Detection On Location Of RhoA Protein In Gastric Cancer Cells And The Effect Of Respective And Associative Action Of CAMP And LPA On The Expression And Activity Of PTEN

ObjectiveTo observe the effect of different fixatives on the location of RhoA protein and find the optimum fixative for the localization of RhoA in SGC7901 cells by immunofluorescence microscopy. In SGC7901 gastric cancer cell, observe the effects of respective and associative actions of cAMP and LPA on the expression of PTEN gene in both mRNA and protein levels and the activity of PTEN protein. To elucidate how the crosstalk between cAMP-PKA mediated signaling and LPA-RhoA mediated signaling influence the expression of PTEN gene and the activity of PTEN protein.Methods(1) Cell culture(2) Fixation of the cells with different fixatives such as 2% paraformaldehyde, 4% formaldehyde, methanol acetone, and 10% trichloroacetic acid and observe the location of RhoA protein by immunofluorescence microscopy.(3) Seed the cells to six well plate. After the respective and associative action of cAMP and LPA on SGC7901 gastric cancer cell, extract RNA and then detect the mRNA expression of PTEN gene.(4) Seed the cells to six well plate. After the respective and associative action of cAMP and LPA on SGC7901 gastric cancer cell, detect the protein expression of PTEN gene by Western blotting.(5) Seed the cells to six well plate. Select one well as control, the other wells were treated with LPA on different acting time point such as 15min,30min,45min,60min and treated with cAMP in advance then treated with LPA for 30min. The change of phosphorylation of Akt protein was detected by Western Blotting.Results(1) Fixation by 2% paraformaldehyde without TritonX-100 treatment showed accumulation of RhoA in nucleus but little distribution in cytoplasm. After penetrating with TritonX-100, RhoA protein could not be detected in cytoplasm and was more accumulated in nucleus. Cells fixed by 4% formaldehyde without TritonX-100 penetrating showed nuclear distribution of RhoA. After penetrating with TritonX-100, the staining became clearer. Fixation of SGC7901 cells with methanol or acetone without TritonX-100 penetrating showed that RhoA distributed mainly in the cytoplasm. After penetrating with TritonX-100, RhoA staining could be seen in nucleus and became weaker in cytoplasm. In addition, in cells fixed by 10% TCA without TritonX-100 penetrating, RhoA was stained blurredly and could be seen in both cytoplasm and nucleus. After TritonX-100 penetrating, the staining became clear and the distribution was reduced in cytoplasm and increased in nucleus.(2) There is no influence on the mRNA expression of PTEN gene by the action of LPA. The expression of PTEN could be decreased slightly by the action of cAMP, it could also be decreased by the action of cAMP plus LPA.(3) There was no influence on the protein expression of PTEN protein by cAMP or LPA treatment.(4) Phosphorylation level of Akt was increased by the action of LPA alone. Action by Y-27632 before LPA treatment blocked the rising up of the level of p-Akt, so does cAMP.Conclusion(1) Different fixatives and penetrating with TritonX-100 or not are important for localizing RhoA protein by immunofluorescence microscopy.(2) Action by LPA can't change mRNA and protein expression of PTEN gene. Using cAMP can decrease mRNA expression of PTEN gene, but can't influence the protein expression of PTEN. Activated RhoA increases the phosphorylation level of Akt by decreasing the activity of PTEN.The cAMP-PKA mediated signal transduction can block activation of PTEN caused by LPA-RhoA mediated signal transduction.