| Background:Renal cell carcinoma(RCC)is one of the most common cancers in the world.The incidence and death rate of this cancer are increasing every year.Renal cell carcinoma(RCC)is the second most common malignancy of the urinary tract,accounting for 3.8% of all new cancers.The median age at diagnosis is 64 years.RCC is a major threat to human health.RCC has more than ten subtypes such as clear cell renal cell carcinoma(cc RCC),papillary renal cell carcinoma(p RCC)and chromophobe renal cell carcinoma(ch RCC).Clear cell renal cell carcinoma is the most common one,accounting for 85-90% of cases.The course of RCC is usually asymptomatic and there are no reliable markers for early diagnosis.As a result,approximately 17% of patients with cc RCC have distant metastases at the time of initial diagnosis.The current treatment modality for kidney cancer is surgery,but clear cell renal cell carcinoma has a high rate of metastasis and recurrence after surgical resection(approximately 30% of patients).And when surgical treatment is no longer feasible,almost all pathological types of kidney cancer are resistant to chemotherapy and radiation therapy.Currently,despite advances in therapies for renal cell carcinoma,such as targeted therapies and immunotherapy,the 5-year overall survival rate for patients with metastatic cc RCC has not improved.A better understanding of the molecular pathogenesis of renal cell carcinoma may provide new opportunities for the discovery of effective tumor biomarkers and therapeutic targets.Genetic studies have found that only 2% of human genomic DNA can be transcribed into m RNA,while the remaining 98% can be transcribed into non-coding RNA(Lnc RNA).With the advancement of technology more and more Lnc RNAs are being discovered.Long-stranded non-coding RNA(Lnc RNA)is an RNA sequence of up to 200 nucleotides in length that does not encode a polypeptide or protein.In recent years,Lnc RNAs function as important regulators of many cellular processes and they are thought to be involved in various physiological or pathological processes.Lnc RNAs can regulate gene expression at three levels: epigenetic,transcriptional and post-transcriptional thus affecting tumorigenesis.Our group initially screened two molecules,DBT and LINC01355,and after comprehensive analysis aimed to investigate the mechanism of LINC01355 in RCC.Its expression was significantly different in RCC tissues and cell lines,and its differential expression was associated with RCC survival prognosis,and pathway enrichment analysis showed that LINC01355 might be related to the development of EMT.Although LINC01355 has been found to be involved in the development of multiple tumors,the mechanism of LINC01355 action in renal cell carcinoma is unclear,and the purpose of this study was to investigate the molecular mechanism by which LINC01355 affects the malignant phenotype of renal cells.The relationship between LINC01355 and clinical stage and pathological grading of RCC,the value of subject characteristic(ROC)curve for RCC diagnosis,pathway enrichment analysis,and the relationship between LINC01355 and immune infiltration of RCC were analyzed using bioinformatics methods.The qRT-PCR assay was then used to detect the expression of LINC01355 in clinical tissue samples and cell lines of RCC patients to verify the predicted results of the previous raw letter analysis.Subsequently,antisense oligonucleotide(ASO)transfection was used to interfere with LINC01355 expression levels in RCC cell lines,and then the effects of low and overexpression of LINC01355 on the malignant phenotype of RCC cells were investigated by in vitro cell function assays.Protein immunoblotting(WB)was used to detect the expression of epithelial mesenchymal transition(EMT)-related proteins,confirming that LINC01355 influences the malignant phenotype of RCC by affecting epithelial mesenchymal transition.Lentiviral transfection was also used to construct a stably transfected RCC cell line that stably interfered with LINC01355 expression.The binding site and target genes of LINC01355 were predicted by the public data network to be probably miR-30a-5p and POSTN,respectively.Dual luciferase reporter gene assays demonstrated that LINC01355 could bind directly to miR-30a-5p,and the same method verified that miR-30a-5p could bind to the POSTN gene.The same qRT-PCR,WB and ASO transfection experiments were used to observe the relationship between LINC01355,miR-30a-5p and POSTN.The relationship between LINC01355 expression levels and the malignant phenotype of RCC was observed by in vivo experiments in tumor-forming nude mice.Part Ⅰ: Expression of LINC01355 and its clinical significanceObjective: To analyze the expression of LINC01355 in renal carcinoma tissues and cells and its clinical significance.Methods:(1)The expression levels of LINC01355 in pan carcinoma as well as renal carcinoma was analyzed using bioinformatics methods.(2)The relationship between LINC01355 expression levels in RCC and tumor characteristics such as clinical stage and pathological grade of RCC.(3)To analyze the value of LINC01355 in the diagnosis of RCC using ROC curves.(4)The value of the column line graph model constructed based on LINC01355 to predict the survival prognosis of RCC patients.(5)Pathway enrichment analysis of potential functional pathways of LINC01355 in RCC;(6)Correlation analysis of LINC01355 expression levels with immune cell infiltration in RCC patients.(7)qRT-PCR detection of LINC01355 expression levels in RCC tissues and individual cell lines.(8)qRT-PCR to detect the expression level of LINC01355 in RCC tissue samples and individual RCC cell lines.(9)Nucleoplasmic separation technique was used to detect the localization of LINC01355 in the cells.Results:(1)LINC01355 was highly expressed in pan carcinoma,RCC tissue paired samples and RCC cell lines.(2)The expression levels in RCC were closely correlated with the clinical stage,relationship of pathological grading,and survival prognosis of RCC.(3)The ROC curve suggested that LINC01355 has some value for the diagnosis of RCC.(4)Univariate and multifactorial analyses showed that LINC01355 was an independent prognostic factor for RCC patients.(5)The column line graph model constructed based on LINC01355 could better predict the survival prognosis of RCC patients.(6)The expression of LINC01355 in RCC patients was associated with multiple immune cell infiltration.(7)Pathway enrichment analysis showed that LINC01355 may be involved in cell proliferation and differentiation and EMT formation in RCC.(8)The expression level of LINC01355 was significantly higher in RCC tissues and cell lines than in paraneoplastic tissues and normal renal proximal tubule cells.(9)Nucleoplasmic separation technique revealed that LINC01355 was distributed in both cytoplasm and nucleus.Conclusion: Bioinformatics analysis revealed that high expression of LINC01355 in RCC tissues and cell lines correlated with clinical staging,grading,and survival prognosis of RCC patients,and was a potential indicator for diagnosing RCC and judging survival prognosis of RCC patients,and was closely associated with immune cell infiltration,cell proliferation and differentiation and EMT occurrence in RCC.LINC01355 is highly expressed in RCC cell lines and tissues,and is distributed in both nucleus and cytoplasm.Part Ⅱ: Validation of LINC01355 and its effect on the biological function of RCCObjective: To explore the effect of LINC01355 on malignant biological function of RCC.Methods:(1)Antisense oligonucleotide(ASO)interfered with the expression of LINC01355 and validated the efficiency.(2)Cell assay CCK8,plate cloning assay to detect the effect of LINC01355 on the proliferation ability of RCC cells.(3)Cell scratch and Transwell assays to detect the effect of LINC01355 on the migration and invasion ability of RCC cells.(4)WB assay was performed to detect the effect of EMT-related proteins.Results:(1)ASO significantly knocked down LINC01355.(2)CCK8 and plate cloning assays revealed that LINC01355 promoted the value-added of RCC cells.(3)Cell scratching and Transwell assays showed that LINC01355 could promote RCC cell migration and invasion.(4)WB experiments revealed that the expression of EMT-related proteins N-cadherin and Vimentin decreased and E-cadherin expression increased after down-regulation of LINC01355,indicating that LINC01355 promoted the EMT process of RCC.Conclusion: In vitro cellular assays demonstrated that LINC01355 promotes RCC cell value addition,migration and invasion.Part Ⅲ: LINC01355 promotes the malignant phenotype of RCC via miR-30a-5p/POSTN axisObjective: To explore the molecular mechanism of LINC01355 in regulating the malignant phenotype of RCC.Methods:(1)Prediction of miRNAs that bind to LINC01355 and downstream target genes using public databases.(2)Dual luciferase reporter gene assays were performed to detect the presence of interactions between LINC01355,predicted miRNAs,and downstream target genes.(3)qRT-PCR and WB to detect changes in the expression levels of LINC01355 at different expression levels of downstream m RNAs and target genes.(4)Rescue experiments to verify whether LINC01355 promotes RCC progression through this signaling axis.(5)Nude mice tumorigenesis assay was performed to verify that LINC01355 promotes the development of renal carcinoma in vivo.Results:(1)miR-30a-5p can bind to both LINC01355 and POSTN m RNA.(2)Dual luciferase reporter gene assay showed that miR-30a-5p can bind directly to LINC01355 and POSTN.(3)qRT-PCR and WB showed that the expression of miR-30a-5p was upregulated after knockdown of LINC01355 expression;POSTN expression was decreased after overexpression of miR-30a-5p.(4)Inhibition of miR-30a-5p expression could partially revert the inhibitory effect of LINC01355 knockdown on RCC cells.(5)In vivo tumorigenic assay in nude mice showed that LINC01355 could promote the growth of RCC in vivo.Conclusion: LINC01355 can promote RCC malignant progression through miR-30a-5p/POSTN signaling axis. |
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