| Objective: Primary bronchial lung cancer,lung cancer for short,includes non-small cell lung cancer and small cell lung cancer.According to the pathological type,non-small cell lung cancer include adenocarcinoma,squamous cell carcinoma,adenosquamous carcinoma and large cell carcinoma.Long-non-coding RNA(lncRNA),microRNA(miRNA)and messenger RNA(mRNA)are DNAs` transcripts.LncRNA and miRNA can participate in the expression of mRNA.In this study,we constructed RNA sequencing of lung adenocarcinoma(LUAD)tumor tissue of TNM I stage,and screened the abnormal expression of lncRNA,miRNA and mRNA.The co-expression network was constructed and the Gene Ontology,(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis was conducted,then we screen out the lncRNA with biological function.Finally,qRT-PCR was performed to determine the lncRNA associated with LUAD.Methods: The TNM I stage Lung adenocarnoma tumor tissues and paired adjacent non-tumor tissues for the experimental were obtained from 18 patients who underwent surgery in the the Thoracic Surgery of the Fourth hospital of Hebei Medical University,from December 29,2015 to February 18,2016。Those patients received none of chemotherapy or radiotherapy before resection surgery。The first 10 cases were selected according to the order of time for RNA high-throughput sequencing,the other 8 cases was used to detect the expression level of RNA by qRT-PCR.The part of lncRNA and mRNA: first,the total RNAs of collected specimens which removed the rRNAs was extracted,then we fragmented the mRNAs and lncRNAs,and then synthesis of cDNAs,finally,we used the Illumina Hiseq4000 for sequencing.The part of MiRNA: the recovered RNAs with 18-30 nt from total RNA was used to reverse transcription to the cDNAs by RT-PCR.After amplified the cDNAs we collected it and then we constructed the small RNAs library,finally we used the Illumina Hiseq2500 for sequencing.The raw image data obtained from RNA-sequencing was translated into raw FastQ sequence data.TopHat was used to align the clean reads of long non-coding RNAs(lncRNAs)and protein coding mRNAs(mRNAs)with the human reference genome,Ensemble GRCh38 v 84(hg19).The obtained sequences were spliced and the positions of lncRNA and mRNA in the human reference genome Ensemble GRCh38 v 84 were obtained.Moreover,the alignment of miRNAs with hg19 was implemented by Bowtie.The differentially expressed lncRNAs(DELs)and differentially expressed mRNAs(DEMs)were identified in tumor tissues compared with adjacent non-tumor tissues by Cuffdiff.Differentially expressed miRNA(DEMIs)in tumor tissues were filtered using DEGseq package.The miRWalk database was used to predict the target genes of DEMIs,then DEMI-DEMs interaction network were constructed and analyzed,and visualized by Cytoscape.Pearson correlation coefficients(PCCs)were calculated for the covariant correlation between the expression levels of DEL and DEM.The DEL-DEM co-expression pair of |PCC≥0.90| is used to construct a co-expression network.The protein coding genes near DEL were screened out in the reference genome of GRCH38,which matched the DEM in LUAD and screened DEM near DEL in LUAD.Then,the DEM near DEL and DEMI negative control of those DEM to take the intersection,thus building DEL-DEMI-DEM co-expression network,and visualized by Cytoscape.In order to understand the potential biological functions and signaling pathways of DELs in tumor tissues,we conducted the Gene Ontology(GO)biological process and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment.Finally,we detected the expression level of DEL / DEMI / DEM by qRT-PCR.Results:1 Respective 15710 lncRNAs and 22528 mRNAs were mapped with the human reference genome Ensemble GRCh38 v 84.Total of 175 differentially expressed lncRNAs(DELs,63 up-and 112 down-regulated)and 1321 differentially expressed mRNAs(DEMs,587 up-and 734 down-regulated)were identified in tumor tissues compared to paired non-tumor tissues.LOC80078 and LOC101930114 were the most significantly up-and down-regulated DELs;EEF1A2 and ANKRD1 were the most significantly upand down-regulated DEMs in tumor tissues compared to paired non-tumor tissues.Total of 94 DEMIs including 87 up-and 7 down-regulated DEMIs were identified in tumor tissues compared with paired non-tumor tissues.hsa-miR-194-5p,hsa-miR-135b-5p and hsa-miR-215-3p were significantly up-regulated in tumor tissues;hsa-miR-486-5p,hsa-miR-338-3p,hsa-miR-7641,hsa-miR-138-5p,hsa-miR-451 a,hsa-miR-486-3p and hsa-miR-139-3p were significantly down-regulated in tumor tissues.2 The targets genes of top 15 up-and down-regulated DEMIs in early stage LUAD were predicted though miRWalk database.Those predicted target genes were overlapped with 1321 DEMs.DEMI-DEM interaction pairs were visualized though Cytoscape.up-regulation DEMIs/down-regulation DEMs regulatory network composed of 395 nodes and 942 edges,which is involved in 15 up-regulated DEMIs and 384 DEMs;hsa-miR-182-5p,hsa-miR-200b-3p and hsa-miR-429 had the highest connectivity with down-regulated DEMs,which interacted with 99,83 and 82 DEMs,respectively.Down-regulation DEMIs/up-regulation DEMs regulatory network composed of 139 nodes and 181 edges,which is involved in 7 down-regulated DEMIs and 132 DEMs;hsa-miR-486-3p,hsa-miR-138-5p and hsa-miR-338-3p had the highest connectivity with up-regulated DEMs,which interacted with 61,45 and 41 down-regulated DEMs,respectively.3 The nearby protein-coding genes of 175 DELs with distance<100kb were identified in GRCH38 reference genome.Total 190 genes,nearby with 175 DELs,were identified.Then190 gene were overlapped with 1321 DEMs in tumor tissues,and 42 DEMs,nearby with 38 DELs,were available.In the DEL-DEM co-expression pairs,CDKN2B-AS1,FENDRR,LINC00312,LINC00515 and LINC00162 respectively co-expressed with 105,63,61,6 and 5 DEMs.DEL-DEMI-DEM network depicted the links among DELs,DEMI and DEMs abovementioned.DEMs were significantly enriched in cell adhesion molecules,focal adhesion and tight junction of Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways;and enriched in cell adhesion,angiogenesis and regulation of cell proliferation of Gene Ontology biological processes.4 q RT-PCR was subjected to validate the dysregulated DEL/DEMI/DEM in 6 stage I LUAD tissues and 6 adjacent non-tumor tissues.hsa-miR-200a-3p(P<0.01),hsa-miR-200b-3p(P<0.05),hsa-miR-200b-5p(P<0.05),hsa-miR-200c-5p(P<0.05)and hsa-miR-429(P<0.01)were obviously up-regulated in and hsa-mi R-338-3p(P<0.05)was obviously down-regulated in tumor tissues compared to control tissues.The expression levels of ADARB1(P<0.01),ADRB2(P<0.05)and ANKRD1(P<0.05)were significantly down-regulated;COL1A1(P<0.05)and MMP13(P<0.05)were significantly up-regulated in stage I LUAD.The expression levels of lncRNA FENDRR(P<0.01)and LINC00312(P<0.01)were significantly down-regulated and lncRNA CDKN2B-AS1 had the up-regulated tendency in tumor tissues.Conclusions: In this study,we use the TNM stage I lung adenocarcinoma as the object of study,through the tumor tissue and adjacent normal tissue RNA sequencing,access to abnormal expression of RNA.LncRNA FENDRR,LINC00162,LINC00515 and LINC00312 were down-regulated in early lung adenocarcinoma.CDKN2B-AS1 was up-regulated in early lung adenocarcinoma.And its expression level may be related to the development and progression of lung adenocarcinoma.This may have an early diagnostic value for lung adenocarcinoma.Our study may lay the foundation for the pathogenesis of lung adenocarcinoma and identify potential therapeutic targets and new early diagnostic biomarkers for patients with lung adenocarcinoma. |
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