Gene specific silencing of mesothelin reduces proliferation of mesothelioma cells.

<p>(A)Anti-mesothelin siRNA was designed to a middle sequence position in mesothelin mRNA. (B)Once electroporated with anti-mesothelin siRNA, the expression levels of mesothelin was significantly reduced in H2373 cells. Negative control siRNA did not cause such reduction. Lower panel shows the results of band-densitometry comparing the intensity of mesothelin expression upon electroporation of H2373 cells with siRNA. (C)Anti-mesothelin siRNA did not affect the expression levels of β-actin, a house-keeping protein, as an evidence for the specificity of this anti-mesothelin siRNA for its target. (D) Proliferation rate of H2373 cells is significantly (p<0.05) reduced at 48 hours post-electroporation to 40% of the values for negative control treated cells. A rebound to higher proliferation rates is observed due to clearance of siRNA from cells at later time points in harmony with our previous studies. Callout panels show the density of cells in each group of the study at 48 hours post-electroporation. (E) NIH3T3 cells are void of mesothelin and their proliferation rate is not affected by exposure to anti-mesothelin siRNA (mouse). Callout panels show the density of cells at 48 hour post-electroporation.</p

Mesothelin silencing reduces proliferation rate of pancreatic and ovarian cancer cells.

<p>(A)Mesothelin protein was detected in pancreatic cancer cell lines, Panc1, Miapaca2 and Bxpc3 and ovarian cancer cells Skov3 and Ovcar3. NIH3T3 and Huvec cells which are void of mesothelin were used to prove the specificity of mesothelin antibody. (B)Skov3 cells had reduced proliferation at day 3 post-electroporation with anti-mesothelin siRNA to about 50% of negative control. Callout panels show the density of cell at each time-point. (C–D)Two pancreatic cancer cell lines, Bxpc3 and Miapaca, were tested for the outcome of silencing mesothelin on their proliferation. In both cases a significant loss of proliferation was observed, however for Bxpc3 the decline initiates at later time points as compared with Miapaca cells. For both cells, once again, a rebound to higher proliferation rates is observed at longer time-points due to the clearance of siRNA from cells. Callout panels show the density of cell at each time-point.</p

Silencing MSLN confers inhibited cell proliferation and invasion.

<p>(A) Ovca429 cell were infected with MSLNmiR3 or Negative Control virus at MOI∼30. Cell proliferation was assessed at the indicated time-points. (B) Photographs of Ovca429 cells expressing EmGFP post-infection at the indicated time-points. 10× objective was used for taking pictures at day 10 and 20× was used at day 20 in order to focus on morphological changes of cells. (C) Left panel: Three-day post infection, Ovca429 cells were transferred and cultured in Boyden assay invasion chambers for 21 hrs. Cells were washed, fixed, and cell nuclei were stained by DAPI. Representative fields from each group were then counted for the number of nuclei and averages were calculated and compared (p<0.05). Numbers of cells invaded for the negative control virus was considered as 100%. Right panel: Photographs of invaded cells nuclei in MSLNmiR3 and negative control virus treated groups. The bar shows 100 µM in length.</p