The Role Of MiR-142-3p In Bleomycin-induced IPF Model And Its Mechanism

Background:Idiopathic pulmonary fibrosis(IPF)is a common interstitial lung disease of respiratory system,which etiology is unknown and morbidity is high among the elderly.Besides,IPF is usually fatal with an average survival of approximately two to three years.IPF patients have multiple clinical symptoms,such as dyspnea,cough,weakness and loss of appetite and weight.At present situation,the problem of the ageing population is getting worse in our country.In addition,the IPF morbidity and mortality rates have been rising in recent years,causing serious damage to human life.But the current methods for the treatment of IPF are still limited,A better understanding of IPF pathogenesis will help us to find a new therapy for IPF.Bleomycin is an anti-tumor drug that has some effect on squamous cell carcinoma and some cancer diseases.It's very common in the tissues of the body.However,the aggregation of bleomycin tends to have a toxic side effect,which can make the cells fibrosis.Therefore,it is limited to use as an anti-tumor drug.MicroRNAs(miRNAs)are approximately 18-26 nucleotide long non-coding RNA molecules which play an important role in cell regulation.In 1993,Lee et al discovered the gene lin-4 associated with embryonic development in Caenorhabditis elegans(C.elegans).Later,researchers found the gene let-7 in C.elegans,and then found some of the same small RNA molecules in many eukaryotes.miRNAs can inhibit the translation of mRNAs or degrade mRNAs by binding to the target mRNA.miRNA can regulate multiple target genes.In addition,one gene can be also regulated by multiple miRNAs.In recent years,miRNAs have been a hotspot in research and studies have reported that miRNAs are associated with a number of diseases,such as cancers,neurodegenerative diseases,diabetes,and inflammation.Furthermore,miRNAs have been successfully applied to the treatment of certain diseases.miR-142-3p is one of the miRNAs.Studies have found that miR-142-3p plays an important role in the inflammatory response.Besides,miR-142-3p can regulate the release of some inflammatory cytokines by high mobility group box 1(HMGB1)and play a vital role in lung cancer cell.But the role of miR-142-3p in idiopathic pulmonary fibrosis(IPF)is not clear.Cyclooxygenase(Cox)also known as prostaglandin synthetase(PGHS),is the main rate-limiting enzyme in the synthesis of prostaglandins.In humans,Cox is able to catalyze the formation of multiple prostaglandins from arachidonic acid(ARA).Cox-2 is one of the Cox isozymes.Furthermore,Cox-2 has close relationship with some of the inflammatory response and high expression in tumor cells,such as lung cancer,gastric cancer,esophagus cancer.In addition,Cox-2 plays an important role in cell cycle,cell apoptosis,tumor angiogenesis and tumor metastasis.In recent years,phosphatidylinositol-3-kinase(PI3K),protein kinase B(Akt)and mammalian target of rapamycin(mTOR)signaling pathway have became a research hotspot and have a close relationship with cell proliferation,differentiation and apoptosis.The blocker of PI3K/Akt/mTOR signaling pathway can be applied to the treatment of diseases.However,the mechanism of effect of PI3K/Akt/mTOR signaling pathway is not very clear.The thorough research about PI3K/Akt/mTOR signaling pathway have a great significance for developing new therapeutic drugs.Objectives:This study was aimed to investigate the effect and mechanism of miR-142-3p on bleomycin-induced IPF.Firstly,lung epithelial MLE-12 cells were treated with different concentrations of bleomycin.In addition,we detected the cell viability,cell apoptosis and the mRNA and protein expression of interleukin-1(IL-1)and tumor necrosis factor-?(TNF-?).Then,we assayed the expression of miR-142-3p in MLE-12 cells.The mRNA and protein expression of interleukin-1(IL-1)and tumor necrosis factor-a(TNF-a)in high/low expression of miR-142-3p cells were detected.Finally,we mearsured the expression of Cox-2 and the key factors of PI3K/Akt/mTOR signaling pathway.Methods:In the IPF model construction experiment,MLE-12 cells were treated with different concentrations of bleomycin,including 0 ?g/ml,10 ?g/ml,50 ?g/ml and 100?g/ml.Thereafter,the cell viability and apoptosis were assessed by Cell Counting Kit-8(CCK-8)assay and flow cytometry,respectively.In addition,we assayed the mRNA and protein expressions of interleukin-1(IL-1)and tumor necrosis factor-a(TNF-a)by Quantitative Real-time PCR(qRT-PCR)and Western blot.In order to explore the role of miR-142-3p in IPF,we firstly assayed the expression of miR-142-3p in different concentrations of bleomycin induced MLE-12 by Quantitative Real-time PCR(qRT-PCR).Then,MLE-12 cells were severally transfected either with miR-142-3p mimic,miR-142-3p inhibitor,miR-142-3p mimic control and miR-142-3p inhibitor control and the cells were exposed to 50 ?g/ml of bleomycin.Thereafter,cell viability,apoptosis and the expression of pro-inflammatory cytokines interleukin-1(IL-1)and tumor necrosis factor-?(TNF-?)were assessed using CCK-8,flow cytometry,qRT-PCR and Western blot analyses,respectively.To investigate the mechanism of miR-142-3p in IPF,firstly,we detected the mRNA and protein expression of Cox-2 using Quantitative Real-time PCR(qRT-PCR)and Western blot respectively.Then the key factors of PI3K/Akt/mTOR signaling pathway in the lung epithelial MLE-12 cells were assayed by Western blot.Results:The IPF model construction experiment:the results showed that the cell viability had no significant difference between this group and control group at concentration of 10 ?g/ml.At concentrations of 50 ?g/ml and 100 ?g/ml,cell viabilities were significantly decreased(P<0.05 or P<0.01);Apoptosis detection experiments showed that no statistical difference was witnessed on cell apoptosis at bleomycin concentration of 10 ?g/ml.At concentrations of 50 ?g/ml and 100 ?g/ml,cell apoptosis were significantly increased(P<0.01 or P<0.001);The qRT-PCR results showed that the mRNA expressions of IL-1 and TNF-a were no statistical difference between the 10 p.g/ml group and control group.At concentrations of 50 ?g/ml and 100?g/ml,the mRNA expressions of IL-1 and TNF-a raised observably compared to that at concentration of 0 ?g/ml(P<0.05).Western blot result demonstrated as concentrations of bleomycin increased,the protein expressions of IL-1 and TNF-a were higher.The protein expressions of IL-1 and TNF-a were both at the highest level at the concentration of 50 ?g/ml.The effect of miR-142-3p on IPF:the results showed that with the increase of concentration of bleomycin,the expression of miR-142-3p was significantly downregulated.At concentration of 10 ?g/ml,50 ?g/ml and 100 pg/ml,the expressions of miR-142-3p raised observably compared to that at concentration of 0?g/ml(P<0.05or P<0.0001).To further investigate cell viability,apoptosis and the expressions of IL-1 and TNF-a found that miR-142-3p overexpression significantly alleviated bleomycin-induced cell viability reduction and apoptotic cells increase(P<0.05).In addition,miR-142-3p overexpression alleviated both IL-1 and TNF-a overproductions induced by bleomycin(P<0.01 or P<0.05).Conversely,miR-142-3p suppression affected MLE-12 cells viability,apoptosis,and IL-1 and TNF-a levels in a completely opposite way.The mechanism of miR-142-3p in IPF:Studies found that bleomycin upregulated both the mRNA and protein expressions of Cox-2 in lung epithelial MLE-12 cells(P<0.05).Furthermore,the mRNA and protein expressions of Cox-2 were significantly down-regulated in the miR-142-3p over-expressed MLE-12 cells.Conversely,the mRNA and protein expression of Cox-2 were significantly increased in miR-142-3p suppressive MLE-12 cells.Moreover,the results demonstrated that the expression of PI3K/Akt/mTOR was suppressed in bleomycin-induced MLE-12 cells.However,the effect was reversed when Cox-2 inhibitor was added.Besides,miR-142-3p increased the protein expression level of PI3K/Akt/mTOR and Cox-2 inhibitor NS398 also upregulated the protein expression level of PI3K/Akt/mTOR.Conclusions and Innovation:In this experiment,we found that bleomycin can affect the cell activity and cell apoptosis.Besides,with the concentration of bleomycin increased,the cell viability was suppressed and the cell apoptosis was expedited.In addition,the mRNA and protein expressions of IL-1 and TNF-a were increased.We successfully established IPF model in vitro in lung epithelial MLE-12 cells by bleomycin.In addition,the expression of miR-142-3p was down-regulated in bleomycin-induced IPF model.Further studies conclusively showed miR-142-3p overexpression alleviated bleomycin-induced cell injury,while miR-142-3p suppression exhibited completely apposite impacts.miR-142-3p strengthened the viability of PI3K/Akt/mTOR signaling pathway by downregulating the expression of Cox-2.These findings might provide a new target for IPF treatment.