| Objective: Pancreatic cancer is a malignancy of the digestive system with insidious onset and rapid development,resulting in delayed and difficult early diagnoses and poor prognosis.The incidence and mortality of pancreatic cancer are rising every year worldwide.The key to improving the prognosis of pancreatic cancer mostly lies in early diagnosis and early treatment,which can be achieved by detection of relevant molecular markers among patients with high risk,followed by early and timely interventions.Circular RNAs(circ RNAs)are a class of noncoding RNAs with continuous,covalently closed circular structures,which have been further found to exhibit species conservation and tissue specificity.Circ RNAs are characterized by stable ring structure formed by a covalently closed continuous loop.Without free 3'and 5' ends,these molecules are not easily degraded by nucleases,which makes them ideal biomarkers for detection of disease.Past researches on pancreatic cancer are mostly confined to DNA level and gene mutation detection,meanwhile,lack of comprehensive and systematic understanding of pancreatic cancer.At present,the research of cancer-related transcriptomes has drawn great attention.The core of the research is the study of protein-encoded m RNAs and non-coding transcripts such as micro RNAs(mi RNAs),pseudogenes,long non-coding RNAs(Lnc RNA),circ RNA and other abnormal expression.Mi RNAs are a kind of non-coding single-stranded RNAs encoded by endogenous genes with about 19-25 nt in length.They play key regulatory roles in the developmental processes of embryonic development,cell differentiation,and organogenesis.Mi RNA networks and RNA regulatory networks are of great significance to understanding the occurrence and development of diseases and exploring potential drug targets.The high-throughput sequencing technology has the advantages of high sensitivity,large sequencing throughput,large data depth and coverage,etc.The analysis of mi RNA expression levels in pancreatic cancer is helpful for finding new species and detecting lower abundance transcripts,which will greatly expand the understanding and cognition of molecular expression profiles of disease differentiation.Pseudogenes,lnc RNAs and circ RNAs can play regulatory roles by competitively binding mi RNAs,also known as competitive endogenous RNAs(ce RNAs).In recent years,ce RNA research on the development of the disease has received widespread attention.The aim of this study was to investigate the expression of circ-MFN2 and circ-LDLRAD3 in pancreatic cancer tissue and the blood of patients with pancreatic cancer to find novel diagnostic biomarkers for pancreatic cancer.On this basis,we use high-throughput sequencing technology to screen mi RNA expression profile,by expanding the sample size to verify the purpose of mi RNA,then combined with bioinformatics analysis and preliminary experimental results,we screened the circ RNA-mi RNA-m RNA regulatory network in pancreatic cancer and verified the role of this regulatory network in the development of pancreatic cancer and its underlying mechanisms.Methods: In this study,we first selected 30 cases of pancreatic cancer tissues and their adjacent tissues,31 cases of pancreatic cancer patients blood and 31 healthy controls blood.Circ-MFN2 and circ-LDLRAD3 expression level were detected by q RT-PCR.The relationship between clinicopathological factors of patient samples and expression of circ-MFN2 and circ-LDLRAD3 in pancreatic cancer was analyzed.The diagnostic value of circ-MFN2 and circ-LDLRAD3 was verified by ROC curve analysis.The mi RNA expression profile of pancreatic cancer was screened by high-throughput sequencing.After extended sample size,mi R-133a-3p was found to be low expressed in pancreatic cancer.Combined with bioinformatics analysis,circ-MFN2-mi R-133a-3p-SLC39A4 regulatory network was obtained.In three representative pancreatic cancer cell lines Bx PC-3,PANC-1,and SW1990,we first identified the location of circ-MFN2 by cell nucleus and cytoplasm separation.After overexpression and knockdown of circ-MFN2,overexpression and knockdown of mi R-133a-3p,co-transfected with circ-MFN2 and mi R-133a-3p,CCK-8 was used to observe their effects on the proliferation of cancer cells.Tranwell test was used to detect the influence of cell invasion.Cell scratch test was used to observe the migration of cancer cells.Flow cytometry was used to detect cancer cell apoptosis and cell cycle,and Western Blot was then usd to observe the expression of apoptosis and invasion related proteins such as MMP9,MMP2,Timp-1,and Cleaved Caspase-3.The dual luciferase reporter assay was used to demonstrate the binding of circ-MFN2,mi R-133a-3p and SLC39A4,and then overexpression and knockdown of SLC39A4 was performed to observe the effect on downstream MAPK/ERK pathway and Smad2/3/Samd4 pathway.Results: 1.The levels of circ-MFN2 and circ-LDLRAD3 in the pancreatic cancer,pancreatic cancer and pancreatic cancer patients were significantly higher than those in the control group(P <0.01)2.The relationship between circ-MFN2 and clinicopathological parameters found that the expression of circ-MFN2 was related to tumor size and T stage in clinical stage,while the expression of circ-LDLRAD3 was correlated with invasion factors.(P <0.01).3.The diagnostic efficiency of using circ-MFN2 as a diagnostic marker independently was 0.7377(0.6093-0.8420)and 0.6066(0.4731-0.7293)for the area under the ROC curve with an AUC of 0.73,a cutoff of 10.9,and a cutoff of 10.9.The diagnostic efficiency of the combination of CA19-9 and circ-MFN2 found that after the combination of the two,the AUC increased to 0.88 and the diagnostic sensitivity and specificity increased to 0.9766 and 0.9836,respectively.The diagnostic efficiency of circ-LDLRAD3 alone as a diagnostic marker was 0.6749 for the area under the ROC curve,9.315 for the cutoff value,0.5738 for the 0.4406-0.6996 range,and 0.7049 for the specificity(0.5743-0.8148).The combined diagnostic efficiency of CA19-9 and circ-LDLRAD3 showed that after the combination of the two,the AUC increased to 0.87 and the diagnostic sensitivity and specificity increased to 0.8033 and 0.9355,respectively.4.High-throughput sequencing results and expanded sample size showed that mi R-133a-3p was lower expressed in pancreatic cancer cells and tissues than the control group(P <0.01).Bioinformatics analysis showed that pancreatic cancer may exist circ-MFN2-mi R-133a-3p-SLC39A4 regulatory network.5.After overexpression of circ-MFN2,in Bx PC-3 and PANC-1 cell lines,the proliferation(P <0.01),invasion(P <0.01)and metastasis(P <0.05)were enhanced,the apoptotic capacity(P <0.01)was weakened,the cell cycle S(P <0.01)phase was increased,and the G0/G1 phase(P <0.01)was decreased.Contrary to the various biological behaviors of knocking down circ-MFN2.However,this effect is not evident in the SW1990 cell line.6.The overexpression of mi R-133a-3p attenuated the proliferation(P <0.01),invasion(P <0.01)and metastasis(P <0.01),increased the apoptotic capacity(P <0.01),decreased the cell cycle S phase(P <0.01)and increased G0/G1 phase(P <0.01)in Bx PC-3 and PANC-1 cells.Contrary to the various biological behaviors of knocking down circ-MFN2.However,this effect is not evident in the SW1990 cell line.7.Circ-MFN2,mi R-133a-3p and SLC39A4 can bind with each other,and the expression of mutual influence.8.After co-transfecting with circ-MFN2 and mi R-133a-3p,there was a negative feedback regulation between them,and over-expression of mi R-133a-3p reversed the biological effect of circ-MFN2.This effect was more pronounced in Bx PC-3 and PANC-1 cell lines but not in SW1990 cells.9.SLC39A4 can affect the expression of p MEK(P <0.01),p ERK(P <0.01),p Smad2 / 3(P <0.01)and Smad4(P <0.01).10.In vitro experiments,knockdown of circ-MFN2 or overexpression of mi R-133a-3p in Bx PC-3 and PANC-1 cells found that tumor growths ignificantly slowed(P <0.05);Co-transfected knockdown circ-MFN2 with mi R-133a-3p overexpression found that tumor growths was further inhibited in Bx PC-3 and PANC-1 cells(P <0.05).However,no obvious tumor growth inhibition was observed in nude mice in SW1990 cell line.Conclusion: 1.Circ-MFN2 is highly expressed in the blood of patients with pancreatic cancer,pancreatic cancer cell lines and pancreatic cancer tissues,and the expression level of circ-MFN2 is closely related to the tumor size.Combined with CA19-9,it can be used as a potential biomarker for the diagnosis of pancreatic cancer.Circ-LDLRAD3 is highly expressed in the blood of patients with pancreatic cancer,pancreatic cancer cell lines and pancreatic cancer tissues,and the expression level of circ-LDLRAD3 is closely related to the tumor size.Combined with CA19-9,it can be used as a potential biomarker for the diagnosis of pancreatic cancer.2.Mi RNA-133a-3p was found to be low expressed in pancreatic cancer by high-throughput sequencing and expanded sample size.Bioinformatics analysis showed that circ MFN2-mi RNA-133a-3pSLC39A4 interaction regulatory network exists in pancreatic cancer.3.Circ-MFN2 plays a role in carcinogenesis of pancreatic cancer.Mi R-133a-3p plays a role of tumor suppressor in pancreatic cancer.Inhibition of circ-MFN2 and increase of mi R-133a-3p can inhibit the growth and biology of pancreatic cancer SLC39A4 can affect the MAPK / ERK pathway and the Smad2 / 3 / Smad4 pathway in pancreatic cancer;the regulatory network of circ-MNF2-mi R133a-3p-SLC39A4 plays an important role in the development of pancreatic cancer and may be a potential target for the treatment of pancreatic cancer. |
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