Screening And Identification Of Circular RNA Molecules In Pancreatic Cancer Based On High-Throughput Sequencing

Objective:Pancreatic cancer,one of the malignancies in digestive system,is characterized by concealed onset,difficulty of early diagnosis,aggressive progression and extremely poor clinical prognosis.Pharmaceutical treatment with gemcitabine and other drugs and surgery have become major therapies for pancreatic cancer for more than a decade.In recent years,the morbidity of pancreatic cancer has presented a significant rising tendency;there are about 200,000 new cases every year,but treatment of pancreatic cancer fails to gain breakthrough progress and clinical options,neoadjuvant therapy or adjuvant chemotherapy,are extremely limited.Regarding the key for diagnosis and treatment of pancreatic cancer,early discovery,precise diagnosis,risk evaluation of relevant molecular markers in pancreatic cancer patients or suspected patients,early and timely intervention for deterioration prevention are required for active function on diagnosis and treatment of pancreatic cancer.Even though an open surgery for excision of pancreatic cancer and clinical pathological diagnosis are golden standards for diagnosis of pancreatic cancer,it is undoubtedly difficult to conduct surgical excision of pancreatic cancer tissue for clear diagnosis and prognosis judgment in suspected pancreatic cancer patients,early-stage pancreatic cancer patients or pancreatic cancer patients incompatible with tumor incision.Ultrasonic endoscopy with a high precision in evaluation of a pancreatic space-occupying lesion is very sensitive to a lesion with a diameter of below 2 cm,and detects even a lesion smaller than 5 mm for its diameter.Besides,endoscopic ultrasonography guided fine-needle aspiration（EUS-FNA）,with high diagnosis sensitivity,has obtained significant effect in diagnosis of pancreatic tumor and prediction of its prognosis.A pathological（liquid-based cytological or histological）examination of punctured tissue sample is significant for differential diagnosis of benign and malignant tumor before an operation,and is valuable for prediction of prognosis after an operation or in late-stage patients.According to the Clinical Guideline of Endoscopic Ultrasonography Guided Fine Needle Aspiration in China（released in 2017）,tissue extracted via EUS-FNA can be used as one of the first choices for pathological diagnosis of pancreatic tumor.Since molecular biological technique is highly sensitive to molecular organism,application of molecular biological technique for detection of tumor aspiration with non-typical cytological manifestation or suspected malignant tumor aspiration avoids multiple puncture examinations in some patients,prevents surgical harms in patients with benign space-occupying lesion,reduces occurrence of postoperative complication,realizes the effect of early pancreatic cancer diagnosis and prognosis improvement,and lowers mortality.Besides,the technique allows targeted detection of genetic and relevant molecular changes in late-stage patients for timely adjustment of therapeutic schedule.EUS-FNA technique provides a first-rate detection tool for early diagnosis of pancreatic cancer and prediction of prognosis,since it can be used to obtain pancreatic tissue cells and interstitial fluid sample directly and improves diagnostic sensitivity and specificity.Therefore,some new and sensitive molecular markers shall be discovered and integrated with EUS-FNA technique for early diagnosis of pancreatic cancer.In recent years,circular RNA has been reported in some researches to possibly provide a new molecular marker for tumor diagnosis.In this paper,whole transcriptome high-throughput sequencings of pancreatic cancer and para-pancreatic cancer tissues were used to screen circRNA expression profile;and further analysis based on interaction network was conducted to find differential circRNA,differential mi RNA and differential mRNA.Additional pancreatic cancer and para-cancer tissue specimens were collected to expand the sample size;real-time quantitative polymerase chain reaction（RT-qPCR）was utilized to preliminarily verify selected circRNA（s）.Based on the expression verification results,regulation relations of hsa_circ₀006215,mi R-378a-3p and SERPINA4 were determined with RT-qPCR,Western blot and other methods,providing a foundation for screening new specific diagnostic markers of pancreatic cancer.Method:Research subjects were divided into two groups,the experimental group（including 4 pancreatic cancer tissue specimens respectively labeled as CA6,CA7,CA8 and CA9）and the control group（including 4 para-pancreatic cancer tissue specimens respectively labeled as NT6,NT7,NT8 and NT9）.Whole transcriptome high-throughput sequencing was used to screen circRNA expression profile,while interaction network was further analyzed to find out differential circRNA,differential mi RNA and differential mRNA.Functional explanation analysis and metabolic pathway analysis of circ RNA were conducted to know circ RNA functions.Based on analyses of differential magnification,statistical significance,relevant encoding gene function,protensity feature,specimen original density and other strategies as well as agreement rate between a candidate gene and pancreatic cancer biological characteristics,the interaction network between circ RNA and circ RNA-miRNA-mRNA was screened out with a top specificity and research values.The sample size was expanded to 30 pancreatic cancer and 30 para-cancer tissue specimens.Trizol one-step method was applied to extract total RNAs and detect the OD values（absorbancy A260/A280 value）in pancreatic cancer and para-cancer tissues respectively.The total RNAs were inversely transcribed into cDNA.A prime was designed and synthesized according to the selected candidate circRNA sequence;an appropriate internal reference sequence was selected;RT-qPCR was used for amplification;and relevant quantitative analysis and statistical analysis were conducted in the amplification products.Based on RT-q PCR results and comparing with bioinformatics analysis results,false positive cases were excluded;expression of the candidate hsa_circ₀006215 was detected;RT-qPCR was used to detect miR-378a-3p expressions in the tissues;and Western blot was applied to detect expression of the target gene protein SERPINA4.A total of 100 blood specimens were collected from normal subjects（n=50）and pancreatic cancer patients（n=50）.And RT-qPCR was used to detect expression of hsa_circ₀006215 in the blood specimens.Culture pancreatic cancer cell line PANC-1,which was divided into three groups—the overexpression group,the silent group and the normal group.RT-qPCR was then utilized to detect hsa_circ₀006215 expression in pancreatic cancer cell line PANC-1;RT-qPCT,Western blot and other methods were applied to detect miR-378a-3p and SERPINA4 expressions in cell groups after upregulation and downregulation of hsa_circ₀006215.CCK-8 was adopted to detect cell proliferation capacity,flow cytometry（FCM）for cell apoptosis rate and Transwell experiment for in vitro invasion ability of cells.Result:High-throughput sequencing was utilized for comprehensive and in-depth sequencing analysis of pancreatic cancer tissues and para-pancreatic cancer tissues;based on the Illumina technological sequencing platform,the valid sequencing depth of every specimen was not less than 200 X.For the sequencing data,the software Target-human-GJT was utilized used for bioinformatics analysis and screening of circ RNA expression profile,and the hsa_circ₀006215-miR-378a-3p-SERPINA4 interaction network with a relatively high specificity was screened out.It is suggested by RT-qPCR verification results that expression of hsa_circ₀006215 is upregulated in pancreatic cancer tissues than in para-cancer tissues;miR-378a-3p expression is downregulated in pancreatic cancer tissues when comparing with para-cancer tissues;upregulation of SERPINA4 expression exists in pancreatic cancer tissues rather than in para-cancer tissues;and,comparing with normal subject,hsa_circ₀006215 expression in blood is upregulated in pancreatic cancer patients,suggesting statistically significant differences（P<0.05）.It is suggested by CCK-8 results that,comparing with the normal group,the cell proliferation capacity is enhanced in the overexpression group and reduced in the silent group.According to Transwell results,the invasion activity is higher in the overexpression group and lower in the silent group than in the normal group.As indicated by FCM results,comparing with the normal group,the overexpression group is weaker while the silent is better in cell apoptosis ability.It is indicated by RT-q PCR,Western blot and other method-based examination results that hsa_circ₀006215 and SERPINA4 are upregulated in the overexpression group but downregulated in the silent group than in the normal group;the miR-378a-3p expression,comparing with the normal group,is downregulated in the overexpression group and upregulated in the silent group;and all the differences are statistically significant（P<0.05）.Conclusion:The latest high-throughput sequencing is used in the paper to detect pancreatic cancer samples,to screen out differential circRNA expression profile in pancreatic cancer,and to find out impacts of circRNA-mi RNA-mRNA interaction relations on pancreatic cancer.With hsa_circ₀006215 as the candidate circRNA,histological verification experiments and in vitro cell examinations are used to verify abnormal expressions in pancreatic cancer.The role of the candidate hsa_circ₀006215-miR-378a-3p-SERPINA4 interaction network on genesis and progression of pancreatic cancer provides a new theoretical perspective for genesis mechanism of pancreatic cancer,and will lay a favorable theoretical foundation for diagnosis and treatment of pancreatic cancer.