SPOP Affects Proliferation And Invasive Migration Of Colorectal Cancer Cells By Regulating HK2

Background:Globally,colorectal cancer(CRC)is a malignancy with a high incidence.Despite the great progress in early diagnosis of colorectal cancer and the continuous improvement of comprehensive treatment methods,the 5-year survival rate after radical surgery has not improved significantly.The mechanisms of its development are complex.The ubiquitin-proteasome pathway is an important pathway for intracellular protein degradation and is essential for maintaining cellular homeostasis,while abnormal protein accumulation may lead to various diseases,including human cancers.Speckle-type POZprotein(SPOP)is a key adapter based on the Cullin3 ubiquitination process and has been newly identified as a member of ubiquitin ligase E3.Several studies have confirmed its important role in gene transcriptional regulation,replication,repair,and ubiquitination and degradation.In recent years,many studies have confirmed that increased glycolysis in tumor cells is associated with enhanced activity of glycolytic rate-limiting enzymes.Among them,hexokinase(HK)is a key enzyme that regulates cellular glycolysis,converting glucose to glucose 6-phosphate,which provides energy to tumor cells and thus stimulates the growth and proliferation of malignant tumors.However,the mechanism of SPOP and HK2 in colorectal cancer is not well understood,while whether SPOP can target HK2 to regulate CRC progression remains to be investigated.Objective:SPOP inhibited To investigate the effects of SPOP regulation of HK2 on proliferation,invasion and migration of colorectal cancer cells,and to provide a new theoretical basis for clinical targeted therapy of colorectal cancer.Methods:Using bioinformatic analysis techniques,based on high-throughput gene expression(Gene Expression Omnibus,GEO)and cancer genome atlas(The cancer genome atlas,TCGA),Genotype-Tissue Expression,GTEx)databases to assess SPOP and HK2 expression levels in colorectal cancer and paracancerous tissues,their correlation with poor diagnosis and prognosis of colorectal cancer,and the interaction between them.The direct protein binding between SPOP and HK2 was clarified by immunoprecipitation(CO-IP).Human colorectal epithelial cells(NCM460)and two strains of colorectal cancer epithelial cells(SW620 and HCT116)were selected as the study subjects.The expression of SPOP and HK2 in the cell lines and the expression correlation were examined by Western Blot and PCR assays;the effects of SPOP and HK2 on the biological functions of colorectal cancer were explored using CCK-8,plate clone formation,transwell and scratch assays.Results:1.SPOP was lowly expressed in colorectal cancer cell lines.analysis of GEO,TCGA,GTEx data and PCR experiments showed that SPOP expression was down-regulated in colorectal cancer tissues and patients in the high SPOP expression group had a good prognosis.The results of bioinformatics analysis indicated the potential of SPOP as a diagnostic for CRC.2.Overexpression of SPOP inhibits the proliferation,invasion and migration of colorectal cancer cells.3.The results of GEO,TCGA,GTEx data analysis and PCR experiments showed that HK2 expression was upregulated in colorectal cancer tissues and patients in the HK2 high expression group had poor prognosis.The results of bioinformatics analysis indicated the potential of HK2 as a diagnostic for CRC.Knockdown of HK2 inhibits proliferation,invasion and migration of colorectal cancer cells.4.Knockdown of HK2 inhibits proliferation,invasion and migration of colorectal cancer cells.5.Bioinformatics analysis showed a negative correlation between HK2 as a target gene of SPOP;immunoprecipitation(CO-IP)experiments showed direct protein binding between them,suggesting that HK2 is a potential protein substrate for the E3 ubiquitinated ligase SPOP.Conclusion:SPOP inhibits the proliferation,invasion,and migration of colorectal cancer cells by inhibiting HK2 expression.The bioinformatics analysis showed that SPOP was downregulated in colorectal cancer tissues and patients with high SPOP expression;instead,the expression of HK2 in the group with high HK2 expression was increased;SPOP and HK2 had some value in the diagnosis of colorectal cancer.