The Study Of Clinical Value And Biological Function Of SR-B1 In Clear Cell Renal Cell Carcinoma

Renal cancer,also is known as renal cell carcinoma(RCC).In urological system,renal cancer is one of the tumors with highest occurrence,high malignancy and strong lethality.However,due to the occurrence of renal cancer is hidden,with no obvious or classical symptoms,when diagnosed,some patients with renal cancer have been in phase of middle or advanced,or have been with local or distant metastasis.Although surgical removement of tumors is the most effective treatment choice,some patients still develop distant metastasis,accompanying poor prognosis.Moreover,as RCC is highly non-sensitive to radiotherapy or chemotherapy,molecular-targeted therapy has limited effectiveness,so,it is of great importance in clinical practice to find and diagnose the renal cancer early,to judge the prognosis of patients with renal cancer early.While the most prevalent type of all renal cancer pathological type is clear cell renal cell carcinoma(ccRCC).The major histopathological character is the lipid accumulating in cell cytoplasm.Recent researches show that lipid metabolism plays vital roles in the carcinogenesis and development of several kinds of cancers.However,the role of lipid metabolism in renal cancer has not been well clarified.Besides,many studies find that high density lipoprotein(HDL),and its receptor(high density lipoprotein receptor,HDL-R)play major roles in lipid accumulation in cells.Scavenger receptor class B type Ⅰ(SR-B1).is the major receptor of HDL,playing important role in mediating up-take of lipid into cancer cells.Therefore,to explore the roles of lipid metabolism in ccRCC,we plan to introduce this study from the following three parts:Part Ⅰ The expression of SR-B1 in clear cell renal cell carcinomaPurpose:To explore the expression pattern of SR-B1 in clear cell renal cell carcinoma,making a basis for subsequent studies.Methods:After the surgery of ccRCC patients,we selected many pairs of ccRCC tissues and adjacent normal kidney tissues,by using real-time quantitative reverse transcription PCR(qRT-PCR)and Western blot assays,we determined the expression of SR-B1 in ccRCC and paired normal kidney tissues;we also carried out oil red o(ORO)staining,hematoxylin-eosin(HE)staining and immunohistochemistry(IHC)staining in these paired tissues.Results:In total 100 pairs of ccRCC and normal kidney tissues determined by qRT-PCR,mRNA of SR-B1 in ccRCC cancerous tissues was markedly up-regulated(P<0.001);moreover,the results of western blot in 10 pairs tissues showed that protein level of SR-B1 in ccRCC cancerous tissues was also much higher than that in normal kidney tissues;ORO and HE staining assays showed that there was much lipied accumulation in ccRCC cancerous tissues rather than adjacent normal kidney tissues.IHC also proved that SR-B1 protein in ccRCC cancerous tissues was much higher than adjacent normal kidney tissues.Conclusion:SR-B1 was up-regulated in ccRCC cancerous tissues;there was much lipid droplets in ccRCC cancerous tissues.Part Ⅱ Clinical value of SR-B1 in clear cell renal cell carcinomaPurpose:We aimed to study the clinical value of SR-B1 in ccRCC,including diagnostic value and prognostic value,to support evidence for early diagnosis,early treatment and early judgement of prognosis.Methods:By using the real-time quantitative reverse transcription PCR(qRT-PCR),we tested the expression of SR-B1 in paired ccRCC cancerous tissues and normal kidney tissues.According to the expression level of SR-B1,we drew the ROC(receiver operating characteristic)curve and got the value of AUC(area under the curve)with the best sensitivity and specificity.In the same way,after expanding the sample size,we tested theSR-B1 expression in paired ccRCC cancerous tissues and normal kidney tissues,analyzed the correlations between SR-B1 expression and clinical variables,conducted Kaplan-Meier analysis and COX regression analysis,and predicted the prognosis of ccRCC patients.Results:According to the expression of SR-B1 in 100 pairs of ccRCC cancerous tissues and adjacent kidney tissues,the ROC curve showed that the value of AUC was 0.8486(95%confidence interval 0.7926-0.9045),with a sensitivity of 0.75(95%confidence interval 0.6535-0.8312)and a specificity of 0.90(95%confidence interval 0.8238-0.9510).By expanding the sample size to 113 pairs,the expression of SR-B1 in ccRCC cancerous tissues exhibited significant correlation with tumor size(P=0.019),nuclear grade(P=0.040)and distant metastasis(P=0.006).Kaplan-Meier curve analysis showed that SR-B1 expression correlated significantly with progression-free survival(PFS)(P=0.0062),and the patients with higher SR-B1 expression had a shorter PFS.When conducting with COX regression analysis,univariate analysis showed that the gender(P=0.029),tumor size(P=0.004),lymph node metastasis(P<0.001).distant metastasis(P=0.001),TNM stage(P<0.001)and SR-B1 mRNA level(P=0.009)were significantly related to the PFS of ccRCC patients,suggesting that these variables can be used as prognostic factors,predicting the length of PFS;multivariate analysis suggested that only high SR-B1 mRNA expression(P=0.021),gender(P<0.001)and tumor size(P=0.002)were independent prognostic factors.Conclusion:SR-B1 expression could be used as a early diagnostic biomarker in ccRCC;high SR-B1 expression related tightly with aggressive features of ccRCC,and could serve as a independent prognostic biomarker.Part Ⅲ Biological function of SR-B1 in clear cell renal cell carcinomaPurpose:We aimed to explore the biological function of SR-B1 in ccRCC,to give support to gene-targeted therapy for ccRCC.Methods:Based on the high expression status of SR-B1,we designed and syntheized specific small interfering RNA(siRNA)of SR-B1 and negative control.We tested the silence effect in 786-0 and Caki-1 ccRCC cell lines.We analyzed the impact on cell proliferation,colony formation,invasion and migration after the interfering of SR-B1 expression,then evaluated its biological function.Results:Specific siRNA could radically decrease the SR-B1 expression in ccRCC cell lines in both mRNA and protein level;MTT[3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide]assays showed that reduction or silence of SR-B1 led to obvious proliferation inhibition;colony formation abilities were greatly inhibited after siRNA interfering to SR-B1;Transwell assays demonstrated that the invasion and migration ability of ccRCC cell lines decreased when SR-B1 expression was interfered;wound healing potention of cells was also attenuated as SR-B1 was specifically down-regulated.Conclusion:The biological behaviors of ccRCC cell lines were highly inhibited when SR-B1 expression was specifically interfered,suggesting that SR-B1 could serve as an oncogene in ccRCC.