| Objective: Salivary adenoid cystic carcinoma (SACC) which can occurin any location of salivary glands is one of the most common malignancies.The tumor was usually with high malignancy, invasive features and growthalong nerves, blood vessels, and common recurrence after surgery. Metastasisof SACC can be involved in lung, bone, brain, and liver, but rarely in regionallymph nodes. It often threatens the survival of patients following its invasivegrowth.B cell lymphoma/leukemia-2(bcl-2) gene is an apoptosis-inhibitingoncogene, which has been intensively investigated among apoptosis-relatedgenes. Located in region21of chromosome18in normal B lymphocytes andhuman follicular lymphoma cells, bcl-2contains three exons and two introns,encoding two proteins, bcl-2α of26kD and bcl-2β of22kD. As an integrationprotein of mitochondrial membrane, bcl-2α plays a major role in the inhibitionof apoptosis. The gene of bcl-2was first found in human follicular lymphoma,and considered as a biomarker of human follicular lymphoma. However,following researches in a diversity of tumor tissues presented remarkablechanges of bcl-2gene expression, which suggested the application of bcl-2inoccurrence, development, and prognosis of tumors.RNAi (RNA interference) is used widely as a genetic technique, whichinduces degradation of mRNA being homologous to particular endogenous orexogenous double-stranded RNA (dsRNA), and thereby inhibits theexpression of corresponding gene. Characterized by high specificity, highefficiency, high stability, hereditary, diffusibility, dependence on time andconcentration, RNAi can specifically inhibit the expression of target genes inRNA level, so as to knock down the gene function, which makes RNAibecome a good tool to study gene functions.In this experiment, RNAi technology was used to specifically knock down bcl-2gene in SACC-83cells derived from human salivary adenoidcystic carcinoma, in order to explore the apoptosis and morphological changesof SACC-83cells after gene silencing.Method:1The construction and identification: Designed and constructedrecombinant adenovirus by the biotechnological company of Wuhan XiMaand identified by double digestion.2Cell culture: RPMI1640contained with15%fetal bovine serum wasused to cultivate SACC-83of human salivary adenoid cystic carcinoma cellline.3Cell transfection and MOI determination: SACC-83cells weretransfected with adenovirus-mediated shRNA eukaryotic expression vector,shRNA-bcl-2(silence group), to silence the expression of bcl-2gene, and withshRNA-HK (blank vector group) as negative control, according to the bestMOI (adenovirus: cell=50:1). SACC-83cells (non-transfection group) wereused as blank control.4Detection of bcl-2gene silencing efficiency: Real-Time PCR was usedto check bcl-2gene silencing efficiency. The expression of bcl-2gene inmRNA level was detected in three groups48h after transfection.5Detection of apoptosis: Using Flow Cytometry, apoptosis was detectedin three groups48h after transfection.Result:1Eukaryotic expression vector shRNA-bcl-2wrapped by recombinantadenovirus was successfully constructed.2The transfection efficiency of adenovirus-mediated shRNA eukaryoticexpression vector shRNA-bcl-2was98.18%, and the transfection efficiency ofshRNA-HK was94.10%.3Real-Time PCR showed that the silencing efficiency of bcl-2gene inSACC-83cells was90.19%after transfection by adenovirus-mediatedshRNA-bcl-2.4Flow Cytometry showed that cell apoptosis rate of SCCC-83-bcl-2was 8.45±0.25, SACC-83-HK was4.26±0.21, SACC-83was1.48±0.23(P＜0.05).Conclusion:1Adenovirus-mediated eukaryotic expression vector of shRNA-bcl-2caneffectively silence the expression of mRNA of bcl-2gene in cell lineSACC-83.2The expression of mRNA of bcl-2gene silenced can induce apoptosis ofSACC-83cells. |
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