The Function And Molecular Mechanism Investigation Of Circular RNA CircPRKCI Promoting Lung Adenocarcinoma Malignant Progression

Background:The incidence and death rate of lung cancer is growing rapidly worldwide.In our country,the incidence of Lung adenocarcinoma(Lung adenocarcinoma,LAC)was higher than squamous cell carcinomas(Lung squamous cell carcinoma,LSCC),active filtering LAC specificity new molecular targets is the current hot spot in the field of Lung cancer.Circular RNAs(circRNAs)due to its unique structure,high stability,RNA enzymes resistance and other properties gradually become a hot research topic.Some studies have found that circRNAs play an important role in the process of tumor development.But its research in lung adenocarcinoma is still in its infancy.Methods:We used circRNA/mRNA co-expression microarray,comparing differentially expressed circRNAs and protein-coding genes in tumor adjacent to normal tissue,GO and KEGG pathway analyses showed that differentially expressed circRNAs and mRNAs were involved in many signaling pathways mainly related to extracellular receptor interactions,focal adhesion and cytokinesis.We further paid attention to the differentially expressed circRNAs with large fold change,small differences within groups and relatively high expression level,and obtained 5 appropriate circRNAs.qRT-PCR revealed that the expression level of the five circRNAs in 20 paired LAC tissues was consistent with the microarray.Then we identify circPRKCI in the LAC tissue samples and cell lines and looking for further verification and biological characteristics.The clinical relevance and prognostic value of circPRKCI were validated,and the potential biological function of circPRKCI was studied through in vivo and vitro experiments.RNA immune coprecipitation(RIP),biotin-based pulldown(biotin-pulldown),dual-luciferase reporter gene system was used to study circPRKCI to sponge miR-545 and mir-R89 to increase E2F7 expression.The rescue experiment confirmed that whether mir-545 and mir-589 could reverse the effects of circPRKCI.Results:We designed two sets of primers for circPRKCI.PCR results indicate the circular form was amplified using the divergent primers in cDNA but not in gDNA.PRKCI copy number amplification may Cause circPRKCI raised in LAC.CircPRKCI was resistant to RNase R treatment and mainly located in cytoplasm.The high expression of circPRKCI was positively associated with lymph node metastasis and TNM staging in LAC patients through qRT-PCR analysis and situ hybridization.Kaplan-Meier survival curves showed that patients with high expression circPRKCI were worse in prognosis.We have designed specific small interfering RNA(siRNA)and construct the expression plasmid of circPRKCI;in vitro experiment results show that the interference circPRKCI can significantly inhibit LAC cell proliferation,migration and invasion ability and leads to the G1 block.In vivo,treatment with siRNA also led to a significant reduction in tumor weight and volume in nude mice.We examined the circPRKCI sequence and found miR-545,miR-589,miR-600,miR-144 and miR-670 binding site by TargetScan and Miranda miRNA prediction programs.We observed that endogenous circPRKCI pulled-down from AGO2 normalized to IgG by qRT-PCR analysis.Biotin-pulldown experiments have established that miR-545 and miR-589 can be combined with circPRKCI,but they do not affect each other expression level.We take the intersection of predicted target genes and upregulated genes in mRNA array getting seven candidate genes.After inhibition of CircPRKCI,E2F7 was obviously downregulated among all the candidate genes.The biotin-pull-down experiment and the dual-luciferase reporter gene confirmed miR-545 and miR-589 could target E2F7.MiR-545 and miR-589 can reduce the expression of E2F7 at the mNRA and protein level.MiR-545 mimic and miR-589 mimic partly reverse the effects of CircPRKCI.Conclusion:In summary,our study revealed the circular RNA profile of LAC tissues and characterized a differentially expressed circRNA circPRKCI derived from the PRKCI gene.We demonstrated that circPRKCI is a new proliferative factor and prognostic marker in LAC.Furthermore,all our results support the hypothesis that circPRKCI acts as a miRNA sponge and positively affect the expression of the miRNA target gene E2F7.Overall,our findings suggest that circRNAs may serve as a novel class of noncoding RNAs that may have potential functions and clinical significance in LAC.