Stable Silencing ROR1 Regulates The Molecular Mechanisms Of Cell Cycle,Apoptosis,and Autophagy In Lung Adenocarcinoma Cell

Objective Lentivirus-mediated shRNA-ROR1(Lv-shROR1)were used to establishstable-silenced ROR1 in lung adenocarcinoma cell lines,and cell models were uesd to study the molecular mechanism of ROR1 regulating cycle,apoptosis,and autophagy of tumor cells,so as to lay a foundation for the follow-up study of ROR1 as the therapeutic target.Methods Firstly,lentivirus-mediated shRNA-ROR1 with green fluorescent protein(GFP)tag was used to infect lung adenocarcinoma cell lines PC9,PC9 erlo,and NCI-H1975.GFP luminescence intensity was detected by fluorescence microscope to measure the efficiency of lentivirus infection,and flow cytometry was used to detect the efficiency of Lv-shROR1 silencing ROR1 protein.Secondly,single-cell clones were selected from Lv-shROR1-infected lung adenocarcinoma cell lines by limited dilution method.Western blot,flow cytometry and RT-PCR were used to detect the expression of ROR1 protein and mRNA in the clone cell lines,and a series of single-cell clones with stable-silenced ROR1 were screened as cell models for follow-up functional studies.Finally,single-cell clone R1 of PC9 erlo was selected to detect the molecules level of cell cycle and apoptosis by western blot;the changes of autophagy molecules,and the phosphorylation level of key kinase in the ROR1-silenced cells were detected by Ray Bio ?C-series Human Autophagy Array 1,western blot and Human Phospho-kinase Array kit.Pre-experiment: Western blot was used to detect the expression of HIF-1 ? before and after ROR1 gene silencing in lung adenocarcinoma cell lines.Results(1)Lentivirus-mediated shRNA-ROR1 was successfully transfected into lung adenocarcinoma cell lines PC9,PC9 erlo and NCI-H1975,in which the ROR1 inhibition rate were 55%,50%,and 35%,respectively.(2)Single-cell clones with stable-silenced ROR1 were successfully obtained from PC9,PC9 erlo and NCI-H1975 cell lines,the inhibition rates of ROR1 in PC9 single-cell clones R3 and R4 were 65% and 89%,and in PC9 erlo R1 and R2 were both 80%,and in NCI-H1975 R3 and R5 were 42% and 33%.The single-cell clone R1 of PC9 erlo was selected for follow-up study.(3)Stable silencing ROR1,the expression of Cyclin E1 and CDK4,which are key regulators of cell cycle,was significantly down-regulated.(4)After stable silencing ROR1,pro-apoptotic molecules Bak,Caspase-3 and Caspase-7 were significantly up-regulated,while anti-apoptotic molecules Bcl-2 and Bcl-XL were significantly down-regulated in R1 with stable silenced ROR1.(5)20 autophagy-related molecules were screened by Human Autophagy Array.It was found that autophagy-related molecules such as ATG7,ATG12,BNIP3 L,LC3 A and LC3 B increased in R1,and the expression level of LC3A/B was conformed upregulated after stable silencing ROR1 by western blot.(6)Our data indicated that blocking ROR1 could deactivate Akt,then activate GSK-3?/? via de-phosphorylation,and finally inactivate m TOR.(7)In the pre-experiment,we found that the expression of HIF-1? decreased after stable silencing ROR1 in lung adenocarcinoma cell lines PC9,PC9 erlo and NCI-H1975.Conclusions We have successfully established a series of single-cell clones with stable-silenced ROR1 from lung adenocarcinoma cell lines PC9,PC9 erlo,and NCI-H1975,which provided an effective cell model for the comprehensive study of the molecular mechanisms of ROR1 in lung adenocarcinoma.Our data show that blocking ROR1 can effectively prevent from entering the proliferation cycle,promote apoptosis and autophagy in lung adenocarcinoma,and Akt/ GSK-3?/? / m TOR signal pathway is one of the main pathway involved in ROR1-mediated tumor pathogenesis.ROR1 may have a potential mechanism in regulating glycolytic pathway,which is worthy for further study.