| Objective: Pancreatic cancer is a fatal disease of digestive system with poor prognosis.Due to its insidious symptoms and rapid development,it is difficult for patients with pancreatic cancer to obtain early diagnosis and timely treatment.Due to the gradual improvement of people’s material standard of living,the incidence and mortality of pancreatic cancer have also increased.With the rise and development of emerging technologies such as individualized molecular diagnosis and targeted therapy of malignant tumors,we have gradually realized that the key to early diagnosis and treatment of pancreatic cancer lies in the detection and risk assessment of molecular markers for patients with suspected pancreatic cancer,timely detection of pancreatic cancer lesions in the early stage,clear diagnosis and staging,monitoring of related gene and molecular changes in patients,and the development of individualized targeted therapy.Therefore,it is significant to elucidate the molecular mechanism of the occurrence and development of pancreatic cancer in order to break through the technical barrier of diagnosis and treatment of pancreatic cancer.In recent years,studies on the effect of competitive endogenous RNAs(ceRNAs),especially circular RNAs(circRNA),on disease progression have attracted extensive attention.Circ RNA is a type of non-coding RNA widely existing in eukaryotic cells with a closed circular structure formed by covalent bonds.Due to the lack of poly A tail,it is not easily degraded by exonuclease and has better stability than linear RNA.Circ RNAs can act as a "sponge" to absorb miRNA and play the role of ceRNA to remove the inhibitory effect of miRNA on target genes,thus indirectly affecting the expression of target genes.A number of studies have proved that such circRNA-miRNA-mRNA regulatory network is closely related to the occurrence and development of a variety of tumors,and can promote or inhibit various biological behaviors of tumors.With the deepening of studies on circRNA and pancreatic cancer,we have gradually realized that circRNA plays an important regulatory role in the occurrence and development of pancreatic cancer and has the potential as a tumor marker for early diagnosis and therapeutic target of pancreatic cancer.In this study,we expect to construct a circRNA-miRNA-mRNA regulatory network that may exist in pancreatic cancer based on the preliminary sequencing data and results of bioinformatics analysis,and verify its role and potential mechanism in the development of pancreatic cancer through cellular functional experiments in vitro and animal experiments in vivo.Methods: Based on the previous sequencing results of our research group,we selected a circRNA molecule with relatively high expression in pancreatic cancer tissue,hsa＿circ＿0002999 as target of the research.First,qRT-PCR was used to verify that hsa＿circ＿0002999 was overexpressed in multiple pancreatic cancer cell lines.The circular structure and circularized site of hsa＿circ＿0002999 were determined by Sanger sequencing,and its subcellular localization was determined by fluorescence in situ hybridization(FISH)assay,and it was mainly expressed in cytoplasm.The downstream binding miRNA(hsamiR-1270)and target gene(CORO2A)were predicted by bioinformatics methods and verified by qRT-PCR.A preliminary conjecture was constructed that the hsa＿circ＿0002999-hsa-miR-1270-CORO2 A regulatory network could affect the progression of pancreatic cancer.Next,we selected paraffin sections of 30 pairs of pancreatic cancer tissue and controll pancreatic tissue.Differential expression of target RNA molecules and target gene protein was verified by FISH and immunohistochemistry,respectively.The correlation between relative expression of target RNA molecules and clinicopathological data was studied to explore their potential as diagnostic markers for pancreatic cancer.Subsequently,we conducted cellular functional experiments in vitro and animal experiments in vivo to explore the roles of the three target molecules in the development of pancreatic cancer,and verified the specific binding and interaction among the three molecules,thus confirming the existence of the circRNA-miRNA-mRNA regulatory network in pancreatic cancer and the mechanism of affecting the development of pancreatic cancer.In pancreatic cancer cell lines Capan-2 and Sw 1990,hsa＿circ＿0002999,hsa-miR-1270 and CORO2 A were overexpressed and knocked down,and hsa＿circ＿0002999 and hsa-miR-1270 were also co-transfected.The effects of transfection of target molecules on the proliferation,migration,invasion and apoptosis of pancreatic cancer cells were studied by cellular functional experiments.CCK8 assay was used to detect the effect on proliferation of pancreatic cancer cells;the effect on migration of pancreatic cancer cells was observed by wound healing assay;the effect on migration and invasion of pancreatic cancer cells was observed by Transwell’s assay;the effect on apoptosis of pancreatic cancer cells was detected by flow cytometry;and the expression variation of apoptosis and invasion-related proteins,Bax,Bcl2,MMP2,MMP9 and Timp-1,were detected by Western Blot.The specific binding between hsa＿circ＿0002999 and hsa-miR-1270 and between hsa-miR-1270 and CORO2 A was demonstrated by dualluciferase reporter assay in 293 T tool cells.qRT-PCR and Western Blot were used to prove the negative-feedback-regulated expression pattern among the three.The role of hsa＿circ＿0002999-hsa-miR-1270 regulatory network on the development of pancreatic cancer was further confirmed by tumorigenesis experiments in nude mouse model.Finally,we conducted bioinformatics analysis of the mechanism of CORO2 A in pancreatic cancer,and Western Blot was used to verify the effects of overexpression and knockdown of CORO2 A on the expression of markers related to mesenchymal-epithelial transition.Results: 1.hsa＿circ＿0002999 is a real circRNA formed by back-splicing of exon 2-7 of BARD1 gene.Its subcellular localization is mainly in the cytoplasm,and its expression level in human pancreatic cancer cells is significantly higher than that in normal pancreatic cells.2.The downstream miRNA that hsa＿circ＿0002999 is most likely to specifically bind to is hsa-miR-1270,and the expression level of hsa-miR-1270 in human pancreatic cancer cells is significantly lower than that in normal pancreatic cells.3.The downstream mRNA that hsa-miR-1270 is most likely to specifically bind and target to is CORO2 A,and the expression level of CORO2 A in human pancreatic cancer cells is significantly higher than that in normal pancreatic cells.4.The expression level of hsa＿circ＿0002999 in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues,and was significantly correlated with tumor diameter,clinical stage,M stage and vascular invasion.5.The expression level of hsa-miR-1270 in pancreatic cancer tissues was significantly lower than that in normal pancreatic tissues,and was significantly correlated with tumor diameter,T stage and vascular invasion.6.The expression level of CORO2 A protein in pancreatic cancer tissues was significantly higher than that in normal pancreatic tissues,and bioinformatic analysis indicated that patients with abnormally high expression of CORO2 A had lower overall survival and disease-free survival.7.Overexpression of hsa＿circ＿0002999 in Capan-2 and Sw 1990 cells can promote cellular proliferation,migration,invasion and inhibit apoptosis,while knockdown of hsa＿circ＿0002999 has opposite effects on cellular biological behaviors.8.Overexpression of hsa-miR-1270 in Capan-2 and Sw 1990 cells can inhibit cellular proliferation,migration,invasion and promote apoptosis,while knockdown of hsa＿circ＿0002999 has opposite effects on cellular biological behaviors.9.Overexpression of CORO2 A in Capan-2 and Sw 1990 cells can promote cellular proliferation,migration,invasion and inhibit apoptosis,while knockdown of hsa＿circ＿0002999 has opposite effects on cellular biological behaviors.10.After cotransfection of Capan-2 and Sw 1990 cells with hsa＿circ＿0002999 and hsa-miR-1270,negative feedback regulation between the two was found.Overexpression of hsa-miR-1270 can reverse the promoting effect of overexpression of hsa＿circ＿0002999 on various biological functions of pancreatic cancer cells.11.Overexpression of hsa-miR-1270 can reverse the promoting effect of overexpression of hsa＿circ＿0002999 on various biological functions of pancreatic cancer cells.Specific binding of hsa＿circ＿0002999,hsa-miR-1270 and CORO2A was observed,and their expression levels were affected by each other.12.Overexpression of CORO2 A can enhance mesenchymal-epithelial transition(MET)of pancreatic cancer cells and affect the expression of MET-related marker proteins(Ecadherin,N-cadherin and Vimentin).13.In animal experiments,the growth rate and weight of tumors developing from Capan-2 cells with hsa＿circ＿0002999 knockdown or hsa-miR-1270 overexpression were significantly reduced.The inhibition of tumor growth was more significant when Capan-2 cells co-transfected by knocked-down hsa＿circ＿0002999 and overexpressed hsa-miR-1270 were used.Conclusion: 1.The expression level of hsa＿circ＿0002999 in human pancreatic cancer cells and tissues was significantly higher than that in normal pancreatic cells and control tissues,and was significantly correlated with tumor diameter,clinical stage,M stage and vascular invasion.2.The expression level of hsa-miR-1270 in human pancreatic cancer cells and tissues was significantly lower than that in normal pancreatic cells and control tissues,and was significantly correlated with tumor diameter,T stage and vascular invasion.3.The expression level of CORO2 A in human pancreatic cancer cells and tissues was significantly higher than that in normal pancreatic cells and control tissues,and bioinformatics analysis indicated that patients with abnormally high expression of CORO2 A had lower overall survival and disease-free survival.4.The hsa＿circ＿0002999-hsa-miR-1270-CORO2 A interactive regulatory network exists in the occurrence and development of pancreatic cancer,in which hsa＿circ＿0002999 and CORO2 A play a carcinogenic role in pancreatic cancer,and hsa-miR-1270 plays a tumor suppressive role in pancreatic cancer.Inhibition of hsa＿circ＿0002999 and simultaneous elevation of hsamiR-1270 can delay the development of pancreatic cancer and inhibit the malignant biological behavior of pancreatic cancer.This circRNA-miRNA-mRNA interactive network may be a potential therapeutic target for pancreatic cancer. |
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