Long-chain Non-coding RNA GSTM3TV2 Regulates The Function And Mechanism Of Chemoresistance In Pancreatic Cancer

BackgroundPancreatic cancer is one of the most deadly malignant digestive tumors,with difficulty in early detection,high malignant degree and poor prognosis.This malignancy is the fourth and sixth leading cause of cancer-related death in the USA and China,respectively,with overall 5-year survival rate lower than 7%.Surgical resection is the only method candidate for healing pancreatic cancer;however,only 15-20%of patients is diagnosed as resectable and then can therefore undergo resection.Chemotherapy could improve the outcome of locally advanced and disseminated pancreatic cancer;however,chemoresistance is a common phenomenon.With single agent chemotherapy or multi-drug chemotherapy on the basis of gemcitabine,the overall survival has not been prolonged obviously.Accordingly,detection of new biomarkers should be applied to increase the early detection rate,explore the molecular mechanism of chemoresistance and then improve the therapeutic efficacy,which is crucial for improving the prognosis of pancreatic cancer.Long non-coding RNAs(IncRNAs)have been confirmed as a class of largely functional transcripts longer than 200 nucleotides,involving in gene regulation at epigenetic,transcriptional,and post transcriptional levels for participating in various biological processes and carcinogenesis,such as tumorigenesis,proliferation,apoptosis,invasion,metastasis,autophagy,chemoresistance,sternness and EMT,dependent on diverse intracellular locations.Meanwhile,several studies have demonstrated that aberrant IncRNAs expression can be potentially used as biomarkers for diagnosis and prognosis of pancreatic cancer based on their tissue-specific expression manner.Moreover,IncRNAs play an irreplaceable role in the initiation and progression of pancreatic adenocarcinoma,such as maintaining cell growth,evasion of apoptosis,promotion of invasion and metastasis,sternness maintenance and EMT.However,mechanisms of IncRNAs in regulation of chemoresistance of pancreatic cancer remain unknown.Therefore,to investigate the underlying mechanisms of IncRNAs in drug resistance of pancreatic cancer would be crucial for indicating the exact molecular mechanisms of chemoresistance.ObjectiveTo detect the differential expression of IncRNA in the parental pancreatic cancer cell lines andgemcitabine-resistant pancreatic cancer cell lines and construct chemoresistance related competing endogenous RNA(ceRNA)network.Then,identify the potential key IncRNA in the ceRNA network,and explore its roles and mechanisms of chemoresistance in pancreatic cancer.At last,to detect the expression level of potential key IncRNA in pancreatic cancer specimens and evaluate its prognosis value.MethodsAberrant expression of IncRNAs,mRNAs and miRNAs in gemcitabine resistant pancreatic cell line(AsPC-1/GR)and parental pancreatic cancer cell line(AsPC-1)were detected by high-throughput gene chip to identify chemoresistance related IncRNAs expression profiling.Next,we used bioinformatics approach to construct chemoresistance related IncRNA-associated competing endogenous RNA(ceRNA)network to characterize the underlying mechanisms of IncRNA mediated gene regulatory network in regulating chemoresistance.Then,we identified the topological key node lncRNA in the ceRNA network and evaluated its expression in 180 pancreatic cancer specimens using ISH to assess the association with survival in patients with pancreatic cancer.Expression level of potential key IncRNA was upregulated or downregulated in pancreatic cancer cell line,then regulation effect of the potential key IncRNA on chemosensitivity,proliferation,apoptosis,invasion and metastasis was explored by adopting CCK-8,flow cytometry,transwell assays and so on.Pancreatic cancer cell lines with stable overexpression of potential key lncRNA and nude mice xenograft tumors were created to evaluate its role in tumor growth and chemotherapy in vivo.At last,we probe into the chemoresistance mechanisms regulated by potential key IncRNA and its potential target genes using RNA immunoprecipitation(RIP)and dual-luciferase reporter gene assay,with the related signaling pathways detected by western blot.Results1.Expression profiling identification of lncRNAs related with gemcitabine resistanceTo screen IncRNAs,mRNAs and miRNAs potentially involved in gemcitabine resistance,microarray IncRNA,mRNA,and miRNA analysis were performed with AsPC-1/GR and AsPC-1 cells.We found that 1897 detected IncRNAs demonstrated>1.5-fold differential expression with 963 IncRNAs showing up-regulated and 934 IncRNAs showing down-regulated.Meanwhile,IncRNA GSTM3TV2(Homo sapiens glutathione S-transferase mu 3,transcript variant 2,non-coding RNA;NR 024537.1)shows significant upregulation(fold change>104.52),based on which ceRNA network related with chemoresistance in pancreatic cancer is created.2.Analysis results of bioinformatics approachWe have identified IncRNA GSTM3TV2 as potential key IncRNA in ceRNA network by analyzing ceRNA network related with chemoresistance.Otherwise,IncRNA GSTM3TV2 might be involved in regulating series of proteins in this network,such as GLO1,KLK6,LAT2,OLR1 and PARM1,by competitively binding let-7,and then mediate chemoresistance of pancreatic cancer.3.Expression levels of IncRNA GSTM3TV2 in pancreatic cancer specimensWe used in situ hybridization(ISH)to determine the GSTM3TV2 expression levels in 180 pancreatic cancer tissue samples and matched tumor-adjacent tissues.ISH staining revealed that GSTM3TV2 was located in the cytoplasm,and the expression of GSTM3TV2 in pancreatic cancer tissues was significantly increased compared with that in tumor-adjacent tissues.The correlation analysis of GSTM3TV2 expression revealed a significant association between GSTM3TV2 expression and lymph node staging(P= 0.007)and TNM staging(P= 0.003).Univariate survival analysis revealed that prognosis significantly correlated with GSTM3TV2 expression level(P= 0.004),differential degree(P= 0.035),T staging(P= 0.008),N staging(P= 0)and TNM staging(P= 0).Multivariable Cox regression analyses revealed that differential degree,TNM staging and high GSTM3TV2 expression could be independent factors for poor prognosis(P=0.008,HR=1.706,95%CI:1.147-2.537;P=0.003,HR=2.786,95%CI:1.149-5.470;P=0.036,HR=1.466,95%CI:1.025-2.096).4.Malignant phenotype regulation of IncRNA GSTM3TV2 in pancreatic cancer cell linesIn vitro,GSTM3TV2 upregulation in AsPC-1 and MIAPaCa-2 could decrease the chemosensitivity of panreactic cancer cells to gemcitabine and nab-paclitaxel,inhibit apoptosis induced by gemcitabine and nab-paclitaxel and promote proliferation,invasion and migration.To contrary,GSTM3TV2 downregulation in AsPC-1/GR and MIAPaCa-2/GR showed the opposite results.In vivo,tumor growth was faster and chemosensitivity to gemcitabine and nab-paclitaxel was higher in mice with stable GSTM3TV2 overexpression(P<0.05).5.LncRNA GSTM3TV2 mediates chemoresistance of pancreatic cancer cells by regulating let-7We examined that let-7 was upregulated in GSTM3TV2 knockdown MIAPaCa-2/GR cells and downregulated in GSTM3TV2 overexpression AsPC-1 cells by real time PCR(P<0.05).Next,we constructed luciferase reporters containing GSTM3TV2,which contains wild type(WT)or mutated let-7 binding sites,for target investigates.We found that the let-7 mimics reduced the luciferase activities of the WT reporter vector but did not reduce the activity of the empty vector and mutant reporter vector,supporting that let-7 are bona fide GSTM3TV2-targeting miRNAs.Then,we performed an RNA immunoprecipitation(RIP)to pull down endogenous miRNAs associated with GSTM3TV2 and demonstrated that the GSTM3TV2 RIP in AsPC-1 and 293A cells were significantly enriched for let-7 mimics compared to IgG,the empty vector(MS2),and GSTM3TV2 with mutations in let-7 targeting sites.All these data suggest that GSTM3TV2 could sponge let-7.In western blot,GSTM3TV2 upregulation or downregulation in pancreatic cancer cell lines could increase or decrease the expression of let-7 targeting genes,including K-Ras,c-Myc and HMGA2,which indicated that GSTM3TV2 are involved in let-7 regulation.Meanwhile,up regulating let-7 in pancreatic cancer cells with stable GSTM3TV2 overexpression could increase the chemosensitivity to gemcitabine(P<0.05),significantly promote apoptosis induced by gemcitabine and inhibit the expression of let-7 targeting genes,including K-Ras,c-Myc and HMGA2.These data indicate that IncRNA GSTM3TV2 mediates chemosensitivity of pancreatic cancer depending on let-7 expression.6.LncRNA GSTM3TV2 mediates chemoresistance of pancreatic cancer by regulating LAT2 and OLR1We examined that downregulating GSTM3TV2 in MIAPaCa-2/GR cells could reduce the expression of LAT2 and OLR1 mRNA by real time PCR(P<0.05).Besides,the expression of LAT2 and OLR1 at protein levels were also upregulated or downregulated through upregulating or downregulating GSTM3TV2 by western blot analysis.Dual-luciferase reporter gene assay indicates that LAT2 and OLR1 are downstream targeting gene of let-7 and GSTM3TV2 can be involved in regulating the expression of LAT2 and OLR1 by competitively binding let-7.Otherwise,chemosensitivity to gemcitabine(P<0.05)and apoptosis induced by gemcitabine(P<0.05)were both increased by suppressing LAT2 and OLR1.Meanwhile,LAT2 and OLR1 downregulation could increase the chemosnsitivity to gemcitabine and the apoptosis induced by gemcitabine in pancreatic cancer cell lines with stable GSTM3TV2 overexpression.The expression of LAT2 and OLR1 in pancreatic cancer cell lines with stable GSTM3TV2 overexpression can be suppressed by let-7 overexpression by western blot analysis,which indicates that IncRNA GSTM3TV2 can mediate chemoresistance of pancreatic cancer by regulating LAT2 and OLR1.7.LncRNA GSTM3TV2 activates PI3K/Akt,MAPK and STAT3 signaling pathways by regulating let-7Upregulating GSTM3TV2 in AsPC-1 abd MIAPaCa-2 could result in series of signaling pathways changes.Firstly,PI3K/Akt pathway which is K-Ras downstream pathway was activated to upregulate and downregulate proteins which could promote proliferation and inhibit apoptosis,such as p-mTOR,p-Ak,Bcl-xl,NF-?B,p-GSK-3?,p-Bad and cleaved caspase-9,p21,p27.Then,cell cycle regulatory proteins including Cyclin D1,Cyclin E1 and CDK6 were upregulated and apoptosis related proteins including cleaved PARP and cleaved caspase-3 were downregulated.Secondly,MAPK pathway which is K-Ras downstream pathway was activated to upregulate c-Raf,p-c-Raf and p-ERKl/2.Thirdly,K-Ras downstream proteins such as Racl and RhoA were upregulated.Fourthly,EMT related proteins such as Vimentin,N-cadherin,ZEB1,Snail and Slug were upregulated.Fifthly,STAT3 pathway was activated and p-STAT3 was upregulated.To contrary,the opposite results were obtained in AsPC-1/GR and MIAPaCa-2/GR.8.LncRNA GSTM3TV2 mediates chemoresistance of pancreatic cancer to erlotinib by regulating EGFRWe examined that upregulating or downregulating GSTM3TV2 in pancreatic cancer cells could suppress or increase the expression of EGFR by western blot and that upregulating or downregulating GSTM3TV2 in pancreatic cancer cells could increase or suppress the expression of EGFR mRNA by real time PCR.These data indicate that GSTM3TV2 modulates EGFR expression post-transcriptional.In RIP,GSTM3TV2 could directly bind to EGFR mRNA and decrease EGFR expression at protein level.Othetwise,upregulating or downregulating GSTM3TV2 in pancreatic cancer cells could decrease or increase the chemosensitivity to erlotinib(P<0.05)and suppress or promote apoptosis induced by erlotinib(P<0.05).Meanwhile,promoting EGFR expression in pancreatic cancer cells with stable GSTM3TV2 overexpression could significantly increase the chemosensitivity to erlotinib(P<0.05)and apoptosis induced by erlotinib(P<0.05).EGFR suppression caused by GSTM3TV2 can be antagonized by EGFR upregulation in western blot,which indicates that LncRNA GSTM3TV2 could mediate chemoresistance of pancreatic cancer to erlotinib by suppressing EGFR.ConclusionLncRNA GSTM3TV2 could competitively bind let-7 to upregulate its target gene including K-Ras,c-Myc,HMGA2,LAT2 and OLR1 through ceRNA mechnism,and then involve in the regulation of chemoresistance to gemcitabine in pancreatic cancer.Otherwise,lncRNA GSTM3TV2 could mediate chemoresistance of pancreatic cancer to erlotinib by regulating EGFR.At last,the expression of IncRNA GSTM3TV2 in pancreatic cancer tissues show a higher level compared to tumor-adjacent tissues and GSTM3TV2 upregulation is an independent factor for poor prognosis of pancreatic cancer.