Identification Of LncRNAs And Their Functional Network Associated With Chemoresistance In SW1990/GZ Pancreatic Cancer Cells By RNA Sequencing

Objective: we performed the high-throughput sequencing technology to screen the differentially expressed lncRNAs(DELs)and differentially expressed mRNAs(DEMs)in chemoresistant pancreatic cancer cells SW1990/GZ in comparison with parental cells SW1990.To investigate chemoresistace-associated lncRNAs initially.Prediction of DEL target genes and construction of the DEL–DEM coexpression network were performed for further research.The results of this study provide insights into the mechanism of chemotherapy resistance in pancreatic cancer as well as potential therapeutic targets for treatment of this disease.Methods: The human pancreatic cancer gemcitabine-resistant cell line SW1990/GZ was constructed by in vitro intermittent concentration gradient method.The proliferation activity of SW1990/GZ and parental cell SW1990 under chemotherapeutic drug culture was detected by MTT method.The growth curve was drawn and the IC50(inhibitory concentration)value and drug resistance index were calculated.The morphology of the two groups was observed under optical microscope;the difference of lncRNA and mRNA expression profiles between pancreatic cancer cell line SW1990/GZ and parental cell SW1990 was analyzed by the second-generation transcript sequencing.And reference-based de novo lncRNA transcript assembly was performed using Cufflinks.Advanced bioinformatics methods were carried out to investigate DELs and DEMs.The expression of 6 randomly selected upregulated and downregulated lncRNAs was evaluated by qRT-PCR(AC006262.5、MIR210HG、RP11-23P13.6 、 LINC00173 、 RP11-497G19.1 、 RP11-1055B8.4 、 RP11-48O20.4 、RP11-442H21.2、EPB41L4A-AS1、SNHG1、SNHG5、RP11-426C22.6).Results:1.A gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was established successfully,SW1990/GZ cell line presented significant large swellings or spindle-shaped cell form in comparison with SW1990 cell line.The IC50 value of SWl990 cells was lower than that of SW1990/GZ cells(2.393 + 0.145 uM vs.234.9 + 4.51 uM),indicating that resistance index of the latter to gemcitabine was 98.2.2.The second-generation transcript sequencing showed that the lncRNA and mRNA expression profiles in the gemcitabine-resistant cell line SW1990/GZ were significantly different from those of the parental cell line SW1990;among them,there were 113 lncRNAs with up-regulated expression more than 2 times(105 annotated)and 92(75 annotated)lncRNAs with more than 2 times down-regulated expression;similarly,391 mRNAs were upregulated and 456 mRNAs were downregulated with more than two fold respectively.3.In contrast to parental cell SW1990,the results of qRT-PCR demonstrated that the expression of selected lncRNAs(AC006262.5、MIR210HG、RP11-23P13.6、LINC00173、RP11-497G19.1、RP11-1055B8.4)in SW1990/GZ was overexpressed and the expression of remaining lncRNAs(RP11-48O20.4 、 RP11-442H21.2 、EPB41L4A-AS1、SNHG1、SNHG5、RP11-426C22.6)was on the opposite.4.mRNAs that were predicted to be related to DELs—that is,they both showed statistically significant changes in expression by RNA-seq—were classified as overlapping mRNAs.About 55 overlapping DEMs(6.5%)were targeted by DELs through cis action,whereas 91(10.7%)were subject to trans regulation.5.The GO analysis revealed that DEL-targeted DEMs were mainly enriched in cellular process,intracellular,or binding processes,KEGG reference pathway indicated that dysregulated transcripts were significantly involved in glucagon signailing pathway,metabolic pathways,bladder cancer,insulin resistance,PI3K/AKt pathway,VEGF signailing pathway,and P53 pathway.6.DEL–DEM interactions with a correlation coefficient >0.995 and p < 0.05 were retained in the coexpression network,which included 1052 nodes and 7049 connections.The top 3 DELs coexpressed with DEMs were MIR210HG、SNHG1 and LOC729970,and the top 3 DEMs coexpressed with DELs were RAB3 D,DDX17 and SPNS2.Conclusion:1.The biological characteristics of the successfully constructed human pancreatic cancer gemcitabine-resistant cell line SW1990/GZ were significantly changed compared with the sensitive cell line SW1990.2.In addition to screening for differentially expressed lncRNA transcripts in pancreatic cancer gemcitabine-resistant cell line,second-generation sequencing technology can also focus on the discovery of some novel lncRNAs.3.The abnormally expressed lncRNA genes in the pancreatic cancer gemcitabine-resistant cell line analyzed by the high-throughput sequencing technology were verified by real-time quantitative PCR,and the consistency of the results further improved the credibility of the sequencing results.The analysis combined with advanced biological information shows that differentially expressed lncRNAs may play an important role in the molecular resistance mechanism of pancreatic cancer,and could become a potential gene target for the treatment of pancreatic cancer chemoresistance in the future.