Study On The Preliminary Mechanism Of CircRNA-1565,A Novel Circrna Identified In Prostate Cancer By RNAseq

Background: Prostate cancer is one of the most common malignant tumors in the male reproductive system,with a global incidence of the 3rd highest incidence of cancer among men and the 6th highest fatality rate.In recent years,the incidence of prostate cancer patients in China has gradually increased,of which in urban areas,the incidence of prostate cancer in men's common tumors accounted for the 5th place and in the urinary system tumor in the 1th place.This poses an extremely serious threat to men's healthy lives and also puts enormous pressure on society.Prostate cancer in China is characterized by late tumor staging and more metastasis in the distance.However,it is still not possible to fully elucidate the molecular mechanism of prostate cancer development.In recent years,with the development of science and Technology,RNA sequencing(RNA-Seq)technology has become increasingly mature,and scientists have found a new type of endogenous non-encoded RNA: Annular RNAs(circular Rna,circrna).It is widely found in the biological world,but there is no in-depth study.The results show that circRNA has the characteristics of stability,spatiotemporal specificity of tissue cells and development,and conservatism.Can participate in the complex mutual regulation network of RNA-RNA,which plays an important role in the post-transcription regulation of genes.This research mainly focuses on prostate cancer related circRNA,the purpose of the study is to explore the biological and molecular mechanisms of circRNA in the development and metastasis of prostate cancer,to find new serological markers that can predict the metastasis of prostate cancer,and to find molecular targets that can treat metastatic prostate cancer,Provide an important theoretical basis for its further precision treatment.Methods: Cultured normal prostate cell RWPE-1,prostate primary focal cell 22RV1,prostate bone metastasis cell PC3.The RNA that collects RWPE-1,22RV1,PC3 cells is sent to biotech companies for sequencing.After mass control of sequencing results,the screening of differential circRNA and the determination of circRNA of purpose were carried out.The circRNA-1565 selected from it were identified,and the intracellular localization was performed.Then,the circRNA-1565 interference and the over-expression vector are constructed,and the efficiency is detected.The apoptosis of cells was detected by flow cytometry,and the effects of circRNA-1565 on cell proliferation were studied by means of CCK8 and plate cloning formation experiments.The effect of circRNA-1565 on cell cycle was detected by flow cytometry.The effects of circRNA-1565 on cell migration and invasion were studied by cell scratches,Transwell,angiogenesis and other experiments.Predict and analyze the possible target miRNA of circRNA-1565.Through RNA Immune co-precipitation experiment and Luciferase experiment,miR-21 and miR-153 were detected as the target genes of circRNA-1565.Then,the cell Rescue experiment and Transwell Rescue experiment were used to detect whether the circRNA-1565 mediated cell phenotype could be miR-21 or miR-153 recovered.Finally,the target gene PI3 k of miR-153 was analyzed and screened.Results: First,normal prostate cells RWPE-1,prostate primary focus cells 22RV1,prostate bone metastasis cell PC3 circRNA expression spectrum is different.Through high-throughput sequencing,we screened a very low expression in normal prostate cell RWPE-1,which was expressed in the 22RV1 of prostate primary focal cells and expressed in the circRNA: circRNA-1565 of prostate bone metastasis cells PC3.It has been identified that circRNA-1565 is a new circRNA with a complete ring structure.CircRNA-1565 is mainly located in the cytoplasm of cells.Although,circRNA-1565 does not affect the proliferation and apoptosis of prostate cancer cells and cell cycle.But circRNA-1565 can promote the invasion and migration of prostate cancer cells.Subsequently,our study concluded that circRNA-1565 could be competitive with miR-21 and miR-153.MiR-153 is involved in the migration and invasion of prostate cancer cells controlled by circRNA-1565.Finally,we find that circRNA-1565 regulates PI3 k expression through competitive combination of miR-153.Conclusion: In this experiment,a new circRNA: circRNA-1565 with function was screened by circRNA high-throughput sequencing.By quantitative detection of peripheral plasma in patients with prostate cancer,it was found that it was related to the metastasis of prostate cancer,and the cell function experiment found that circRNA-1565 could promote the migration and invasion of prostate cancer cells.Further mechanism studies have found that circRNA can play a biological role by competing with miR-153 and silencing downstream target protein PI3 k.Through this study,in order to find a new plasma target for prostate cancer,to find a potential molecular target for the treatment of metastatic prostate cancer provides a new important theoretical basis...