Hsa＿circ＿0001666 Promotes EMT In Pancreatic Ductal Adenocarcinoma Through MiR-1251/SOX4 Axis

Background and objectivePancreatic cancer is one of the most lethal malignant tumors.The overall median survival time is 8.6 months,and the 5-year survival rate is 9.7%[1].In recent years,the incidence of pancreatic cancer in China shows an increasing trend year by year and has become the top 10 tumor in mortality.The most ordinary type of pancreatic cancer is pancreatic ductal adenocarcinoma（PDAC）,accounting for approximately 90%of pancreatic exocrine tumors.The absence of typical clinical symptoms in the early stage of PD AC makes it unmanageable to achieve inchoate diagnosis.PD AC is highly prone to invade surrounding tissue organs through nerves,blood vessels,and rapid distant metastasis through lymphatic vessels.Due to difficulties in early diagnosis,the diagnosis of pancreatic cancer is mostly established after the occurrence of metastasis in clinic,and only 10-20%of patients have the possibility of surgical resection at the time of diagnosis.Even with resection operation,about 90%of patients experience recurrence within 12-18 months after surgery[2].Therefore,the search for new biological indicators of pancreatic carcinogenesis and development,early diagnosis and prognosis,and revealing the regulatory network of pancreatic carcinogenesis and development can lead to the search for new therapeutic targets for pancreatic cancer prevention and treatment,and also provide the basis for early diagnosis and prognosis evaluation of pancreatic cancer,which is of great clinical significance.Circular RNAs,a type of circular non coding RNA molecules commonly found in mammals,are capable of transcriptional or post transcriptional level regulation of the expression of genes.Studies have shown that circular RNAs can promote or inhibit the proliferation,apoptosis,migration and invasion of malignant cancer cells and participate in biological behaviors such as tumor growth,invasion and metastasis by regulating the expression levels of downstream genes through direct or indirect effects.In recent years,a large number of studies have shown that circular RNAs with miRNA molecules can act through the mechanism of sponge adsorption to produce inhibition of the functional activity of miRNAs,thereby regulating miRNAs at the transcriptional level.However,most of the current studies on the functional roles played by circular RNAs in pancreatic cancer progression and the mechanisms still remain at the detection level of gene expression,and only a few of the more thorough studies are related to the function and mechanism of circular RNAs in pancreatic cancer progression.In this study we used bioinformatics analysis combined with classical molecular biology techniques etc.,aiming to explore the effects and mechanisms of aberrantly expressed circular RNAs affecting the biological behavior of pancreatic cancer at the levels of molecular cellular,experimental animals and clinical samples.It is expected that we unearth potential markers for pancreatic cancer and provide new targets and theoretical data for targeted therapy of pancreatic cancer.Method1.Through the microarray data of pancreatic ductal adenocarcinoma（PDAC）downloaded by gene expression omnibus,the differential transcriptional profiles of 26 pairs of PD AC and adjacent normal tissues were analyzed.High throughput sequencing was used to detect the expression level of circRNAs in the cancer and adjacent tissues of 3 patients with pancreatic cancer,and then the results of circRNAs expression profile of pancreatic cancer reported by other scholars were combined to analyze the differential circRNAs to determine the research object.The research group took the cancer tissues and adjacent tissues of 50 patients with pancreatic cancer as experimental samples,and used qPCR to detect the screened circular RNA in pancreatic cancer tissues and cells,and found the relationship between the expression of hsa_circ₀001666 and the clinicopathological characteristics of patients with pancreatic cancer.2.after overexpression of hsa_circ₀001666 with plasmid in pancreatic cancer cell lines AsPC-1 and PaCa-2,CCK-8 assay was used to detect the change of proliferation level of pancreatic cancer cells after overexpression or knockdown of hsa_circ₀001666,annexin-v-pi flow cytometry was used to detect the effect of hsa_circ₀001666 on apoptosis level of pancreatic cancer cells,cell scratch assay was used to detect the effect of hsa_circ₀001666 on migration of pancreatic cancer cells,and transwell assay was used to detect the effect of hsa_circ₀001666 on invasion of pancreatic cancer cells.3.Multiple bioinformatics software such as Circinteractome,Circbank and Miranda were used to predict the miRNA that may be associated with hsa_circ₀001666.The miRNA that is predicted to be regulated by a was further analyzed and verified by double luciferase reporting experiment and rip experiment.The expression of mir-1251 was detected by qPCR,and the relationship between the change of mir-1251 expression and the clinicopathological characteristics of patients with pancreatic cancer at different stages was analyzed.The overexpression plasmid of circ₀001666 and mir-1251 mimics were transfected into pancreatic cancer cell line AsPC-1,and the interfering RNA of circ₀001666 and mir-1251 inhibiter were transfected into pancreatic cancer cell line PaCa-2.CCK-8,clone formation test,annexin-v-pi flow cytometry,scratch test and transwell test were used to study the interaction between them and the change of pancreatic cancer cell function.4.Multiple bioinformatics prediction software such as Miranda,Pictar and Targetscan were used to analyze and predict the possible target genes of mir-1251.Double Luciferase Report experiment and multiple cell function related experiments were used to further study and analyze the targeting effect of mir-1251 on SOX4,and the reversal experiment was carried out to verify it.5.The effect of hsa_circ₀001666 on pancreatic cancer through mir-1251 in vivo was confirmed by heterotopic tumor implantation experiment in nude mice.Result1.Through the microarray data of pancreatic ductal adenocarcinoma（PDAC）downloaded by gene expression omnibus,the differential transcriptional profiles of 26 pairs of PD AC and adjacent normal tissues were analyzed,and a total of 256 differentially expressed circRNAs were obtained.Combined with high-throughput sequencing,the expression levels of circRNAs in cancer and adjacent tissues of 3 patients with pancreatic cancer were detected.According to the results of qPCR experiments in pancreatic cancer tissues and various pancreatic cancer cell lines,hsa_circ₀001666 with the most significantly up-regulated expression was selected as the research object.hsa_circ₀001666 was significantly correlated with the differentiation level of pancreatic cancer,TNM stage of pancreatic cancer patients and lymph node metastasis.2.CCK-8 experiments showed that compared with the negative control group,the proliferative capacity of hsa_circ₀001666 overexpressed pancreatic cancer cell line AsPC-1 was enhanced,and the proliferative capacity of ahsa_circ₀001666 knockdown pancreatic cancer cell line PaCa-2 was decreased;Annexin-v-pi flow cytometry showed that the apoptosis of pancreatic cancer cell line AsPC-1 with overexpression of hsa_circ₀001666 decreased,and that of pancreatic cancer cell line PaCa-2 with hsa_circ₀001666 knockdown increased;Cell scratch test showed that the migration ability of hsa_circ₀001666 overexpressed pancreatic cancer cell line AsPC-1 was enhanced,while that of hsa_circ₀001666 knockdown pancreatic cancer cell line PaCa-2 was decreased;Transwell experiment showed that the invasive ability of hsa_circ₀001666 overexpressed pancreatic cancer cell line AsPC-1 was enhanced,and that of hsa_circ₀001666 knockdown pancreatic cancer cell line PaCa-2 was weakened.3.Bioinformatics software combined with literature analysis predicted the combined targeted miRNA of hsa_circ₀001666 It was found that hsa-mir-144-3 p,hsa-mir-1251,hsa-mir-451a,hsa-mir-148a-3p,hsa-mir-557 were correlated with the progression of pancreatic cancer.RT-qPCR test detected the expression level changes of five candidate miRNAs in pancreatic cancer cell line PaCa-2 when the expression of hsa_circ₀001666 was disturbed.The results showed that hsa_circ₀001666 had the most obvious effect on the expression changes of mir-1251.Dual luciferase assay confirmed that there was a binding site between a and mir-1251.Overexpression of a could inhibit the expression of mir-1251 in pancreatic cancer cell lines AsPC-1 and PaCa-2.Pancreatic cancer patients with low expression of mir-1251 have the characteristics of poor cell differentiation,late tumor TNM stage and high lymph node metastasis rate.The results of CCK-8 and clone formation experiments showed that,compared with the negative control group,mir-1251 simulant in pancreatic cancer cell line AsPC-1 could partially reverse the proliferation promoting effect caused by hsa_circ₀001666 overexpression,and mir-1251 inhibitor in pancreatic cancer cell line PaCa-2 could partially reverse the proliferation inhibiting effect caused by hsa_circ₀001666 knockdown;Annexin-v-pi flow cytometry showed that mir-1251 mimetic could partially reverse the inhibition of apoptosis caused by overexpression of hsa_circ₀001666 in pancreatic cancer cell line AsPC-1,and mir-1251 inhibitor could partially reverse the promotion of apoptosis caused by knockdown of hsa_circ₀001666 in pancreatic cancer cell line PaCa-2;The results of cell scratch test showed that mir-1251 simulant in pancreatic cancer cell line AsPC-1 could partially reverse the migration promotion caused by hsa_circ₀001666 overexpression,and mir-1251 inhibitor in pancreatic cancer cell line PaCa-2 could partially reverse the migration inhibition caused by hsa_circ₀001666 knockdown;The results of transwell experiment showed that mir-1251 simulant in pancreatic cancer cell line AsPC-1 could partially reverse the invasion promoting effect caused by overexpression of hsa_circ₀001666,and mir-1251 inhibitor in pancreatic cancer cell line PaCa-2 could partially reverse the invasion inhibiting effect caused by knockdown of hsa_circ₀001666.4.RT-qPCR was used to detect the expression changes of five tumor related mRNA IL22RA2、SOX4、ETV5、SLC2A4、MEGF9 predicted by bioinformatics software in pancreatic cancer cell line PaCa-2 with mir-1251 overexpression.The results showed that SOX4 was most significantly affected by mir-1251.The double luciferase reporter gene confirmed that there was a binding site between mir-1251 and SOX4.The expression level of SOX4 in pancreatic cancer was significantly higher than that in adjacent normal tissues.The expression of SOX4 and mir-1251 was negatively correlated in pancreatic cancer tissues and pancreatic cancer cell lines.CCK-8 results showed that upregulation of SOX4 could not reverse the proliferation inhibition caused by miR-1251 upregulation:annexin-v-pi flow cytometry showed that upregulation of SOX4 could not reverse the promotion of apoptosis caused by miR-1251 upregulation;Cell scratch test showed that up regulating the expression of SOX4 could reverse the role of miR-1251 in refraining invasion;Transwell experiment showed that up regulating the expression of SOX4 could reverse the role of miR-1251 in refraining invasion.5.In the tumorigenesis experiment in nude mice,the volume and weight of xenograft tumor in PaCa-2 group with silenced expression of hsa_circ₀001666 were smaller than those in the control group,and the reduction of xenograft tumor volume and weight was reversed by inhibiting the expression of mir-1251.HE staining showed that the number of tumor microvessels decreased when a was low expressed and mir-1251 was overexpressed.Immunohistochemical results showed that Ki67 staining decreased when hsa_circ₀001666 was silent,and Western blotting showed hsa_circ₀001666 expression silencing decreased the expression of EZH2,SOX4 and vimentin,increased the expression of E-cadherin,inhibited the expression of mir-1251,and partially reversed the expression changes caused by hsa_circ₀001666 silencing.ConclusionHsa_circ₀001666 is highly expressed in pancreatic cancer tissues and cells,which can significantly promote the proliferation,migration,invasion and EMT function of pancreatic cancer cells,and inhibit apoptosis.Hsa_circ₀001666 can combine with mir-1251 to play the role of competing endogenous RNA.SOX4 is significantly up-regulated in pancreatic cancer tissues and can be targeted combined with mir-1251.This study found for the first time that hsa_circ₀001666 indirectly regulates SOX4 through competitive binding with mir-1251,which has an impact on a variety of biological behaviors of pancreatic cancer cells,and can provide new enlightenment for the study of the mechanism of occurrence and development of pancreatic cancer.