The Experimental Study On The Effect And Mechanism Of RNA-edited IGFBP7 In Promoting Progression And Angiogenesis In Lung Adenocarcinoma

Objective:Insulin-like growth factor binding protein 7(IGFBP7)is a secreted protein with multiple biological functions.At present,many studies have shown that IGFBP7can exert a tumor suppressor effect by inhibiting proliferation,inducing cell apoptosis or senescence,and inhibiting tumor angiogenesis.RNA editing is a widespread post-transcriptional modification mechanism.The most common type of RNA editing in humans is from adenosine to inosine catalyzed by the adenosine deaminases acting on RNA(ADAR)family.IGFBP7 is one of the genes edited by ADAR2,a member of the ADAR family.In lung adenocarcinoma,the edited level of IGFBP7 is as high as 10%,but the role of edited IGFBP7 in lung adenocarcinoma is still unclear.Therefore,this article intends to clarify the biological regulation of edited IGFBP7 on lung adenocarcinoma through in vivo and in vitro experiments.Methods:1.We used Western blot to detect the protein expression of IGFBP7 in lung adenocarcinoma cell lines.2.We constructed the stable control group,IGFBP7 wild type overexpression group and IGFBP7 edited type overexpression group of H1975with lentiviral transfection.3.Western blot was used to detect the protein expression of IGFBP7 in the stably transfection cells and the culture supernatant of cells.4.CCK-8,plate clone formation and flow cytometry were used to detect the effect of edited IGFBP7 on the proliferation and cell cycle of H1975.5.We used transwell and wound healing experiments to detect the influence of edited IGFBP7 on the migration of H1975.6.The chemotherapy drug paclitaxel was used to treat cells,and Western blot was used to detect the expression of cleaved-Caspase3 and cleaved-PARP.7.Using a nude mouse transplanted tumor animal model,we subcutaneously inoculated 3×10~6cells of H1975,and measured the tumor volume every two days after tumor formation.8.We used Micro-CT to detect the distribution of blood vessels in the tumor.9.We extracted the conditional medium(CM)of the control,IGFBP7 WT and edited groups,and cultured human umbilical vein endothelial cells(HUVEC)with CM.10.CCK-8was used to detect the effect of CM on HUVEC proliferation in each group.11.Transwell experiment and wound healing experiment were used to detect the influence of each group of CM on the migration ability of HUVEC.12.We used the tube formation experiment to compare the effects of CM on the tube formation of HUVEC.13.We subcutaneously inoculated 3×10~6 cells of H1975 and 6×10~5 cells of HUVEC together,and measured the tumor volume every two days after tumor formation.14.We used Micro-CT to detect the distribution of blood vessels in the mixed injection tumor.15.The expression of PEDF and VEGF-A in CM of each group was detected by ELISA.Results:1.Edited IGFBP7 promoted the proliferation of H1975.2.Edited IGFBP7promoted the migration of H1975 cells.3.Edited IGFBP7 reduced the sensitivity of H1975 cells to paclitaxel.4.The volume of transplanted tumor and the density of blood vessels in the edited IGFBP7 group were larger than those in the wild group.5.The CM of the edited IGFBP7 group promoted the proliferation,migration and angiogenesis of HUVEC.6.The volume of transplanted tumors which co-inoculated with H1975 of edited group and HUVEC was larger than the wild co-transplantation group and higher than that of the single-vaccination group.7.The density of blood vessels in transplanted tumors was higher in edited co-injection group than the wild co-transplantation group.8.The cells in the edited IGFBP7 group secreted higher levels of VEGF-A and lower levels of PEDF.Conclusion:RNA-edited IGFBP7 increased the proliferation and migration ability of H1975 cells,and reduced the sensitivity of H1975 cells to paclitaxel.RNA-edited IGFBP7 also promoted angiogenesis possibly by up-regulating the expression of VEGF-A and down-regulating the expression of PEDF.