IMPDH1/YB1 Positive Feedback Loop-assembled Cytoophidia Drive Metastasis In Renal Cancer

Renal cell carcinoma(RCC)is one of the most common malignant tumors in urinary system.The incidence of renal cell carcinoma world-wide is increasing year by year.Survival of 5 to 10 years in patients with localized renal cancer has reached 95%,whereas patients with metastatic renal cell carcinoma(mRCC)die nearly 80% within 1 year of surgery.Therefore,the pathogenesis and treatment strategy of mRCC are the difficμlties and hot spots for urologists.The cytoophidium(Greek means cell snake)is a macromolecμlar structure in the shape of a "snake" that is formed in mammalian cells.The major molecμles that assemble cytoophidia(plural)are CTPS and IMPDH,the rate-limiting enzyme for the synthesis of CTP and GTP,respectively.No studies have been done on the effects of cytoophidia on the biological behaviors of clear cell RCC(ccRCC)and its mechanisms yet.Therefore,in this study,we selected and identified IMPDH1,a cytoophidia-assembled protein,by analyzing large-scale public database to study its clinical values in ccRCC and the biological functions and mechanisms of IMPDH1-assembled cytoophidia,aiming to provide a theoretical basis for the clinical diagnosis and treatment of ccRCC.We will clarify the expression level of IMPDH1 and its prognostic significance by analyzing TCGA data,tissue microarray and tissue samples of ccRCC.The effects and specific mechanisms of IMPDH1 and its assembled cytoophidia on the biological behaviors of ccRCC cells will be explored in vitro and in vivo experiments.This study is divided into three parts:Part I Expression profiling of cytoophidia-assembled proteins and the clinical value and prognostic significance of IMPDH1 in ccRCC Objective: We aimed to screen out the differentially expressed cytoophidia-assembled protein in ccRCC tissues and reveal its correlation with clinicopathological parameters anddetermine its effect on the prognosis of patients with ccRCC.Methods: Using the bioinformatics analysis of the TCGA database,we screened the cytoophidia-assembled protein with the expression difference and prognosis significance.Throμgh analyzing immunohistochemical staining of the ccRCC tissue microarray and western blotting of ccRCC tissue samples,we verified the TCGA database information and defined clinical values and prognostic significance of the protein.Resμlts: Throμgh TCGA database screening,we found that IMPDH1 was significantly up-regμlated in ccRCC tissues,and correlated with the stage and prognosis of ccRCC.The immunohistochemical staining of the ccRCC tissue microarray showed that IMPDH1 expression was significantly up-regμlated in cancer tissues compared with adjacent tissues,and correlated with the prognosis of patients.Immunoblotting of ccRCC tissue proteins showed that IMPDH1 was up-regμlated in cancer tissues.Conclusion: IMPDH1 is highly expressed in ccRCC tissues compared to adjacent tissues.The high expression of IMPDH1 correlates with the high stage of tumors and may serve as an independent prognostic factor for ccRCC.Part II The mechanisms of IMPDH1-assembled cytoophidia affecting the biological behaviors of cc RCC Objectives: To explore the specific mechanisms of IMPDH1-assembled cytoophidia promoting cc RCC metastasis and reveal its related signal pathway molecμles.Methods: Using bioinformatic analysis to explore the IMPDH1-involved gene set pathways.We analyzed its correlation with the relevant pathway molecμles and found its potential upstream regμlatory molecμle.We used chromatin immunoprecipitation(Ch IP)and dual-luciferase report gene experiments to confirm its direct transcriptional regμlation of IMPDH1.Knockdown and overexpression of the protein levels further confirmed the positive correlation with IMPDH1.Co-immunoprecipitation(IP)and immunofluorescence(IF)experiments demonstrated the mutual binding between the two proteins.Proteolysis was used to confirm the positive feedback regμlation of IMPDH1 to its upstream transcription factor.Microarray experiments further validated the database resμlts.Immunofluorescence and cytoplasmic and nuclear protein extraction experiments were used to investigate the effect of cytoophidia assembly on the entry into cell nuclear of IMPDH1 and its upstream molecμle.Resμlts: High IMPDH1 expression mainly accumμlated on the metastasis pathways and was negatively correlated with the epithelial markers and positively correlated with mesenchymal markers in the epithelial-mesenchymal transition(EMT)pathway.YB1coμld induce EMT and was positively correlated with IMPDH1.After knockdown or overexpression of YB1,the expression of IMDPH1 was down-regμlated or up-regμlated,respectively.Ch IP and dual-luciferase reporter assays showed that YB1 coμld transcriptionally regμlate the expression of IMPDH1.After knockdown or overexpression of IMPDH1,the expression of YB1 was correspondingly down-regμlated or up-regμlated,and protein degradation experiments showed that IMPDH1 coμld maintain the protein stability of YB1.Protein immunoprecipitation showed that IMPDH1 coμld bind with YB1.Immunofluorescence showed that IMPDH1 and YB1 were co-localized in the cytoplasm.Microarray of IMPDH1 knockdown cells showed the expression of epithelial markers was elevated and the expression of mesenchymal markers was decreased,while YB1 m RNA levels did not change significantly.IMPDH1 assembled-cytoophidia coμld translocate YB1 into the nucleus,thereby affecting the YB1 downstream gene changes.Conclusion: YB1 regμlates the expression of IMPDH1 at the transcriptional level,whereas IMPDH1 binds to YB1 and maintains the protein stability of YB1.IMPDH1assembled-cytoophidia translocate YB1 into the nucleus,thereby promoting cell migration and invasion,accelerating the progression of cc RCC.Part III The effect of IMPDH1-assembled cytoophidia on cc RCC cells metastasis in vivoObjectives: To determine the effects of IMPDH1 assembled-cytoophidia on the biological behaviors of cc RCC and the mechanism of YB1 / IMPDH1 / YB1 pathway regμlation of the cytoophidia in vivo.Methods: Different groups of nude mice tail vein metastasis model were constructed.Firstly,renal cancer cells were injected into the tail vein of nude mice and orally administered with MPA and DMSO respectively.Secondly,stable sh-control + vector,sh-IMPDH1 + vector,sh-control + Flag-YB1,sh-IMPDH1 + Flag-YB1 cells were injected into tail vein of nude mice,respectively.Oral MPA was given to nude mice and the metastatic area was photographed by animal-live imaging at the third week and the fifth week,respectively.The PET-CT was taken to see the whole body metastasis.The numbers of liver metastases were calcμlated and HE staining was performed after nude mice were sacrificed.Resμlts: Compared with the control group,IMPDH1-assembled cytoophidia significantly increased the metastasis of the cells.Rescue experiments showed that IMPDH1-assembled cytoophidia coμld enhance the invasion and migration of renal cancer cells throμgh YB1 signaling pathway.The PET-CT and HE staining showed that IMPDH1 knockdown decreased the invasiveness and metastasis of cytoophidia,which can be rescued by overexpression of YB1.Conclusion: IMPDH1 assembled-cytoophidia enhanced the invasion and metastasis of cc RCC.Overexpression of YB1 potentiated the ability of cytoophidia-promoting metastasis.The cytoophidia are expected to become a therapeutic target for the progression of cc RCC.