Molecular Mechanisms Of PBX1/RNF6 Axis In The Development Of Non-small Cell Lung Cancer

ObjectiveUbiquitination is a key post-translational modification in the regulation of numerous biological processes in eukaryotes.Ubiquitination plays an important role in the localization,metabolism,function,regulation and degradation of proteins.During protein ubiquitination,substrate specificity is mainly determined by E3 ubiquitin ligase(E3).Ring finger protein 6(RNF6)belongs to the ring finger protein family and acts mainly as a E3 ubiquitin ligase,PBX1 is an upstream gene of RNF6,it modulated RNF6 expression by binding to the specific PRE.There are many studies have shown that PBX1/RNF6 were involoved in the development of prostate cancer,leukemia and other diseases.However,there are few investigations between PBX1/RNF6 axis and the development of non-small cell lung cancer(NSCLC).In the current study,we first examined the expression of PBX1 and RNF6 in tumor tissues and para-cacinoma tissues of non-small cell lung cancer patients and in several lung cancer cell lines,also,we used PBX1/RNF6 plasmid and siRNA/shRNA to overexpressed or interference PBX1 or RNF6 to detect their role in non-small cell lung cancer,which can clarify the functions and mechanisms of PBX1/RNF6 axis in non-small cell lung cancer.Therefore,our findings expected to find a new therapeutic intervention for treating non-small cell lung cancer patients.Methods1.Tumor tissue and para-carcinoma tissue samples from forty-five pairs non-small cell lung cancer patients among 2012 to 2016 were selected.Western Blot and RT-PCR were used to compare the expression level of PBX1 or RNF6 between lung cancer tissue and para-carcinoma tissues,the grayscale analysis was used to make an intuitive comparison chart.2.Non small cell lung cancer cell lines H1299,H460 and A549 cells were infected with PBXl or RNF6 plasmids followed by 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolimbromide assay(MTT)to explore the effect of PBX1 or RNF6 in non small cell lung cancer cell line proliferation and cisplatin drug effect.3.NSCLC cell lines H1299,H460 and A549 cells were infected with shRNAs to interference the expression of PBX1 or RNF6,then followed by 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyltetrazolimbromide assay(MTT)to explore the effect of PBX1 or RNF6 interference in non small cell lung cancer cell line proliferation and cisplatin drug effect.4.NSCLC cell line H1299 was transfected with PBX1/RNF6 plasmid or shRNF6 to overexpressed or interference the expression of PBX1 or RNF6.For cell cycle,flow cytometry was carried out.5.PBX1 or RNF6 plasmids were transfected into H1299 and H460 cell lines,the effect of PBX1 or RNF6 on cell cycle proteins such as CCDN2,p-Rb were detected by Western blotting and RT-PCR.6.H1299 cells were infected with PBX1/RNF6 plasmids or shRNAs,the effect of PBX1/RNF6 overexpressed or knockdown on the migration of lung cancer cell lines was examined by wound-healing assay.7.RNF6 and CCND2 plasmids were co-transfected into NSCLC cell line H1299,the ubiquitination of CCND2 was detected by co-immunoprecipitation.Results1.Western bolt results showed that the protein expression level of PBX1 in lung cancer tissues was lower than it in para-carcinoma tissues in 22(91.7%)of 24 patients,while the expression level of RNF6 in lung cancer tissue was also lower than that it in para-carcinoma tissues in 22(91.7%)patients.The results of RT-PCR showed that the mRNA expression of RNF6 in lung cancer tissues was lower than that in para-carcinoma tissues in 15 of 18 patients(83.3%),too.Furthermore,the grayscale analysis of Western bolt from 45 pairs of tumor tissues and adjacent tissues showed that the expression level of PBX1 and RNF6 in tumor tissues was significantly lower than that in para-carcinoma(P<0.001).2.PBX1/RNF6 overexpressed significantly inhibited the proliferation of NSCLC cells.This indicates that PBX1/RNF6 may be closely related with the development of non-small cell lung cancer.At the same time,PBX1 overexpressed can significantly reduce the IC50 of cisplatin in non-small cell lung cancer cell lines and enhance the drug sensitivity.Moreover,upregulated RNF6 protein levels in A549 and H1299 cells.In contrast,knock down RNF6 gets the opposite effect,improve the IC50 of cisplatin in non-small cell lung cancer cell lines and enhance the drug resistance.It means that PBX1/RNF6 may involve in CDDP drug resistance in NSCLC cells;3.these demonstrated that RNF6 may also involved in the drug resistance of NSCLC cells.Knock down RNF6 gets the opposite effect.4.PBX1/RNF6 overexpressed can significantly reduce the wound healing rate in H1299 cells compared to control cells,indicating that RNF6 also affected the cell motility ability of non-small cell lung cancer.5.PBX1/RNF6 overexpressed can arrest cell cycle of NSCLC cells in the G1/GO phase,however,knock down RNF6 can accelerate cell cycle process,making it into the S phase.In addition,PBX1/RNF6 decreased the expression of cell cycle related proteins CCND2,p-RB.These results indicated that PBX1/RNF6 can inhibit cell cycle progression in non-small cell lung cancer.6.RNF6 can promote CCND2 ubiquitination,decrease CCND2 protein level through the ubiquitin proteasome pathway,which further explanation that RNF6 can inhibit cell cycle progression in non-small cell lung cancer.ConclusionsIn our studies,we found that PBX1/RNF6 axis can inhibit cell proliferation and migration of non-small cell lung cancer.Moreover,reduce the IC50 of cisplatin in non-small cell lung cancer cell lines,enhance the drug sensitivity and arrest cell cycle in G1/GO phase,which might help to find the new strategies of non-small cell lung cancer treatment.