The Functional Mechanism Of STC2 In The Interactive Regulatory Network Of Transcription Factors Nrf1 And Nrf2

Cancer ranks as a leading cause of death and an important barrier to increasing life expectancy in every country of the world^[1].However,China is a populous country^[2]and the proportion of aging population is gradually increasing.In the future,China’s cancer burden will further increase.Liver cancer is a deadly malignancy with poor prognosis^[3].Because the mechanism of the development of liver cancer is not clear,as well as the lack of effective biomarkers,the prevention and treatment of liver cancer has been slow,and the mortality rate of liver cancer has been high for a long time.With the advancement of liver cancer research,new problems are constantly discovered.For example,the knockout of Nrf1a（NFE2L1）in Hep G2 promoted the growth of bearing-tumor in nude mice,while the knockout of Nrf2（NFE2L2）of the same family significantly inhibited the growth of bearing-tumor^[4].To clarify the reason for this difference May contribute to understanding of liver cancer.TCGA contains a variety of data on various human cancers and is an important database in cancer research.Therefore,we will use the useful information of liver cancer data mining in TCGA to explore the reasons for the difference between Nrf1a knockout and Nrf2 knockout,and to explore the potential biomarkers of liver cancer,which may help the prevention and treatment of liver cancer.We use the liver cancer（LIHC）transcriptome data in the TCGA database as a starting point to explore the difference in gene expression between liver cancerous tissues and normal liver tissues,explore potential biomarkers of liver cancer,and establish a prognostic model of liver cancer.Then combined with the clinical data of liver cancer to explore the difference between the differentially expressed genes（DEGs）caused by Nrf1a knockout or Nrf2 knockout in Hep G2.The prognostic model was used to predict the risk changes of Nrf1a knockout and Nrf2 knockout in Hep G2,and then the relationship between Nrf1/Nrf2 and STC2,a potential biomarker of liver cancer,was explored.Finally,CRISPR/Cas9 technology^[5]and lentiviral technology were used to establish a Hep G2 cell line with STC2 knockout or stable overexpression.Through cell experiments,animal experiments and RNA-Seq technology,the function of STC2in liver cancer cells was thoroughly and comprehensively explored.The research got the following results:（1）After cancer,the cell division ability is stronger,and the risk of chromosomal aberration is higher;at the same time,it disturbs the normal liver function;and finally leads to the malignant proliferation and heterogeneity of liver cancer.The DEGs caused by the knockout of Nrf1a in Hep G2 are more in line with the trend of cancerous genes than the DEGs caused by the knockout of Nrf2.The COX model based on TCGA-LIHC predicts that the risk of Nrf1a knockout in Hep G2 is significantly higher than that of Nrf2 knockout.STC2 is a biomarker of liver cancer.It is up-regulated in liver cancer tissues,which can reduce the overall survival rate of patients.The rapid growth of bearing-tumor of Hep G2 with Nrf1a knocking out may be caused by the high expression of STC2.（2）The up-regulation of STC2 in Hep G2 with Nrf1a knocked out is mainly caused by Nrf2;however,the increasion of STC2 in(ca Nrf2^（35）N),Hep G2 cells with knocked out of KEAP1 binding site on Nrf2 protein,is caused by both Nrf2 and HIF1A,and Nrf2can indirectly promote STC2 expression in a way independent of HIF1A.STC2 may negatively regulate KEAP1 to promote the function of Nrf2,and it can also positively regulate Nrf2 in a manner independent of KEAP1;these functions of STC2 may be mediated by calcium ion signals.（3）Knockout or stable overexpression of STC2 in Hep G2 cells can slightly enhance the migration ability;STC2 promotes the clonal formation ability of liver cancer cells and the growth of bearing-tumor in nude mice.（4）Transcriptome analysis found that after knocking out or overexpressing STC2,differential genes were enriched in various membrane components,extracellular matrix,protein binding,various enzyme activities,angiogenesis,MAPK pathways,cancer-related pathways,and metabolic pathways.Therefore,the secreted protein STC2can promote the malignant proliferation of liver cancer through two aspects;on the one hand,STC2 affects the protein structure and enzyme activity by regulating the ion level of cells or the body;On the other hand,by binding to receptors to conduct signals,it affects itself or other cells.（5）The protein with the smallest molecular weight of STC2 is not translated from the first AUG;the translation start site of this small protein may be before the 268th base of the corresponding CDS region.