Molecular Evolution And Proteomics Studies On The Regulation Of The Proliferation And Migration Of Non-small Cell Lung Cancer Cells By TRIM17

Background:Malignant tumors are one of the main causes of death in China.How to find effective drug targets for the treatment of malignant tumors is one of the challenges that people are still exploring.With the development of genomics sequencing technology,scientists have discovered that some mammals do not develop cancer including two long-lived hypoxic rodents,blind mole rats and naked mole rats,that live underground.This discovery has initiated the study on the cancer suppressing mechanism in anti-cancer species.Based on the current studies,most anti-cancer species are in hypoxic environment for a long time or periodically.However,at present,the research on the anti-cancer mechanism of mammals with hypoxic tolerance is relatively superficial.There is a lack of comprehensive and in-depth studies on the genetic basis and molecular level.As one of the molecular mechanisms to adapt to the hypoxia environment,gene loss has the strongest phenotypic effect.Earlier study in our research group revealed that the loss of TRIM17 in naked mole rats and blind mole rats may be one of the genetic causes of their adaptation to hypoxia.In order to further investigate whether the loss of TRIM17 contributes to the low occurrence of tumors,we use evolutionary analysis,functional experiments,and proteomics to verify the function of TRIM 17 on the proliferation and migration of tumor cells and to explore the underlying molecular mechanism.Significance:Discovery of the potential tumor-related genes in anti-cancer mammals is a new idea for studying the mechanism of cancer.We verify whether the comparative genomics and gene screening from molecular evolution are feasible to discover new anti-tumor genes.It provides a reference for studying the anti-cancer phenotype of wild animals.At present,there is no in-depth study on TRIM17 gene in human tumors.Therefore,exploration of the relationship between TRIM17 loss and tumor development is a new direction in cancer biology.Methods:To explore the genetic causes that mammals adapt to hypoxia,we used comparative genomics to compare the genomes of currently known hypoxic-tolerant animals with those of normal-oxygen-surviving animals and identified the genes that were lost in mammals living in hypoxia.We then analyzed the expression of these missing genes in human tumors through the TCGA database and choose the most relevant gene TRIM17 to carry out the functional experiments.We further used molecular biology techniques to construct TRIM17 knockout plasmids,generated stable TRIM 17 knockout non-small cell lung cancer A549 cells,and examined the proliferation,migration,and apoptosis of the control and knockout cells under normoxic and hypoxic conditions.In addition,we constructed TRIM17 expression plasmids,generated TRIM17 stable expressing cell lines,used label-free quantitative proteomics to identify proteins that are regulated by TRIM17,and verified their regulation by immunoblotting analysis.Results:Bioinformatics analysis found that TRIM17 was lost in naked mole rats,blind mole rats,whales,and other animals.By analyzing the data in the TCGA database,we found that TRIM17 was highly expressed in six types of human tumors,and its high expression reduced the survival time of lung cancer patients and gastric cancer patients.Tumor biology experiments found that TRIM17 knockout did not exhibit obvious inhibitory effect on the proliferation of lung cancer cells under normal oxygen.However,under hypoxic conditions,TRIM17 knockout inhibited the proliferation and migration of A549 cells.Furthermore,proteomics analysis discovered 24 proteins that were differentially regulated by TRIM17.Biological function analysis revealed that these proteins are involved in hypoxia response and glucose starvation biological progress.Conclusions:Molecular evolution analysis discovered that TRIM17 was lost in hypoxic and anti-cancer animals.Experiments verified that the downregulation of TRIM17 in hypoxic environment reduces the viability and migration of non-small cell lung cancer cells.In addition,label-free quantitative proteomics and bioinformatics analyses discovered that TRIM17 regulated proteins are involved in hypoxia response biological processes,which suggests that TRIM17 may affect the signaling pathways under hypoxic environment.Biochemical methods confirmed that the tumor suppressor gene p53 was positively regulated by TRIM17.The inhibition on the proliferation and migration of non-small cell lung cancer may not work through the p53 pathway,and the specific molecular mechanism needs to be further explored.