Comprehensive Bioinformatics Analysis And Experimental Validation Reveal The Function And Clinical Significance Of DIPAH3 In Cervical Cancer

Objective: Cervical cancer is one of the common cancers among women worldwide,seriously endangering their health.Therefore,the early diagnosis of cervical cancer is particularly important,and the discovery of a sensitive and specific biomarker for cervical cancer is currently an extremely urgent need.This study aims to identify a potential biomarker for cervical cancer through bioinformatics analysis,understand its expression in cervical cancer,analyze its relationship with immune cell infiltration in cervical cancer,and reveal its regulatory role in the proliferation,migration,and invasion ability of cervical cancer cells.Methods:(1)The differentially expressed genes related to cervical cancer were screened from GEO(Gene Expression Omnibus)database and TCGA(The Cancer Genome Atlas)database by bioinformatics technology.GO(Gene Ontology)enrichment analysis and KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis of these differentially expressed genes were used to understand the distribution of these differentially expressed genes in cells,cell function and the signal pathways that these differentially expressed genes may participate in regulation.Through LASSO regression analysis,building the risk models for these genes,and then COX regression analysis and survival curve analysis were used to find out the key gene related to the prognosis of cervical cancer——DIAPH3.(2)Pan-cancer analysis was used to determine the expression of DIAPH (Diaphanous Related Formin 3)in cervical cancer tissues and normal cervical tissues in TCGA database and GTEx(Genotype-Tissue Expression)database.Then 20 cervical cancer tissues and normal cervical tissues were collected from patients with cervical cancer in the Affiliated Hospital of Guizhou Medical University.None of these patients received adjuvant therapy such as radiotherapy or chemotherapy before operation(see the Table 1 for specific collection criteria).The expression of DIAPH3 in cervical cancer and normal cervical tissues was detected by real-time fluorescence quantitative polymerase chain reaction(RT-q PCR)and immunoblotting technique(Western-blot).The localization of DIAPH3 in cervical cancer cells was detected by immunohistochemical technique.Subsequently,the relationship between the expression of DIAPH3 and immune cell infiltration in cervical cancer was analyzed through the TIMER(Tumor Immune Estimation Resource)database.(3)Through the transient transfection technique to constructed DIAPH3 knock-down cervical cancer cells.Western blotting was used to detect the efficiency of DIAPH3 knock-down cervical cancer cells.Then in DIAPH3 knock-down cervical cancer cells(He La and Si Ha),through the CCK-8(Cell Counting Kit-8)test to detected the proliferation of cervical cancer cells,the migration ability of cervical cancer cells was detected by scratch test,and the invasive ability of cervical cancer cells was detected by Transwell test.The downstream molecules SLC31A1(Solute Carrier Family 31 Member 1)and ATP7B(ATPase Copper Transporting Beta)of DIAPH3 were found by bioinformatics method.The expression levels of SLC13A1 and ATP7 B in cervical cancer tissues and normal cervical tissues were detected by immunohistochemical experiment,and the results were compared with those in cervical cancer tissues and normal cervical tissues in HPA(Human Protein Atlas)database.Finally,detected the expression levels of SLC31A1 and ATP7 B in DIAPH3 knock-down cervical cancer cells(He La and Si Ha).Results:(1)The 146 common differentially expressed genes associated with cervical cancer were screened from GEO database and TCGA database.These 146 differentially expressed genes were mainly concentrated in the chromosome region,centrosome region and spindle region,which participated in the process of organelle division and nuclear division,and affected cell microtubule binding and ATP enzyme activity.And these genes may be involved in the regulation of cell cycle,cell meiosis and cell senescence.The results of LASSO regression analysis and risk model showed that among the 146 differentially expressed genes,13 genes were associated with the prognosis of patients with cervical cancer.The high expression of these 13 genes in cervical cancer predicted the shortening of overall survival time.Multivariate COX regression analysis showed that 6 of the 13 genes(DIAPH3,IQGAP3,TYMS,BLM,SLC24A3,SPON1)may be independent risk factors for the prognosis of cervical cancer.The results of the nomogram showed that the survival rate of patients with cervical cancer decreased gradually with the increase of the expression of DIAPH3 in these six genes.The results of survival analysis showed that the high expression of DIAPH3 was a poor prognostic factor in patients with cervical cancer(HR=2.3,P < 0.05).(2)The results of pan-cancer analysis showed that DIAPH3 was highly expressed in cervical cancer tissues in TCGA database and GTEx database.The results of clinical samples from cervical cancer patients also showed that the expression of DIAPH3 in cervical cancer tissues was higher than that in normal cervical tissues significantly(P < 0.001),and DIAPH3 protein was mainly expressed in the cytoplasm of cervical cancer cells.The results of immune cell infiltration analysis showed that the expression of DIAPH3 in cervical cancer was negatively correlated with the infiltration of B cells and CD4+T cells.(3)Western blotting showed that the DIAPH3 knock-down cell in cervical cancer(He La and Si Ha)were successfully constructed by transient transfection technique.The results of CCK-8 cell proliferation assay showed that the proliferation level of cervical cancer cells in the DIAPH3 knock-down group was lower than that in the control group significantly(P < 0 05).The results of scratch test showed that the migration ability of cervical cancer cells in the DIAPH3 knock-down group was lower than that in the control group significantly(P < 0 01).The results of Transwell assay also showed that the invasive ability of cervical cancer cells in the DIAPH3 knockdown group was lower than that in the control group significantly.(4)The results of bioinformatics analysis based on TCGA database showed that the expression level of SLC31A1 and ATP7 B in cervical cancer decreased with the decrease of the expression level of DIAPH3.The results of immunohistochemical experiment showed that the expression of SLC31A1 and ATP7 B in cervical cancer was higher than that in normal cervical tissue,which was consistent with the immunohistochemical results of cervical cancer in HPA database.Western blotting was used to detect the expression of SLC31A1 and ATP7 B in cervical cancer cells(He La and Si Ha)in DIAPH3 knock-down group.The results showed that knocking down the expression of DIAPH3 could down-regulate the expression of SLC31A1 and ATP7 B in cervical cancer cells.Conclusions:DIAPH3 is a potential biomarker of cervical cancer,and its expression level is up-regulated in cervical cancer tissue,and it is related to the inhibition of immune cell infiltration of cervical cancer.SLC31A1 and ATP7 B are downstream molecules of DIAPH3.Knocking down the expression level of DIAPH3 in cervical cancer cells may reduce the proliferation ability of cervical cancer cells by down-regulating the expression levels of SLC31A1 and ATP7 B,and slow down the migration and invasion of cervical cancer cells.