The Role Of Nr5a2 And Cdh1 In The Self-renewal Of Lung Adenocarcinoma Stem Cells And Its Mechanism Research

Part? Isolation,culture and characterization of mouse Lewis lung adenocarcinoma stem cellsObjective: To establish the lung adenocarcinoma stem cell model with stem cell characteristics from mouse Lewis lung adenocarcinoma LLCParental cells.Methods:(1)The LLC-Parental cells were collected and suspended in a normal culture condition with serum.After eight consecutive rounds of collecting floating cells in culture medium with serum,cells stably growing in suspension were isolated,then passaged by stem cell culture medium without serum and repeated five times of 96-well plate monoclonal formation assay to establish a stable LLC-SD cell model.(2)The stem cell characteristics of LLC-SD were examined by the functional assay in vitro,including RT-q PCR,soft agar colony formation assay,96-well plate monoclonal formation assay,and the detecting of stem cell surface marker CD133 with flow cytometry.(3)The subcutaneous tumor transplantation assay in BALB/c nude mice and orthotopic tumor transplantation of C57BL/6 mice were used to examined the tumor formation and metastasis ability in vivo.Results:(1)Using RT-q PCR,we found the expression of embryonic stem-cell markers(Nestin,CK-18,Aldh1a1,Klf4,Nanog)were significantly increased in the LLC-SD cells compared to that in the LLC-Parental cells.(2)The number of clonogenic spheroids rate of LLC-SD was significantly higher than that of LLC-P cells.And single cell cloning rate of LLC-SD was also significantly elevated than that of LLC-P cells.(3)The expression of stem cell surface marker CD133 was significantly higher in LLC-SD cells than in LLC-P cells.(4)Animal experiments show that LLC-SD cells have higher tumorigenic ability and tumor metastasis ability in situ than LLC-P cells.Conclusion: Eight-rounds of selection for stable spheroid-forming floating cells followed by five successive rounds of single-cell cloning assay resulted in the isolation and purification of spheroid-forming LLC-SD cells with high tumorigenicity from the LLC-Parental cells culture.This research established foundation to further explore the molecular mechanism and biological characteristics of CSCs self-renewal.Part? Nr5a2 promotes cancer stem cell properties and tumorigenesis in non-small cell lung cancer by regulating NanogObjective: To explore the regulation mechanism of Nr5a2 in lung adenocarcinoma stem cells and its biological characteristics.Methods:(1)The clinicopathologic and prognostic importance of Nr5a2 expression in human lung adenocarcinoma was analyzed by bioinformatics analysis.(2)The m RNA expression of Nr5a2 in LLC-SD was detected by RT-q PCR.(3)The ability of Nr5a2 to promote self-renewal of LLC-SD cells was verify by the functional assays in vitro,including si RNAs transient interference assay,the detecting of stem cell surface markers CD133 with flow cytometry,serial spheroid formation assay,soft agar colony formation assay and 96-well plate monoclonal formation assay.(4)After construction of Lentiviral short-hairpin RNA(sh RNA)with Nr5a2 stably knocked down,the subcutaneous tumor transplantation assay in BALB/c nude mice and orthotopic tumor transplantation of C57BL/6 mice were used to verify the effect of Nr5a2 on the tumor formation and metastasis ability of LLC-SD cells in vivo.(5)Ch IP assay and Nanog rescue experiment were used to verify the direct regulation of Nr5a2 on Nanog.(6)The expression and correlation of Nr5a2 and Nanog in paraffin-embedded sections of clinical human lung adenocarcinoma were detected by RT-q PCR.Results:(1)The results of bioinformation analysis showed that Nr5a2 is significantly elevated in human lung adenocarcinoma than in normal lung tissues and Nr5a2 overexpression shortens the overall survival and progression-free survival of patients with lung adenocarcinoma.(2)Nr5a2 expression is elevated in LLC-SD CSC cells by RT-q PCR.(3)We first inhibited Nr5a2 expression by transient si RNA-1 and si RNA-2 interference and verified its down-regulation at both transcriptional and translational levels.The flow cytometry results revealed that the LLC-SD-si Nr5a2-1 and LLC-SD-si Nr5a2-2 cells have lower level of CD133 compared with the LLC-SD-N.C.cells.Both of the si Nr5a2(si Nr5a2-1/si Nr5a2-2)interference altered the size of the spheroids and cell morphology,and greatly reduced the number of spheroids.The number of clonogenic spheroids in soft agar was significantly lower in LLC-SD-si Nr5a2-1 and LLC-SD-si Nr5a2-2 cells than that in the negative control.Similarly,single cell cloning rate of LLCSD-si Nr5a2-1 and LLC-SD-si Nr5a2-2 cells was also significantly lower than that of the control cells.(4)Nr5a2 promotes lung CSCs tumorigenesis and cancer metastasis in vivo.(5)Ch IP assay showed that Nr5a2 directly regulates Nanog,and overexpression of Nanog after Nr5a2 inhibition restores the self-renewal phenotype of LLC-SD in vitro,further indicating that Nr5a2 directly regulates Nanog to promote LLC-SD self-renewal.(6)The results of clinical paraffin-embedded tissues showed that Nr5a2 and Nanog were significantly higher in patients with advanced lung adenocarcinoma than in early patients,and there was a significant positive correlation between Nanog and Nr5a2 expression.Conclusion: Nr5a2 promotes the stemness and tumorigenicity of lung adenocarcinoma stem cells by directly regulating the expression of Nanog.Part? Cdh1 functions as an oncogene and induces selfrenewal of lung cancer stem-like cells via PI3 K and MAPK pathwayObjective: To investigate the regulation mechanism of Cdh1 as an oncogene in lung adenocarcinoma stem cells.Methods:(1)The expression and prognosis of Cdh1 in a variety of cancers,including human lung adenocarcinoma were analyzed by bioinformation analysis in multiple human cancer databases.(2)The m RNA expression of Cdh1 in LLC-SD cells was detected by RT-q PCR.(3)The ability of Cdh1 to promote self-renewal of LLC-SD cells was verified by the functional assay in vitro,including si RNA transient interference assay,soft agar colony formation assay and 96-well plate monoclonal formation assay.(4)After construction of Lentiviral short-hairpin RNA(sh RNA)with Cdh1 stably knocked down,the subcutaneous tumor transplantation assay in BALB/c nude mice and orthotopic tumor transplantation of C57BL/6 mice were used to detect the effect of Cdh1 on the tumor formation and metastasis ability of LLC-SD cells in vivo.(5)Multiple database integration biological information analysis,including PPI,GO,GSEA,etc.,were used to predict the signal transduction pathway and its potential regulatory mechanisms of Cdh1 in lung adenocarcinoma.(6)The expression of predicted signaling pathways in LLC-P and LLC-SD cells were detected by RT-q PCR and Wb,respectively.CCK-8 proliferation assay confirmed the main proliferative regulation signaling pathway in LLC-P and LLC-SD cells respectively.Inhibitor blockade and si RNA transiently interfered with the highlighted pathway in LLC-SD to observe the differential expression of Cdh1 and multiple pluripotency genes in LLC-SD cells.Soft agar colony formation assay and 96-well plate single cloning assay were applied to verify the regulation mechanism of Cdh1 in LLC-SD cells.(7)After construction of Lentiviral short-hairpin RNA(sh RNA)with related signaling pathway blocked,the subcutaneous tumor transplantation assay in BALB/c nude mice and orthotopic tumor transplantation of C57BL/6 mice assay were used to verify the effect of signaling pathway-mediated changes in Cdh1 expression on the tumorigenic and cancer-promoting ability of LLC-SD cells in vivo.Results:(1)The bioinformation analysis results indicated that the Cdh1 expression was increased in 17 types of cancers,including lung adenocarcinoma.The Cdh1 expression level was much higher in patients at advanced stage(stage?~?)compared with those at early stage(stage?~?).And Patients with LUAD with a low Cdh1 expression exhibited a better overall survival(OS),progression-free survival(PFS)and post-progression survival(PPS).(2)LLC-SD cells had elevated Cdh1 RNA and protein expression in comparison with the LLC-Parental cells.(3)The expression of Cdh1 was effectively inhibited by si RNA.The soft agar clone formation rate of the LLC-SD-si Cdh1 experimental group was significantly lower than that of the LLC-SD-N.C.control group.The single cell cloning formation of 96-well plates showed that the morphology of single-cell cloned spheres of LLC-SD cells became loose and irregular after Cdh1 was interfered by si RNA,and the single cell cloning rate was significantly lower than that of control cells.(4)Subcutaneous tumor formation in nude mice and left lung in situ tumor implantation in C57BL/6 mice showed that Cdh1 promoted the tumorigenic and metastasis effects of LLC-SD cells in vivo.(5)The results of database integration analysis predicted that PI3 K and MAPK transduction pathways are the most relevant regulatory pathways for Cdh1 related enrichment in human lung adenocarcinoma.(6)The result of RT-q PCR and Wb analysis showed that the expression of Mapk1,Mek1 and Eif4 e was robustly upregulated in LLC-SD cells,while the expression of PI3 K,Akt and Mtor was elevated in LLC-Parental cells.The CCK-8 assay showed that the PI3 K signaling pathway plays a major regulatory role in proliferation of LLC-P cells,and the MAPK signaling pathway plays a major regulatory role in proliferation of LLC-SD cells.In the LLC-SD cells,the expression of Cdh1 and pluripotent genes including Nanog,Nr5a2 and Tbx3 were downregulated after inhibitor LY294002 blocking the PI3 K pathway,while the expression of Cdh1 and pluripotent genes including Sox2,Nanog,CD133,Aldh1a1,Nr5a2 and Tbx3 were significantly elevated after inhibitor PD98059 blocking the MAPK pathway.After the inhibitors effectively blocked the PI3 K and MAPK signaling pathways,the single cell cloning rate of the LLC-SD-LY294002 group and the LLC-SD-PD98059 group were significantly lower than that of the control group.However,the morphology of the single-cell cloned sphere of the LLC-SD-LY294002 group became loose and irregular,and the structure of the single-cell cloned sphere of the LLC-SD-PD98059 group became compact and dense.After si RNA effectively blocking the PI3 K and MAPK signaling pathways,the cloning rate of the LLC-SD-sip110 and LLC-SD-si Mek1/2 experimental groups were significantly lower than that of LLC-SD-N.C.control group in the soft agar colony formation assay and the 96-well plate single cell colony formation assay.However,in the 96-well plate single cell colony formation assay,the morphology of the single-cell cloned sphere of the LLC-SD-sip110 group became loose and irregular,and the structure of the single-cell cloned sphere of the LLC-SD-si Mek1/2 experimental group became compact and dense,which was consistent with the inhibitor results.(7)Subcutaneous tumor formation in nude mice and left lung in situ tumor implantation in C57BL/6 mice indicated that Cdh1 promoted the tumorigenic and metastasis effects of LLC-SD cells in vivo through the regulatory mechanisms mediated by PI3 K and MAPK transduction pathways.Conclusion: The positively regulation of PI3 K and negatively regulation of MAPK on Cdh1 as an oncogene to promote self-renewal of lung CSCs in vitro and in vivo.