The Significance And Mechanism Of Phospholipase C-like Protein 1 In Abnormal Lipid Metabolism Of Clear Cell Renal Cell Carcinoma

In the urinary system,renal cell carcinoma(RCC)is one of the solid tumors with the highest incidence,accounting for about 80%-90% of all malignant tumors of the kidney.Among the pathological types,clear cell renal cell carcinoma(cc RCC)is the most common,accounting for about 70%-80% of renal cell carcinoma.At present,the treatment of clear cell renal cell carcinoma is based on a comprehensive treatment method combining surgery and molecular targeted drugs.Although advances in treatment methods have improved the prognosis of patients to a certain extent,the huge fluctuations in survival rates and the resistance of targeted drugs make the treatment of clear cell renal cell carcinoma still an important clinical challenge.Metabolic abnormality is a hallmark biological feature of cc RCC,and lipid metabolism abnormality is the most significant.Previous studies have shown that there is a large accumulation of lipid droplets in cc RCC cells,and more and more evidences show that such abnormal accumulation of lipids plays a key role in the occurrence and progression of cc RCC.Nevertheless,the specific regulation and mechanism of lipid metabolism in cc RCC is still unclear.Based on the lipid metabolism of cc RCC,this study found and clarified the metabolism-related biomarker Phospholipase C-like Protein 1,PLCL1(Phospholipase C-like Protein 1,PLCL1)through comprehensive bioinformatics analysis.At the same time,restoring the expression of PLCL1 in cc RCC cells can significantly inhibit tumor progression and significantly reduce lipid accumulation.Further studies have shown that PLCL1 in cc RCC cells affects the expression of uncoupling protein 1(UCP1),thereby affecting the progress of cc RCC and the metabolic state of cells.The main purpose of this study is to explore the expression of PLCL1 in cc RCC and its correlation with the clinical prognosis of cc RCC,and to clarify the specific role of PLCL1 in the occurrence and development of cc RCC,cell metabolism and other biological processes,and to analyze the specific mechanism by which PLCL1 affects the progress of cc RCC.Part I: The expression level of PLCL1 in cc RCC and the relationship between PLCL1 and the clinical prognosis of kidney cancerObjective: Explore the differential expression of PLCL1 in cc RCC and analyze the relationship between the expression level of PLCL1 and the clinical prognosis of cc RCC.Methods: 1.First,conduct a comprehensive bioinformatics analysis based on the relevant data from the public database Oncomine,the public database EMBL-EBI(European Bioinformatics Institute)and the public database TCGA(The Cancer Genome Atlas)of cc RCC,to clarify the expression of PLCL1 in cc RCC.2.Further analyze the relevant clinical information in the TCGA database,and analyze the relationship between the expression level of PLCL1 and the clinical characteristics of cc RCC patients,disease stages and patient prognosis.3.Use real-time quantitative PCR,Western blot,immunohistochemistry and other molecular biology experimental techniques to verify the differential expression of PLCL1 in cc RCC in clinical tissue specimens and specific cell lines of cc RCC.Results: 1.Based on the screening results of cc RCC lipid metabolism-related differential genes in the public database Oncomine and the public database EMBL-EBI,PLCL1 was found to be an important lipid metabolism-related biomarker in cc RCC.2.Analysis based on the TCGA database shows that PLCL1 is significantly lower expressed in cc RCC tissues compared with adjacent tissues.At the same time,bioinformatics analysis based on five independent cc RCC-related data subsets in the Oncomine database(Beroukhim Renal,Lenburg Renal,Gumz Renal,Jones Renal,Yusenko Renal)also confirmed that PLCL1 is significantly low in cc RCC.3.Further bioinformatics analysis based on the clinical information of the TCGA database shows that lower PLCL1 expression levels in cc RCC indicate shorter overall survival time OS(Overall Survial)and shorter disease-free progression survival time DFS(Disease Free Survival).Related ROC curve analysis shows that the expression level of PLCL1 has obvious diagnostic value for cc RCC.At the same time,the results of bioinformatics analysis also showed that the expression level of PLCL1 gradually decreased with the increase of cc RCC clinical stages(TNM stage,G stage).And based on the results of COX regression,PLCL1 is considered to be an independent risk factor for the prognosis of cc RCC patients.4.Molecular biology experiments based on cc RCC clinical tissue specimens and specific cell lines showed that the m RNA and protein levels of PLCL1 were significantly lower in cc RCC than the control group.Conclusion: PLCL1 is an important lipid metabolism-related differentially expressed gene in cc RCC.It is significantly low expressed in cc RCC and is highly correlated with the staging and clinical prognosis of cc RCC.The expression level of PLCL1 in cc RCC can be used as a new molecular marker related to the diagnosis and prognosis of renal cancer.Part II: In vivo and in vitro study of the effect of PLCL1 on the biological function of clear cell renal cell carcinomaObjective: Explore the significance of the expression level of PLCL1 on the biological function of clear cell renal cell carcinoma.Methods: 1.Construct cc RCC cell lines which stably overexpresses PLCL1 and stably silences PLCL1 by using PLCL1 overexpressing lentivirus and specifically silenced sh RNA.Real-time quantitative PCR and western blot were used to evaluate the silencing and overexpression efficiency of PLCL1 at the m RNA level and protein level,respectively.2.Carry out a series of molecular biology experiments such as CCK8 proliferation experiment,clone formation experiment,cell scratch experiment,transwell experiment,etc.in the constructed cc RCC cell lines with PLCL1 stably overexpressed and stably silenced to evaluate the effect of PLCL1 on the proliferation and growth of cc RCC cells,Migration,invasion and many other aspects of the basic biological functions of tumors.3.Use oil red staining,triglyceride content detection and other molecular biology experimental methods to detect changes in lipid content in cc RCC cell lines with PLCL1 stably overexpressed and stably silenced to assess the effect of PLCL1 on cc RCC lipid metabolism.4.Select 6-week-old BALB/c male nude mice,use PLCL1 overexpressing clear cell renal cell carcinoma CAKI cell line and corresponding control cells as tool cells,and construct subcutaneous xenografts model in nude mice by subcutaneous injection and metastasis model by tail vein injection.5.The subcutaneous xenograft tumor model constructed above was tested every four days to evaluate the weight and volume of the xenograft of the nude mice,and anatomy was performed on the 44 th day.The corresponding xenograft tumor was weighed,frozen and fixed.Subsequently,oil red staining and immunohistochemical analysis were performed on the above-mentioned transplanted tumor specimens to evaluate the effect of PLCL1 on the growth,proliferation and lipid metabolism of cc RCC cells in vivo.6.For the tail vein metastasis model constructed above,the body weight of the nude mice was also assessed on the basis of four days and dissected on the 44 th day.Subsequently,the relevant main organs(liver,spleen,kidney and lungs)were weighed,and the tumor metastasis of the relevant organs was evaluated.At the same time,the corresponding tissue samples were saved for immunohistochemistry,H&E staining and other molecular biological tests to evaluate the effect of PLCL1 on the migration and invasion ability of cc RCC cells in vivo.Results: 1.Real-time quantitative PCR and western blot detection showed that when the cc RCC cell line was transfected with PLCL1 specific overexpression lentivirus,the m RNA level and protein level of PLCL1 were significantly improved compared with the corresponding control cell line.At the same time,when cc RCC was transfected with sh RNA specific to PLCL1,compared with the corresponding control cell line,the m RNA level and protein level of PLCL1 were significantly reduced.The above results indicated that the experimentally constructed cc RCC cell lines with PLCL1 stably overexpressed and stably silenced were successful and effective.2.CCK8 cell proliferation experiments and clone formation experiments showed that PLCL1 overexpressed could obviously suppress the proliferation and growth of cc RCC cells.At the same time,knockdown of PLCL1 could significantly promote the proliferation and growth of cc RCC cells.In addition,the results of cell scratch experiments and transwell experiments showed that PLCL1 overexpressed could obviously suppress the migration and invasion of cc RCC cells,while silencing the expression of PLCL1 could significantly promote the migration and invasion of cc RCC cells.3.Cell oil red staining and triglyceride content determination showed that overexpression of PLCL1 could significantly reduce lipid accumulation in cc RCC cells,while silencing PLCL1 could significantly promote lipid accumulation in cc RCC cells.4.The evaluation of the subcutaneous transplanted tumor model showed that compared with the control group,the growth rate of the subcutaneous transplanted tumor was significantly reduced after PLCL1 overexpression,and the corresponding malignant indicators were significantly reduced.At the same time,the results of oil red staining showed that the overexpression of PLCL1 could significantly reduce Lipid accumulation in subcutaneous xenografts.5.The evaluation of the tail vein metastasis model of nude mice showed that the tumor metastasis of various organs was significantly reduced after PLCL1 overexpression compared with the control group.Conclusion: PLCL1 is an important tumor suppressor of cc RCC.Overexpression of PLCL1 can significantly inhibit the progress of cc RCC and significantly reduce the lipid accumulation of cc RCC.Part III: Study on the mechanism of PLCL1 affecting the biological function of clear cell renal cell carcinoma by up-regulating UCP1Objective: Explore the specific molecular mechanism of PLCL1 affecting the occurrence and development of cc RCC and lipid metabolism.Methods: 1.Perform whole-transcriptome sequencing analysis on the cc RCC cell line stably overexpressing PLCL1 to analyze the metabolic pathways and potential molecular mechanisms involved in PLCL1 in cc RCC.2.Further conduct comprehensive bioinformatics analysis on whole transcriptome sequencing data,analyze the relationship between PLCL1 and lipid browning-related pathways,and use western blot to clarify the regulation of PLCL1 on key lipid browning genes(PGC1A,CIDEA,DIO2,UCP1).3.Use immunofluorescence,western blot and real-time quantitative PCR and other molecular biology experimental techniques to detect the expression of UCP1 in the cc RCC cell lines with PLCL1 stably overexpressed and stably silenced,and clarifies the specific regulation mode and regulation level of PLCL1 on UCP1.4.Using bioinformatics analysis,western blot,real-time quantitative PCR and immunohistochemistry and other experimental techniques to clarify the expression of UCP1 in cc RCC tissues and cell lines.5.UCP1 specific overexpression plasmid and si RNA were used to construct cc RCC cell lines with PLCL1 overexpressed and knocked down,and used CCK8 proliferation experiment,transwell experiment,cell oil red staining and triglyceride content determination and other molecular biology experiments methods to clarify the influence of UCP1 on the progress of cc RCC and lipid accumulation.6.Use specific si RNA to knock down UCP1 in the cc RCC cell line stably overexpressing PLCL1 to construct a functional recovery model,and use CCK8 proliferation experiment,transwell experiment,cell oil red staining and other molecular biology experimental methods to detect the progress of cc RCC and changes in lipid accumulation in the above model,so as to clarify the importance of UCP1 in PLCL1 regulating the progress and metabolism of cc RCC.7.Use cycloheximide(CHX)to construct a protein half-life experimental model in cc RCC cell lines with PLCL1 stably overexpressed and stably silenced.Western blot technology was used to detect the expression of UCP1 in the above models,and to evaluate the influence of PLCL1 on the protein stability of UCP1.8.Add MG132 and chloroquine to the cc RCC cell line with PLCL1 stably silenced,and perform ubiquitination-related immunoprecipitation to clarify the specific molecular biological mechanism of PLCL1 regulating UCP1.9.Use the cc RCC cell line stably overexpressing PLCL1 and the corresponding control cell line to construct a nude mouse subcutaneous xenograft model in BALB/c male nude mice.Use immunohistochemistry to detect the expression of UCP1 in subcutaneous xenograft tumors to verify the regulation of UCP1 by PLCL1 in vivo.Results: 1.The results of whole transcriptome sequencing based on PLCL1 overexpression show that PLCL1 highly affects fatty acid metabolism and cholesterol metabolism pathways in cc RCC.2.Further analysis of the whole transcriptome sequencing data shows that PLCL1 does not directly affect the m RNA expression of key lipid browning genes in cc RCC,and the results of western blot show that among the key lipid browning genes,PLCL1 directly regulates UCP1 expression.3.UCP1 test results show that overexpression of PLCL1 in cc RCC can significantly increase the expression of UCP1,while silencing PLCL1 can significantly down-regulate the expression of UCP1,and this regulation is limited to the protein level of UCP1 and does not affect the m RNA level of UCP1.4.The results of bioinformatics analysis and related experimental tests show that UCP1 is also significantly under-expressed in cc RCC tissues and cell lines,and overexpression of UCP1 in cc RCC can significantly inhibit the proliferation,migration,invasion and lipid accumulation of cc RCC.At the same time,silencing UCP1 in cc RCC can also significantly promote the proliferation,migration,invasion and lipid accumulation of cc RCC.5.The test results based on the functional recovery model show that UCP1 in cc RCC can significantly reverse the inhibition of tumor proliferation,migration,invasion and lipid accumulation caused by overexpression of PLCL1.6.Protein half-life experiments indicate that PLCL1 can significantly improve the protein stability of UCP1 in cc RCC,and the results of ubiquitination immunoprecipitation show that overexpression of PLCL1 can significantly reduce the ubiquitination level of UCP1,while silencing PLCL1 can significantly increase the ubiquitination level of UCP1.7.The evaluation of the nude mouse subcutaneous xenograft tumor model showed that the expression level of UCP1 in the subcutaneous xenograft tumor was significantly increased compared with the control group after PLCL1 overexpression.Conclusion: The influence of PLCL1 on the occurrence,development and lipid accumulation of cc RCC is achieved by up-regulating the expression of UCP1.Its mechanism is to directly affect the ubiquitination level of UCP1,which in turn affects the protein stability of UCP1,thereby regulating its expression.