Study On The Mechanisms In The Development Of The GA6- Adjusted Autophagy In Human Cancer Research

Objective:Autophagy begins with the formation of an autophagosome, when the cells under the harmful stress, such as, lacking of growth factors, hunger, low oxygen, they will use the lysosome to damaged organelles itself or the macromolecular substances. Most of the long half-life protein in the cell protein could degradate by the autophagy pathway, so as to maintain stability in the cell, as well as a response to a wide range of stimuli that endanger the cell. Autophagy is a unique intracellular protein degradation phenomenon in eukaryotic cell and is also highly conserved in organic evolution. There is two types of programmed cell death, autophagy and apoptosis. Autophagy is proved to have a close relationship with tumor. GA6 is a relatively new gene, and its role in the tumor is in its infancy, the study of it is also very limited. Depending on GA6, The objective of these present studies towards autophagy related gene is to elucidate the molecular mechanisms of GA6 involved in crosstalk of signaling pathways in autophagy, simultaneously promoting new therapies able to clinical GA6 targeted drugs application.Methods:We use the growth-measuring, colony-forming, scratch test to verify GA6's effect on tumor cell growth, proliferation and invasion. Human coding region and the segments was amplified from human mammary gland cDNA by PCR. and was inserted into the prokaryotic expression vector pGEX-KG, pET-28a or GST to obtain the recombinant plasmids. Western blot to validate the effect of over expression and knock down GA6 to autophagy. Autophagy inductions were given to cancer cells in vitro, to observe the co-localization of GA6 and LC3B in cells through immunofluorescence experiments. Immunoprecipitation test to verify its interaction. The function of GA6, Atg4B, LC3B was detected by GST pull-down assay.Results:GA6 can promote the tumor cell growth, proliferation and invasion. It is essential to autophagy and has the co-localization with LC3B. Proteins of GA6 have interaction with both LC3B and Atg4B, The former is better than the latter. None interaction with P62?BNIP3?Atg3?Atg5?Atg7?Atg12. GA6 have competitive inhibition of the effect of Atg4B to LC3B.Conclusion:GA6 have the competitive inhibition of the effect of Atg4B to LC3B and hinder the process of LC3B precursor protein Pro-LC3B converting to LC3B-I, which is cleaved by Atg4B, thereby inhibiting autophagy, GA6 can promote tumor cell growth, proliferation and invasion, which is a cancer gene.