| Part One The TUG1 expression level and the clinical data of NSCLC patients in the resistant and sensitive groups.Objective: To study the TUG1 expression level and the clinical data of NSCLC patients in the resistant and sensitive groups.Methods: RTq-PCR was used to detect the TUG1 expression level in the resistant and sensitive group.The Kaplan-Meier was used to plot the survival curves.Results:1.The TUG1 expression level of NSCLC patients in the resistant and sensitive groups.According to the qRT-PCR detection,the TUG1 expression of the patients in the resistant group was significantly lower than that in the sensitive group(P < 0.01).2.The clinical data of NSCLC patients in the resistant and the sensitive groups.More patients with smoking history,poor differentiation and lymph node metastasis were observed in the resistant group than the sensitive group(P <0.05).The total survival time of the sensitive group was significantly higher than that of the resistant group.Conclusions: The TUG1 expression in the resistant group was significantly lower than that in the sensitive group.TUG1 might be involved in the occurrence of chemotherapy-resistant.Part Two The effect of TUG1 on cell biological characteristics and che-mosensitivity of NSCLC cells.Objective :To detect the effect of TUG1 on cell biological characteristics and chemosensitivity of NSCLC cells.Methods:1.Cell culture,transfection and transcription analysisThe lung cancer cell line SPC-A1 were cultured in high-glucose DMEM and the cell lines NCI-H1650,NCI-H520 and NCI-H1299 in RPMI-1640 medium.The 16 HBE cells were cultured by high-glucose DMEM containing10% FBS,100U/ml penicillin and 100mg/L streptomycin.TUG1 was knocked down by si-RNA and overexpressed by transfection using a TUG1 overexpression vector in SPC-A1,NCI-H520,SPC-A1/DDP and NCI-H520/DDP cells.The transfection efficiency of TUG1 was detected by qRT-PCR.2.The chemosensitivity of SPC-A1 cells and NCI-H520 cells transfected with si-TUG1 and SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNA-TUG1 detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT).3.Colony forming ability of SPC-A1 cells and NCI-H520 cells transfected with si-TUG1 and SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNA-TUG1 detected by colony formation assay.4.The migration ability and invasive ability of SPC-A1 cells and NCI-H520 cells transfected with si-TUG1 and SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNA-TUG1 detected by scratch test and Transwell assay.5.The cell cycle distribution and apoptosis ability of SPC-A1 cells and NCI-H520 cells transfected with si-TUG1 and SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNA-TUG1 detected by flow cytometry.6.The autophagy ability of SPC-A1 cells and NCI-H520 cells transfected with si-TUG1 and SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNA-TUG1 detected by GFP-LC fusion protein,MDC staining and Western blot analysis.7.SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNA-TUG1 or pc DNA-NC were injected subcutaneously to establish a xenogenic transplantation model.8.The apoptosis ability of NSCLC mice detected by TUNEL staining.9.The autophagy ability of NSCLC mice detected by Western blot analysis.Results:1.Compared with the si-NC group,si RNAs significantly down-regulated the expression of TUG1 in SPC-A1 and NCI-H520 cells.The expression of TUG1 in SPC-A1/DDP and NCI-H520/DDP cells transfected with pc DNATUG1 was significantly up-regulated.2.TUG1 inhibited proliferation,migration and invasion ability in NSCLC.TUG1 enhanced apoptosis,autophagy ability and the sensitivity to DDP in NSCLC.3.TUG1 promoted sensitivity of xenograft tumors to DDP in vivo.TUG1 enhanced apoptosis and autophagy ability in NSCLC.Conclusions: TUG1 inhibited proliferation,migration and invasion ability in NSCLC,while enhanced apoptosis,autophagy ability and the sensitivity to DDP in NSCLC.Part Three TUG1 regulates NSCLC chemosensitivity via the miR-221/PTEN axisObjective :A mechanistic study of TUG1 affecting the chemosensitivity of NSCLC.Methods:1.Dual luciferase reporter assay was used to verify the relationship between miR-221 and PTEN.Dual luciferase reporter assay and RIP assay was used to verify the relationship between TUG1 and miR-221.2.RTq-PCR was used to detect the miR-221 and PTEN expression level in the resistant and sensitive group.RTq-PCR was performed to analyze miR-221 expression after knocking down or overexpressing TUG1 and detect PTEN expression after knocking down or overexpressing miR-221/TUG1.3.The colony forming ability,migration ability,invasive ability,apoptosis ability and autophagy ability of SPC-A1/NCI-H520 cells transfected with si-NC,si-TUG1,si-TUG1+miR-NC or si-TUG1+miR-221 and SPC-A1/DDP or NCI-H520/DDP cells transfected with pc DNA-NC,pc DNA-TUG1,pc DNA-TUG1+NC inhibitor or pc DNA-TUG1+miR-221 inhibitor detected by colony formation assay,scratch test,Transwell assay,flow cyto-metry and Western blot analysis.4.The colony forming ability,migration ability,invasive ability,apoptosis ability and autophagy ability of SPC-A1/NCI-H520 cells transfected with sh-RNA,sh-PTEN,sh-RNA+pc DNA-TUG1 or sh-PTEN+pc DNA-TUG1 and SPC-A1/DDP or NCI-H520/DDP cells transfected with pc DNA-NC,pc DNA-PTEN,pc DNA-NC+si-TUG1 or pc DNA-PTEN + si-TUG1 detected by colony formation assay,scratch test,Transwell assay,flow cytometry and Western blot analysis.Results:1.The expression level of miR-221 in the sensitive group was significantly lower than that in the resistant group(P<0.01)and the expression level of PTEN in the sensitive group was significantly higher than that in the resistant group(P<0.01).Dual luciferase reporter assay revealed that PTEN is a direct target of miR-221 and miR-221 is a direct target of TUG1.The expression level of miR-221 was increased when TUG1 was inactivated,and the level of miR-221 was decreased when TUG1 was overexpressed(P<0.01).Transfection of miR-221 mimics or si-TUG1 reduced the expression level of PTEN,while transfection of miR-221 inhibitor or pc DNA-TUG1 increased the expression level of PTEN(P<0.01).TUG1 positively regulated PTEN and miR-221 negatively regulated PTEN.2.The overexpression of miR-221 further enhanced the effect of TUG1down-regulation on promoting proliferation,migration and invasion while inhibiting apoptosis,autophagy and senescence in NSCLC.The downregulation of miR-221 further enhanced the effect of TUG1 overexpression on inhibiting proliferation,migration and invasion while promoting apoptosis,autophagy and senescence in NSCLC.3.The overexpression of TUG1 partially reversed the effect of PTEN down-regulation on promoting proliferation,migration and invasion while inhibiting apoptosis,autophagy and senescence in NSCLC.The down-regulation of TUG1 partially reversed the effect of PTEN overexpression on inhibiting proliferation,migration and invasion while promoting apoptosis,autophagy and senescence in NSCLC.Conclusions: TUG1 enhances NSCLC chemosensitivity via the miR-221/PTEN axis. |
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