Screening And Validation Of Differentially Expressed LncRNA In Coal Worker’s Pneumoconiosis

Objective:The differentially expressed lnc RNA and m RNA were screened by transcriptome sequencing,and further population validation and establish an in vitro pulmonary fibrosis cell model to verify the differentially expressed lnc RNA,providing ideas for the related research on biomarkers of coal workers’pneumoconiosis（CWP）.Methods:（1）Four patients diagnosed as coal workers’pneumoconiosis and four healthy controls（HC）were selected as the study subjects.Blood were collected from two groups and peripheral blood mononuclear cell（PBMC）RNA was extracted.The differentially expressed lnc RNA and m RNA were screened by transcriptome sequencing.The bioinformatics method was used to predict the differentially expressed lnc RNA target gene and to analyze the GO function enrichment and KEGG signal pathway.（2）Based on the expression quantity,fold change,P value and related literature,two highly expressed lnc RNAs（LINC01359,LINC01503）and one low expressed lnc RNA（LINC01303）were selected.The expanded sample size was verified by real-time fluorescent quantitative PCR in 30 CWP patients and 30 healthy controls.Analyze the correlation between clinical indicators and target lnc RNAs,and explore the diagnostic value of target lnc RNA expression level in CWP using ROC curve.（3）Use TGF-β1 intervention of BEAS-2B cell and A549 cell to establish an in vitro pulmonary fibrosis model.The indicators established by the model were detected by real-time fluorescence quantitative PCR and Western Blot.Real-time fluorescence quantitative PCR was used to verify the differential expression of lnc RNA in the pulmonary fibrosis model and control cells.Results:（1）According to the screening conditions（P-value<0.05 and|log₂FC|>1）,584 kinds of m RNA with significant difference in expression were screened,of which 242 kinds of m RNA were significantly up-regulated and 342 kinds of m RNA were significantly down-regulated;A total of 248 lnc RNAs with significant differences in expression were screened,of which 138 were significantly up-regulated and 110 were significantly down-regulated.（2）The results of GO enrichment analysis and KEGG pathway analysis of differentially expressed lnc RNA-related target genes showed that they were involved in P53 signal pathway,NF-kappa B signal pathway,TNF signal pathway,AMPK signal pathway,MAPK signal pathway,and chemokine signal pathway;Trans results suggested that the role of a large number of differentially expressed lnc RNA was related to the transcription factor CTCF.（3）The expression level of LINC01359 and LINC01503 in CWP group was higher than that in HC group,and the expression level of LINC01303 in CWP group was lower than that in HC group,the difference was statistically significant（P<0.01）.（4）LINC01359 was positively correlated with SBP（r=0.354,P<0.01）,WBC（r=0.368,P<0.01）and PLT（r=0.357,P<0.01）;LINC01303 was negatively correlated with LDLC（r=-0.261,P<0.05）,SBP（r=-0.300,P<0.05）,TCHO（r=-0.401,P<0.01）,ALT（r=-0.287,P<0.05）and PLT（r=-0.287,P<0.05）;There is no correlation between target lnc RNA and other clinical indicators.（5）The value of the relative expression of LINC01359,LINC01503 and LINC01303 in the diagnosis of CWP was evaluated by using receiver operating characteristic curve（ROC）curve.The area under the curve of LINC01359 AUC=0.871（95%CI:0.783～0.959）,sensitivity was 76.7%,specificity was 83.3%;The area under the curve of LINC01503 AUC=0.744（95%CI:0.613～0.876）,sensitivity 60.0%,specificity 70.0%;The area under the curve of LINC01303 AUC=0.888（95%CI:0.798～0.978）,sensitivity 76.7%,specificity 96.7%;The area under the curve AUC of the three combined diagnosis was 0.977（95%CI:0.946～1.000）.（6）Use 2ng/ml TGF-β1 induce BEAS-2B treatment for 48 hours to induce pulmonary fibrosis cell model in vitro;Use 5ng/ml TGF-β1 induce A549 treatment for48 hours to induce pulmonary fibrosis cell model in vitro.（7）In vitro pulmonary fibrosis cell model,the expression levels of LINC01359,LINC01503 in the model group were higher than those in the control group,the expression levels of LINC01303 in the model group were lower than those in the control group,the difference was statistically significant（P<0.05）.Conclusion:（1）A total of 248 kinds of lnc RNAs with significant expression differences were obtained through transcriptome sequencing,of which 138kinds of lnc RNAs were significantly up-regulated and 110 kinds of lnc RNAs were significantly down-regulated,providing a theoretical basis for subsequent research.（2）LINC01359 and LINC01503 were highly expressed in the validation of CWP population,and LINC01303 was low expressed in the validation of CWP population;The expression of LINC01359 and LINC01503 in the model group of pulmonary fibrosis in vitro was higher than that in the control cell group,the expression of LINC01303 in the model group of pulmonary fibrosis in vitro was lower than that in the control cell group.（3）The ROC curve results suggest that LINC01359,LINC01503 and LINC01303 can be used as potential biomarkers of coal workers’pneumoconiosis.