The Study Of The Mechanism Of Malignant Biological Behavior Mediated By CircRNA RHOT1 In Pancreatic Cancer Cells

background:As one of the important members of non-coding RNAs,circular RNAs play a significant role in various diseases and tumors.In pancreatic cancer,the abnormal expression of circular RNAs is closely related to the formation and development of tumor.In this study,we observed that highly expressed circ RHOT1 is involved in malignant biological behaviors such as proliferation and apoptosis of pancreatic cancer cells.Although circ RNAs can also function by binding to target m RNAs or proteins,as well as protein translation at specific RNA sites such as IRES and m6 A modification,the ce RNA mechanism is still the main way of circ RNA functions.After further research of literature and website prediction,we found that circ RHOT1 has a binding site with mi R-125a-3p,suggesting that circ RHOT1 may adsorb and regulate the expression level of mi R-125a-3p through a ce RNA mechanism,while E2F3,as a key downstream target gene of mi R-125a-3p,its expression and function are regulated by mi R-125a-3p.Therefore,this study aimed to explore the main mechanism of circ RHOT1 regulating the malignant biological behaviors in pancreatic cancer cells,and to clarify the role of circ RHOT1/mi R-125a-3p/E2F3 signaling pathway in the formation and development of pancreatic cancer,to provide experimental theoretical basis for further understanding the pathogenesis of pancreatic cancer and searching for new therapeutic targets.Part I.The differential expressions of circ RHOT1,mi R-125a-3p and E2F3 in pancreatic cancers and adjacent tissuesObjective: To detect the differential expressions of circ RHOT1,mi R-125a-3p and E2F3 in pancreatic cancer and adjacent tissues,and to analyze the correlation between their expression levels and clinical characteristics of pancreatic cancer patients.Methods: RNA was extracted from 28 pairs of pancreatic cancer and adjacent nontumor tissues by TRIzol reagent,and RT-q PCR was performed to detect the expression levels of circ RHOT1 and mi R-125a-3p.The patients were divided into the circ RHOT1 high expression group and the low expression group by comparing with the related adjacent noncancerous tissues,and the correlation between circ RHOT1 expression level and clinicopathologic feature of patients was analyzed by statistical methods.The expression of E2F3 in normal pancreatic tissues and pancreatic cancer was analyzed by TCGA and GTEx databases,and the correlation between E2F3 and prognosis of pancreatic cancer was analyzed by survival analysis and univariate COX regression analysis.Results:(1)Circ RHOT1 expression was significantly up-regulated in pancreatic cancer tissues compared to the related adjacent noncancerous tissues,while the expression of mi R-125a-3p was down-regulated(P<0.01 and P<0.05).(2)The expression level of circ RHOT1 was correlated with lymphatic metastasis(P<0.05),but there was no statistical significance with tumor stage and size(P>0.05).(3)The expression of E2F3 in pancreatic cancer was significantly higher than that in normal pancreatic tissue(P < 0.01).(4)Survival time of pancreatic cancer patients was correlated with the expression of E2F3(P < 0.05).Conclusion: The expressions of circRHOT1 and E2F3 are up-regulated and mi R-125a-3p expression is reduced in pancreatic cancer.The high expression level of circ RHOT1 was correlated with lymphatic metastasis,while E2F3 is associated with survival time in patients with pancreatic cancer.Part II.The differential expressions of circ RHOT1,mi R-125a-3p and E2F3 in pancreatic cancer cell lines and their function of regulating the malignant biological behaviorObjective: To investigate the differential expression levels of circ RHOT1,mi R-125a-3p and E2F3 in pancreatic cancer cell lines and the pancreatic duct epithelial cell.And to explore their roles in regulating the malignant biological function of pancreatic cancer cells.Methods: Four pancreatic cancer cells(SW1990,PANC1,COLO357 and CF-PAC1)and one pancreatic duct epithelial cell(HPDE)were collected,and the expression levels of circ RHOT1,mi R-125a-3p and E2F3 were detected by RT-q PCR,and the protein expression of E2F3 was detected by Western Blot.Then sh RNA was enveloped by lentivirus to knock down the expression of circ RHOT1,mi R-125a-3p mimic and inhibitor were designed to adjust its expression level,and si RNA was used to knock down the expression of E2F3.Cell proliferation ability was detected by CCK8 and Ed U,apoptosis and cycle level were measured by flow cytometry,cell clonal formation ability was assessed by plate clonal formation assay,and cell invasion ability was evaluated by Transwell chamber assay.Results:(1)The expression levels of circ RHOT1 and E2F3 were significantly upregulated(P<0.01),while the mi R-125-3p expression was reduced in pancreatic cancer cell lines compared with those of HPDE(P<0.01).(2)Knocking down the circ RHOT1 expression level resulted in suppressed PANC1 cell proliferation,invasiveness and colony forming capacity.Additionally,decreased circ RHOT1 level promoted PANC-1 cell apoptosis and reduced cell cycle in S phase arrest(P<0.05).(3)Overexpression of mi R-125a-3p inhibited PANC1 cell proliferation,invasiveness and colony forming capacity.Moreover,upregulated mi R-125a-3p promoted cell apoptosis and reduced cell cycle in S phase arrest.In contrast,decreased mi R-125a-3p expression level induced PANC1 cell proliferation,invasiveness and colony forming capacity.In addition,increased cell cycle in S phase arrest(P<0.05).(4)After E2F3 knockdown,the proliferation,colony forming capacity and invasiveness of PANC1 cells are inhibited,and the apoptosis level is promoted,and the distribution ratio of cells in S phase is reduced(P<0.05).Conclusion:(1)The expression levels of circ RHOT1 and E2F3 were increased in pancreatic cancer cells,while the expression levels of mi R-125a-3p were decreased.(2)circ RHOT1,mi R-125a-3p and E2F3 can regulate the proliferation,invasion and clonal formation capacity of PANC1 cells,as well as affecting apoptosis and cell cycle.Part III.The study of the mechanism of circ RHOT1/ Mir-125A-3p/E2F3 regulating the malignant phenotype of pancreatic cancerObjective: To verify the relation among circ RHOT1,mi R-125a-3p and E2F3.Methods: Cells were transfected with lentivirus,si RNA,mi RNA mimic and inhibitor,RT-q PCR and Western Blot were performed to test the expression level of circ RHOT1,mi R-125a-3p and E2F3.Dual-luciferase assay and rescue experiment were used to verify the correlation between circ RHOT1 and mi R-125a-3p,E2F3 and mi R-125a-3p.Results:(1)After knocking down the expression level of circRHOT1,the expression of mi R-125a-3p was increased,while that of E2F3 was decreased(P<0.05).(2)When the expression of mi R-125a-3p was up-regulated,the expression levels of circ RHOT1 and E2F3 were both decreased.Then inhibited the expression of mi R-125a-3p,the expression levels of circ RHOT1 and E2F3 were both increased(P<0.05).(3)When both knockdown the expression levels of circ RHOT1 and mi R-125a-3p,the expression level of E2F3 was higher than that of circ RHOT1 knockdown alone(P<0.01).(4)Dual-luciferase assay showed that luciferase activity of circ RHOT1WT+ mi R-125a-3p mimic co-transfection group was lower than that of circ RHOT1MUT+ mi R-125a-3p mimic co-transfection group(P<0.05).And the luciferase activity of the E2F3 WT+ mi R-125a-3p mimic co-transfection group was lower than that of the E2F3 MUT+ mi R-125a-3p mimic co-transfection group(P<0.05).(5)E2F3 cooperated with CDK family,RB family,DNMT1,OSMR,CCNA2 and other proteins,and synergistically participated in regulating FOXO,p53 and other signaling pathways.Conclusion:(1)circ RHOT1 can regulate the expression levels of mi R-125a-3p and E2F3.(2)circ RHOT1 has a direct targeted binding relation with mi R-125a-3p and can regulate the expression level of each other through competing endogenous RNA mechanism.(3)mi R-125a-3p can bind to the 3’UTR region of E2F3,thus regulating the expression of m RNA and protein levels of E2F3.(4)circ RHOT1 can regulate the expression of E2F3 through mi R-125a-3p.(5)E2F3 regulates cell cycle,FOXO,p53 signaling pathways by interacting proteins and co-expressed genes in pancreatic cancer.