| Part ⅠEstablishment and Characteristics of Drug-resistantColorectal Cancer Cell linesObjective: To establish a stable oxaliplatin-resistant human colorectalcell lines, and to detect some of their characteristics, and to lay thefoundation for the subsequent experiments.Methods:(1) Oxaliplatin-resistant cells (SW480/OxR and SW620/OxR,they are called as drug-resistant cells) were generated by culturing cell lines(SW480, SW620, they are called as parental cells) in the presence ofincreasing concentrations of oxaliplatin.（2）Compared with their parentalcells, using the following methods to detect some biological characteristicsof the drug-resistant cell lines: the sensitivity to drugs (Oxa or5-FU) weredetected by using the WST-1reagent; the mRNA expression levels ofMDR-1, Survivin and Oct4were analyzed by real-time quantitative PCR(RT-qPCR); the percentage of drug-resistance associated proteins (P-gp and Survivin) positive cells were detected by indirect method of flowcytometry; the percentage of cancer stem cell markers (CD44and CD133)positive cells were detected by direct method of flow cytometry; thetumorigenicity of drug-resistant cells and the parental cells weredistinguished by inoculated cells into NOD/SCID mice; the protein levelsof epithelial-mesenchymal transition markers (E-ca, N-ca, Vim) and Oct4were detected by Western blot.Results:72h after exposure to drugs (Oxa or5-FU), the number of theparental cells significantly reduced (P <0.01), while the number of thedrug-resistant cells has not apparent changed (P>0.05). In thedrug-resistant cells, the mRNA expression levels of MDR-1, Survivin andOct4in the drug-resistant cells significantly increased (P<0.01), thepositive cells percentage of P-gp, Survivin, CD44and CD133remarkablyincreased, the tumorigenicity in NOD/SCID mice significantly enhanced,the mRNA and protein levels of Oct4significantly increased (P<0.01),when compared with their parental cells. The protein expression levelsmesenchymal markers (N-ca and Vim) in SW620/OxR cells weresignificantly higher than those in SW620cells (P<0.05and P<0.01), whilethe protein expression levels mesenchymal markers (E-ca) in SW620/OxRcells were significantly lower than those in SW620cells (P<0.01).Conclusions: The drug-resistant cells displayed the followingproperties: multidrug resistance characteristics; induction of EMT (SW620/OxR cells); overexpression of CSCs markers (CD133and CD44);enhanced tumorigenicity in NOD/SCID mice; overexpression oftranscription factor Oct4. These results suggest that the chemoresistantcolorectal cancer cells with Oct4overexpression enriched for CSCs. Part IIConstruction and Identification of Oct4-shRNAlentiviral vectorObjective: To construct Oct4-shRNA lentiviral vector which caneffectively silence the Oct4gene, and to investigate the effects of silencingOct4gene on apoptosis in human colorectal cancer SW480cell lines andits potential mechanism.Methods: Overexpression lentiviral vector plasmid of Oct4-GFPfusion and four Oct4-shRNA lentiviral vector plasmids (their interferencesequences were called respectively as pSC-1, PSC-2, PSC-3, and PSC-4)were constructed, and each of the four Oct4-shRNA lentiviral vectors andOct4-GFP overexpression plasmid were co-transfect respectively into293Tcells (called these experiments as exogenous sieving target), then the GFPprotein expression in the cells was detected by western blot. Through theprevious experiment, it was determined which one of the four Oct4-shRNAlentiviral vectors was the best one in terms of interference effect, then theOct4-shRNA lentiviral vector was transfected into the packaging cells293T.(2)Oct4-shRNA lentiviral vectors silences Oct4gene and induces cellapoptosis in colorectal cancer SW480cell lines (called experiment asendogenous authentication). There were three groups in the experiment: I).the control group (Con): untransfected lentiviral vector plasmids, II). negative control group (NC): transfected with negative control lentiviralvector plasmids, III). RNA interference group (RNAi): transfected withOct4-shRNA lentiviral vector plasmids; in the above three groups, thelevels of Oct4-mRNA was detected by RT-qPCR, the protein levels of Oct4and p-Akt were detected by Western blot, the percentage of Oct4+andCD44+cells and cell apoptosis were analyzed by flow cytometry.Results:(1) Oct4-shRNA lentiviral vector containing pSC-2sequencewas the most effective to silence the Oct4gene, which made GFP proteinexpression in the293T cells most significantly decreased.(2) Comparedwith the NC group, the Oct4mRNA levels (P<0.01), the Oct4proteinlevels (P<0.01), the p-Akt protein levels (P<0.01), and the percentage ofOct4+and CD44+cells in the RNAi group were significantly reduced,while cells apoptosis in the RNAi group was significantly increased(P<0.01).Conclusions: We successfully constructed Oct4-shRNA lentiviralvector silencing effectively the Oct4gene. Silenced the Oct4gene inSW480cell with Oct4-shRNA lentiviral vector could significantly reducethe percentage of CD44+cells, and obviously increase apoptosis, theseevents may be related to the PI3K/Akt pathway. Part IIIOct-4is Required for an Antiapoptotic Behavior ofChemoresistant Colorectal Cancer Cells Enriched for CancerStem Cells: Effects Associated with STAT3/SurvivinObjective: To identify whether Oct4play anti-apoptotic role inchemoresistant colorectal cancer cells enriched for cancer stem cells, and toexplore its possible mechanisms.Methods:(1) the protein levels of STAT3, phosphorylation of STAT3(P-STAT3) and Survivin in the drug-resistant cells and their parental cellswere detected by Western blot; immunofluorescence was used to detectco-expression of Oct4and phosphorylation of STAT3in the drug-resistantcells.（2）In the experiments of Oct4-shRNA lentiviral vector silencingOct4gene in the two drug-resistant cells (SW480/OxR SW620/OxR),therewere the following two groups: I). negative control group (NC): transfectedwith the negative control lentiviral virus, II). RNA interference group(RNAi): Oct4-shRNA lentiviral vector transfection. The changes of themRNA levels of Oct4, STAT3and Survivin were analyzed by RT-qPCR,the changes of the protein levels of Oct4, STAT3, P-STAT3and Survivinwere detected by Western blot, the changes of the percentage of CD133+and CD44+cells and cell apoptosis were analyzed by flow cytometry, andthe changes of tumorigenicity in NOD/SCID mice were observed. Results: The protein levels of STAT3, P-STAT3and Survivin in thetwo drug-resistant cells were significantly higher than those in theirparental cells (P <0.01or P <0.05); using immunofluorescent microscopy,we observed a co-localization of Oct4and P-STAT3proteins in the tworesistant cell lines. Oct4and P-STAT3proteins were mainly expressed inthe nucleus of the resistant cells. When compared with the NC group, theresults in the RNAi group are as follows: the mRNA levels of Oct4, STAT3and Survivin significantly decreased (P <0.01), the protein levels of Oct4,STAT3, P-STAT3and Survivin remarkable decreased (P <0.01), thepercentage of CD133+and CD44+cells significantly decreased, apoptosissignificant increased, tumorigenicity in NOD/SCID mice significantlyweakened, the growth of tumors were significantly inhibited (P <0.05).Conclusions: Oct4plays an important role in maintaining the survivalof CRC drug-resistant cells enriched for CSCs, which may be, at least inpart, attributed to the STAT3/Survivin pathway. |
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