Function And Mechanism Study Of Hypoxia-associated NR2F1-AS1 In Pancreatic Cancer By Regulating The NR2F1/AKT Pathway

Objective:Pancreatic cancer(PC)is a highly malignant solid tumor with insidious onset,difficult early diagnosis,poor patient prognosis,and a five-year survival rate of less than 9%.In the early stage of pancreatic cancer,surgical resection is the main method,which is insensitive to radiotherapy and chemotherapy and lacks effective adjuvant treatment measures.In recent years,targeted therapy has become a hot topic.The invasion,metastasis and recurrence of pancreatic cancer are important factors affecting the prognosis of patients.Therefore,studying the mechanism of occurrence,invasion and metastasis of pancreatic cancer and finding effective predictive markers and key therapeutic targets have important clinical significance for the early diagnosis and treatment of pancreatic cancer.Studies have shown that long non-coding RNAs(lncRNAs)are involved in the occurrence and progression of many diseases,including pancreatic cancer.lncRNA NR2F1 antisense 1(NR2F1 antisense 1,NR2F1-AS1)is abnormally expressed in a variety of human tumors,but the study of NR2F1-AS1 in pancreatic cancer has not been reported.Therefore,this study intends to explore the expression and prognostic value of NR2F1-AS1 in pancreatic cancer;explore the effect of NR2F1-AS1 on the biological behavior of pancreatic cancer;explore the mechanism of action of NR2F1-AS1 in pancreatic cancer and the reasons for its abnormal expression.To provide a new theoretical basis for the diagnosis,prognosis and treatment of pancreatic cancer patients.Methods:1.Bioinformatics methods were used to screen out the abnormally highly expressed lncRNA NR2F1-AS1 in pancreatic cancer tissues from GEO and TCGA databases,and Kaplan-Meier analyzed its prognostic value for pancreatic cancer patients.Quantitative real-time polymerase chain reaction(q RT-PCR)was used to detect the expression level of NR2F1-AS1 in 90 pairs of PC and its adjacent tissues,and used to detect the expression of NR2F1-AS1 in PC cell lines PANC-1,MIA Pa Ca-2,SW1990,CFPAC-1,Capan2,BXPC-3 and normal human immortalized pancreatic duct epithelial(HPDE).In situ hybridization(ISH)assay was used to detect the expression level of NR2F1-AS1 in PC tissues and adjacent non-cancerous tissues,and to analyze the correlation between the expression level of NR2F1-AS1 and clinicopathological characteristics.The subcellular localization of NR2F1-AS1 in PC cells was detected by fluorescence in situ hybridization(FISH).The distribution level of NR2F1-AS1 in the nucleus and cytoplasm of PC was detected by q RT-PCR.2.Two PC cells with high expression of NR2F1-AS1,MIA Pa Ca-2 and PANC-1,were selected for in vivo and in vitro experiments to synthesize and construct a cell model of lentivirus-mediated knockdown and overexpression of NR2F1-AS1.The effects of NR2F1-AS1 on the biological function of PC cells were studied by CCK-8,plate cloning,Ed U,cell scratch,Transwell,glucose consumption,lactate production and ECAR assays.The expression levels of E-cadherin,Vimentin,GLUT1,PKM2and LDHA were detected by Western blot,immunofluorescence(IF)and q RT-PCR.Subcutaneous tumor formation in nude mice was used to observe the effect of knockdown of NR2F1-AS1 on the growth of transplanted tumors in vivo.The expression levels of PCNA and Ki67 in the transplanted tumor was detected by immunohistochemical(IHC)assay.3.The potential target genes of NR2F1-AS1 were predicted by bioinformatics,and analyzed the genomic position correlation of the target genes.The expression level of target genes in PC and its correlation with NR2F1-AS1 expression were analyzed by GEO and TCGA database,and NR2F1 might be the key target gene of NR2F1-AS1.To analyze whether there is a regulatory relationship between NR2F1-AS1 and NR2F1,q RT-PCR was used to detect the expression level of NR2F1 in PC tissues.The expression level of NR2F1 in PC cell lines was detected by q RT-PCR and Western blot.Pearson correlation coefficient was used to analyze the correlation between NR2F1-AS1 and NR2F1 expression in PC.The effect of NR2F1-AS1knockdown on NR2F1 expression in PC cells was analyzed by q RT-PCR,Western blot and IF.To further explore whether NR2F1-AS1 regulates the biological functions of pancreatic cancer through NR2F1,two si NR2F1s were designed and synthesized and transfected into MIA Pa Ca-2 and PANC-1 cells.CCK-8,plate cloning and Ed U assays were used to detect the proliferation ability of PC cells,while cell scratch and Transwell assays were used to detect the migration and invasion ability of PC cells.The downstream signaling pathways of NR2F1-AS1 and NR2F1 were analyzed by KEGG enrichment.The expression levels of key molecules in the signaling pathway and downstream targets were detected by Western blot.Moreover,functional rescue experiments were performed to verify the relationship between NR2F1-AS1,NR2F1and the signaling pathway.CCK-8,plate cloning,Ed U and Transwell experiments were used to detect the changes in PC cell proliferation and migration and invasion ability.Western blot was used to detect the expression levels of NR2F1 and key molecules in the signaling pathway and downstream targets.4.To further explore the reasons for the abnormal expression of NR2F1-AS1 in PC,Gene Ontology(GO)was used to analyze the biological processes that NR2F1-AS1might be involved in.The correlation between NR2F1-AS1 and HIF-1?expression in PC was analyzed by TCGA database.Use the JASPAR website to predict whether there are putative hypoxia response elements(HREs)upstream of the NR2F1-AS1promoter.Simulate the hypoxic microenvironment of the tumor,and explore the changes in the expression of NR2F1-AS1 and HIF-1?under hypoxia(5%CO₂,1%O₂,94%N₂)or HIF-1?stabilizer Co Cl₂.q RT-PCR and FISH assay were used to detect the effect of HIF-1?knockdown on NR2F1-AS1 expression,and luciferase reporter assay was used to investigate whether HIF-1?transcriptional regulation of NR2F1-AS1 expression.To investigate whether NR2F1-AS1/NR2F1 pathway is involved in hypoxia-induced malignant phenotype of PC cells by knocking down or overexpressing NR2F1-AS1 and interfering HIF-1?.The expressions of NR2F1,E-cadherin and vimentin were detected by Western blot and IF.The changes of migration and invasion ability of PC cells were detected by cell scratch assay and Transwell assay.Results:1.The expression profile results of GEO database(GSE15471,GSE16515,GSE58561,GSE91035)were analyzed to identify the significantly overexpressed lncRNA NR2F1-AS1 in PC tissues.TCGA database results further showed that NR2F1-AS1 was up-regulated in pancreatic cancer tissues.Kaplan-Meier survival curve analysis showed that NR2F1-AS1 was correlated with overall survival(p=0.029)and disease-free survival(p=0.045)of pancreatic cancer patients,suggesting that high expression of NR2F1-AS1 was significantly positively correlated with poor prognosis of pancreatic cancer patients.The expression of NR2F1-AS1 in metastatic tumor tissues from PC were significantly higher than that primary tumor.Furthermore,both q RT-PCR and tissue microarray ISH results showed that NR2F1-AS1 was upregulated in PC tissues compared with adjacent pancreatic tissues.Compared with HPDE,the expression of NR2F1-AS1 was significantly increased in PC cell lines.FISH and cytoplasmic separation assay showed that NR2F1-AS1 was mainly localized in the cytoplasm of MIA Pa Ca-2 and PANC-1 cells.2.To investigate the effect of NR2F1-AS1 on the biological function of PC cells,q RT-PCR results showed that lentiviral expression vector targeting NR2F1-AS1 was successfully constructed.CCK-8 and plate cloning experiments showed that NR2F1-AS1 knockdown inhibited the proliferation of PC cells.The results were further verified by Ed U assay.Cell scratch assay and Transwell migration assays showed that NR2F1-AS1 knockdown inhibited the migration ability of PC cells.Transwell invasion assay showed that NR2F1-AS1 knockdown could significantly inhibit the invasion ability of PC cells.After knockdown of NR2F1-AS1 in PANC-1and MIA Pa Ca-2 cells,Western blot and IF assays revealed that the expression of E-cadherin was up-regulated and Vimentin was down-regulated.Moreover,NR2F1-AS1 knockdown significantly inhibited the glucose consumption and lactate production of PC cells,and effectively inhibited the extracellular acidification rate(ECAR)of PC cells.Overexpression of NR2F1-AS1 promoted glucose consumption,lactate production and ECAR in PC cells.q RT-PCR and Western blot results demonstrated that down-regulation of NR2F1-AS1 inhibited the m RNA and protein expressions of GLUT1,PKM2 and LDHA,while overexpression of NR2F1-AS1showed the opposite effect.Subcutaneous tumor-forming experiments in nude mice showed that NR2F1-AS1 knockdown significantly inhibited tumor growth compared with negative control group.3.Bioinformatics analysis showed that NR2F1 may be a potential target gene of NR2F1-AS1.Ensembl and UCSC website analysis revealed that NR2F1-AS1 is located in the antisense chain of NR2F1 coding genes.The results of GEO databases(GSE15471,GES91035)and TCGA database showed that NR2F1 was highly expressed in PC tissues.Moreover,q RT-PCR results further confirmed the high expression of NR2F1 in PC tissues.The results of q RT-PCR and Western blot showed that NR2F1 was elevated in PC cell lines.Pearson correlation coefficient analysis showed that NR2F1 and NR2F1-AS1 were positively correlated in TCGA database and GSE15471 and GES91035 expression profiles.Pearson correlation coefficient further analyzed NR2F1-AS1 and NR2F1 in PC and paracancerous tissues detected by q RT-PCR,and the results showed that the expressions of the two were positively correlated.These results suggested that NR2F1-AS1 may interact with NR2F1 to regulate the biological function of PC cells.In order to explore the interaction relationship between NR2F1-AS1 and NR2F1,knockdown of NR2F1-AS1 reduced the expression of NR2F1 m RNA and protein levels,and the above results were further confirmed by IF assay.In addition,CCK-8,plate cloning and Ed U experiments showed that down-regulation of NR2F1 could significantly inhibit the proliferation of PC cells.Cell scratch and Transwell experiments showed that knockdown of NR2F1could significantly inhibit the migration and invasion ability of PC cells.KEGG analysis showed that NR2F1-AS1 and NR2F1 were significantly correlated with PI3K/AKT signaling pathway.TCGA database analysis showed that NR2F1-AS1 and NR2F1 were positively correlated with AKT.Western blot results showed that down-regulation of NR2F1-AS1 significantly reduced the protein expressions of p-AKT,p-m TOR,p-p70 S6K and HIF-1?in PC cells.Overexpression of NR2F1-AS1performed in the opposite result.The results of functional recovery assays showed that down-regulation of NR2F1 or inhibition of AKT activation could significantly inhibit the proliferation,migration and invasion ability of PC cells mediated by NR2F-AS1.4.GO analysis showed that NR2F1-AS1 was involved in biological processes such as hypoxia and glycolysis.TCGA analysis showed a positive correlation between NR2F1-AS1 and HIF-1?expression in PC.Genomic analysis revealed two potential HREs in the promoter region of NR2F1-AS1 gene.After PC cells were treated with hypoxia or Co Cl₂,the expression of NR2F1-AS1 was up-regulated and the protein expression of HIF-1?was increased.PC cells cultured in normoxic or hypoxic conditions showed that HIF-1?knockdown significantly reduced NR2F1-AS1expression.FISH analysis further confirmed that hypoxia-induced NR2F1-AS1expression was inhibited by HIF-1?knockdown.To further verify the transcriptional activation of HIF-1?on the NR2F1-AS1 promoter,luciferase reporter assay results showed that hypoxia enhanced the activity of NR2F1-AS1 promoter,and knockdown of HIF-1?significantly inhibited luciferase activity in PC cells.In addition,Western blot or IF results showed that hypoxia promoted the up-regulation of NR2F1 and Vimentin protein expressions and down-regulation of E-cadherin in PC cells.NR2F1-AS1 knockdown significantly reversed hypoxia-promoted NR2F1 Vimentin and E-cadherin expression.In addition,hypoxia-induced migration and invasion were inhibited by NR2F1-AS1 knockdown.Knockdown of HIF-1?could significantly inhibit the malignant phenotype of PANC-1 and MIA Pa Ca-2 cells caused by hypoxia,but overexpression of NR2F1-AS1 could rescue the malignant phenotype of PC cells caused by knockdown of HIF-1?.These results suggested the role of NR2F1-AS1/NR2F1 pathway in hypoxia-induced malignant progression of PC cells.Conclusion:1.Compared with adjacent tissues,the expression level of NR2F1-AS1 was increased in pancreatic cancer tissues,and the high expression of NR2F1-AS1 was associated with poor prognosis of pancreatic cancer,suggesting that NR2F1-AS1 is one of the oncogenes of pancreatic cancer.NR2F1-AS1 was mainly localized in the cytoplasm of pancreatic cancer cells.2.NR2F1-AS1 could promote the proliferation,migration,invasion and glycolysis of pancreatic cancer cells.Knockdown of NR2F1-AS1 could inhibit the growth of transplanted tumors in vivo.3.NR2F1-AS1 interacted with NR2F1 and promoted the biological functions of pancreatic cancer cells by regulating the AKT/m TOR signaling pathway.4.Hypoxia was one of the reasons for the increased expression of NR2F1-AS1 in pancreatic cancer,and HIF-1?could transcriptionally activate the expression of NR2F1-AS1.NR2F1-AS1 was involved in the regulation of hypoxia-induced invasion and metastasis of pancreatic cancer cells.