The Role And Related Mechanism Of ROR α Gene In Non-small Cell Lung Cancer

Object:Lung cancer is one of the worldwide serious threat to human health of malignant tumor,the incidence and mortality of malignant tumor was the first.A recent study found that lung cancer is a kind of aging related diseases,including a onset time is about 70 years old,and the incidence rate increased with age gradually increased.Lung cancer is a disease of high consumption.The need to provide abundant nutrients for the growth of tumor cells,and the metabolic level of the elderly is low,relative to non-elderly patients in elderly patients with lung cancer slowly.Studies have shown that in hepatoma,ROR alpha by inhibiting the growth of tumor cells to provide energy for the cell glycolysis activation of the P53/P21 pathway,thereby reducing tumor cell proliferation in addition.In recent years,ROR was found in colorectal cancer,breast cancer,prostate cancer and so on.However,the relationship between ROR alpha and lung cancer is not clear,and the related regulatory mechanism needs to be specific for further study.P53/P21 signal pathway is one of the classic study of aging related pathways,activation of this pathway can promote cell senescence and inhibit cell proliferation.At the same time,P53,a key transcription factor P21 or regulate cell cycle activity,cell cycle arrest and cell senescence typical features.This study uses the Western blot method was applied to analyze the expression of ROR alpha in lung cancer tissues and adjacent normal lung tissues,with in vitro experiments to further explore ROR alpha on the proliferation viability of lung cancer cell,colony forming ability,cell cycle distribution,senescence associated beta galactosidase staining experiments,in order to clarify the relationship between ROR alpha and lung cancer.Method:1.Western blot method was used to detect the expression of ROR alpha in 6 paired human non-small cell lung cancer(NSCLC)tissues and adjacent normal tissues;2.ROR alpha knock down plasmid and over-expression plasmid were constructed and therefore used to verify the knockdown and overexpression of ROR alpha,then to screen the NSCLC cell lines A549 and PC9 which stably expressing ROR alpha;3.MTT method and clone formation assay were used to examine the effect of ROR alpha on the growth viability of A549 and PC9 cell lines;4.Western blot method was used to detect the effect of ROR alpha on P53/P21 signaling pathway;5.Senescence associated beta galactosidase staining and cell cycle assay to evaluate the effect of ROR alpha on cell senescence.Results:1.The expression of ROR alpha in lung cancer tissues was significantly lower than that in adjacent normal tissues,and the expression of ROR alpha 4 was the main subtype ROR alpha gene;2.ROR alpha knockdown plasmids can significantly promote the proliferation of NSCLC cells,on the contrary,over-expression and its agonist significantly inhibited cell proliferation activity;3.In A549 cells,compared with control group,the SA-beta-gal staining of ROR alpha over-expression group was significantly increased,G2 cell cycle arrest,but in P53 mutant PC9 cells,cells of SA-beta-galstaning positive rate and cell cycle distribution has no significant difference,indicating that cell senescence and P53 activity was closely related;4.In A549 cell line,ROR alpha over-expression and the agonist significantly increased the expression of P53,P21,and decreased the expression of CyclinB1;5.In the A549 cell line which stably over-expressing ROR alpha 4,P53 knockdown inhibited cell senescence induced by ROR alpha 4.Similarly,the P53 inhibitor PFT showed similar results,indicating that ROR alpha 4 induced cell senescence was dependent on P53 activity.Conclusion:ROR alpha 4 is the main subtype of ROR gene expression in human lung tissues,and the expression of ROR alpha 4 in NSCLC tissue was significantly lower than that of adjacent tissues.ROR alpha 4 can activate the P53/P21 pathway to induce cell senescence and inhibit the proliferation of NSCLC cells.This investigate enriched the study of lung cancer and cell senescence,and provided new ideas of potential treatment for clinical lung cancer.Background:Lung cancer is one of the most common malignant tumor with high prevalence and motality rate.With the increase of age,its incidence increased year by year.With China’s population aging intensifies,social burden caused by lung cancer increased dramatically,become an huge obstacle for people’s health and quality of life.In recent years,with the development of cancer treatment technology,the effect of the treatment of lung cancer has significantly improved,but the 5 year survival rate is still less than 15%,therefore,a new direction of study on treatment of lung cancer is necessary.Retinoid related orphan nuclear receptor alpha(ROR alpha)gene shared a closed relationship with tumor,especially in digestive system and breast cancer,and play the role of tumor suppressor genes.But its relationship with lung cancer is still not clear.We previously found that,compared with the surrounding normal tissues,reduced expression of ROR alpha 4 protein was shown in lung cancer tissues,it can suppress the proliferation activity of lung cancer cells,like A549,PC9.In A549 cells,it could inhibit the proliferation of tumor cells through cellular senescence which was P53 gene dependent.Therefore,we hypothesized that in nude mice,ROR alpha 4 gene may inhibit A549 cells in vivo.Objective:In order to study the effect of tumorigenicity of NSCLC cells in nude mice of ROR alpha 4 gene,we constructed ROR alpha 4 expression plasmid,infection of lung adenocarcinoma cell line A549,selected stable transfected cell lines,and tumor formation in nude mice.Method:1.A ROR alpha 4 gene over-expression plasmid was constructed,and Western gene by PCR and blot were uused to verify the expression of ROR alpha 4 gene;2.A549 cells which transfected with ROR alpha 4 gene were selected with G418 and the stable expression cell lines;3.The nude mice were divided into ROR alpha 4 group and GV144 group,6 mice in each group,then the mice were inoculated subcutaneously in stable expression cell strain.Dynamic observation of tumor growth,tumor size,draw the growth curve,30 days after the experiment was end.Western blot was applied for the detection of tumors P53,P21,CyclinB1 protein expression.Results:1.The A549 cell line stably expressing ROR alpha 4 gene was successfully screened and verified by qRT-PCR and Western blot;2.Subcutaneous tumor formation after 30 days,ROR alpha 4 group the tumor growth rate and size was less than that of GV144 group(P<0.05);Western blot showed the expression of ROR alpha 4 gene,P53,P21 expression increased,CyclinB1 expression decreased in nude mice.Conclusion:ROR alpha 4 gene over-expression plasmid could stably transfected NSCLC cell line A549.In nude mice,the tumor formation ability of A549 cells was decreased expressing ROR alpha 4 gene over-expression,and the expression of P53 and P21 in tumor was increased,while the expression of CyclinB1 protein was decreasedObject:Cisplatin is the standard first-line chemotherapeutic agent for the treatment of non-small cell lung cancer(NSCLC).However,resistance to chemo-therapy has been a major obstacle in the management of NSCLC.Aldehyde dehydrogenase 1A1(ALDH1A1)overexpression has been observed in a variety of cancers,including lung cancer.The purpose of this study was to investigate the effect of ALDH1A1 expression on cisplatin resistance and explore the mechanism responsible.Method:Reverse transcriptase-PCR was applied to measure the messenger RNA expression of ALDH1A1,while Western blot assay was employed to evaluate the protein expression of ALDH1A1,B-cell lymphoma 2,Bcl-2-like protein 4,phospho-protein kinase B(p-AKT)and AKT.A short hairpin RNA was used to knockdown ALDH1A1 expression.A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to determine the effect of ALDH1A1 decrease on cell viability.The cell apoptotic rate was tested using flow cytometry assay.Results:ALDH1A1 is overexpressed in cisplatin resistant cell line A549/DDP,compared with A549.ALDH1A1 depletion significantly decreased A549/DDP proliferation,increased apoptosis,and reduced cisplatin resistance.In addition,the phosphoinositide 3-kinase(PI3K)/AKT pathway is activated in A549/DDP,and ALDH1A1 knockdown reduced the phosphorylation level of AKT.Moreover,the combination of ALDH1A1-short hairpin RNA and PI3K/AKT pathway inhibitor LY294002 markedly inhibited cell viability,enhanced apoptotic cell death,and increased cisplatin sensitivity.Conclusion:These results suggest that ALDH1A1 depletion could reverse cisplatin resistance in human lung cancer cell line A549/DDP,and may act as a potential target for the treatment of lung cancers resistant to cisplatin.