| Part Ⅰ The significance and correlation of TROP2and CD133inNon-small Cell Lung CancerObjective To investigate the expression of TROP2in Non-small Cell Lung Cancer(NSCLC)tissues and cell lines. To explore the significance and correlation of TROP2and CD133in NSCLC.Methods TROP2expression was detected by flow cytometry in five NSCLC cell lines and oneimmortalized human bronchial epithelial cell line HBE. TROP2mRNA was examined in22patientswith NSCLC and its tumor-adjacent tissues. The significance and correlation of TROP2and CD133inNSCLC was detected by immunohistochemistry and double-labelling immunofluorescence.Furthermore, the correlations between TROP2and prognosis of NSCLC were analyzed．Results TROP2was detected in H1975, SPCA-1and PC-9, but not in A549, H1299and HBE.TROP2mRNA expression in NSCLC tissues detected by RT-PCR was68.2%（15/22）. The expressionof TROP2and CD133detected by immunohistochemistry were42.86%（42/98）and29.59%（29/98）in98NSCLC tissues respectively, which were significantly higher than that in the tumor-adjacentnormal tissues or benign lesion tissues (P<0.01). CD133-positive expression was significantly higher inpoor differentiated than that in the well differentiated (P=0.02). Positive TROP-2, CD133expression inthe lymph node metastasis group was significantly higher than that in the non-lymph node metastasisgroup（P=0.02, P=0.03）. The positive expression rate of TROP2, CD133increased with the increasingTNM stage （P=0.045, P=0.045）. TROP2expression was correlated with CD133expression(P=1.87E-05). And there was collocation between the TROP2and CD133proteins. TROP2tended to beexpressed in cases with an unfavorable outcome. But no difference was found the correlation betweenTROP2expression and months after surgery of NSCLC（P=0.076）.Conclusions TROP2is overexpressed in NSCLC tissues and cell lines. TROP2may be involvedin carcinogenesis of NSCLC. The collocation of TROP2and CD133was detected in NSCLC. TROP2 and CD133may be used as two important markers in judging the CSC of NSCLC．Part ⅡDesign and packaging of the small hairpin RNA targetingagainst TROP2Objective To construct the lentiviral expression vector mediated TROP2-shRNA.Methods Four virus plasmids vectors coding for shRNA targeting TROP2gene sequence wereconstructed．The recombinant virus plasmids were identified by PCR and sequencing. The TROP2genesilencing effect was measured by Western blotting. Then, recombinant lentivirus carryingTROP2-shRNA was produced by293T cells following by co-transfection of GV248（hU6-MCS-Ubiquitin-EGFP-IRES-puromycin） and packaging plasmids pHelper1.0andpHelper-2.0, and the virus titer was measured.Results The shRNA fragment targeting against TROP2was successfully packaged into thelentivirus，and the titer was approximately1E+9TU/ml.Conclusions The lentiviral vector of LV-TROP2-shRNA was successfully constructed.Part Ⅲ Transfection in NSCLC cell line PC-9and observe the effects ofLV-TROP2-shRNA on NSCLC in vitroObjective To transfect the lentivirus mediated TROP2-shRNA to NSCLC cell line PC-9andobserve the change of PC-9in vitro.Methods Flow cytometry was used to measure the transfection efficiency after the LV-TROP2-shRNA transfection into PC-9. TROP2gene knockdown were observed by Real-time PCR. Threegroups were created for this study. PC-9group(PC-9cells alone), PC-9/Con group(native control,transfected with LV-EGFP), PC-9/shRNA group(knock down, transfected with LV-TROP2-shRNA).We applied the test of CCK-8, scratch and transwell to investigate the role of TROP2in the proliferation,migration and invasion of PC-9.Results The transfection efficiency was above90%when enhanced green fluorescent protein(EGFP) stably expressed in PC-9cell. The mRNA inhibitory rates of TROP2were more than65%. Incontrast to PC-9group and PC-9/Con group, an inhibit on the proliferation, migration and invasion of PC-9/shRNA group were showed.Conclusions PC-9/shRNA was successfully constructed with the silencing of TROP2in NSCLCcell line PC-9. The over expression of TROP2in NSCLC cells can improve proliferation, migration,invasion ability.Part IV The effects of LV-TROP2-shRNA on NSCLC in vivoObjective To observe the effects of LV-TROP2-shRNA on NSCLC in vivo.Methods To construct NSCLC xenograft models, PC-9group, PC-9/Con group and PC-9/shRNAgroup were injected subcutaneously into SCID murine respectively. Tumor growth were observed.TROP2expression in tumor were observed by RT-PCR and Western blot. TROP2and Cyclin D1protein expressions were detected by immunohistochemical method.Results No difference was found among the weight of the three groups of SCID murine.PC-9/shRNA group, compared with the PC-9group and PC-9/Con group, tumor growth ofsubcutaneous xenograft significantly decreased in three experimental groups（P<0.05）. The mRNA andprotein expression of TROP2in PC-9/shRNA group was significantly decreased. TROP2ans Cyclin D1protein expressions were decreased in PC-9/shRNA group detected by IHC.Conclusions The mRNA and protein expression of TROP2were decreased in PC-9/shRNAgroup tumors. In vivo experiments showed that TROP2-RNAi tumors displayed a slower growth rate.Perhaps the decreased expression of Cyclin D1protein caused by TROP2-shRNA was the reason. |
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