The Function And Mechanism Of CircZMYM4 In Lung Cancer

Objective Circular RNA(circular RNA,circRNA)is a special endogenous non-coding RNA.A lot of studies have shown that large amounts of circular RNA are dysregulated in lung cancer and affect the progression and prognosis of the disease.However,the molecular mechanism of circular RNA in lung cancer has not been clearly elucidated.This article aims to explore the role of circZMYM4 in lung cancer,clarify the biological function and the molecular mechanism of circZMYM4,so as to provide biomarkers for the prognosis of lung cancer and potential intervention targets for the treatment of lung cancer.Method(1)Screening and identification of circRNA related to lung cancer: find and screen the circRNA(circZMYM4)with significant differential expression in lung cancer and adjacent tissues from public database,and perform reverse transcription PCR with specific reverse primers.Agarose gel electrophoresis,Sanger sequencing,and exonuclease(RNase R)tolerance experiments were used to test the circular structure,loop formation site and stability of circZMYM4.Real-time quantitative PCR was used to detect the expression of circZMYM4 in lung cancer and adjacent tissues,lung cancer and normal lung epithelial cells,to determine whether circZMYM4 is dysregulated in lung cancer.The localization of circular RNA in cells can be tested by fluorescence in situ hybridization.Explore the biological function of circZMYM4: Overexpress circZMYM4 in A549 lung cancer cell line,and detect cell migration,proliferation and apoptosis by Transwell,MTT and EdU,respectively.A model of lung cancer subcutaneous transplantation tumor in nude mice was established.By monitoring the size and weight of the transplanted tumor,it was analyzed whether overexpression of circZMYM4 could affect the growth of subcutaneous transplantation tumor in nude mice.(3)Explore the mechanism of circZMYM4 in lung cancer: Use the specific circZMYM4 probe for RNA pulldown experiment and mass spectrometry identification.Then we find the target protein(YBX1)that can be combined with circZMYM4,and reversely we verify thebinding ability of circZMYM4 and YBX1 through RNA-IP experiment.Change the expression level of circZMYM4 in A549 cells,and check whether the expression level of YBX1 changes by Western blot.At the same time,whether the localization of YBX1 in the cells is changed by immunofluorescence experiment.Perform immunofluorescence experiments on tumors of nude mice lung cancer subcutaneous transplantation tumor models and detect the location change of YBX1 in the cell,verify whether circZMYM4 affects the development of lung cancer through YBX1.Results(1)Screening and identification of target circRNA: By screening 5 pairs of circular RNAs of lung cancer and adjacent tissues in the tissue chip,it was found that the expression difference of circZMYM4 was the most obvious between lung cancer and adjacent tissues.In RT-PCR and natural gel electrophoresis experiments,using cDNA as a template,amplification products can be obtained by reverse primers,but amplification products cannot be obtained using genomic DNA(gDNA)as a template,which proved that circZMYM4 has a circular shape structure.Sanger sequencing results showed the gene sequence in loop site.RNase R experiments showed that circZMYM4 is more stable than linear mRNA.The results of qRT-PCR confirmed that circZMYM4 was significantly low expression in lung cancer tissues(37/38,97.4%).Compared with BEAS-2B,circZMYM4 was significantly decreased in A549,HCC2279,H460,H3255,H1299,HCC4006 and PC9.FISH experiments found that circZMYM4 is mainly located in the cytoplasm of lung cancer cells.(2)Explore the biological function of circZMYM4:Through a series of biological function tests,we found that after overexpressing circZMYM4 at the cellular level,the proliferation and migration ability of A549 cells decreased significantly.At the animal level,a nude mouse subcutaneous xenograft model shown that overexpression of circZMYM4 can significantly inhibit the growth of lung cancer.(3)Explore the mechanism of circZMYM4 in lung cancer: biotin-labeled circZMYM4 pulldown probe can specifically bind circZMYM4 and identify the binding protein through mass spectrometry experiment,finally determine the target protein is YBX1.RNA-IP experiments further verified that circZMYM4 and YBX1 bind to each other.Western blot results showed that the change of circZMYM4 expression in lung cancer cell lines did not affect the degradation of YBX1 protein.Through immunofluorescence experiment,it has been demonstrated in cells and animals that increasing the expression of circZMYM4 in lung cancer cell lines can promote the transfer of YBX1 from the nucleus to the cytoplasm,thereby inhibiting thecancer-promoting function of YBX1 in the nucleus.Conclusion(1)This study explored the bio-function and mechanism of circZMYM4 in the development of lung cancer by using human lung cancer samples,lung cancer cell lines,and animal tumor models.The experimental results show that: circZMYM4 is a highly stable circular RNA,which is mainly located in the cytoplasm of the cell.Its expression in lung cancer tissues and cells is significantly down-regulated,which suggesting that circZMYM4 may play an important role in the development of lung cancer.(2)Overexpression of circZMYM4 can significantly inhibit the proliferation and migration of lung cancer cells,and inhibit the growth of solid tumors.(3)circZMYM4can inhibit the biological function of YBX1 by binding YBX1 and inhibiting its nuclear metastasis,which ultimately hinders the progress of lung cancer.This study provides new ideas and theoretical basis for the treatment of lung cancer by exploring the function and potential mechanism of circRNA in lung cancer.