The Study Of RhoA-GTP Signaling Pathway Activation Manners By Galectin-3and Phosphatase Of Regenerating Liver-3in Lung Adenocarcinoma Cell Line

Objective:To investigate the possible mechanism of interaction between Galectin-3and Phosphatase of Regenerating Liver-3during activition of RhoA-GTP signaling pathway by molecular biology techniques, analysis and verify these protein molecules by functional experiments in lung adenocarcinoma cell line. Our study will contribute to better understanding of the molecular mechanisms involved in lung adenocarcinoma and be helpful to development of new approaches for cancer molecular therapy.Methods:1. Gal3-overexpressed LTEP-a2lung adenocarcinoma cell lines was constructed using GV208lentiviral vector containing Galectin-3mRNA exon fragment.2. lipid-mediated transfection with Lipofectamine2000Transfection Reagent and targeting specific siRNA were used to transfect LTEP-a2and A549cell lines, expression of Galectin-3, PRL-3and RhoA were blocked respectively in these cells.3. The varying mRNA levels of Galectin-3, PRL-3and RhoA were assessed by one-step Real-time Quantitative Reverse Transcription-Polymerase Chain Reaction and further protein expression levels were assessed by Western blot.Results:1. Western blot and RT-qPCR results showed that after specifically blocking Galectin-3, expression levels of PRL-3and RhoA were significantly down-regulated (P<0.01), indicate that PRL-3and RhoA were positively correlated with expression level of Galectin-3.2. Single-blocking PRL-3resulted in decrease level of RhoA but not Galectin-3, implying that PRL-3regulated expression of RhoA directly and Galectin-3might had an indirect mechanism.3. Expression of PRL-3and RhoA were significantly up-regulated in LTEP-a2cell line after transfected with GV208-Gal3vector, the results further confirmed Galectin-3had positive effection on the two downstream effector proteins.4. RhoA was down-regulated after block PRL-3with specific siRNA in Gal3-over-expressed LTEP-a2cells, but the reduction rate of RhoA is less than PRL-3.Conclusions:1、These results suggest that Galectin-3palys important roles during activation of RhoA-GTP signaling pathway including direct activation manner and indirect manner through PRL-3, however, there might be some indirect ways activating RhoA through unkonwed molecules, we would find more evidences in further studies.2. Interaction between Galectin-3and PRL-3involved in proliferation, invasion and metastasis of lung adenocarcinoma, these biological effects related to activation of RhoA-GTP signaling pathway.3. Blocking expression of Galectin-3and PRL-3, interfering their multiplex effects and RhoA-GTP deactivation method might be new areas in lung adenocarcinoma therapy and have potential application prospects. Further studies would be helpful to finding new biological therapy targets, development of new approaches for cancer individual diagnosis and personalized molecular therapy.