| The development and metastasis of tumor is a multi - step process and involves a lot of genetic alterations. As a cell adhesion molecule, E - cadherin is capable of maintaining the polarity of epithelial cells and cell junction. De-cressed expression of E - cadherin has been regarded as one of the main molecular events involved in dysfunction of the cell - cell adhesion system, triggering cancer invasion and metastasis. Loss of E - cadherin expression or function is observed in many gastric cancer cells. A direct role for E - cadherin in the suppression of tumor invasion has been demonstrated by the reversion of undifferenti-ated, invasive tumors to a differentiated phenotype after the transfection of E -cadherin cDNA in cell culture models. To further explore whether E - cadherin contributed to the invasion, RNAi was performed to investigate changes in invasion capacity of cancer cells by inhibiting expression of E - cadherin in MKN45 cell line.Materials and metoedsGastric cancer cell line MKN45 (derived from poorly - differentiated ade-nocarcinoma) was a kind gift from Professor Yokoyama, Japan Physical and Chemical Institute.siRNA according to 5'non - coding or coding sequence of E - cadherin was designed and the inhibition of siRNA was analyzed. 21 - nuecleotide dssiRNA was synthesized by in vitro transcription with Ambion SilencerTM siRNA Construction Kit. siRNA was transfected into MKN45 cells using TransMessengertransfection Kit. RT - PCR and immunofluoresent assay were used to investigate the inhibition of the expression of E — cadherin . Invasive capacity of the cells was determined by Transwell assay.ResultsThe results showed that the synthesis of E - cadherin mRNA rather than protein expression was suppressed dramatically after 7 days of interference. On the 11th day of interference, E - cadherin protein expression was downregulated and invasive ability of cancer cells was upregulated. Cells penetrating filter membrane were counted before (51±6) and after interference (78±8). The difference between two groups was significant( p <0.05) , which indicated that siRNA targeted at non — coding and coding sequence of E — cadherin showed significant inhibition on both mRNA and protein expression. Inhibited E - cadherin expression resulted in increased invasion ability of cancer cells, which suggested E - cadherin can inhibit tumor metastasis.ConclusionE - cadherin gene is capable of inhibiting the metastasis of gastric cancer by siRNA.Gastric cancer is a common human malignancy and remains an important cause of death in China. Although many genes were to be related to gastric cancer, such as P53 , APC , MCC , ERBB2 , RB , P16 , P21 , P27 , FHIT , PTEN , CD44 , CDH 1 etc, the molecular mechanism of gastric cancer was far from clear. Our previous study displayed that DICER 1 gene were down - regulated in gastric paracancerous, cancer tissue and lymph node metastases compared to normal tissue by cDNA chip. It implied that DICER 1 gene is one of important genes related to gastric carcinogenesis and progression.The RNaseIII - containing enzyme, DICER 1, plays a central role in the RNA interference machinery, which is associated with epigenetic regulation. DICER 1 expresses in many tissues intensively, the function of which contributes to processing micro RNA precursors or double stranded RNA into mature effector micro RNA (miRNA) or small, 21 24nt, short interfering RNA(siR-NA) molecules. DICER is also required for development and participates in multiple, fundamental biological processes in a mammalian organism, ranging from stem cell differentiation to the maintenance of centromeric heterochromatin structure and centromeric silencing. Gastric cancer originates from abnormal differentiation of proliferation. If hyperproliferation is accompanied by downregula-tion of DICER 1, the cells will ultimately lead to stomach cancer. In order to explore the suggestion, the function of DICER 1 gene in gastric cancer was analyzed by RNAi in the sudy.Material and methodsTissues and cell lineAll gastric cancer tissue and matched normal tissues confirmed pathologically were obtained from the first affiliated hospital of China Medical University. Gastric cancer cell line MKN45 was generously given by professor YokoYaMa, Japan physical chemical institute and pNeo - RNAi was kindly gifted by Dr. Song Jun, UCL, USA.MethodsExpressions of DICER 1 were detected by RT - PCR and immunohistochem-ical staining.pNeo - siRNA - DICER 1 was constructed and sequenced .Interference effects targeted at DICER 1 were analyzed by RT - PCR, Western Blot and Cell Immunofluorescent Chemistry.pNeo — RNAi — E - cadherin and siRNA - E — cadherin were transfected into DICER 1 - silenced MKN45 to study the processing function of DICER1.The survival rate of cells was measured by MTT assay and Colony - forming assay after RNAi targeting at DICER 1.Karyotypic analysis: For assessment of chromosome numbers, 50 meta-phase cells were analyzed.The total proteins of MKN45 cells were separated through 2D - electropho-resis after RNAi targeting at DICER1.ResultsExpression of DICER 1 gene in gastric tissues21 of 32 (65. 6% ) advanced carcinoma was down - regulated by RT -PCR. 13 of 33(39.4%) early gastric cancer and 19/30(63.3%) advanced gastric cancer were down - regulated by immunochemistry, which showed the expression of DICER 1 gene in gastric cancer was down - regulated.DNA of vector sequencingpNeo - RNAi - DICER 1 construct were confirmed by DNA sequencing.Effects of RNAi on DICER 1 expressionThe results showed obvious suppression of DICER 1 mRNA expression on the fifth, seventh or 12th d of interference, and suppression of DICER1 protein expression occurred clearly on the seventh or 12th d of interference compared to control group.The function of DICER1 enzyme processing shRNA into siRNAThe results showed significant inhibition of E - cadherin expression in MKN45 cells transfected by siRNA - E - cadherin and no difference in those transfected by pNeo - RNAi - E - cadherin compared to control group. E -cadherin was suppressed in unprocessed MKN45 cells ( DICER 1 expressing) with pNeo - RNAi - E - cadherin transfection.Results of MTT and Colony - forming assayMTT assay was performed to determine the cell growth rate. Cell growth rates were 15% and 24% (P <0. 05) respectively on the seventh d and 12th d of interference, which cell growth rates increased in cells in the absence of DIC-ER1.Colony - forming rate of MKN45 cells with DICER 1 expression (12. 3% ± 3.4% ) was lower than that without DICER 1 expression (20. 6% ± 5. 7% , P < 0. 01) on the 11th d of interference,Results of Karyotypic analysisThe main numbers of chromosomes focused on 40 -44(50) : 40 (1) , 41 (1), 42(7), 43(36), 44(5) in MKN45 cells, which showed hypodiploid. After interference, the karyotype of MKN45 cells changed to hyperdiploid and the chromosome numbers were 37-61(50) : 37(2),38(3), 39(4), 41(5), 42(5), 43(20), 44(5), 47(2), 48 (1) , 57(1) , 58(1) , 61(1).Results of 2D - electrophoresisResults of 2D - electrophoresis showed that the expression of total proteins in MKN45 cells markedly changed after DICER 1 was inhibited. 28 protein spots were up - regulated for 5 times or more and 8 protein spots were down - regulated for 5 times or more.Conclussion1. DICER 1 is one of important gastric cancer - associated genes.
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