| Objective:Pancreatic cancer（PC）is one of the leading causes of cancer-related death among all cancers worldwide.In recent years,the development of molecular targeted drugs and immunotherapy has slightly improved the 5-year overall survival rate of patients with PC,which highlights the importance of clarifying the molecular mechanism of PC and finding more effective therapeutic targets.Circular RNA,as a class of non-coding RNA molecules commonly expressed in eukaryotes,are widely involved in the biological process of PC and other tumors.Therefore,this study aims to screen differentially expressed circular RNAs in PC,understand the expression and clinical significance of circular RNA hsa＿circ＿0006117 in PC.Then,its biological function and molecular mechanism were explored to provide theoretical basis for finding more accurate and effective therapeutic targets for PC.Methods:1.The expression profiles of circular RNAs in PC were retrieved from GEO（https://www.ncbi.nlm.nih.gov/geo/）database,then the microarray datasets GSE69362（including 6 pairs of PC and para-cancerous tissues）and GSE79634（including 20 pairs of PC and para-cancerous tissues）were included and downloaded for screening potential differentially expressed circular RNAs,respectively.Venn diagram analysis was performed to overlap and focus candidate differentially expressed circular RNAs.And real-time quantitative reverse transcription PCR（RT-q PCR）was further performed to detect their relative m RNA expression in PC tissues and cell lines,and circular RNA hsa＿circ＿0006117 was identified as the focus of this study.Subsequently,PCR amplification（divergent primers）,agarose gel electrophoresis,Sanger sequencing,RNase R,and actinomycin D assay were used to determine whether circular RNA hsa＿circ＿0006117 exists in a circular form in PC and how it forms a loop.Next,the subcellular distribution of circular RNA hsa＿circ＿0006117 in PC cells was further determined by separation of cytoplasmic and nuclear fractions and Fluorescent In Situ Hybridization Kit（FISH）.And receiver operating characteristic curves（ROC）were drawn based on the relative m RNA expression of circular RNA hsa＿circ＿0006117 in PC tissues and para-cancerous tissues to evaluate its potential diagnostic value for PC.2.Here,three small interfering RNAs（si RNAs）were designed to target the particular back-splicing site of circular RNA hsa＿circ＿0006117,and RT-q PCR was used to ascertain whether transfection of these si RNAs decreased the expression of circular RNA hsa＿circ＿0006117 and its parent gene protein tyrosine phosphatase receptor type A（PTPRA）.The two si RNAs with the most significant interference efficiency were transfected into PANC-1 and MIA Pa Ca-2 cells for functional experiments in vitro,such as cell counting kit-8（CCK-8）,colony formation,wound healing,transwell migration and invasion,to explore their effects on proliferation,migration,and invasion of PC.Then,these two si RNA sequences were selected to construct short hairpin RNAs（sh RNAs）for xenograft tumor formation in nude mice,further exploring the effect of circular RNA hsa＿circ＿0006117 on the proliferation of PC in vivo.Meanwhile,IVIS Spectrum In Vivo Imaging System was used to collect fluorescence images after tumor formation in nude mice,and immunohistochemistry（IHC）was used to detect the positive expression of proliferation-related specific markers Ki67 and PCNA in tumor tissues.3.Bioinformatics databases such as CSCD,circ BANK,Target Scan,mi RDB,and mi RTar Base were used to predict the micro RNAs（mi RNAs）binding to circular RNA hsa＿circ＿0006117 and their corresponding target messenger RNAs（m RNAs）.R（4.0.2）program was applied for gene ontology（GO）and Kyoto Encyclopedia of Genes and Genomes（KEGG）enrichment analysis,and Gene Expression Profiling Interactive Analysis（GEPIA）was performed to screen target m RNAs with high expression in PC,which was closely related to prognosis and might be enriched in circular RNA hsa＿circ＿0006117 regulatory signaling pathway.Through experimental verification such as RT-q PCR and western blot,KRAS and mi R-96-5p were identified as downstream target genes that may be regulated by circular RNA hsa＿circ＿0006117.Then,the expression of KRAS and mi R-96-5P in PC tissues was detected by RT-q PCR,and the correlation between them was analyzed by Pearson’s correlation analysis.Next,circular RNA hsa＿circ＿0006117 wild type（WT）and mutant（MUT）pmi R-RB-Report TM dual-luciferase vectors,as well as KRAS 3′UTR wild type（WT）and mutant（MUT）pmi R-RB-Report TM dual-luciferase vectors were constructed to conduct dual-luciferase reporter assays to verify whether mi R-96-5p has a direct binding relationship with them.In addition,KRAS-OE plasmid or mi R-96-5p inhibitor was co-transfected into circular RNA hsa＿circ＿0006117-silenced PANC-1 and MIA Pa Ca-2 cells,and CCK-8,colony formation,wound healing,transwell migration and invasion rescue assays were performed to explore whether circular RNA hsa＿circ＿0006117 could promote the proliferation,migration,and invasion of PC by regulating the KRAS/MAPK signaling pathway and adsorption of mi R-96-5p.Results:1.Bioinformatics analysis showed that there were 115 and 41 differentially expressed circular RNAs in microarray datasets GSE69362 and GSE79634,of which81 and 15 were upregulated,respectively.Venn diagram analysis suggested that the overlapping circular RNAs（circular RNA hsa＿circ＿0006117&circular RNA hsa＿circ＿0029634）in two datasets were both elevated in PC tissues.RT-q PCR analysis indicated that circular RNA hsa＿circ＿0006117 was significantly upregulated in 20 pairs of PC tissues（t=4.661,P<0.001）.At the same time,circular RNA hsa＿circ＿0006117expression was upregulated in PANC-1,MIA Pa Ca-2,As PC-1,and Bx PC-3 cells compared with that in HPDE cells（P<0.001）.PCR amplification（divergent primers）and agarose gel electrophoresis showed that circular RNA hsa＿circ＿0006117 could only be amplified from c DNA using divergent primers.Next,Sanger sequencing of PCR amplicons validated that circular RNA hsa＿circ＿0006117 is derived from 5′of exon 8 to 3′of exon 9 of PTPRA by back-splicing,and its back-splicing site is CAGATA.After that,RNase R assay revealed that circular RNA hsa＿circ＿0006117had strong tolerance to RNase R digestion（P>0.05）,whereas the linear form of PTPRA was degraded immediately（P<0.001）.A stability assay using actinomycin D treatment also verified that circular RNA hsa＿circ＿0006117 was substantially more stable than linear PTPRA in PC cells（P<0.001）.Furthermore,separation of cytoplasmic and nuclear fractions and FISH assays suggested that the subcellular localization of circular RNA hsa＿circ＿0006117 was mainly in the cytoplasm of PANC-1 and MIA Pa Ca-2cells.And ROC curve suggested that the Area Under Curve（AUC）was 0.810,and the95%confidence interval was 0.653-0.967.2.All designed si RNAs effectively interfered with the expression of circular RNA hsa＿circ＿0006117（P<0.001）,but not that of its parent gene PTPRA（P>0.05）.Among them,circ RNA-si#1 and circ RNA-si#2 elicited the best results.The results of CCK-8and colony formation assay after transfection of circ RNA-si#1 and circ RNA-si#2 into PANC-1 and MIA Pa Ca-2 cells showed that compared with the NC group,circ RNA-si#1 and circ RNA-si#2 groups inhibited the proliferation of PANC-1 and MIA Pa Ca-2cells（P<0.05）,respectively.The results of wound healing assay,transwell migration and invasion assays showed that interfering with circular RNA hsa＿circ＿0006117inhibited the migration and invasion of PC（P<0.001）.Subsequently,circular RNA hsa＿circ＿0006117-silenced lentiviruses were successfully constructed and transfected into nude mice for xenograft tumor formation experiment.The results showed that circ RNA-sh#1 and circ RNA-sh#2 groups significantly inhibited the subcutaneous tumor formation of PC compared with the NC group（P<0.001）.And IVIS Spectrum In Vivo Imaging System showed that the fluorescence intensity of the NC group was significantly stronger than that of circ RNA-sh#1 and circ RNA-sh#2 groups.Further,IHC results indicated that the expression of Ki67 and PCNA was decreased after circular RNA hsa＿circ＿0006117 was knocked down（P<0.001）.3.Bioinformatics analysis suggested that MAPK and RAS signaling pathways were most likely to be enriched in the downstream targets of circular RNA hsa＿circ＿0006117,and KRAS,GRB2,IGF2BP2,and RAP1A were the most likely target m RNAs to be regulated.Then,KRAS was determined to be the downstream target m RNA regulated by circular RNA hsa＿circ＿0006117 in RT-q PCR and western blot assays,and the m RNA expression of circular RNA hsa＿circ＿0006117 and KRAS was positively correlated（r=0.5511,P<0.05）.The rescue assay of KRAS-OE plasmid co-transfection suggested that circular RNA hsa＿circ＿0006117 promoted the phosphorylation of mitogen-activated extracellular signal-regulated kinase 1/2（MEK1/2）and extracellular signal-regulated kinases 1/2（ERK 1/2）in a KRAS-dependent manner,and activated the KRAS/MAPK signaling pathway to facilitate the proliferation,migration,and invasion of PC.Next,through the following bioinformatics analysis,RT-q PCR,and dual-luciferase reporter assay,mi R-96-5p was identified as the mi RNA absorbed by circular RNA hsa＿circ＿0006117 and was low expressed in PC.And mi R-96-5p was negatively related to the expression of circular RNA hsa＿circ＿0006117（r=-0.7402,P<0.001）and KRAS（r=-0.5085,P<0.05）,and could directly bind to circular RNA hsa＿circ＿0006117 and KRAS 3′UTR.Furthermore,a series of rescue experiments with mi R-96-5p inhibitor co-transfection showed that circular RNA hsa＿circ＿0006117 acted as a sponge for mi R-96-5p to promote the proliferation,migration,and invasion of PC in a mi R-96-5p/KRAS-dependent manner.Conclusions:1.Circular RNA hsa＿circ＿0006117 is highly expressed in PC tissues and cell lines,and mainly exists in the cytoplasm of PC cells in a closed-loop form,which has moderate diagnostic value.2.Circular RNA hsa＿circ＿0006117 facilitates the proliferation of PC in vitro and in vivo,as well as migration and invasion of PC.3.The specific and highly expressed circular RNA hsa＿circ＿0006117 facilitates the proliferation,migration,and invasion of PC via the regulation of the mi R-96-5p/KRAS/MAPK signaling pathway and might be a promising therapeutic target for PC. |
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