Study On The Role Of Ubiquitin-ligase HECTD3 In Lung Adenocarcinoma Based On High Expression Of UBE2

Objective:By looking for the differentially expressed ubiquitin-conjugating enzyme(E2),the associated ubiquitin ligase(E3)was screened out to study the expression level and clinical significance of the E3 in lung adenocarcinoma,and to verify the role of this E3 in lung adenocarcinoma.Methods1.Through Oncomine,Ualcan,HPA,The Kaplan-Meier plotter[Lung],Cbioportal and GEPIA databases,we screened out the differentially expressed E2 and verified the clinical significance of this E2.The E2 was used to search for the neighbour genes that altered with it in several different studies,so as to determine which E3 needs to be studied.2.The clinical significance and function of the E3 ubiquitin ligase were verified from the public database and clinical specimens in our department.3.The expression of E3 in wild-type lung cancer cell lines was detected,and one of the cell lines was selected for lentivirus infection to construct the E3 overexpression stable cell lines and CRISPR/Cas9 knockout cell lines.4.CCK-8 assay,Transwell assay and Wound Healing assay were used to detect the effects of E3 on the proliferation,invasion and migration of lung adenocarcinoma.Results1.The mRNA and protein expressions of UBE2C and UBE2S in lung adenocarcinoma were higher than those in normal lung tissue and were related to the clinical stage of the patients.Higher UBE2C and UBE2S mRNA levels in patients with lung adenocarcinoma predicted worse overall survival and progression-free survival.The neighbour gene most frequently altered with UBE2C and UBE2S in lung adenocarcinoma is HECTD3.2.The mRNA and protein expressions of HECTD3 in lung adenocarcinoma tissue were significantly higher than those of normal tissue,and the expression of HECTD3 showed a trend of increase with the rise of stages.An overexpression of HECTD3 mRNA predicts worse overall survival(HR=1.65,95%CI:1.31-2.09)and progression-free survival(HR=1.68,95%CI:1.23-2.31)in patients with lung adenocarcinoma.Transcriptome sequencing results of clinical specimens showed that the expression of HECTD3 in lung adenocarcinoma tissues was higher than that in adjacent normal tissues,which was correlated with histological subtypes of lung adenocarcinoma,high-risk factors(Invasion of blood vessels and/or pleura?spread through air spaces and cancer embolus),and lymph node metastasis.3.The results of GO analysis suggested that HECTD3 was closely related to gene expression,protein metabolism,enzyme binding,protein complex binding,and ubiquitin protein ligase binding.KEGG analysis showed that HECTD3 was associated with pathways in cancer,p53 signalling pathway,prostate cancer,colon cancer,pancreatic cancer,thyroid cancer and apoptosis.4.The expression of HECTD3 was the highest in wild-type A549 cells.The mRNA and protein levels of HECTD3 were up-regulated in the constructed HECTD3 overexpression and stable transduction cell line.The HECTD3 protein was completely knocked out due to the insertion mutation of the 282 of exonl of the HECTD3 gene in the constructed stable transfer cell line of CRISPR/Cas9 knockout.5.The results of the CCK-8 experiment showed that the proliferation rate of wild-type A549 cells was 100%,47.6%in the HECTD3 knockout group and 125.8%in the HECTD3 overexpression group.The Transwell results showed that compared with wild-type A549 cells,the number of cells per field decreased significantly in the HECTD3 knockout group and increased significantly in the HECTD3 overexpression group.The percentage of wound healing distance between scratches in HECTD3 overexpression group was significantly higher than that in wild-type A549 cells.The percentage of wound healing distance between scratches of HECTD3 knockout group was significantly lower than that of wild-type A549 cells.Conclusion1.The expressions of UBE2C and UBE2S in lung adenocarcinoma were significantly up-regulated compared with the normal group.The expression of UBE2C and UBE2S was positively correlated with that of HECTD3,2.The expression of HECTD3 was significantly up-regulated in lung adenocarcinoma tissues compared with adjacent normal tissues and correlated with N stage,prognostic risk factors and pathological subtypes of lung adenocarcinoma patients.3.The overexpressed and stably transfected A549 cell lines of HECTD3 and the knockout and stably transfected A549 cell lines of HECTD3 were successfully constructed.4.HECTD3 promotes the proliferation,invasion and migration of lung adenocarcinoma cells in vitro.