| Background and PurposeRenal cancer is one of the ten common human tumors,and its morbidity and mortality are increasing year by year.Clear cell Renal Cell Carcinoma(ccRCC)is the most common pathological type of renal cancer,and its prognosis is often poor.The pathogenesis of renal cancer is still unclear,and VHL gene mutation is believed to be closely related to the occurrence and development of ccRCC.With the development of autophagy research,it has been found that there is a close relationship between renal cancer and autophagy regulation,which provides a new research direction to explain the pathogenesis of renal cancer.In the past decade or so,renal cancer has moved from early non-specific immunotherapy(the cytokine era)to targeted inhibition of vascular endothelial growth factor(VEGF)therapy,and is now entering a new era of immunotherapy.Although these advances have improved the prognosis of renal cancer patients,there are still many patients with advanced and metastatic kidney cancer who are insensitive or resistant to these drugs.Early diagnosis and timely surgical treatment are still the keys to the treatment of localized renal cancer.However,due to the lack of reliable biomarkers for the diagnosis and prognosis of renal cancer,it is very important to find biomarkers that can contribute to the specific diagnosis of renal cancer and evaluate the prognosis of patients.At present,a number of studies have shown that lysosomal membraneassociated protein 1(LAMP1)is differentially expressed in a variety of tumors and plays an important role in tumors,which is expected to become a potential prognostic factor.However,there are few reports on LAMP1 expression in renal carcinoma and its relationship with prognosis of patients.The purpose of this study was to analyze the expression of LAMP1 in ccRCC and its clinical significance,and to study the role and possible mechanism of LAMP1 in ccRCC.MethodsFirstly,bioinformatics analysis was conducted,and the expression of LAMP1 gene in different tumor tissues and normal tissues was analyzed by using TIMER database,and the expression difference of LAMP1 in ccRCC tissues and normal tissues and the relationship between the expression level and clinicopathological features were analyzed by using TCGA database.The expression difference of LAMP1 at ccRCC protein level was also analyzed using ULCAN database.At the same time,the differentiation effect of LAMP1 between tumor tissue and normal tissue was evaluated by ROC curve.Single sample gene enrichment(ssGSEA)was used to analyze the relationship between LAMP1 and immune infiltration.Kaplan-Meier analysis and COX regression analysis were used to evaluate the correlation between LAMP1 and survival.Based on the results of multivariate COX regression analysis,the effect of LAMP 1 on the prognosis of patients was predicted by using rosette.Next,we also constructed a clinical specimen bank by collecting 60 pairs of cancer tissues and adjacent tissues from clinical ccRCC patients.Weston blot technique was used to detect the expression difference of LAMP1 protein in these tissues,and the correlation between LAMP1 expression and clinical related indicators and prognosis was statistically analyzed.Finally,through molecular cell biology experiments,we transfected lentivirus into renal cancer cell lines to overexpress LAMP 1 gene,and then used siRNA knockdown to do rescue experiments after overexpression.The effects of LAMP1 on cell proliferation,invasion and migration were investigated by cell scratches assay,CCK8 assay,Transwell assay and colony formation assay.At the same time,the effects of the key gene VHL and autophagy related gene LC3C on the expression of LAMP1 were explored through molecular biology experiments,and the upstream and downstream regulatory pathways of LAMP1 were explained.ResultsThrough the analysis of LAMP1 gene in TIMER database,it was found that the high and low expression of LAMP1 gene was different in different tumors.LAMP1 gene was significantly underexpressed in ccRCC tissues(P<0.001).Patients with low LAMP1 expression had higher T stage,clinicopathological stage,and histological grade(P<0.01),LAMP1 expression level in male was lower than that in female(P<0.05).ssGSEA analysis showed that LAMP1 was associated with multiple immune cell infiltration(P<0.05).Patients with low LAMP1 expression were associated with poorer overall survival(OS),disease-specific survival(DSS),and progression-free survival(PFI)(P<0.01),and low LAMP1 expression was also associated with poor OS in T3&4,T3,M1,G3&4,Stage Ⅲ&Ⅳ and Stage Ⅳ patients(P<0.05).Multifactorial survival analysis showed that LAMP1 was an independent prognostic indicator of OS(P<0.05).The nomogram based on multivariate analysis showed that LAMP1 had a good predictive ability for ccRCC patients.In conclusion,LAMP1 plays an inhibitory role in renal carcinoma.Analysis of cancer and paracancer tissue samples from 60 patients with ccRCC showed that LAMP1 protein expression level in cancer tissue was significantly lower than that in paracancer tissue(P<0.001).Clinical correlation analysis showed that LAMP1 was associated with age(P<0.001),gender(P<0.001)and T stage(P=0.039),but not with M stage,clinical stage and nuclear grade(P>0.05).Further survival analysis showed that LAMP1 expression level(P=0.019),T stage(P=0.040),clinical stage(P<0.001)and nuclear grade(P=0.043)were independent factors affecting the prognosis of ccRCC patients.Cytological experiments were carried out to verify the significance of LAMP1 on renal carcinoma cells.The high expression of LAMP1 in renal carcinoma cell lines inhibited the proliferation,migration and invasion of the cells,while rescue experiments restored the proliferation,migration and invasion of the cells.Studies on the upstream and downstream regulatory pathways of LAMP 1 found that when autophagy was activated or blocked,LAMP1 protein expression did not change significantly.However,when LC3C gene was knocked down,The expression of LAMP1 protein decreased,while the expression of LC3C protein decreased after overexpression of LAMP 1 gene.Meanwhile,we found that the expression of LAMP1 in VHL(+)cells was lower than that in VHL(-)cells.ConclusionThe above studies indicated that database and clinical specimen studies showed that LAMP1 was low expression in ccRCC,and patients with low expression of LAMP 1 had a poor prognosis.Cell experiments showed that LAMP1 could inhibit the proliferation,migration and invasion of renal cancer cells.Therefore,LAMP1 is a tumor suppressor and can be used as a biomolecular marker to evaluate tumor stage and prognosis.Studies on the mechanism showed that VHL gene may affect LAMP1 expression through regulating LC3C mediated autophagy,which may affect the efficacy of targeted drugs for renal cancer and may serve as a marker protein to evaluate the efficacy of drugs. |
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