| The most common histological subtype of renal cell carcinoma is clear cell renal cell carcinoma(ccRCC).Up till now,radical nephrectomy remains the primary surgical intervention for localized renal cell carcinomas.In recent years,multiple targeted agents have been developed and used,only with limited beneficial therapeutic effects.The mortality associated with ccRCC metastasis and recurrence remains high,making it an urgent need to conduct more research on ccRCC to improve its prognosis.Renal clear cell carcinoma is an immunogenic tumor,involving various immune cells in its microenvironment.T cells recruited by the presentation of tumor antigens can induce an anti-tumor immune response.But immune checkpoints on the surface of immune cells can inhibit immune responses.Based on this,immune checkpoint inhibitors emerge and become one of the main treatments for ccRCC.However,ccRCC is also a highly heterogenous tumor with a complex immune microenvironment,which provides many opportunities for immune escape,leading to disparate responses of ccRCC to immunotherapy.Therefore,the characteristics of the immune microenvironment of ccRCC need to be further explored to discover more molecules that can affect tumor immunity.A large number of cells die every day in the body by the mechanism of cell death,a significant biological process regulating homeostasis.Widely occurring in inflammatory and neoplastic diseases,abnormalities in cell death play a pathogenic role that can not be ignored.Unlike cell necrosis,programmed cell death is a regulated and active form of cell death with the merit of controllability,making it a prospective and significant direction in oncological researches.Recent researches on cell death identified more types of programmed cell death,including apoptosis,pyroptosis,necroptosis,ferroptosis,and PANoptosis.These cell death types have their respective molecular mechanisms and morphological characteristics.PANoptosis is a newly defined type of programmed cell death with strong immunogenicity,which involves the cross-talk and co-activation of apoptosis,pyroptosis,and necroptosis.Currently,research efforts on PANoptosis and its related biomolecules are mainly directed toward infectious and autoimmune diseases.But its role in tumors,especially in immunogenic tumors like renal clear cell carcinoma,remains unclear.This study aims to explore the clinical and biological value of PANoptosis-related long non-coding RNA(lncRNA)in ccRCC.First,the roles of apoptosis/pyroptosis/necroptosis-related lncRNAs in ccRCC were investigated.Subsequently,the clinical value of PANoptosis-related lncRNAs in ccRCC was determined.Finally,the most representative PANoptosis-related lncRNA was selected and its biological characteristics were then explored.In general,our research fall into the following three parts:Part Ⅰ The correlation between apoptosis/pyroptosis/necroptosis-related lncRNAs and the progression,prognosis,and immune infiltration of ccRCCObjective:To investigate the correlation between apoptosis-related lncRNA,pyroptosis-related lncRNA,and necroptosis-related lncRNA with the progression,prognosis,and immune infiltration of ccRCC.Methods:1.Gene expression data and clinical data of 611 patients with ccRCC were obtained from the Cancer Genome Atlas(TCGA)database.2.Apoptosis/pyroptosis/necroptosis-related genes were obtained from previous studies,and the expression of these genes in ccRCC samples was explored.3.LncRNAs significantly associated with these three cell death types were identified by correlation analysis,and lncRNAs with remarkable prognostic value were screened by univariate COX regression analysis.4.Based on these three cell death-related lncRNAs,the ccRCC samples were clustered,among which,the differences in clinicopathological factors and prognosis were determined.5.Tumor immune microenvironment scores,immune checkpoint gene expression,and immune cell infiltration levels were compared among different clusters.Results:1.The number of apoptosis/pyroptosis/necroptosis lncRNAs with significant prognostic values was 54,147,and 39,respectively.2.Significant differences were found in the survival rate and clinical stage of ccRCC patients among the clusters obtained by three clustering methods.3.There are significant differences in the expression of immune checkpoints among different clusters.4.The infiltration of immune cells and the tumor microenvironment’s immune score significantly differed among clusters.Summary:Apoptosis/pyroptosis/necroptosis-related lncRNAs are associated with the progression and prognosis of ccRCC;These three types of cell death-related lncRNAs are associated with immune cell infiltration and the immune microenvironment in ccRCC.Part Ⅱ Comprehensive analysis of the role of immune-related PANoptosis(apoptosis,pyroptosis,and necroptosis)lncRNAs in ccRCCObjective:To comprehensively analyze the role of immune-associated PANoptosis lncRNAs in ccRCC and construct a new immune-associated PANoptosis lncRNAs signature.Method:1.Through correlation analysis,genes significantly associated with apoptosis,pyroptosis and necroptosis were obtained(PANoptosis-related genes).2.We clustered PANoptosis-related genes with similar expression patterns into different gene modules.Subsequently,we explored the correlation between these gene modules and various immune cell infiltration and then selected one module for subsequent analysis.3.lncRNAs were extracted from the selected module genes and lncRNAs with the maximal prognostic value were identified through univariate COX regression analysis.4.Consistent clustering was performed based on the obtained lncRNAs,and differences in patient prognosis,clinicopathological parameters,functional enrichment,and tumor immunity among different clusters were analyzed.5.Lasso regression analysis was used to construct a risk model based on immune-related PANoptosis lncRNAs.6.Differences in functional enrichment and immune infiltration between ccRCC samples at different risks were analyzed and determined.7.Differences in immune efficacy and drug sensitivity among ccRCC samples at different risk levels were analyzed and measured.8.Collect 60 patients with ccRCC undergoing surgical treatment in clinical practice,use qRT-PCR to detect the expression level of model-related lncRNA,and calculate the risk score for each patient based on the model formula.Analyze the clinical characteristics and prognostic differences of patients with different risk scores.Results:1.The results showed that these PANoptosis-related gene modules were strongly or weakly associated with various immune cell infiltration.2.All immune-related PANoptosis lncRNAs we screened were highly expressed in ccRCC tumor tissues and were associated with poor prognosis.3.Through analysis,we found significant differences in the pathological grade,clinical stage,and overall survival rate of ccRCC among these clusters.The enrichment analysis showed that the differences were mainly related to cytokine activity,T cell receptor complex,T cell receptor signaling pathway,and antigen processing and presentation.We found higher levels of immune cell infiltration,lower tumor purity,and higher expression of immune checkpoint genes and antigen presentation-related genes in Cluster 2 samples than in Cluster 1 samples.4.Five immune-associated PANoptosis lncRNAs were selected to construct a novel risk model,which enables the classification of ccRCCpatients into two groups with significantly different clinical outcomes.The results of the ROC curves indicate that the risk model has high prediction accuracy.The results of univariate and multivariate COX regression analysis suggest that the risk score calculated by the model may be an independent prognostic factor for ccRCC.Combining the risk score with two other independent prognostic factors,we constructed a nomogram that can predict patient survival and has good predictive efficacy.In addition,using the principal component analysis and survival analysis,we verified the sample division ability and applicability of the risk model we built.The verification of the model’s predictive capabilities in another cohort of patients with renal cell carcinoma demonstrated the reliability of the risk model.5.The risk score of renal cancer patients was significantly related to the infiltration of immune cells,the enrichment of immune-related functions,and the extensive activation of immune-related pathways.6.The expression of immune checkpoint genes and TCIA scores were higher in high-risk ccRCC samples.In addition.We have also obtained several treatment drugs suitable for patients with different risk stratification.Summary:Immune-related PANoptosis lncRNAs are associated with the progression,prognosis,microenvironment immune infiltration,antigen presentation,and immunotherapy response of ccRCC;The risk model we constructed based on immune-related PANoptosis lncRNAs can be used to predict the prognosis of ccRCC patients and evaluate the efficacy of immunotherapy.PartⅢ Exploration of the biological value of immune-related PANoptosis lncRNA LINC00944Objective:To explore the biological value of immune-related PANoptosis lncRNA LINC00944 in ccRCC.Method:1.We first analyzed the differences in LINC00944 expression in 611 TCGA-ccRCC tumor samples and adjacent samples.Afterward,we detected the expression of LINC00944 in ccRCC clinical samples and cell lines through qRT-PCR.2.We explored the correlation between LINC00944 expression and various clinical pathological features in the TCGA patient cohort and 60 clinical samples.3.We explored the prognosis and clinical diagnostic value of LINC00944 in the TCGA cohort and clinical samples.4.We examined the migration and invasion of renal carcinoma cell lines after overexpression and knockdown of LINC00944 by scratch and Transwell test.5.Obtain LINC00944-related signaling pathways through enrichment analysis.Detect the expression changes of JAK-STAT pathway proteins after overexpression and knockdown of LINC00944 through Western blot experiments.6.The interference of AG490 with LINC00944 regulating the JAK pathway was detected by western blot.Functional recovery experiments were designed to verify whether LINC00944 could affect ccRCC migration and invasion through the JAK-STAT pathway.7.The correlation of LINC00944 expression with immune cell infiltration,immune score,immune checkpoint gene expression,antigen presentation gene expression,and TCIA score was analyzed.8.To explore the expression of LINC00944 at the single-cell level and to verify the correlation between LINC00944 and T-cell infiltration by immunohistochemistry.Results:1.LINC00944 was highly expressed in ccRCC tumor tissues and cells.2.In the TCGA patient cohort,high expression of LINC00944 is significantly correlated with older age,later clinical staging,and higher pathological grading.In 60 clinical samples,high expression of LINC00944 was significantly correlated with later T and M stages in patients.3.In the TCGA cohort,patients with high expression of LINC00944 had worse overall and progression-free survival.And LINC00944 had high clinical diagnostic value.In 60 clinical samples,patients with high expression of LINC00944 had lower overall survival with no significant difference.4.Overexpression of LINC00944 can promote the migration and invasion of renal cancer cells while knocking down LINC00944 can inhibit the migration and invasion of renal cancer cells.5.Pathway enrichment analysis indicates that LINC00944 is related to the activation of the JAK-STAT pathway.Western blot results showed that the expression of p-JAK and p-STAT3 protein decreased after LINC00944 knockdown while increasing after overexpression.6.Overexpression of LINC00944 increased the expression of p-STAT3 protein,while the addition of AG490 reversed the regulation of p-STAT3 protein expression by LINC00944.Overexpression of LINC00944 increased the migration and invasion ability of renal cell carcinoma cells,while the addition of AG490 partially recovered the effect of LINC00944 on the migration and invasion of cancer cells.7.The results of immune correlation analysis indicate that the expression of LINC00944 is related to T cell infiltration.Renal cancer samples with high expression of LINC00944 have higher immune scores,such as intra-tumor heterogeneity,CTA score,and TCIA score.LINC00944 expression is significantly correlated with antigen-presenting genes and immune checkpoint genes.8.The exploration of LINC00944 showed that the lncRNA was significantly overexpressed in epithelial and T cells.In addition,the expression of LINC00944 in tumor epithelial cells was significantly higher.The apoptosis/pyroptosis/necroptosis gene set enrichment score was also generally higher in epithelial cells with high LINC00944 expression.The results of cell communication analysis show that T cells mainly receive signals from epithelial and endothelial cells.The MIF signal pathway is the main signal pathway in the interaction between epithelial cells and T cells.The immunohistochemical results showed that the positive rate of T cells in ccRCC clinical specimens with high expression of LINC00944 was higher.Summary:LINC00944 is significantly overexpressed in ccRCC tissues and cells;LINC00944 is associated with the progression and prognosis of ccRCC and has a high diagnostic value;LINC00944 can affect the migration and invasion of renal cancer cells through the JAK-STAT pathway;The expression of LINC00944 is related to the response of ccRCC patients to immunotherapy;The expression of LINC00944 is significantly correlated with the T-cell infiltration of ccRCC.Conclusion:1.Apoptosis/pyroptosis/necroptosis-related lncRNAs are associated with the progression,prognosis,and microenvironmental immune infiltration of ccRCC.2.Immune-related PANoptosis(apoptosis,pyroptosis,necroptosis)lncRNAs are significantly associated with the progression,prognosis,immune cell infiltration,and immunotherapy response of ccRCC.3.A novel risk model based on immune-related PANoptosis lncRNAs was constructed.4.LINC00944 is overexpressed in ccRCC tissues and cells and is associated with the progression of ccRCC.LINC00944 has critical prognostic and clinical diagnostic value.5.High expression of LINC00944 can promote the migration and invasion of ccRCC cells;Knocking down LINC00944 can inhibit the migration and invasion of ccRCC cells.6.LINC00944 can affect the invasion and migration of ccRCC cells by regulating the activity of the JAK-STAT pathway.7.LINC00944 is related to the response of ccRCC patients to immunotherapy.The high expression of LINC00944 is significantly correlated with the high T cell infiltration in the ccRCC microenvironment. |
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