The Function Of Circular Rna Circ NDUFB2 In The Tumorigenesis Of Lung Cancer

Background and significanceThe incidence of lung cancer is the highest in the world and my country.Despite tremendous progress in diagnosis and treatment in recent years,due to the higher metastasis and recurrence rates of lung cancer,the overall prognosis is still poor,and the five-year survival rate is still low(about 17%).Therefore,it is of great significance to explore the pathogenesis of lung cancer and find new prognostic indicators and therapeutic targets.Circular RNA(circular RNA,circRNA)is a newly-discovered endogenous non-coding RNA(nc RNA),which is a relatively new research hotspot in the field of RNA.Studies have shown that circRNA is widely involved in the normal physiological processes of the human body and the occurrence and development of diseases,and plays an important role in diseases such as tumor,cardiovascular system and nervous system.We plan to use GEO data to screen out circRNA molecules associated with lung cancer through bioinformatics analysis,analyze their effects on the proliferation,growth and apoptosis of lung cancer cells at the cellular level,and observe their role in the growth of transplanted tumors in animals.Finally,the molecular mechanism by which circRNA affects the occurrence of lung cancer through miRNA is clarified.This will provide a theoretical basis for elucidating the pathological mechanism of circRNA in lung cancer,as well as searching for early diagnosis markers and potential therapeutic targets of lung cancer.Methods(1)Screen abnormally expressed circRNA molecules through the GEO database.After realtime fluorescence quantitative PCR(RT-qPCR)verification in tissue samples,it was identified that circNDUFB2 is highly expressed in tissues and chips and the difference is most significant.Therefore,circNDUFB2 was used as the target molecule in this study.(2)RT-qPCR detection of circNDUFB2 expression in lung cancer tissue specimens,combined with clinical case factors,to clarify the potential relationship between circNDUFB2 and clinical pathological factors.(3)The nucleocytoplasmic distribution of circNDUFB2 in lung cancer cells was determined through the experiment of nucleoplasmic separation.(4)Design and synthesize si RNA and overexpression plasmid for circNDUFB2: Transfect si RNA and overexpression plasmid into lung cancer cells,and detect the proliferation,growth and cell growth of lung cancer cells by circNDUFB2 through cloning,MTT,flow cytometry and other methods Effects of apoptosis.(5)The miRNAs molecules that are potentially bound to circNDUFB2 are predicted from the database,and circNDUFB2 is knocked down and overexpressed,and the expression of miRNAs is detected by RT-qPCR.The dual luciferase experiment further verified the targeting relationship between circNDUFB2 and miR-625-5p.The expression level of miR-625-5p in lung cancer tissues was detected by RT-qPCR.Pearson correlation analysis of the correlation between circNDUFB2 and miR-625-5p in tissue expression.(6)Co-transfected overexpressed circNDUFB2 plasmid and miR-625-5p mimics in lung cancer cells,and tested their effects on lung cancer cell proliferation,growth and apoptosis by cloning,MTT,flow cytometry and other methods.(7)The regulation of circNDUFB2 on target genes Akt2 and IGF1 R and downstream signaling pathways were verified by RT-qPCR,luciferase experiment and Western Blot experiment.(8)Animal experiment analysis of tumor transplantation in nude mice circNDUFB2 affects tumor growth in vivo.Results(1)Based on the results of bioinformatics analysis of GEO database,several differentially expressed circRNAs were obtained.After verification,circNDUFB2 with the most obvious differential expression was selected.Further analysis showed that circNDUFB2 is a closed circular RNA molecule formed by reverse splicing of exons 2 and 3 of ubiquinone oxidoreductase subunit B2(NDUFB2).(2)The expression of circNDUFB2 is significantly up-regulated in lung cancer tissues.Analysis with clinical case factors revealed that the expression of circNDUFB2 was related to tumor size.(3)Overexpression of circNDUFB2 can significantly promote lung cancer cell proliferation,growth and inhibit cell apoptosis.Knocking down circNDUFB2 expression can significantly inhibit lung cancer cell proliferation,growth and promote cell apoptosis.(4)The nucleoplasm separation experiment results show that circNDUFB2 is mainly located in the cytoplasm of lung cancer cells.(5)There is a direct targeting relationship between circNDUFB2 and miR-625-5p.Detection of lung cancer tissue samples found that miR-625-5p was significantly down-regulated in lung cancer tissues compared with normal paracancerous tissues,and was negatively correlated with the expression of circNDUFB2.(6)Through database prediction and dual luciferase experiment results,miR-625-5p has direct targeting relationship with IGF1R and Akt2 3’UTR.At the same time,the expression levels of IGF1R and Akt2 in lung cancer tissue samples were significantly up-regulated,and were negatively correlated with the expression of circNDUFB2.It is positively correlated with the expression of circNDUFB2.(7)Overexpression of miR-625-5p can restore the inhibitory effect of circNDUFB2 on the proliferation and growth of lung cancer cells and the promotion of apoptosis.Western Blot results showed that the increased expression of IGF1 R and Akt2 caused by high expression of circNDUFB2 can be restored by up-regulating miR-625-5p.(8)In the nude mice subcutaneous tumor formation experiment,compared with the control group,the miR-625-5p group overexpressed the nude mice subcutaneous tumor formation ability was significantly reduced.At the same time,the ability to overexpress circNDUFB2 and miR-625-5p in nude mice was partially restored.Conclusions(1)Compared with normal paracancerous tissues,circNDUFB2 is highly expressed in lung cancer tissues and is related to the tumor size of tumor patients.(2)At the cellular level,circNDUFB2 can promote the proliferation and growth of lung cancer cells and inhibit apoptosis.(3)In animals,circNDUFB2 promotes tumor growth in vivo by regulating miR-625-5p.(4)Mechanistically,circNDUFB2 mediates the regulation of the expression of target genes Akt2 and IGF1R through competition with miR-625-5p,which in turn affects the expression of downstream GSK-3βandβ-catenin,thereby affecting the occurrence of lung cancer.Therefore,circNDUFB2 may be used as a marker for early diagnosis of lung cancer and a potential target for precise treatment.