| Objective: To investigate value of KLF4and β-catenin for theoccurrence and developments of the gastric carcinomas (GC) and define thepossible relationships between KLF4and β-catenin, lay the foundation forfurther study. The mRNA and protein expression of KLF4and β-cateninwere detected in GC and matched adjacent noncancerrous tissues.Methods: The mRNA and protein of KLF4and β-catenin wereexamined by immunohistochemical staining, RT-PCR and western blottingdetection in GC tissue and paired adjacent noncancerrous tissues. Therelationships between KLF4and β-catenin proteins were confirmed byimmunoprecipitation techniques in GC tissues.Results: Compared with paired adjacent noncancerous tissue, thelevels of KLF4mRNA and protein expression were significantly decreased; however expressions of β-catenin were significantly increased in GC.Positive expression rate of KLF4protein was8.3%(7/84) in GC andsignificantly lower than the positive rate of81%(68/84) in adjacent tissues.There was statistically significant differences between them (χ2=44.08,P<0.05). However, the positive expression rate of β-catenin protein was76.2%(64/84) in GC, higher than that in matched adjacent noncanceroustissues (15.48%,13/84). There was statistically significant differencesbetween them (χ2=26.35, P<0.05). In addition, the relationship betweenprotein expression of KLF4and β-catenin in GC tissues was negativelycorrelated (rs=-0.672, P<0.05). Compared with paired adjacentnoncancerous tissue, KLF4mRNA expression was decreased (P<0.05), andβ-catenin mRNA expression was significantly increased (P<0.05) in GCspecimens. Comparet with the macthed adjacent noncancerous tissue, KLF4protein expression was shartly reduced in GC specimens, and there werestatistically significant difference between them (P<0.05); however,β-catenin protein expression was significantly increased (P<0.05), whichwas consistent with the mRNA expression trence of this two genes.Co-immunoprecipitation showed that goat anti-human polyclonal KLF4antibody could precipitate β-catenin protein in GC tissues, and rabbitanti-human monoclonal β-catenin antibody also precipitated the KLF4protein in GC tissues.Conclusion: KLF4expression significantly reduced but β-catenin expression increased, and the negative expression tendency between KLF4and β-catenin was observed in GC tissues. KLF4was related with clinicalstage, differentiation, invasion depth of GC, and β-catenin was closelyrelated with lymph nodes in GC. KLF4interacts β-catenin each other in GCtissues. Objective: To further clarify the exogenous KLF4gene on malignantand mechanism of action in gastric cancer cells, building KLF4overexpression lentiviral vectors.Method: Exogenous KLF4gene was transferred into the emptylentiviral vector pLv-UbC-IRES2-EGFP through eukaryotic expression ofpcDNA3.1IE-KLF4-EGFP plasmid for building pLv-KLF4-IRES2-EGFPrestructuring lentiviral vector. Recombinant plasmid was transfected intogastric cancer cells and KLF4mRNA expression changes was detected byRT-PCR after restriction enzyme digestion and sequencing. The rate of transfection was observed by fluorescence microscopy, and KLF4proteinwas detected by western blotting after packaged lentiviral vector wastransfected into the same gastric cancer cells.Results: Correctly inserted target gene in the recombinant plasmid wasconfirmed by restriction enzyme digestion and DNA sequencing. ElevatedmRNA expression of KLF4was found after pcDNA3.1IE-of KLF4-EGFPwas transfected into gastric cancer cells. Packaged lentiviral vector wastransfected into gastric cancer cell-lines, and95%cells was greenfluorescence staining by fluorescence microscopy observation; the targetprotein of KLF4was consistent with the theoretical proteins by westernblotting.Conclusion:"three-plasmid vector" was an effective method to buildlentiviral vector; successful construction of KLF4overexpression lentiviralvector was confirmed after detection of KLF4mRNA and proteinexpression was measured in gastric cancer cells was transfected bylentiviral vector. Objective: Building KLF4gene overexpression lentiviral vectors toclear the effects of exogenous KLF4gene on malignant biological behaviorsand possible mechanism in gastric cancer cells in vitro and in vivo.Methods: β-catenin mRNA and protein expression were detected inparental and KLF4over-exprssion gastric cancer cell-lines byRealTime-PCR and western blotting, respectively; cell cycle andproliferation levels were detected in parental and KLF4over-exprssiongastric cancer cell-lines by cytometry; protein of Bcl-2, Bax, β-catenin,KLF4and E-cadherin were analysed by western blotting; the capability ofinvasion and migration were detected in gastric cancer cell-lines MKN-45and45-KLF4by Transwell; the construction of nude mice transplatationmodal confirmed the results in vitro, protein of Bcl-2, Bax, β-catenin,KLF4and E-cadherin were detected in nude mice transplatation modal bywestern blotting and immunohistochemistry.Results: To compared with the parental gastric cancer cell-lines,β-catenin mRNA and protein expression were decreased sharply in KLF4over-expression cell-lines (P<0.05); To compared with the parental gastriccancer cell-lines MKN-45, the proliferation speed of45-KLF4cells weredecreased, and G1/S ratio was increased (P<0.05); the apoptosis rate of cellincreased (P<0.05); the ability of invasion and metastasis in45-KLF4cellsdecreased (P<0.05); colony forming ability decreased (P<0.05). Tocompared with the parental gastric cancer cell-lines MKN-45, the growth speed of45-KLF4cells in nude mice transplatation modal was decreased,and the volume was smaller; the volume of experimental group was(464.4±12.5)mm3, but the volume of control group was (1161.0±41.5)mm3(P<0.05) after injection in twenty-first day; the weight of experimentalgroup and control group were (348.46±50.76)mg,(924.0±151.78)mg,respectively (P<0.05). The proteins of Bcl-2, β-catenin, Bax andE-cadherin were detected by western blotting and immunohistochemistry innude mice transplatation modal; to compared with MKN-45cells group,decreased expression of Bcl-2and β-catenin proteins (P<0.05), increasedexpression of Bax and E-cadherin proteins (P<0.05) were discoved in45-KLF4cells group.Conclusion: KLF4inhibits β-catenin expression and regulatesβ-catenin-mediated biological behaviors of gastric cancer cells. Decreasedproliferation of gastric cancer cell-lines was related to increased Bcl-2expression; inducitive apoptosis of gastric cancer cells was associated withincreased Bax expression; decreased capability of gastric cancer cells wasrevelant to E-cadherin expression. Modulation of KLF4expression mayrepresent a novel therapeutic approach for β-catenin-driven malignancies.
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