The Clinical Study Of Serum Exosomal MiRNAs As Novel Molecular Markers For Clear Cell Renal Cell Carcinoma

BACKGROUD : Renal cell carcinomas(RCC)are common adult urinary system malignancies.The latest epidemiological data shows that the incidence of RCC accounts for about 3.8% of all malignancies,while,the mortality rate is as high as 24%,and its incidence rate of 2% to 4% of the rate of growth year by year.Clear cell renal cell carcinoma(ccRCC)is the most common and aggressive pathological type of RCC and accounts for 80~90% of all cases of RCC.ccRCC pathogenesis is unknown and approximately 30% of patients are diagnosed at an advanced stage when the tumor metastasis has occurred,which leading to a poor prognosis.Currently,surgical excision is the mainstay to cure ccRCC for resisting to both chemotherapy and radiation therapy.Clinical data shows that the 5-year survival rate of early ccRCC patients is up to 75%,so early diagnosis has great clinical significance for cure ccRCC.Unfortunately,clinical diagnostic examinations,such as general examinations,ultrasound scans,CT scans and X-ray angiography cannot achieve early diagnosis yet.Therefore,it is urgent to find out early markers of ccRCC for early diagnosis and early treatment.Exosomes are 30-150 nm nanovesicles which are actively secreted by different types of cells under normal or pathological conditions and are stable in circulating body fluids.Exosomes play a key role in cell-cell communication by delivering biologically active molecules(including signal peptides,lipids and nucleic acids),particularly by carrying micro RNA(miRNA)to participate the progression of diseases.miRNA are small non-coding RNAs in the size range of 19-24 nucleotides(nt),which could lead to the degradation or translational suppression of target genes in post-transcriptional level.miRNAs are closely related to a variety of pathological activities,especially tumors.Recent studies have found that urinary exosomal miRNAs of patients with ccRCC were significantly elevated,indicating that the potential of urinary exosomal miRNAs as non-invasive diagnostic biomarkers for ccRCC.However,the expression and clinical significance of serum exosomal miRNAs in ccRCC patients have not been brought to light yet.OBJECTIVE: To analyze the serum levels of exosomal miRNAs in patients with ccRCC and to screen specifically expressed miRNAs in patients with ccRCC,and to evaluate their diagnosis value to ccRCC,especially to diagnosis early ccRCC.METHODS: Serum exosomes from 126 ccRCC patients without any treatment were selected as study specimens.At the same time,124 age and sex matched normal human serum exosomes were collected as controls.First,nanoparticle tracking analysis technology(NTA)and western blotting technology(WB)were used to identify exosome size and surface characteristic proteins.Then,768 miRNA expression levels in two mixed serum exosomes from 20 patients with ccRCC and 20 normal controls were measured using Taq Man low density array(TLDA)technology,to screen the significantly up-regulated serum exosomal miRNAs in ccRCC patients.Next,in training set(27 cases of ccRCC and 26 cases of normal controls),using real-time fluorescent quantitative PCR technology(q RT-PCR)to detect selected miRNAs from TLDA,in order to screen significantly increased serum exosomal miRNAs.Then again by performing q RT-PCR,miRNAs were validated in the validation set(72 cases and 72 normal controls)to obtain stable elevated miRNAs in ccRCC.In testing set(27 cases and 26 normal controls),miRNAs were re-validated.In addition,miRNAs were also detected in 40 ccRCC patients who had undergone surgery.Finally,ROC curve,Risk score analysis and logistic regression analysis were applied to evaluate the clinical diagnostic value of miRNAs and miRNA panels for ccRCC.RESULTS: NTA showed that the diameter of exosomes was about 100 nm and consistent with previous reports.WB indicated the expressions of CD63 and CD9 in exosomes were detected in samples,which are specific markers of exosomes.TLDA showed that the miRNAs expression profile of serum exosomes in ccRCC patients were significantly different from normal controls,44 miRNAs in ccRCC were significantly upregulated(more than 4-fold),then 6 miRNAs with cycle threshold(Ct)<30(including miR-28-3p,miR-200 a,miR-1826,miR-103,miR-1249 and miR-640)were selected for further analysis.Firstly,q RT-PCR technology was used to verify the expression levels of these miRNAs and results showed that all 6 miRNAs were significantly higher than those in normal controls(fold change ranged from 2.90 to 11.43 times,P<0.05).Then in validation set and testing set,data indicated that levels of all 6 miRNAs were stable and significantly elevated in ccRCC(fold change ranged from 1.71 to 3.44,P<0.05;2.17 to 8.24,P<0.05,respectively.).Statistical analysis showed that 6 miRNAs expression in early stage(Grade I and II)were significantly higher than that in normal controls.In addition,the expression levels of the 6 miRNAs were significantly decreased after surgery.ROC curve analysis(AUC)showed that the range of AUC for 6 miRNAs in the training set,validation set and testing set were 0.751~0.893,0.620~0.808,0.744~0.920.According to Risk score analysis to compare combinatorial analysis of 6 miRNAs,statistical results showed that the 6-miRNA panel owned the better diagnostic efficiency,the AUC of the 6-miRNA panel in the training,validation set and testing set were 0.751(95%CI=0.619~0.884),0.804(95%CI=0.729~0.878)and 0.887(95%CI=0.791~0.984),respectively,and superior to other miRNA panels.Based on the selected cut off value,the negative predictive value(NPV)and positive predictive value(PPV)of the 6-miRNA panel to diagnosis ccRCC in three groups were 73% and 73%,72% and 75%,92% and 92%,respectively.Besides,the AUC of 6-miRNA panel for early diagnosis ccRCC was 0.832(95% CI=0.774~0.889),according to the selected cut off value,the NPV and PPV of the 6-miRNA panel in the early diagnosis of ccRCC was 77% and 73%.Logistic regression analysis showed the OR values of the six miRNAs and 6-miRNA panel were statistically significant.CONCLUSIONS: Serum exosomal miRNA expression profiles of patients are significantly different from normal controls.Compared with normal controls,the concentration of six miRNAs(miR-28-3p,miR-200 a,miR-1826,miR-103,miR-1249 and miR-640)were significantly elevated in preoperative samples from patients with ccRCC and in early stage of ccRCC 6 miRNAs has shown a significant upward trend,and their expression level were significantly reduced in patients after surgery,so they have the value to evaluate surgical treatment.In addition,6-miRNA panel have high accuracy in the diagnosis of ccRCC and early ccRCC,therefore,they can be used as potential biomarker for ccRCC.