The Study Of Effects And Mechanisms Of ShRNA Mediated Gene Silencing Of β-catenin On Biological Behaviors Of Esophageal Carcinoma Cells

IntroductionHuman esophageal carcinoma is one of the most aggressive malignant tumors with a generally poor prognosis.Esophageal squamous cell carcinoma(ESCC)is the most common subtype of esophageal carcinoma and has a higher incidence than esophageal adenocarcinoma in China.Despite significant advances in screening,surgical care,and chemoradiotherapy techniques,the prognosis of this disease is still poor, with an overall 5-year survival rate for patients with esophageal carcinoma of less than 10%.Therefore,identification of the genetic defects responsible for the development of esophageal carcinoma and the development of therapies that reverse these defects represent potentially useful adjuncts to the therapy of this disease.Wnt signaling pathway plays an important role in differentiation, morphogenesis and tumorigenesis.One of the key mediators of this pathway is B-catenin,which plays a pivotal role in Wnt signal transduction in addition to its function as a cell-adhesion component. Studies of ESCC have demonstrated that accumulation ofβ-catenin in cytoplasm and nucleus are frequent events during the carcinogenesis of the squamous epithelium of esophagus.By binding to T-cell factor-lymphoid enhancer factor(Tcf-Lef)family transcription factors,β-catenin could regulate transcription of a number of target genes such as c-jun,c-myc and cyclin D1 et al.So,the key step in activating the Wnt signaling pathway is the stabilization ofβ-catenin in cytoplasm and its translocation into the nucleus,which may serve as a potential target for esophageal carcinoma therapy. In recent years,RNA interference(RNAi)has emerged as a powerful method of gene therapy,and has been widely used for silencing malignant cellular and viral genes.In this study,we employed RNAi-mediated suppression ofβ-catenin gene to evaluate its effects on esophageal carcinoma cells growth and investigate its preliminary mechanisms.ObjectiveOur current investigation attempts to study the effects and mechanisms of short harpin RNA(shRNA)mediated gene silencing ofβ-catenin on biological behaviors of esophageal carcinoma cells and the role ofβ-catenin gene in esophageal carcinogenesis,and to provide theoretical and experimental evidences for the gene therapy of ESCC through specifically turning offβ-catenin gene.MethodsA single strand DNA was synthesized according to the hair loop RNA sequence,and subcloned into eukaryotic expression vector pGenesil-3 to construct a shRNA-expression pDNAs driven by human U6 promoter ofβ-catenin(pGen-3-CTNNB1),then transformed into DH5αcompetent cells.One additional construct of random siRNA (pGen-3-con)not homologous to any human genes was made in the same way as control.Positive clones were identified and verified by using restrictive cleavage and sequence.pGen-3-CTNNB1 and pGen-3-con were transfected into esophageal carcinoma cell line Eca-109 with liposome,respectively.Positive colonies were selected with G418.Expression of B-catenin protein and mRNA in the transfected and nontransfected Eca-109 cells was examined by Western blotting,immunofluorescence and RT-PCR,respectively.Cell growth ability of three different cells(pGen-3-CTNNB1, pGen-3-con and nontransfected Eca-109 cells)in vitro was evaluated by MTT and clone formation assay.Xenograft cancer model was used to compare the tumorigenicity of three different cells.Tumor volume curves were generated with data from 5 mice in three different groups,and the weights of tumor were measured after termination of mice.Expressions ofβ-catenin and PCNA in all tumor tissues were examined by immunohistochemistry(IHC)staining.Cell cycle and apoptosis were evaluated by analyzing tumor cells DNA content by PI staining and Flowcytometry.Transmission electron microscopy(TEM)was used to examine the changes of ultrastructure in three different cells.Expression of Cyclin D1 protein and mRNA in three different cells was detected by Western blotting,real-time and RT-PCR, respectively.We investigated the changes of the PCNA and ERK1/2 phosphorylation state by Western blotting.PCNA Label index(PCNA-LI) was also determined by immunocytochemistry staining.IHC staining was used to examine the expression ofβ-catenin protein in 65 cases of formalin-fixed,paraffin-embeded ESCC tissue containing area of tumor(n=65)and normal esophageal mucosa(n=20).RT-PCR was used to determine the expression ofβ-catenin in 31 cases of fresh ESCC tissue containing area of tumor(n=31)and normal esophageal mucosa(n=31).Resultsβ-catenin expression levels were found to markedly decrease in Eca-109 cells transfected with pGen-3-CTNNB1.Down-regulation ofβ-catenin was accompanied with growth inhibition of tumor cells and reduced colony formation of Eca-109 cells in vitro.In vivo,transfection withβ-catenin siRNA greatly impeded tumor growth. IHC staining showed significantly decreased expression ofβ-catenin and PCNA protein inβ-catenin siRNA transfected cells than in random siRNA transfected and nontransfected cells.By analyzing tumor cells DNA content by PI staining and Flowcytometry,we found that there was an increased fraction of cells in the G0/G1-phase and subsequent decrease in S-phase after down-regulatingβ-catenin expression,but results failed to reveal a significant differences in apoptosis.TEM detection revealed that the ultrastructure of Eca-109 cells had undergone a significant change after being transfected with pGen-3-CTNNB1,the volume of nucleolus and the nucleo-cytoplasmic ratio lessened,the nuclear shape became regular,euchromatin in nucleus decreased while heterochromatin increased,and more cell organelle appeared.The expression ofβ-catenin downstream target gene cyclin D1 was decreased,and simliar to cyclin D1,the expression of PCNA and ERKI/2 phosphorylation state was also down-regulated in pGen-3-CTNNB1 transfected cells.At the same time,PCNA Label index decreased accordingly.Immunohistochemistry showed 41 tumor samples(63.1%)had cytoplasmic accumulation ofβ-catenin and 20 cases(30.8%)had nuclear staining.The cytoplasmicβ-catenin was significantly associated with lymphatic node metastases(P=0.005),but not with the patient's age, gender,tumor grade or differentiation grade.RT-PCR showed expression ofβ-catenin mRNA was elevated in 67.7%(21/31)of ESCC compared to adjacent normal esophageal mucosa. Conclusion1.For the first time,we demonstrated that there existed abnormal expression ofβ-catenin and activation of Wnt signaling pathway in human esophageal carcinoma cell line Eca-109.RNAi plasmid targeted againstβ-catenin,pGen-3-CTNNB1,was successfully constructed and transfected into Eca-109 cells,and cell model steadily suppressingβ-catenin was obtained.2.We established successfully Xenograft cancer model ofβ-catenin steadily suppressed and demonstrated for the first time that RNAi plasmid targeted against B-catenin could obviously suppress the proliferation of Eca-109 cells in vitro and growth of xenograft tumor in nude mouse.3.Inhibition of tumor cells proliferation by down-regulatingβ-catenin expression could improve cell ultrastructure by mediating blockade in the G0/G1 through inhibiting DNA replication(PCNA)and MAPK pathway(p-ERK).These findings suggested that down-regulatingβ-catenin expression might reverse partly malignant phenotype of Eca- 109 cell.4.There exists an abnormal expression ofβ-catenin in ESCC,which may be associated with the carcinogenesis and progression of esophageal carcinoma.