The UFC1 mRNA expression level of shRNAl and shRNA2 were both lower than Controls 1,and its cell growth rate and proliferation activity decreased,apoptotic rate increased too,the difference compared to Controls 1 had statistical significance(P<0.01).4 Correlation study of UFC1 and miR-34a expression in OA chondrocytesThe miR-34a mRNA expression level of OA chondrocytes was higher than the healthy chondrocytes(P<0.05),and the further analyse showed that there was negative relevance berween UFCI and miR-34a in UA cnondrocytes.5 Interaction of UFC1 and miR-34a in OA chondrocytesWe down regulated UFC1 mRNA expression through RIP experiment,and found the RIP level of OA chondrocytes was higher than the controls,and at miR-34a' effect target,UFCI got mutation(UFCI-mut).Luciferase receptor analysis results showed miR-34a over expression reduce original receptor's luciferase activity,but UFCI-mut or the controls.The down regulated UFCI by AG02 increased in miR-34a over expression cells,and the original UFCI over expression suppress miR-34a,but UFCI-mut.6.UFC1 regulation the behavioristics of OA chondrocytes through miR-34aCompared to Controls,OA chondrocyte grow rate and proliferation activity of shRNAl and shRNA2 decreased,while apoptotic rate increased.After given miR-34a inhibitor,OA chondrocyte grow rate and proliferation activity increased,while apoptotic rate decreased.Compared to Controls,OA chondrocyte grow rate and proliferation activity of UFC1 over expression increased,while apoptotic rate decreased.After given miR-34a,OA chondrocyte grow rate and proliferation activity decreased,while apoptotic rate increased.And there was statistical difference between groups(P<0.01).7.Mechanism research of UFC1 to regulate behavioristics of OA chondrocytes through miR-34aThere was no significant difference between Controls 1 and Controls 2 in the indexes(P>0.05),the DAMTS-4 mRNA and protein expression level of shRNA increased,which decreased after given miR-34a inhibitor,and there was statistical difference between groups(P<0.05).The MMP-3,MMP-9,MMP-13 mRNA and protein expression level of shRNA increased too.which decreased after given miR-34a inhibitor,and there was statistical difference between groups(P<0.01).Conclusion1.This study compared UFCI expression between OA chondrocytes and normal chondrocytes,and analyzed the influence of UFC1 to the behavioristics of OA chondrocytes.We found that UFCI can promote cell proliferation and inhibit cell apoptosis,and is benefit for the growth of OA chondrocytes,which may be paly important role in the pathogenesis of OA.2.The regulation ability of UFC1 on OA pathological of may be associated with miR-34a,and UFC1 can make miR-34a silence by combination,so as to restrain its function,as a results,the cell proliferation was promoted and the cell apoptosis was inhibit.Futher study of the mechanism is expected to provide new targets for the clinical diagnosis and treatment of OA.3.The combination of UFC1 and miR-34a has ability to inhibit the expression of downstream factors,such as ADAMTS-4?MMP-3?MMP-9 ? MMP-13,so as to alleviate extracellular matrix degradation and cartilage destruction,which may be one of the important mechanisms of OA.