| BackgroundOsteoarthritis(OA)is a common chronic osteoarthritis disease characterized by reduced chondrocyte and destruction of articular matrix.It is also one of the most common causes of pain and disablLity in middle-aged and elderly people.Epidemiological investigation shows that the total prevalence of osteoarthritis in the knee joint of middle-aged and elderly people over 40 years old in China is 17.0%.The prevalence rate of the aged population over 65 years old can reach 60-70 with the increase of age.With the increase of aging population,osteoarthritis has become a worldwide problem affecting human health.Studies have found multiple factors leading to articular cartILage degeneration,including age,obesity,strain,trauma,and inflammation.The pathogenesis of osteoarthritis is still improving.More and more studies have proved that immune response is involved in the pathogenesis of osteoarthritis,including cellular immunity,abnormal humoral immunity,activation of autoimmune signal pathway and so on.The mechanism of articular cartlLage degeneration needs more clinical and experimental research.Non-coding RNA(non-coding RNAs,ncRNAs)refers to a RNA,that cannot be translated into a protein.The length of which is larger than 200nt is called long chain noncoding ma(long non-coding rnas,lncRNAs.In recent years,a large number of studies have shown that IncRNAs can regulate gene expression in many aspects,such as epigenetics,transcriptional regulation,posttranscriptional regulation,and so on.Thus,the important processes of life,such as embryonic development,cell differentiation,metabolism and disease occurrence,can also regulate the differentiation of immune cells by interacting with protein complexes or transcription factors.And by regulating the expression of inflammatory factors in inflammatory response,participate in the regulation of inflammatory response.It is closely related to the occurrence and development of rheumatic diseases,immune response,immune cell differentiation and dynamic balance of immune system.LncRNAs plays an important role in the pathogenesis of OA by regulating the proliferation,differentiation and apoptosis of chondrocytes,affecting the secretion and degradation of extracellular chondrocytes,regulating the release of inflammatory factors.MicroRNAs(miRNAs)are another non-coding RNAs in eukaryotic cells with around 22 nucleotides(nt).LncRNAs can regulate the expression of miRNAs in eukaryotic cells.miRNA-19b(miR-19b)has been found to participate in the regulation of multiple inflammatory response by modulating NF-κB pathway.Zhang et al.demonstrated that miR-19b inhibited intestinal inflammation by reducing,olonic suppressor of cytokines signaling 3(SOCS3)expression in Crohn ’s disease.However,it stILl remains unclear that whether miR-19 has a simlLar anti-inflammatory role in osteoarthritis.More studies are also needed to explore the effects of miR-19b on osteoarthritis.Therefore,in this research,we used LPS,also known as endotoxin,to stimulate murine chondrogenic ATDC5 cell inflammatory injury model.Then,the potential effects of MALAT1 and miR-19b on LPS-induced ATDC5 cell viabILity inhibition,apoptosis induction and pro-inflammatory cytokines expressions were measured.Furthermore,the effects of MALAT1 and miR-19b on LPS-induced activation of Wnt/p-catenin and NF-κB pathways were also analyzed.This finding wIL1 be helpful for further understanding the critical roles of MALATI and miR-19b in osteoarthritis and may provide possible therapeutic and diagnostic targets for osteoarthritis treatments.ObjectivesThis study aimed to investigate the effects of long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1(IncRNA MALAT1)on lipopolysaccharide(LPS)-induced murine chondrogenic ATDC5 cell inflammatory injury,as well as underlying potential mechanism.Methods1、LPS induced ATDC5 cell apoptosis and inflammatory injury.ADTC5 cells were cultured in DMEM/F12.ViabILity of ATDC5 cells after treated with different concentration of LPS was measured using cell counting kit-8(CCK-8)assay.Apoptosis of ATDC5 cells after LPS treatment was assessed using Annexin V-PE staining.Western blotting was performed to analyze the expressions of Bcl-2,Bax,Pro-caspase 3,Cleaved-caspase 3,Pro-caspase 9 and Cleaved-caspase 9 in ATDC5 cells after LPS treatment.The mRNA and protein expressions of IL-1β,IL-6,IL-8 and TNF-a in ATDC5 cells after LPS treatment were detected using quantitative reverse transcription PCR(qRT-PCR)and western blotting,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the concentrations of IL-1β,IL-6,IL-8 and TNF-a in culture supernatant of ATDC5 cells after LPS treatment.2.Expression of MALAT1 in LPS-stimulated ATDC5 cells.Quantitative reverse transcription PCR(qRT-PCR)was performed to measure the expression of MALAT1 in ATDC5 cells after LPS treatment.3、Effection of MALAT1 on LPS-induced ATDC5 cell inflammatory injury.After pEX-MALAT1 or sh-MALAT1 tansfection,the expression of MALAT1 in ATDC5 cells was determined using quantitative reverse transcription PCR(qRT-PCR).ViabILity and apoptosis of ATDC5 cells after LPS treatment and/or pEX-MALAT1(sh-MALAT1)transfection were assessed using cell counting kit-8(CCK-8)assay and Annexin V-PE staining,respectively.Western blotting was performed to analyze the expressions of Bcl-2,Bax,Pro-caspase 3,Cleaved-caspase 3,Pro-caspase 9 and Cleaved-caspase 9 in ATDC5 cells after LPS treatment and/or pEX-MALAT1(sh-MALAT1)transfection.The mRNA and protein expressions of IL-1β,IL-6,IL-8 and TNF-a in ATDC5 cells after LPS treatment and/or pEX-MALAT1(sh-MALAT1)transfection were detected using quantitative Reverse Transcription PCR(qRT-PCR)and western blotting,respectively.Enzyme-linked immunosorbent assay(ELISA)was used to analyze the concentrations of IL-Iβ,IL-6,IL-8 and TNF-α in culture supernatant of ATDC5 cells after LPS treatment and/or pEX-MALATI(sh-MALAT1)transfection.4.Regulation of the expression of miR-19b in ATDC5 cells by MALAT1.After pEX-MALAT1 or sh-MALAT1 transfection,the expression of miR-19b in ATDC5 cells was measured using quantitative reverse transcription PCR(qRT-PCR).5.MALAT1 controlled LPS-induced ATDC5 cell inflammatory injury by regulating miR-19b.The expression of miR-19b in ATDC5 cells after miR-19b inhibitor transfection was assessed using quantitative reverse transcription PCR(qRT-PCR).After LPS treatment and/or pEX-MALAT1(miR-19b inhibitor)transfection,the viabILity of ATDC5 cells,apoptosis of ATDC5 cells and expressions of Bcl-2,Bax,Pro-caspase 3,Cleaved-caspase 3,Pro-caspase 9 and Cleaved-caspase 9 in ATDC5 cells were measured using cell counting kit-8(CCK-8)assay,Annexin V-PE staining and western blotting,respectively.After LPS treatment and/or pEX-MALATl(miR-19b inhibitor)transfection,the mRNA and protein expressions of IL-1β,IL-6,IL-8 and TNF-a in ATDC5 cells,as well as concentrations of IL-1β,IL-6,IL-8 and TNF-α in culture supernatant of ATDC5 cells,were evaluated using quantitative reverse transcription PCR(qRT-PCR),western blotting and Enzyme-linked immunosorbent assay(ELISA),respectively.6、The relationship between MALAT1 suppressed and LPS-induced Wnt/β-catenin and NF-κB pathways activation in ATDC5 cells Western blotting was conducted to analyze the expressions of Wnt3a,β-catenin,t-IκBα,p-IιBα,t-p65 and p-p65 in ATDC5 cells after LPS treatment and/or pEX-MALAT1(miR-19b inhibitor)transfection.Results1.LPS induced ATDC5 cell apoptosis and inflammatory injuryLPS treatment significantly inhibited the viabILity of ATDC5 cells in a dose-dependent manner.The rate of apoptotic cells was remarkably increased after 2.5 or 5 μg/ml LPS treatment.The apoptotic rate of 5μg/ml was significantly higher than that of 2.5μg/ml.LPS treatment up-regulated the expressions rpf Bax,Cleaved-caspase 3 and Cleaved-caspase 9,as well as down-regulated the expression of Bcl-2.Moreover,the mRNA expressions of IL-1β,IL-6,IL-8 and TNF-α in ATDC5 cells were remarkably increased after 5μg/ml LPS treatment.5 μg/ml LPS treatment up-regulated the expressions of IL-1β,IL-6,IL-8 and TNF-α in ATDC5 cells.5 μg/ml LPS also enhanced the concentrations of IL-1β,IL-6,IL-8 and TNF-α in culture supernatant of ATDC5 cells.2.MALAT1 was up-regulated in LPS-stimulated ATDC5 cellsAfter 2.5 or 5 μg/ml LPS treatment,the expression of MALAT1 in ATDC5 cells was up-regulated.3.MALAT1 exerted protective effects on LPS-induced ATDC5 cell inflammatory injurypEX-MALAT1 transfection significantly increased the expression of MALAT1 in ATDC5 cells and sh-MALAT1 transfection remarkably decreased the expression of MALAT1.LPS-induced ATDC5 cell viabILity inhibition and apoptosis induction were markedly suppressed by pEX-MALAT1 transfection and obviously exacerbated by sh-MALAT1 transfection.The LPS-induced expressions increases of Bax,Cleaved-caspase 3 and Cleaved-caspase 9,as well as expression decrease of Bcl-2,were also suppressed by pEX-MALAT1 transfection and exacerbated by sh-MALAT1 transfection.In addition,the LPS-induced expressions(concentrations)increases of IL-1β3,IL-6,IL-8 and TNF-a in ATDC5 cells or culture supernatant of ATDC5 cells were curbed by pEX-MALAT1 transfection and aggravated by sh-MALAT1 transfection.4.MALAT1 positively regulated the expression of miR-19b in ATDC5 cellspEX-MALAT1 transfection significantly up-regulated the expression of miR-19b in ATDC5 cells,whILe sh-MALAT1 transfection obviously down-regulated the expression of miR-19b in ATDC5 cells.5.MALAT1 alleviated LPS-induced ATDC5 cell inflammatory injury by up-regulating miR-19bThe expression of miR-19b in ATDC5 cells was significantly decreased after miR-19b inhibitor transfection.MiR-19b inhibitor transfection remarkably reversed the protective effects of MALAT1 on LPS-induced ATDC5 cell viabILity inhibition and apoptosis induction.Compared to LPS+pEX-MALAT1 group,the expressions of Bax,Cleaved-caspase 3 and Cleaved-caspase 9 were increased,as well as the expression of Bcl-2 was decreased,in LPS+pEX-MALAT1+miR-19b inhibitor group.Furthermore,miR-19b inhibitor transfection reversed the effects of pEX-MALAT1 on LPS-induced ATDC5 cell pro-inflammatory cytokines expressions.6.MALAT1 suppressed LPS-induced Wnt/β-catenin and NF-κB pathways activation in ATDC5 cells by up-regulating miR-19bLPS treatment significantly activated Wnt/β-catenin and NF-κB pathways in ATDC5 cells by up-regulating the expressions of Wnt3α,β-catenin,p-IκBα and p-p65 in ATDC5 cells.pEX-MALAT1 transfection remarkably reversed the LPS-induced activation of Wnt/β-catenin and NF-κB pathways in ATDC5 cells by down-regulating LPS-induced Wnt3α;β-catenin,β-IκBα and p-p65 expressions increases.More importantly,miR-19b inhibitor transfection obviously reversed the effects of pEX-MALATl on LPS-induced activation of Wnt/β-catenin and NF-κB pathways.ConclusionLPS treatment remarkably induced ATDC5 cell apoptosis and inflammatory injury.MALAT1 was up-regulated in LPS-stimulated ATDC5 cells.Overexpression of MALAT1 significantly reversed the LPS-induced ATDC5 cell inflammatory injury,whILe suppression of MALAT1 had opposite effects.Further results showed that MALAT1 positively regulated the expression of miR-19b in ATDC5 cells.Knockdown of miR-19b obviously reversed the protective effect of MALATI on LPS-induced ATDC5 cells.In addition,MALATI reduced LPS-induced Wnt/β-catenin and NF-κB pathways activation in ATDC5 cells by up-regulating miR-19.Our research verified that MALAT1 alleviated LPS-induced ATDC5 cell inflammatory injury by up-regulating miR-19b and inactivating Wnt/β-catenin and NF-κB pathways. |
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