| Objective: Renal clear cell carcinoma is one of the three major urinary tract tumors,and its incidence is on the rise.The role of microRNA in tumorigenesis and development has also attracted attention.In this study,microarray screening of microRNA in renal clear cell carcinoma tissues and adjacent renal tissues was performed to find differentially expressed microRNA.Combined with the literature,target microRNA that may affect the occurrence and development of renal clear cell carcinoma were selected 。 Further validation analysis was performed to explore the relationship between target microRNA and clinical factors.Bioinformatics analysis tools were used to analyze the possible modes of action and mechanisms of the target microRNA.Finally,cell experiments were performed to analyze the effects of target microRNA on proliferation,apoptosis and invasion of renal clear cell carcinoma cells.We hope to find microRNA that can help in the prevention,diagnosis,prognosis judgment,treatment and other aspects of renal clear cell carcinoma,so as to provide clues and directions for subsequent studies.Methods:(1)The carcinoma and paracancerous tissues of 6 patients with renal clear cell carcinoma were analyzed by Agilent human microRNA microarray.Fluorescent labeling was performed according to the standard labeling method of Agilent microRNA.The fluorescence-labeled samples and microarray were hybridized and cleaned.The scanning images and data of each array of fluorescent dyes were obtained by the biochip scanner,and the data were extracted and analyzed by the number extraction software of the gene chip to obtain the microRNA genes with different expressions.(2)According to the literature,miR-15b-5p,miR-21-5p and miR-155-5p were selected as the target microRNA in this study.148 pairs of renal clear cell carcinoma tissues and adjacent normal renal tissues were collected,and 27 pairs of them were randomly selected as the research objects.The expression levels of miR-155-5p,miR-15b-5p and miR-21-5p in 27 pairs of renal clear cell carcinoma tissues and adjacent normal kidney tissues were detected by q RT-PCR,and the differences between the two groups were compared and analyzed.The relationship between the expression of miR-155-5p,miR-15b-5p and miR-21-5p and the clinical features and prognosis was further analyzed.(3)The target gene of the target microRNA was predicted by bioinformatics method,and the possible mechanism of action of the target microRNA target gene was further analyzed.(4)Cell experiment: The expression of miR-15b-5p in renal clear cell cancer cells was inhibited,and its effects on proliferation,apoptosis,migration and invasion of renal clear cell cancer cells were observed.Results:(1)Compared with normal renal tissue,165 microRNA were abnormally regulated in renal clear cell carcinoma,among which 42 microRNA were up-regulated and 123 microRNA were down-regulated.Preliminary results showed that the expression of the two microRNA,hsa-miR-454-3p and hsa-miR-363-3p,was higher in the low-nuclear grading group than in the high-nuclear grading group.(2)The expression level of miR-15b-5p in renal clear cell carcinoma was(1.618±1.004).The expression level of miR-15b-5p in the adjacent normal kidney tissues was(1.119±0.507),and the difference was statistically significant(P=0.025).The expression level of miR-15b-5p was not significantly correlated with the gender,age,smoking history,body mass index,T stage,and pathological nuclear grade of the patients(P > 0.05).Kaplan-Meier survival analysis showed that patients with upregulated miR-15b-5p had a lower rate of disease progression and a longer progression-free survival(PFS).However,further Cox multivariate analysis showed that miR-15b-5p upregulation was not a risk factor for disease progression in patients(P>0.05).The expression level of miR-155-5p in renal clear cell carcinoma was(1.89±1.19).The expression level of miR-155-5p in adjacent normal kidney tissues was(1.16±0.52),and the difference was statistically significant(P=0.014).The expression level of miR-155-5p in renal clear cell carcinoma tissues was not significantly correlated with gender,age,smoking history,body mass index,T stage,and pathological nuclear grade(P > 0.05).Cox multivariate analysis showed that downregulation of miR-155-5p was an independent risk factor for disease progression(P<0.05).Patients with downregulation of miR-155-5p had a higher risk of disease progression and a shorter progression-free survival(PFS)(RR=13.195).The expression level of miR-21-5p in renal clear cell carcinoma tissues was(2.118±1.402).The expression level of miR-21-5p in adjacent normal kidney tissues was(1.246±0.711),and the difference was statistically significant(P=0.014).The expression level of miR-21-5p in renal clear cell carcinoma tissues was not significantly correlated with gender,age,smoking history,body mass index,T stage,hypertension,diabetes and pathological nuclear grade(P > 0.05).Kaplan-Meier survival analysis suggested that there was no statistical difference in the rate of disease progression between the up-regulated and down-regulated expression of miR-21-5p in renal clear cell carcinoma tissues(P > 0.05).(3)Bioinformatics analysis suggested that miR-15b-5p plays an important role in renal clear cell carcinoma through nucleoplasm signal pathways,miR-21-5p plays an important role in renal clear cell carcinoma through blood vessel remodeling and miR-155-5p plays an important role in renal clear cell carcinoma through nucleus and other signaling pathways.(4)Proliferation experiment: the OD values of blank control group,control group,and miR-15b-5p-inhibitors group were 0.788±0.050,0.726±0.041,0.500±0.057,respectively.The cell proliferation of miR-15b-5p-inhibitors group was significantly lower than that of the other two groups,P < 0.05.Apoptosis experiment: Apoptosis rate(%)of blank control group,control group,and miR-15b-5p-inhibitors group were 7.290±0.616,7.827±0.701,and 12.897±0.508,respectively.Apoptosis rate of miR-15b-5p-inhibitors group was significantly higher than the other two groups,P<0.05.Cell migration and invasion detection: Cell migration in the blank control group,the control group,the inhibitors group and the miR-15b-5p-inhibitors group were 122.778±16.476,112.000±9.695,68.000±14.714,respectively.Cell migration in the miR-15b-5p-inhibitors group was significantly lower than that in the other two groups,P<0.05.The cell invasion of blank control group,control group,miR-15b-5p-inhibitors group was 116.444±17.155,113.556±12.914,65.778±13.160,respectively.The cell invasion of miR-15b-5p-inhibitors group was significantly lower than that of the other two groups,P<0.05.Conclusion:(1)We found that 165 microRNA were abnormally regulated in CCRCC,suggesting the existence of clear cell carcinoma specific expression profile.Hsa-miR-454-3p and hsa-miR-363-3p were helpful in distinguishing different nuclear classifications.(2)The expression of miR-15b-5p in renal clear cell carcinoma tissues is higher than that in normal renal tissues.Bioinformatics analysis suggested that miR-15b-5p has an influence on renal clear cell carcinoma through nucleoplasm pathway,providing information for further mechanism research.(3)The expression of miR-155-5p in renal clear cell carcinoma tissues was higher than that in normal renal tissues,and down-regulation of miR-155-5p was an independent risk factor for disease progression in patients.Bioinformatics analysis suggested that miR-155-5p could affect renal clear cell carcinoma through nucleus and other signaling pathways,which could provide clues for further mechanism research.(4)The expression of miR-21-5p in renal clear cell carcinoma tissues was higher than that in normal renal tissues.Biochemistry analysis suggested that miR-21-5p could exert influence on renal clear cell carcinoma through blood vessel remodeling and other signaling pathways,which could provide clues for further mechanism research.(5)Inhibition of miR-15b-5p expression can inhibit the growth,induce apoptosis,inhibit migration and inhibit invasion of renal clear cell cancer cells.The tumor-promoting effect of miR-15b-5p in renal clear cell carcinoma may make it a new target for the treatment of clear cell carcinoma. |
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