CUL4 E3 Ligase Regulates The Proliferation And Apoptosis Of Lung Squamous Cell Carcinoma And Small Cell Lung Carcinoma

Objective: About 80% of the proteins in the cell are degradated by the ubiquitin proteasome system(UPS).In the UPS,E3 ligases act as scaffold to get ubiquitin and substrate closer,it determines the specificity of substrate.CUL4 A and CUL4 B as members of CUL4 families,although two molecules homology reached nearly 80%,but they have distinguishing features from the cell positioning to functions.At the same time,for CRL4 s receptors present different substrates,CUL4 A and CUL4 B play different roles in different biological stimuli and different cell positioning,which strengthen the biological diversity of CRL4 s.In recent years,researches have shown that CUL4 involved in a variety of biological processes,such as cell cycle regulation,apoptosis,transcriptional regulation,DNA damage repair and epigenetics stability.CUL4 abnormally high expressed in many kinds of tumors,and was associated with tumor progression,metastasis and poor prognosis in breast cancer,prostate cancer,squamous carcinoma,adrenal cortical carcinoma and liver cancer.Our previous study found CUL4 high expression in lung cancer,and was associated with smoking index,high-level clinical stage and poor prognosis.In addition,tumor growth is restrained obviously with lower CUL4 expression in vivo.The research results show that the CUL4 in lung squamous carcinoma and small cell lung cancer has played a very important role in cancer progress.This study aims to explore the regulation mechanism of CUL4 in lung squamous carcinoma and small cell lung cancer growth and apoptosis,whcich would provide significant evidence of potential therapeutic targets in lung cancer therapy.Methods: 1.Western Blot to screen high CUL4 A and CUL4 B expression lung squamous carcinoma and small cell lung cancer cell lines,then build stable low expression CUL4 A and CUL4 B cell lines model by the application of lentivirus plasmid packaging system.Using Western Blot and Real-time PCR to validate the expression of CUL4 A and CUL4 B.And using cell counting method to observe the change of cell proliferation rate after the decrease expression of CUL4 A and CUL4 B.2.Through the PI single staining to detect the change of cell cycle after the decrease expression of CUL4 A and CUL4 B.At the same time,to test P21 and cyclin E1 protein levels changed after knock out CUL4 A.Further,to screen the dissolution of cell cycle arrest after the silence of P21 by flow cytometry methods.3.Through the Annexin V-FITC/PE double staining method to detect the change of cell apoptosis after the decrease expression of CUL4 A and CUL4 B.IHC method to detect the correlation between CUL4 B and FOXO3 A in lung squamous carcinoma and small cell lung cancer tissues.And using Western Blot to detect the changes of FOXO3 A,apoptosis related proteins and antiapoptotic proteins after the decrease expression of CUL4 A and CUL4 B.Silencing FOXO3 A to observe the change of cell apoptosis on the basis of the CUL4 B knockout,which in order to determine if FOXO3 A is the key regulation factor of cell apoptosiss in lung cancer.4.Using Western Blot to detect the change of the activity of ERK pathway in CUL4 B knockout,and through the PD98059 inhibition experiment to verify P-FOXO3 A was phosphorylated by ERK pathway.MG132,MLN4924 and silencing UBC12 were applied to determine P-FOXO3 A degradation through UPS,and non CUL4 B mediated.5.Through the literature and cytological experiments exclude LATS1 was the main element in how CUL4 B regulate ERK activity in lung squamous carcinoma and small cell lung cancer.Then,combined with document,mass spectrum detection and cytology,we found P16 was transcriptional inhibited by CUL4 B.At the same time,silencing P16 would recover ERK pathway,that verified CUL4 B regulated the activity of ERK pathway through transcription inhibition of P16.Results: 1.The proliferation rate of lung squamous carcinoma and small cell lung cancer was slow down while knockdown CUL4 A and CUL4 B.2.In lung squamous carcinoma and small cell lung cancer,CUL4 Aknockdown cause G1 phase blocking.After double knockdown CULA and P21,G1 phase block was released.3.In lung squamous carcinoma and small cell lung cancer,CUL4 Bknockdown led to an increase of cell apoptosis.Double knockdown CUL4 B and FOXO3 A could reverse the phenomenon of apoptosis increase.CUL4 Bknockdown upregulated FOXO3 A and downregulted P-FOXO3 A.Upregulation of FOXO3 A was not affected by transcriptional regulation of CUL4 B,but because CUL4 B improved ERK activity and promoted FOXO3 A to degraded.Subsequent P16 m RNA detection and double knockdown experimental results shown that the CUL4 B probably by inhibiting the transcription of P16,lessened the P16 ERK inhibition,increase activity of ERK.In conclusion,in lung squamous carcinoma and small cell lung cancer,CUL4 B probably by inhibiting the transcription of P16,increased activity of ERK,prompting FOXO3 A phosphorylation and degradation,thus reducing the cell apoptosis.Conclusion: 1.CUL4 A and CUL4 B involve in cell proliferation and apoptosis regulation in the lung squamous carcinoma and small cell lung cancer.2.CUL4 A affects cell cycle through regulating its ubiquitin substrates P21 in lung squamous carcinoma and small cell lung cancer.3.CUL4 B affects cell apoptosis through transcriptional inhibition of P16 to raise ERK pathway activity,and promotes the FOXO3 A phosphorylation and degradation through UPS in lung squamous carcinoma and small cell lung cancer,to enhance the viability of the lung cancer.