Effects Of Mesothelin RNAi On Cell Growth Characteristics As Well As Adhesion, Invasion Ability In Human Ovarian Cell

Objective:Mesothelin is a tumour differentiation antigen that is normally present on the mesothelial cells lining the pleura,peritoneum and pericardium.It is,however,highly expressed in several human cancers including malignant mesothelima,pancreatic,and epithelial ovarian carcinoma(EOC). The MSLN precursor protein is cleaved by furin to yield the 40-kDa fragment ,mesothelin, attached to the cell membrane by a glycosyphosphatidy inositol linkage and a smaller 32-kDa fragment named megakaryocyte-potentiating factor, that is released from the cell. Analysis the gene expression profiles between EOC and normal ovarian tissue by the cDNA microarray, the result showes that the mesothelial which has potential biological markers up-regulated the expression significantly. Recent studies have shown that CA125 binds to mesothelin in a specific manner with the two-mediated interaction between the cell adhesion,which may play a role in the peritoneal spread of EOC suggesting that mesothelin may be a promising tumor markers and therapeutic targets. But, so far, the function study of the mesothelial is still limited to cell adhesion,and the knowledge about the other mesothelial-function is still very poor.RNAi can silence gene expression in long-term, that can be used for the study of gene function and gene therapy, and so on. Using lentiviral pRNAT-U6.2/Lenti vector was constructed five recombinant plasmid on preliminary studies, preparation of the recombinant plasmid cutting and sequencing proved successful reorganization. And packaging the virus particles,the virus particles were conformed to the following experiment.To test whether this RNAi strategy can silence MSLN gene expressed in many tumors, following investigations were conducted in this study.Methods:1 Observe the infection efficiency by the laser scanning confocal microscope after the SKOV-3 cells was infected with the virus which respectively carry pMSLN-negative, pMSLN-shRNA1, pMSLN-shRNA2, pMSLN-shRNA3, pMSLN-shRNA4 plasmid for 96 hours.2 Using Western blotting methods to detect the expression of mesothelin protein,and choosing the high-efficiency cells carried out the following experiment.3 Using indirect immunofluorescence staining to detect recombinant lentivirus-mediated RNAi of mesothelin expression.4 The proliferation of cells changes was evaluated by proliferation experiments and clone formation methods.5 Cell adhesion ability were examined by EDTA-induced cell detachment assay and cell adhesion test. 6 Cell invasion and migration assay to detect the invasion ability .Results:1 Using the laser scanning confocal microscope to observe the efficiency of infection,found that the infection rate was 80% -90%.2 Western blotting results showed that, compared with OVCAR-3 , OVCAR-3/neg, OVCAR-3/(shRNA1 ~ shRNA4) protein expression were reduced by 10%, 9%, 53%, 84%, 90%, that means the maximum interference efficiency is OVCAR-3/shRNA4, it is 90%.3 It can be seen that MSLN marked as red fluorescence, mainly distributed in the membrane, SKOV-3/shRNA group MSLN protein fluorescence intensity was significantly lower than SKOV-3, SKOV-3/neg groups.4 Cell proliferation results: The amount of cells were (9.89±2.0)×10~5,(18.9±2.24)×10~5,(19.81±2.5)×10~5,respectively, the results had a significant difference(F=29.76,P<0.05).The proliferating ability of SKOV-3/shRNA cells was significantly lower than the latter two groups (P<0.05), and the negative control group and the blank control group were no significant difference in the proliferation rate (P=0.531).Cloning experiment results:The colony forming rate were (15.85±2.5) %in interference group, (28.3±2.7)% in the negative control group , (29.13±1.98)% in the blank control group , the results had a significant difference (F=47.62 ,P<0.05).SKOV-3/shRNA colony formation was significantly lower than the negative control group (SKOV-3/neg) or the blank control group (SKOV3) (P<0.05). Negative control group (SKOV-3/neg) and the blank control group (SKOV3) between cloning was no significant difference (P=0.612).5 Adhesion testThe detachment rate of interference group (SKOV-3-shRNA) in time points was (13.86±1.05)%,(34.44±2.72)%,(56.71±3.4)% ,(83.76±4.37)%,respectively.They were significantly higher than that of the negative control group(SKOV-3/neg)and the blank control group(SKOV)(P<0.05), the negative control group and the blank control group were no significant difference in the cell detachment rate(P>0.05).Cell adhesion test shown that the adhesion rate were (12.12±2.21)% in interference group, (49.78±3.2)% in the negative control group(50.74±2.65)% in the blank control group , the results had a significant difference( F=327.87,P<0.05).SKOV-3/shRNA cell adhesion rate was significantly lower than that of the negative control group(SKOV-3/neg)and the blank control group(SKOV3)( P<0.05), the negative control group and the blank control group were no significant difference in the cell adhesion rate(P=0.587).6 Cell invasion and migration assay to detect the invasive ability of cellsThe cell counts in assay of invasive ability were(7.5±1.78), (27.54±3.39), (29±2.29),respectively. The results had a significant difference( F=208.96,P<0.05).There were significant differences compared the interference group with the latter two groups(P<0.05),while there was no difference between the latter two(P=0. 227).The cell counts of migratory test were (18.7±2.11), (44.7±4.57) and (47±4.14), respectively.The results had a significant difference( F=174.51,P<0.05).There were significant differences compared the interference group with the latter two group( P<0.05),while there was no difference between the latter two(P=0. 183).Conclusion:1 Virus vector systems can integrate the target cell genome effectively, and achieve stable expression, and have high transfection efficiency.2 Confirmed by Western blotting detection MSLN RNAi can effectively inhibit protein expression,and chang the characteristis of cell growth.3 MSLN RNAi can decrease cell adhesion, invasion and migration ability significantly. Mesothelin impact of the ovarian cancer cells and the basement membrane of tumor cell adhesion and migration function of the invasion.The above results are very helpful for the further study of MSLN function and treating the malignant tumor by blocking the mesothelin gene expression.