Effect Of Hsa＿circ＿0074298 In Regulating Pancreatic Cancer Progesssion And Gemcitabine Resistance

Pancreatic cancer（PC）is now widely reckoned as an important cause of malignant tumorrelated death.Due to its rapid progression and early metastasis,only about 15%of the diagnosed PC patients have the opportunity to receive radical tumor resection.Moreover,there is no systematic and effective treatment for postoperative recurrence of PC,and the 5-year survival rate is still less than 10%.circRNA,as an important member of non-coding RNA（ncRNA）,has been proved to play an important regulatory role in a variety of malignant tumors by the competitive endogenous RNA（ceRNA）mechanisms,and is involved in malignant tumorigenesis and progression.We plan to explore the effect of circRNAs on the tumorigenesis,progression and the chemo-resistance of PC.Part Ⅰ Expression and clinical outcomes of hsa_circ₀074298 in human pancreatic tissuesOBJECTIVE:This study aimed to explore the expression of these up-regulated circRNAs in PC tissues and the sublocalization in tumor cells,and to explore the relationship between the most differentially expressed circRNA and the clinicopathological characteristics of PC patients.METHODS:The expressions of hsa_circ₀037207,hsa_circ₀005109,hsa_circ₀003251,hsa_circ₀048232,hsa_circ₀074298,hsa_circ₀089762 were detected by qRT-PCR in 30 pairs of pancreatic cancerous and adjacent normal tissues from 30 patients in Zhongda Hospital affiliated to Southeast University from January 2018 to June 2019.RNA in situ hybridization（ISH）technique was applied to attain the subcellular location of the most differentially expressed circRNA.Significance of the particular circRNA in predicting the clinical outcomes of PC was explored.The ROC curve was used to evaluate the selected circRNA as a biomarker for the diagnosis of PC.RESULT:Hsa_circ₀074298 showed the most differentially expressed tendency in PC tissues than normal tissues（Fold Change=5.23,P<0.01）.RNA-ISH confirmed that hsa_circ₀074298 existed mainly in the cytoplasms.Fisher’s precision probability test,Kendall correlation analysis and logistic multivariate analysis suggested that the expression level of hsa_circ₀074298 was significantly correlated with tumor size（P<0.001）,lymphatic metastasis（P<0.001）and pathological grades（P<0.001）.,and the area under the ROC curve was 0.676（P=0.023）.CONCLUSION:Hsa_circ₀074298 was the most up-regulated in PC tissues and was located in cytoplasms to play its biological role.Analysis of clinical data suggested that the expression of hsa_circ₀074298 was correlated with tumor size,lymphatic metastasis and pathological grades and could be considered as an auxiliary diagnostic biomarker of PC.Part Ⅱ Effect of hsa_circ₀074298 regulating the biological behavior of pancreatic cancer cell linesOBJECTIVE:To explore the pro-tumor effect of hsa_circ₀074298 on PC cell lines,and to further confirm the molecular mechanism of hsa_circ₀074298 regulating biological behavior of PC cell lines.METHODS:qRT-PCR was used to detect hsa_circ₀074298 expression in PC cell lines.The regulatory mechanism of hsa_circ₀074298 was assessed by means of bioinformatics analysis,Western Blot（WB）,qRT-PCR and dual-luciferase assay.The effects of hsa_circ₀074298 exerted on the proliferation,migration,invasion,cell cycle and apoptosis of PC cells were evaluated by flow cytometry,CCK8,colony formation assay and transwell assay.RESULTS:The expression of hsa_circ₀074298 in PC cell lines（ASPC-1,PANC-1,BXPC3,SW1990）was up-regulated compared with normal pancreatic cells（HPDE）.Downregulation of hsa_circ₀074298 resulted in limited proliferation,migration,invasion,colony formation,cell cycle arrest,and increased apoptosis rate of pancreatic cancer cells.Bioinformatic analysis and dual-luciferase assays showed that hsa_circ₀074298 served as sponge of miR-519d and that miR-519d could bind to the 3’-UTR region of the predicted target gene SMOC2.miR-519d inhibition could promote the expression of SMOC2 in vitro.SMOC2 was up-regulated in cancerous tissues and PC cell lines.Down-regulation of miR-519d or upregulation of SMOC2 could respectively reverse the anti-tumor effect caused by downregulation of hsa_circ₀074298.CONCLUSION:Hsa_circ₀074298 could promote PC progression by the miR-519d/SMOC2 axis in vitro.Part Ⅲ Mechanism of hsa_circ₀074298 regulating biological behavior of pancreatic cancer cells in vivoOBJECTIVE:To establish a subcutaneous transplantation tumor model of PC in BALB/c/nu mice,and to validate the tumorigenesis effect of hsa_circ₀074298 on PC.METHODS:Transfection of sh-circ0074298,sh-circ0074298+miR-519d inhibitor,shcirc0074298+SMOC2 overexpression plasmids and sh-NC in PANC-1 cells were performed,respectively.Subcutaneous pancreatic tumor model of male and female immunodeficient mice was established.Tumor growth and weight were thereafter recorded.The expressions of hsa_circ₀074298 and miR-519d in tumor tissues were detected by qRT-PCR,and the expression of SMOC2 was detected by WB.RESULTS:The down-regulation of hsa_circ₀074298 significantly inhibited the tumorformation ability of PANC-1 cells in vivo,while down-regulation of miR-519d or up-regulation of SMOC2 could reverse the inhibited tumor-formation ability of PANC-1 cells.Downregulation of hsa_circ₀074298 could promote miR-519d expression and inhibit SMOC2 expression in subcutaneous tumor tissues.CONCLUSION:Hsa_circ₀074298 could promote the tumor formation of PC cells in immunodeficient mice by the miR-519d/SMOC2 axis.Part Ⅳ Relationship between hsa_circ₀074298 and gemcitabine resistance in pancreatic cancerOBJECTIVE:To establish the gemcitabine-resistant PC cell lines in vitro,and to develop the molecular biological function of hsa_circ₀074298 during the process of acquiring gemcitabine resistance within PC cells.METHODS:By means of gemcitabine concentration recursion method,gemcitabine-resistant cancer cell line（PANC-1-GEM）was established.Cell proliferation was detected by CCK8 assay.The expression of hsa_circ₀074298 in parent cells and drug-resistant cells was detected by qRT-PCR.The expression of p-GP and protein product of SMOC2 was detected by WB.After transfected with sh-NC,sh-circ0074298,miR-519d inhibitor or SMOC2 overexpression plasmids,CCK8 assay was used to detect the resistance of PANC-1-GEM to gemcitabine,and flow cytometry was used to detect the apoptosis of the cells.PANC-1-GEM cells transfected the interfering plasmids were subcutaneously transplanted to immunodeficiency nude mice.All mice were then inoculated with gemcitabine or PBS regularly.Tumor weight and growth were recorded.WB was applied to detect the expression of MDR1 and SMOC2 in all subcutaneous tumors.RESULTS:PANC-1-GEM cell line was successfully established,showing higher gemcitabine resistance comparing with parental PANC-1 cells.Hsa_circ₀074298,MDR1 and SMOC2 were all up-regulated in PANC-1-GEM cells.The gemcitabine chemo-resistance of PANC-1GEM was inhibited by hsa_circ₀074298 down-regulation both in vivo and in vitro.miR-519d inhibitor or SMOC2 overexpression plasmids could reverse the gemcitabine chemo-resi stance inhibition.CONCLUSION:Hsa_circ₀074298 and SMOC2 was up-regulated in PANC-1-GEM cells.Gemcitabine chemo-resi stance of PC cells could be regulated by hsa_circ₀074298 through the miR-519d/SMOC2 axis in vitro and in vivo.