Preliminary Study On Expression Profile Of Long Non-coding RNA In Gemcitabine-resistant Pancreatic Cancer Cell Line

Objective:We investigated differences in lnc RNA(long non-coding RNA)and m RNA expression profiles between parental cell line SWl990 and gemcitabine-resistant pancreatic cancer cell line SWl990/GZ with high-throughput lnc RNA microarray assays. To investigate how gemcitabine regulate the expression of lnc RNA in SW1990. Gene Ontology analysis and Pathway analysis were performed for further research. We need a new angle to explain the mechanism of pancreatic cancer chemotherapy resistance. And provide a novel approach reversing pancreatic cancer drug resistance from new perspectives.Methods:Gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was obtained by treating parental cell line SWl990 in vitro with increasing dosage of gemcitabine in culture medium intermittently for ten months. Cellular morphology was observed by histology microscopy. The drug resistance of SWl990/GZ cells was evaluated using MTT assay. The doubling time was calculated and the growth curve was observed.Flow cytometry assay was used to evaluate the cell cycle distribution in this two cell lines.We investigated differences in lnc RNA and m RNA expression profiles between parental cell line SWl990 and gemcitabine-resistant pancreatic cancer cell line SWl990/GZ with high-throughput lnc RNA microarray assays. Gene Ontology analysis and Pathway analysis were performed for further research. Quantitative real time PCR confirmed the changes of six lnc RNAs(RP11-58D2.1, linc RNA-ZNF532, AP000221.1, CTC-338M12.5, CR619813, DDX6P) and nine m RNAs(SYT1, FAM171 B, ZNF331, FAM187 B, CYP1A1, SRXN1, HIST1H2 BL, TOMM40 L and SPP1) in SW1990 and SW1990/GZ. In order to investigate how gemcitabine regulate the expression of linc RNA-ZNF53 and DDX6 P, we determined the level of linc RNA-ZNF53 and DDX6 P in SW1990 cells when treated by gemcitabine at different concentration(0,1,2,4,8,16 μM) and different time(0,1,2,4,8,16 d) using quantitative real time PCR.Results:1. A gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was established successfully.2. Gemcitabine-resistant cell line SWl990/GZ underwent obvious morphological and functional changes. Compared with the parental cell line SW1990, SWl990/GZ cell was small and tumed into round shape. The IC50 of SW1990 and SWl990/GZ were 3.03±0.27μmol/L,230.46±0.31μmol/L, and the drug resistance indexes of cell line SW1990/GZ to gemcitabine was 71.6(P<0.01). Compared with the SW1990 cell,SW1990/GZ exhibited a lower growth rate, and their population doubling time were 32.80±2.12 h and 22.36±1.78h(P<0.05),meanwhile cell distribution increased in G1 phase and decreased in G2 phase(P<0.05),but no difference in S phase.3. We show that SW1990/GZ cells exhibit a considerable dysregulation of the lnc RNA and m RNA profile as compared with their parental lines SW1990 among, We identified 4983 of 13310 detected lnc RNAs demonstrated > 2-fold abnormally expressed in response to the gemcitabine-resistant, among of them, 1993 and 2990 lnc RNAs were upregulated and downregulated. Meanwhile, 4759 m RNAs exhibited at least a 2-fold, of these, 2671 and 2088 m RNAs were upregulated and downregulated. GO and pathway analysis indicated that the identified m RNAs were mainly related to the new supersedes old,cell cycle process,molecular binding,nucleotide binding,kinase activity, Toll-like receptor signaling pathway, small cell lung cancer, chronic myeloid leukemia, pancreatic cancer, DNA replication, bladder cancer, non-small cell lung cancer, glycolysis/ Gluconeogenesis, p53 signaling pathway.4.The results demonstrated that RP11-58D2.1(p< 0.01),linc RNA-ZNF532(p< 0.01)and AP000221.1(p< 0.01)were upregulated and that CTC-338M12.5(p< 0.05), DDX6P(p< 0.05) and CR619813(p< 0.01) were downregulated in the SW1990/GZ cells compared with SW1990 cells. For m RNA, SYT1, FAM171 B, ZNF331, FAM187 B and CYP1A1 were upregulated(p< 0.01) and that SRXN1, HIST1H2 BL, TOMM40 L and SPP1 were downregulated(p< 0.01)in the SW1990/GZ cells compared with SW1990 cells.The q RT-PCR results are consistent with microarray data. We also found that the upregulating of gemcitabine on the expression of linc RNA-ZNF532 was time-dependent. Gemcitabine at a range from 1.0 μM to 16.0 μM induced a increase of linc RNA-ZNF532 in SW1990 cells. The relative level of DDX6 P is opposite to that of linc RNA-ZNF53 in the same circumstancece.Conclusions:1. A gemcitabine-resistant pancreatic cancer cell line SWl990/GZ was established successfully. Compared with the SW1990, SW1990/GZ showed great changes in biological characteristics;2.The high throughput lnc RNA microarray can be an effective tool detected deregulation of lnc RNA and m RNA expression in diseases;3. Our results were the first to reveal differentially expressed lnc RNAsand m RNA in gemcitabine resistant pancreatic cancer cell line and some of the differentially expressed lnc RNAs and m RNAs were validated by q RT-PCR to promote the credibility of results. Prelininary bioinformatic analysis revealed that m RNAs was involved in the occurrence and development chemoresistance;4. The expression of lnc RNA was impacted by gemcitabine concentration and different time. The dysregulated lnc RNAs may represent good candidates for future diagnostic and potential targets for gene therapy in drug-resistance of pancreatic cancer.