Function Of MAP3K7(TAK1) Isomers In Lung Adenocarcinoma

Objective: Lung cancer is the second largest cancer threatening human health,ranking the first among male patients and the third among female patients.The overall five-year survival rate is 45%,and the mortality rate ranks the first.In-depth research on the pathogenesis of lung cancer is urgently needed to provide a new direction for the precise targeted treatment of clinical lung cancer.MAP3K7 is a member of the MAPK kinase kinase(MAPKKK)family,which can be activated by various extracellular stimuli.Its downstream can activate the MAPK cascade(ERK,JNK,P38MAPK)and NF-κB survival pathway,as well as cell death(apoptosis,pyrodeath,necrotic apoptosis)pathway.MAP3K7 is one of the key genes that control cell survival and death.MAP3K7 as an oncogene in lung cancer,MAP3K7 inhibition in various ways has shown good therapeutic effect in the preclinical studies of lung cancer treatment,but its non-specificity and side effects limit its clinical application.MAP3K7 has four RNA splicing isomers,whose functions have been less studied.MAP3K7 can promote cell survival and cell death,and its "contradictory" functions make us focus on the differences in the functions of its isomers.In this study,we investigated the functional differences of MAP3K7 isomers in lung adenocarcinoma(the most common histological type of lung cancer)cells,providing new information and ideas for the targeted treatment of lung adenocarcinoma with this gene.Methods: Firstly,RT-QPCR and RT-PCR were used to detect the expression level of MAP3K7 total RNA and the splicing changes of MAP3K7 RNA in 52 lung adenocarcinoma clinical samples and their adjacent tissues in 2018.Immunohistochemistry(IHC)assessed the total amount of MAP3K7 protein from clinical samples in 2021 while MAP3K7 isomer protein from clinical samples in 2019 was assessed by Western Blot.Then,lung adenocarcarcinoma cells A549(TP53+)and H1299(TP53-)were infected with lentivirus expressing different isomers of MAP3K7 with 3FLAG tag.The in vitro proliferation,clonformation,migration,and invasion ability of lung adenocarcinoma cells were detected using CCK-8(Cell Counting Kit-8),plate cloning,Transwell without or with matrix glue,respectively.Tumor formation experiments were performed in nude mice,and the activation of downstream signaling pathways was detected by Western Blot.Precipitation of the MAP3K7 isomers and its binding partner TABs was investigated using Immunoprecipitation(IP).After overexpression of 3flag-tagged MAP3K7 splicing factor(predicted by website combined with experimental screening),the in vitro proliferation,clonogenesis,migration and invasion of lung adenocarcinoma cells,as well as their xenograft formation ability in nude mice were detected.Finally,RNA Sequence was used to detect the potential of overexpressed MAP3K7 isomer d as a targeted therapy for lung adenocarcinoma.Results: In 52 clinical samples of lung adenocarcinoma,the transcription level of MAP3K7 was unchanged in cancer tissues,but the proportion of RNA splicing isomers was different,and the proportion of b isomers was increased in cancer tissues compared with paracancer tissues.MAP3K7 protein expression was elevated in clinical samples,and the prognosis was poor.Compared with the normal lung epithelial cell line(BEAS-2B),the transcription level of MAP3K7 was not increased in lung adenocarcinoma cell lines,but the total protein of MAP3K7 was increased.The expression of endogenous MAP3K7 protein in two lung adenocarcinoma cell lines was not successfully down-regulated by sh RNA.Overexpression of four isomers of MAP3K7 in two lung adenocarcinoma cell lines(A549 and H1299)resulted in different malignant phenotypes.In A549(TP53+)cells,isomer b enhanced the phosphorylation of ERK and JNK and maintained the phosphorylation of e IF4 E to increase cell migration,invasion and the formation of transplanted tumor.In H1299(TP53-)cells,isomer b enhanced the phosphorylation of ERK and JNK better than isomer a.However,isomer a can increase the ability of cell migration,invasion and tumor formation.Isomers c and d(especially d)failed to activate the MAPK pro-survival signaling pathway,which significantly reduced the proliferation,clonogenesis,migration,invasion and transplantation tumor formation of the two lung adenocarcinoma cells.Different isoforms of MAP3K7 form different activation complexes with TABs.Three splicing regulatory factors of MAP3K7 were screened by website prediction combined experiment: SLM2 and SAM68 promoted the removal of its variable exon 12 and 16,and hn RNPF inhibited the removal of its variable exon 12.Overexpression of the three splicing regulatory factors changed the RNA splicing changes of endogenous MAP3K7 in lung adenocarcinoma cells,but did not cause activation of the MAPK pro-survival signaling pathway,and greatly reduced the proliferation,clonogenesis,migration,invasion and transplant tumor formation of the two cells.Compared with hn RNPF,SLM2 and SAM68 showed stronger ability to reduce malignant phenotype.RNA transcriptomic sequencing results of overexpression of MAP3K7 isomer d showed that MAP3K7 isomer d down-regulated the RNA levels of proto-oncogene Jun in two lung adenocarcinoma cell lines,which provided the basis for MAP3K7 targeted therapy for lung adenocarcinoma.Conclusion: The elevated translation level of MAP3K7 in lung adenocarcinoma is the main cause of carcinogenesis,and the prognosis is poor.Isomer b increased the malignant phenotype of cancer cells in A549 cells,but isomer a increased the malignant phenotype of cancer cells in H1299 cells.Different isoforms of MAP3K7 form different activation complexes with TABs.Overexpression of splicing regulators of MAP3K7 variable exons 12 and 16 can reduce the malignant phenotype of cancer cells.Increasing the proportion of isomer d has the potential to target lung adenocarcinoma with MAP3K7.