Screening,Verification And Functional Analysis Of MicroRNAs As COPD Classification Biomarkers

Background:Chronic obstructive pulmonary disease(COPD)is a disease characterized by persistent airflow limitation,which has been an important public health problem in the world.In China,the prevalence rate is high and up to 13 ~ 30% over 60 years old.With the advent of the aging society,the incidence will continue to rise.The clinical feature of COPD is different,its severity mismatches with the pulmonary function.The disease is characterized by progressive aggravation and many complications,such as pulmonary and extrapulmonary complications,which seriously affects the quality of life and survival period of patients.However,the current treatment can not prevent lung function from declining,so it is urgent to develop new treatments to delay the progress of the disease.It plays an important guiding role in the individualized treatment of patients with COPD that screening biomarkers and therapeutic targets are associated with classification(severity)of the disease.COPD is a multifactorial disease caused by environmental determinants as well as genetic risk factors.The pathogenesis is complex and the disease progresses slowly.It is widely believed that COPD is related to inflammation caused by monocytes/macrophages,neutrophils and T lymphocytes,oxidative stress and imbalance between protease and antiprotease,but recently the genetic susceptibility of COPD has been paid much attention.Mi RNA,non coding RNA with regulatory function,is involved in many important biological processes such as growth,cell proliferation,cell differentiation and apoptosis,and is closely related to the occurrence and development of lung diseases,involved in the pathogenesis of many pulmonary diseases.Mi RNA is characterized by its good stability,convenient detection,strong specificity and high sensitivity in the blood,therefore it is possible to become a new molecular marker of COPD.The result of searching for mi RNA associated with COPD classification(severity)may provide new targets for therapy.Furthermore,mi RNA is carried out the functional analysis involved in the biological process of the disease,which wiil provide a new direction for the study about the pathogenesis of COPD,and provide the theoretical basis for individualized treatment plan in future.Objective:1.Via high-throughput sequencing of small RNA,screening the differential expression of mi RNAs associated with the classification(severity)of COPD;verifying whether it could be the biological marker and therapeutic target.2.Clarify the function of candidate mi RNA in the biological process of COPD in vivo cellular level,and explore regulatory mechanism of its target gene.And to explore the role of mi RNA in the pathogenesis of COPD and provide a new target for the treatment of the disease.Methods:1.Between January 2014 and December 2014,141 patients were spirometry-diagnosed COPD patients,using combined assessment according to the GOLD guideline of 2013 in the department of respiration Shanxi Dayi Hospital.20 stable COPD patients were selected,5 patients per group(A/B/C/D groups,).The control group was consisted of six patients.peripheral blood of all the participants was collected,isolating peripheral leucocytes and extracting total RNA.Via the high-throughput sequencing of small RNA and bioinformatics,differentially expressed mi RNAs were analysed between ABCD four groups and the two clinical models of different disease progression,meanwhile GO and KEGG pathway analysis were also carried out.Using real-time fluorescent quantitative PCR method(RT-PCR),the candidate mi RNAs were validated in 80 local patients with COPD.2.The expression level of validated mi RNA in THP-1 was analysed at the cellular level by RT-PCR.Construction of lentivirus infected cells and screening of mi RNAs function by MTS activity assay.he function of mi RNAs was further confirmed by cell functional experiments,including CCK-8,PI-FACS cell cycle and apoptosis of Annexin V-APC.Lucifersae assay was used to determine whether there was a direct regulatory role between target mi RNA and its target genes.Results:1.(1)Compared with the healthy control,there were 1715 differentially expressed mi NRAs,including 51 upregulated and 18 downregulated significantly mi NRAs in COPD.(2)All COPD groups had 19 differentially expressed mi RNA in common.Of these,17 mi NRAs were upregulated in all groups.The expression level of mi R-106b-5p in A group was the highest,in D group was the lowest(A>B>C>D).Mi R-125a-5p was upregulated in all groups,its expression level gradually decreased in ABC group,but the expression level elevated again in D-group.(3)Mi R-183-5p was found to be continually downregulated in clinical models of disease progression form A group to B group to D group.(4)GO analysis showed that most of the gene functional annotation was focused on COPD biological processes,such as cell cycle,apoptosis,metabolism,adhesion,and so on.(5)KEGG pathway analysis showed that mi RNA targeted genes were associated with many important signaling pathways and complications of COPD,such as Regulation of autophagy,Focal adhesion,Toll-like receptor signaling pathway,Pathways in cancer,Non-small cell lung cancer Erb B,signaling pathway and p53 signaling pathway,and so on.(6)The expression levels of mi R-106b-5p and mi R-125a-5p in the local COPD samples were validated by real-time fluorescent quantitative PCR,consistent with the sequencing results.2.(1)The expression level of mi R-106b-5p and mi R-125a-5p in the THP-1 was low by RT-PCR.(2)Constructing mir-up lentivirus infected cells,the expression of mi R-106b-5p was 7.636 times higher than that of the control group,and the expression of mi R-125a-5p was 59621.539 times higher than that of the control group by q PCR.(3)MTS activity test results showed that:compared to the control group NC,there were significant changes in cell proliferation multiples of hsa-mi R-125a-5p group(P<0.05),and the proliferation of hsa-mi R-106b-5p cells is not obvious.(4)CCK-8 cytotoxicity test showed that compared to the control group NC,the mir-125a-5p group had a significant change in the absorption rate of 5 days.(5)PI-FACS cell cycle analysis showed that in the mi R-125a-5p-UP group the number of cells in S phase reduced(P<0.05),the number of cells in G2/M phase increased(P<0.05),which accelerated the process from S phase to G2/M phase and promoted cell proliferation.(6)The detection of Annexin V-APC cell apoptosis showed that compared to the control group,the number of apoptotic cells in mir-125a-5p group had no significant difference(P>0.05).(7)Luciferase showed that mi R-125a-5p was combined with 3’UTR of target gene IL-16,inhibiting the expression of IL-16.mi R-125 a and its target gene IL-16 had direct regulatory relationship.Conclusion:1.Mi R-106b-5p,mi R-125a-5p and mi R-183-5p may play a vital role in the pathogenesis of COPD,especially mi R-125a-5p and mi R-106b-5p were associated with the disease severity of COPD.2.The function of mi R-125a-5p was obvious in the THP-1 cell,which can promote the proliferation of mononuclear phagocyte system,cause its target gene IL-16 down regulation,and become a protective factor of COPD by enhancing anti-inflammatory effect.3.Mi R-125a-5p may become a marker associated to COPD classification(severity)and therapeutic target in the future.