Mechanism Of GRP78 Promoting Proliferation,Migration And Invasion Of Hepatocellular Carcinoma

Objective: The 78 KDa glucose-regulated protein GRP78 play key role in the development of tumors.Based on the overexpression of GRP78 in Huh7 and Hep1-SK cells,the expression of GRP78 on proliferation,migration and invasion of HCC was investigated.Furthermore,according to the structure of GRP78 protein,the molecular mechanism of GRP78 promoting the proliferation,migration and invasion of hepatoma cells was discussed,which laid a foundation for further study of GRP78 as a therapeutic target for liver cancer.Moreover,the anti-GRP78 rabbit polyclonal antibodies and ascites monoclonal antibodies were prepared and characterized successfully,and a double-antibody sandwich ELISA method was established to detect GRP78 protein..Methods:(1)RT-PCR and Western blot were utilized to verify the overexpression of GRP78 recombinant adenovirus in Huh7 and Hep1-SK cells by mRNA and protein levels,and the interference effect of GRP78 siRNA.(2)The effects of GRP78 overexpression and interference on theproliferation,migration and invasion of HCC were detected by long-term dynamic cell imaging and data analysis system.(3)Through the nude mouse tumor formation model,the effect of GRP78 overexpression on tumor growth was confirmed in vivo.(4)GRP78 signal peptide deletion adenovirus,site mutants in ATPase domain T38 A,T229A,and S300 A mutant adenovirus,and site mutants in peptide binding domain,R492 E and Y570 F adenovirus were constructed successfully,and overexpression of mutant adenovirus were confirmed by sequencing and Western blot.(5)The effects of GRP78 signal peptide deletion,GRP78 ATPase mutant and GRP78 peptide domain mutant on the proliferation,migration and invasion of HCC were detected by long-term dynamic cell imaging and data analysis system.(6)GRP78 rabbit polyclonal antibody and ascites monoclonal antibody were successfully obtained with high potency,sensitivity and specificity.And a double antibody sandwich ELISA system was established preliminarily..Results:(1)The effect of GRP78 overexpression and GRP78 siRNA interference were successfully verified in both Huh7 and Hep1-SK cells.(2)After overexpression of GRP78 in Huh7 and Hep1-SK cells,the proliferation,migration and invasion of HCC were significantly enhanced.When Huh7 and Hep1-SK cells were transfected with GRP78 siRNA,the proliferation,migration and invasion of HCC were inhibited.(3)In vivo,the nude mice model further confirmed that the overexpression of GRP78 promoted the growth and proliferation of liver cancer.(4)According to the spatial structure and functional domain of GRP78 protein,GRP78 signal peptide deletion adenovirus,GRP78 protein ATPase domain T38 A,T229A,S300 A mutant adenovirus,GRP78 protein peptide domain R492 E,Y570F mutation adenovirus were designed and successfully constructed.The adenovirus were tested and the overexpression effect was verified in the liver cancer cell line.(5)Long-term dynamic cell imaging and data analysis system was used to detect the proliferation and migration of liver cancer cells what GRP78 protein functional structure mutant ATPase domain mutant T229 A inhibited,while GRP78 signal peptide deletion,and other ATPase structures Domain mutants,peptide domain mutants had no effect on the tumorigenic effect of GRP78.(6)Eight mouse monoclonal antibodies against GRP78 protein and two rabbit polyclonal antibodies were successfully prepared and purified,and a double-antibody sandwich ELISA immunoassay for detecting GRP78 protein was successfully established.Conclusion: Both in vitro and in vivo experiments showed that the proliferation,migration and invasion of hepatoma cells after overexpression of GRP78 were enhanced,and the proliferation and migration ability of HCC were weakened after interfering with theexpression of GRP78.It was concluded that GRP78 promotes liver cancer and played an important role in the growth and development of HCC.At the same time,From the perspective of GRP78 protein functional structure,the specific functional regions of GRP78 that play a role in promoting liver cancer were explored.We had taken the conclusions as follows: Site mutants in GRP78 peptide binding domain R492 E and Y570 F mutations had no effect on GRP78 tumor-promoting function;and Site mutants in GRP78 ATPase domain T38 A and S300 A site mutations also had no effect on GRP78 tumor-promoting function.While,Site in GRP78 ATPase domain T229 A after point mutated,the activity of both Huh7 and Hep1-SK cells were significantly reduced,which affected its tumor-promoting function.