The Relationship Between Telomere Length And Risk Of Idiopathic Pulmonary Fibrosis And Mechanism Study

Telomere is a ribonucleoprotein complex at the end of a cell’s chromosomes,consisting of an oligonucleotide sequence and corresponding proteins.The shortening of telomere length(TL)can cause cell aging,which is related to the occurrence and development of human diseases,especially aging diseases.The main mechanism for maintaining telomere length is to rely on telomerase to extend the telomere repeat sequence.Idiopathic pulmonary fibrosis(IPF)is an age-related chronic progressive aging diseases.Due to unclear etiology and no effective treatment measures,human health is seriously endangered.The study found that the shortened telomere length in patients with IPF may be a risk factor for the occurrence and prognosis of IPF.However,there are few studies on the shortening of telomere length and IPF,and they are mainly concentrated in Caucasians.There is insufficient evidence in the Chinese,so further research is needed.In this study,the case-control design was first used to detect the telomere length in peripheral blood of IPF patients and healthy controls,and the correlation between the risk of IPF and clinical parameters was analyzed.Secondly,single nucleotide polymorphisms(SNPs)assay were used to investigate the relationship between two candidate polymorphisms of telomere reverse transcriptase(TERT)and human mucin 5B(MUC5B)and the pathogenesis and prognosis of IPF.Finally,by constructing an in vitro aging model of alveolar epithelial cells(HPAEpiC),the possible regulatory mechanisms of telomerase and telomere length in IPF were further studied.This study provide a basis for understanding the role of telomere length in IPF and the possible mechanisms regulating the development of IPF.1.Telomere Length and the Risk of IPFIn this study,the case-control design was used to detect the telomere length in peripheral blood of 90 patients with idiopathic pulmonary fibrosis and 126 healthy people in Chinese Han population by PCR.To analyze the correlation between telomere length and the risk of IPF and clinical parameters.Each sample was provided with three replicates to ensure the accuracy of the test results.The results showed that the telomere length was significantly shorter in patients with idiopathic pulmonary fibrosis than in healthy controls(P<0.05).Telomere shortening was positively correlated with the severity of pulmonary fibrosis(R~2=0.348,P<0.05).2.TERT and MUC5B SNPs and the Risk of IPFAt present,the genetic variation associated with IPF in Chinese Han population is still unclear.This study selected TERT and MUC5B as candidate genes,detecting by sequencing method.To explore the relationship between SNPs and the susceptibility of IPF in Chinese population.Results showed that MUC5B rs868903 mutant allele was significantly elevated in patients with IPF(P<0.05).The MUC5B rs868903 mutation carriers were 2.47 times more likely to have IPF than the wild type(P<0.05).The mutation in the TERT rs2853676 site was not found to have an impact on the risk of IPF(P>0.05).The Kaplain-Meier method was used to analyze and compare the TERT rs2853676 and MUC5B rs868903 polymorphisms with the survival time of IPF patients.There was no statistical difference in the survival time between wild-type and mutant carriers of TERT rs2853676 and MUC5B rs868903(P>0.05).This study suggests that MUC5B rs868903 polymorphism can be used as a screening for high-risk patients with pulmonary fibrosis.3.Study on the related mechanism of IPF telomere lengthIn this study,HPAEpiC cells were first treated with bleomycin and evaluated by cell senescence specificβ-galactosidase detection reagents to simulate an in vitro cell senescence model of idiopathic pulmonary fibrosis.When the concentration of bleomycin was 12.25μg/mL,pulmonary fibrosis aging model was successfully established after continuous exposure for 48hours.Further Wnt 3a treatment of senile alveolar epithelial cells mimics the abnormal activation of Wnt/β-Catenin signaling pathway in IPF patients.When Wnt 3a concentration is100 ng/mL,the expression of GSK-3β,a key protein of the pathway,decreases after treatment for 48 hours.The expression of GSK-3βsignificantly decreased,P-GSK-3β,β-Catenin total protein,andβ-Catenin nucleoprotein were significantly increased(P<0.05),and the Wnt/β-Catenin pathway was successfully activated.The expression of TERT and the change of telomere length were detected by RT-qPCR and Western Blot.It was found that the expression of TERT decreased significantly after Wnt/β-Catenin pathway activation,and the length of telomere was significantly shortened(P<0.05).This indicates that the Wnt/β-Catenin pathway in senescent HPAEpiC can reduce telomere length by inhibiting the expression of TERT.To investigate its regulatory mechanism,we detected the expression of two transcription factors,KLF4 and CBP downstream of Wnt/β-Catenin,and found that the expression of KLF4 and CBP in senescent cells was increased by 3.44 and 2.12 folds respectively,after activation of Wnt/β-Catenin signaling pathway(P<0.05).It is suggested that Wnt/β-Catenin may regulate TERT expression through the KLF4 or CBP transcriptional pathway.Further suppression of KLF4 and CBP expression by small interfering RNA(siRNA)technology,compared with Wnt3a alone in senescent cells,TERT expression was upregulated after KLF4 was inhibited,and telomere length was increased(P<0.05).It is suggested that Wnt/β-Catenin in senile alveolar epithelial cells mainly regulate TERT and telomere length through the transcription factor KLF4 pathway.In summary,the case-control design of the study found that the telomere length in IPF patients in Chinese Han population was significantly shorter,and the telomere length was negatively correlated with the degree of pulmonary fibrosis in IPF patients.;MUC5B rs868903mutant genotype may increase the risk of IPF;the abnormal activation of Wnt/β-Catenin pathway in senile alveolar epithelial cells may shorten the telomere length by inhibiting the expression of TERT,and selectively inhibit KLT4 and upregulate the expression of TERT and repair the abnormal activation pathway of Wnt pathway.The resulting telomere shortening indicates that regulation of telomeres by the Wnt/β-Catenin/KLF4 pathway may be one of the mechanisms that influence the occurrence of IPF.The study provides a basis for further understanding the causes and mechanisms of IPF and provides a new direction for the prevention and treatment of IPF.