Effect Of Prognosis Autophagy Model Based On CASP4 And BIRC5 On Overall Survival Rate And Biological Characteristics Of Renal Cancer

Section 1 Identification and validation of a prognostic autophagy model to predict overall survival in patients with renal cancer◆ ObjectiveAutophagy is a mechanism by which cellular materials are transported to lysosomes for degradation,thereby fulfilling the metabolic needs of the cell itself and the renewal of certain organelles.Studies at this stage have confirmed that autophagy disorders are related to a variety of diseases,but its specific mechanism in the occurrence and development of renal cancer(RC)is still unclear.This study aims to use bioinformatics analysis to screen the data of tumor tissue and adjacent tissues in the The Cancer Genome Atlas(TCGA)database,combined with the human autophagy database,to identify the key autophagy-related genes(ARGs)in renal cancer,and finally construct the prognosis model and establish a nomogram.At the same time,it is supplemented by the verification of clinical samples to explore its possible role and prognostic value in RC patients.◆ Methods1.Download the transcriptome data and clinical information of RC and paracancerous tissues from the TCGA database,and download autophagy-related genes from the human autophagy database and then take the intersection,that is,only retain the data of ARGs.2.The ARGs in the dataset with an absolute log2 fold change(FC)>1 and an adjusted P value of<0.001 were considered eligible for subsequent analysis.To better understand the role of differentially expressed ARGs,we use the R "cluster Profler" package for enrichment analysis.3.The data downloaded from the TCGA database is randomly divided into a train set and a test set.The patients in the training set are analyzed by univariate,Lasso and multivariate regression analysis,and a prognostic model is constructed using the final autophagy-related differential genes.The mRNA expression level of the obtained gene was investigated by Oncomine database.4.The prognostic model was used to evaluate the train set and test set respectively,and the risk score of each sample was calculated.The patients were divided into high-and low-risk groups based on the median value of the risk score,and a K-M survival curve was drawn to compare the survival differences between the two groups.5.The clinical and pathological data of patients who underwent nephrectomy in Beijing Hospital from October 2012 to April 2019 were collected retrospectively.The paraffin sections were stained with immunohistochemistry and the paired fresh samples were quantified by quantitative real-time PCR,to explore the expression level of genes contained in the prognostic model in clinical RC patients(recorded as Beijing Hospital set).The sections were scored by immunohistochemistry,the patients were divided into high and low risk groups based on the median score,and the K-M curve was drawn to compare survival differences.6.The prognosis model and other clinical variables(including age,gender,etc.)were used as independent variables,and the overall survival rate of the patient was used as the dependent variable.Univariate and multivariate Cox analysis were performed.All the obtained independent prognostic factors were combined to construct a nomogram to predict the 3-and 5-year overall survival rate of RC patients.◆Result1.According to the above conditional analysis,there were 38 autophagy-related differential genes,including 33 up-regulated and 5 down-regulated.Functional enrichment analysis suggests that autophagy may play an important role in the initiation of RC.2.We constructed a prognostic model based on CASP4 and BIRC5.At the same time,we searched the Oncomine database and found that the expression levels of CASP4 and BIRC5 mRNA in RC tissues were significantly higher than those in normal tissues(all P<0.05).3.According to the median risk value calculated by the prognostic model,the RC patients were divided into high-risk group and low-risk group.The OS of high-risk group was significantly lower than that of low-risk group.Then we used the test set and the Beijing hospital set of RC patients to verify the prognostic model and the expression levels of the two genes(all P<0.05).4.Multivariate Cox regression analysis showed that age and prognostic model were independent prognostic factors of RC patients.In addition,considering the prognostic significance of clinical stage to RC patients,we constructed a nomogram based on age,clinical stage and prognostic model.◆Conclusions1.Autophagy-related genes CASP4 and BIRC5 are highly expressed in RC patients and are related to disease progression and poor prognosis.2.CASP4 and BIRC5 may be potential therapeutic targets for RC,but further experiments are needed.Part II The effect of silencing the expression of CASP4 and BIRC5 on the biological behavior of renal cancer cells and its mechanism◆BackgroundThe cystatin family,including human cystatin-4(CASP4),plays a key role in the innate immune response,including promoting the fusion of phagosomes carrying pathogenic bacteria and lysosomes,preventing pathogens from intracellular replication,and promoting the maturation and secretion of pro-inflammatory cytokines,etc.Although previous studies have suggested that CASP4 is dysregulated in certain cancers including esophageal cancer,breast cancer,and prostate cancer.There are few studies on CASP4 in renal cell carcinoma,and it has not been fully studied at this stage.Survivin(encoded by the BIRC5 gene)is a small protein in the apoptosis inhibitor protein family.Compared with adult differentiated tissues,it is highly expressed in tumors,and it has been reported that its overexpression is related to the poor prognosis of patients with many different types of tumors.It is reported that this apoptosis inhibitor plays an important role in promoting cancer cell survival and inhibiting cell death.Therefore,the abnormal expression of BIRC5 can accelerate tumor progression and is significantly related to the increase of tumor drug resistance.Current studies have confirmed that Survivin/BIRC5 can be used as a potential biomarker and therapeutic target in renal cancer,but its specific mechanism remains to be further explored.◆ObjectiveIn recent years,the complex role of autophagy in tumor has been paid more attention by researchers.However,some studies have suggested that the level of autophagy in renal cancer cells is inhibited,and the reduced level of autophagy is related to the increased stage and grade of renal cell carcinoma,that is,the level of autophagy affects the occurrence and development of renal cell carcinoma.Our previous studies suggest that CASP4 and BIRC5 may affect the occurrence and development of renal cell carcinoma by affecting the level of autophagy.Therefore,this study intends to further clarify the influence of the expression of CASP4 and BIRC5 on the biological behavior of renal cell carcinoma cells through cell experiments,and explore whether CASP4 and BIRC5 affect the occurrence and development of renal cell carcinoma by regulating tumor autophagy.◆Methods1.After cell culture,the expression of CASP4 and BIRC5 in normal renal cortical proximal convoluted tubule epithelial cell line(HK-2)and five renal carcinoma cell lines(A498,ACHN,SN12C,786-O,769-P)were detected by real-time quantitative PCR and western blot techniques.2.The RNA knockdown plasmids of CASP4 and BIRC5 were constructed respectively to package the lentivirus.3.According to the results of background expression,two renal cancer cell lines with the highest expression were selected,and the selected cell lines were transfected with the packaged lentivirus to construct stable down-expression cell lines that knock out CASP4 and BIRC5.4.The knockout efficiency of different shRNA was detected by real-time quantitative PCR,western blot and immunofluorescence detection.5.The cell cycle and apoptosis of each group of cells were detected by flow cytometry.6.The migration level of each group of cells was detected by cell scratch test and Transwell invasion and migration test.7.The level of cell proliferation in each group of cells was detected by CCK-8.8.The constructed knockdown cell models of CASP4 and BIRC5 were divided into(1)control group,(2)knockdown group,(3)knockdown+autophagy inhibitor(3-methyladenine)group.9.Western blot technology was used to detect the expression of CASP4,BIRC5 and autophagy-related protein LC3 in each group of cells;10.CCK-8 was used to detect the activity of each group of cells,and TUNEL staining was used to compare the changes of cell survival in each group before and after autophagy inhibition.◆Results1.qRT-PCR and western blot were used to verify the expression of CASP4 and BIRC5 in five renal cancer cell lines.Human renal tubular epithelial cells(HK-2 cells)were used as negative control.The results showed that the expression levels of CASP4 and BIRC5 increased in different degrees in renal cancer cell lines,especially in SN12C and 786-O cells.2.Based on the background expression results,this study chose to infect SN12C and 786-O cells with lentivirus to construct a stable down-expressing cell line that knocked out CASP4 and BIRC5.3.qRT-PCR,western blot and immunofluorescence were used to detect the knockout efficiency of different shRNAs.The results showed that compared with other groups,the knockdown efficiency of shBIRC5-3 group and shCASP4-1 group was the best.Therefore,shBIRC5-3 and shCASP4-1 were used to construct knockdown plasmids to infect SN12C and 786-O cells.4.In this study,flow cytometry was used to detect the level of cell cycle and apoptosis.The results showed that after knocking down BIRC5 and CASP4,the level of proliferation of renal cancer cells decreased significantly and the level of apoptosis increased significantly.5.Scratch and Transwell experiments showed that the migration ability of renal cancer cells was significantly reduced after knocking down BIRC5 and CASP4.6.The results of CCK-8 assay showed that the proliferation ability of renal cancer cells decreased significantly after down-regulation of BIRC5 and CASP4.7.Compared with the control group,the expression of LC3 protein in the knockdown group increased,suggesting that the autophagy ability of the cells was enhanced after the knockdown of CASP4 and BIRC5.8.Compared with the knockdown group,the expression of LC3 protein in the knockdown+autophagy inhibitor group was reduced,that is,autophagy was inhibited;at the same time,the cell activity of the knockdown+autophagy inhibitor group was significantly higher than that of the knockdown group.9.TUNEL staining showed that the level of apoptosis in the knockdown group was higher,and it could be reversed by autophagy inhibitors.◆Conclusions1.The expression of mRNA and protein of and in renal cancer cell lines was significantly higher than that in normal renal cells.2.Knockout of CASP4 and BIRC5 can affect a variety of biological behaviors of renal cancer cells,including reducing the level of proliferation and migration,and increasing the level of apoptosis.3.The autophagy of renal cancer cell lines expressing CASP4 and BIRC5 was significantly inhibited,and knocking out CASP4 and BIRC5 can relieve the inhibited state of autophagy.After applying autophagy inhibitors on this basis,renal cancer cells have a tendency to transform into a more aggressive phenotype.4.The effects of CASP4 and BIRC5 on the biological characteristics of renal cancer cells may be achieved by affecting the autophagy level of renal cancer cells.Part III Caspase 4 Overexpression as a Prognostic Marker in Clear Cell Renal Cell Carcinoma◆ObjectiveThe dysregulation of caspase 4(CASP4)expression is related to the occurrence,development,and outcome of many malignant tumors;however,its role in clear cell renal cell carcinoma(ccRCC)remains unclear.Herein,we investigated the expression of CASP4 in tumor tissues and its relationship with clinical prognosis,immune infiltration,and drug sensitivity status of ccRCC patients.◆Methods1.Prediction of the expression level of target gene(CASP4)in ccRCC tissues in Oncomine database;2.The Cancer Genome Atlas(TCGA)databases were used to determine CASP4 mRNA expression in ccRCC patients.The correlation between CASP4 expression and disease prognosis was evaluated using Kaplan-Meier analysis.3.Related pathways were obtained from TCGA database via gene set enrichment analysis(GSEA)and gene set variation analysis(GSVA).4.Using ggplot2 and circlize package in R software to explore the genes co-expressed with CASP4 in ccRCC and visualize them.5.Finally,we analyzed the proportion of tumor-infiltrating immune cells(TICs)using the CIBERSORT computational method and assessed CASP4 methylation and its relationship with drug sensitivity.◆Results1.According to the analysis in Oncomine and TCGA databases,the expression level of CASP4 mRNA in ccRCC tissues was significantly higher than that in the normal tissues(all P<0.001).2.The expression of CASP4 was positively correlated with clinicopathological features(clinical stage and pathological grade),and negatively correlated with patient overall survival(OS).The same conclusion was also verified in the external dataset(P<0.05).3.GSEA and GSVA showed that the genes in the CASP4-high expression group were primarily enriched in immune-related activities.4.Moreover,CIBERSORT analysis of TIC proportions revealed that activated CD4 memory T cells were positively correlated with CASP4 expression.5.Notably,methylation analysis revealed that the abnormal upregulation of CASP4 might be caused by hypomethylation.6.Finally,we found that the abnormal expression of CASP4 may be related to tumor drug resistance.Conclusions1.Overall,our study shows that CASP4 is overexpressed in ccRCC and is an important factor affecting disease prognosis.2.This study highlights the relevance of CASP4 in the occurrence and development of ccRCC,particularly with respect to the TME,methylation and drug resistance.3.CASP4 may serve as a potential prognostic biomarker and therapeutic target in ccRCC.