| Objective:Transcriptomic sequencing and bioinformatics techniques were performed to screen out the differentially expressed lncRNA associated with idiopathic pulmonary fibrosis.To verify whether it could be a molecular target related to IPF.To further study the function of this lncRNA at animal and cellular levels and to explore the possible mechanism in IPF.Methods:1.（1）According to the diagnostic criteria,4 peripheral blood samples of IPF patients（aged with older than 60 years）and 4 peripheral blood samples of healthy controls（aged with older than 60 years）were selected.LncRNAs in the peripheral blood were detected by transcriptome sequencing,and differentially expressed lncRNAs and mRNAs were analyzed by bioinformatics techniques.（2）30 IPF patients and 30 healthy controls were collected again.Peripheral blood was collected.RNA was extracted from peripheral blood.LncRNAs were verified by PCR and the expression of lncRNAs was compared between the two groups.The lncRNA with the relatively larger changes between the two groups was selected.At the same time,the expression of this lncRNA and its associated m RNA was verified.（3）100 cases of peripheral blood samples were collected from healthy people of different ages in this study,such as,20 cases were 20-30 years old,20 cases were 20 to 40years old,20 cases were 40 to 50 years old,20 cases were 50 to 60 years old,20 cases were more than 60 years old.The expression of AP003419.16 and RPS6KB2 in different age groups was examined by PCR.2.Animal level:It was revealed that AP003419.16 and RPS6KB2 were homologous between human and mouse by bioinformatics analysis.6 week old Male C57BL/6 mice and6 month old Male C57BL/6 mice were chosen in this study.Six-week-old mice were randomly divided into normal group（QC group）,young pulmonary fibrosis group（QM group）,and young lung fibrosis+rapamycin group（QL group）（n=12 per group）.The6-month-old mice were randomly divided into normal aging group（LC group）,old pulmonary fibrosis group（LM group）,old lung fibrosis+rapamycin group（LL group）（n=12 per group）.The injury of lung tissue in each group was observed by HE staining.The pulmonary fibrosis in each group was observed by Masson staining.The expression of AP003419.16 in lung tissue was detected by in situ hybridization.The expression of AP003419.16 and RPS6KB2 in peripheral blood and lung tissues were detected by PCR.The expression of P-P70S6K protein in lung tissue was detected by Western blot.3.Cell levels:（1）A549 cells were cultured and divided into groups:A549 group,TGF-β1 group,A549（7d）group,and TGF-β1（7d）group.Cell senescence was detected by theβ-galactosidase assay.PCR was performed to examine the expression of AP003419.16 and RPS6KB2.Western blot was performed to examine the expression of P-P70S6K.（2）AP003419.16 was interfered with by si RNA.There were 6 groups in our study,A549 group,TGF-β1 group(A549TGF-β1group),negative si RNA group(A549 negative siRNAgroup),negative siRNA+TGF-β1 group(A549 negative siRNA+TGF-β1group),positive siRNA group(A549 positive siRNAositive siRNA group)Positive si RNA+TGF-β1 group(A549 positive siRNA+TGF-β1group).The expression of AP003419.16 and RPS6KB2 was examined by PCR.Results:1.（1）The results showed that there was differentially expressed lncRNAs in IPF by transcriptome sequencing.Compared with the healthy control,there were 1699 differentially expressed lncRNAs in IPF.GO analysis found that the functions of target genes in IPF,such as the regulation of B cell receptor signaling pathway,regulate calcium channel regulator activity,etc.Pathway analysis showed that target genes in IPF associated with many pathways,such as,mTOR signaling pathway and ErbB signaling pathway,etc.The positional relationship mostly was the exon sense-overlapping,natural antisense and intergenic between the differentially expressed lncRNAs and adjacent protein-coding genes in IPF.Based on the sequencing results and the screening conditions,we selected three lncRNAs:AP003419.16,RASSF1-AS1,and RP11-20I23.8.（2）The three lncRNAs were validated in 30 IPF patients and 30 healthy controls.It was found that AP003419.16 had the largest changes in the three lncRNAs.We would use AP003419.16 for the further study.It revealed that the differentially expressed mRNA was RPS6KB2 by bioinformatics analysis.Theneighboring gene of AP003419.16 is RPS6KB2.It was found that lncRNA AP003419.16 and the mRNA RPS6KB2 were highly expressed in the IPF patients but lowly expressed in the control group（P<0.05）.（3）It was found that AP003419.16 and RPS6KB2 are also increasing with age increases in healthy populations in this stduy（P<0.05）.2.The degree of interstitial thickening and the degree of fibrosis in the lung was most evident in the LM group by HE staining and Masson staining.The QL group was better than the QM group,and the LL group was better than the LM group.According to the result of in situ hybridization,the expression of AP003419.16 was higher in the LC group than in the QC group（P<0.05）.the QM group was higher than the QC group,the LM group was higher than the LC group,the highest in LM group（P<0.05）,the QL group was lower than the QM group,the LL group was lower than the LM group（P<0.05）.The result of PCR in C57BL/6lung tissues and peripheral blood showed that the expression of AP003419.16 and RPS6KB2 in the LC group was higher than in the QC group,the QM group was higher than the QC group,the LM group was higher than the LC group,the highest in LM group（P<0.05）,the QL group was lower than the QM group,the LL group was lower than the LM group（P<0.05）.According to the result of Western Blot,the expression of P-P70S6K in lung tissue of each group was consistent with the result of gene RPS6KB2（P<0.05）.3.We observed the A459 cells by an inverted phase contrast microscope,A459 cells was changed from the original polygonal shape to long spindle shape when cultured in the presence of 10ng/ml TGF-β1 for 7 days.And it was found that the number of positive cells stained byβ-galactosidase was significantly increased when cultured in the presence of10ng/ml TGF-β1 for 7 days.（1）The result of PCR showed that the expression of AP003419.16 and RPS6KB2 in TGF-β1 group was higher than that in A549 group（P<0.05）,and the expression of AP003419.16 and RPS6KB2 in A549 group（7d）was higher than that in A549 group（P<0.05）.According to the result of Western Blot,the expression of P-P70S6K in lung tissue of each group was consistent with the result of gene RPS6KB2（P<0.05）.（2）We found AP003419.16 and RPS6KB2 were mostly overlapping by bioinformatics techniques.We interfered with AP003419.16 by siRNA and found that RPS6KB2 was almost undetectable by PCR.Conclusion:1.The results showed that there was differentially expressed lncRNAs in peripheral blood of IPF by transcriptome sequencing.It was found that lncRNA AP003419.16 and the mRNA RPS6KB2 were highly expressed in the IPF patients butlowly expressed in the control group.It was found that AP003419.16 and RPS6KB2 are also increasing with age increases.2.AP003419.16 and RPS6KB2 were found to be associated with aging and IPF in peripheral blood and lung tissues in animal models.3.AP003419.16 and RPS6KB2 were mostly overlapping by bioinformatics techniques.We interfered with AP003419.16 by siRNA and found that RPS6KB2 was almost undetectable at cell levels.It is speculated that lncRNA AP003419.16 may play a potential molecular target in aging-associated IPF by regulating the adjacent gene RPS6KB2. |
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