| Object : 1.Screen differentially expressed miRNAs in clear cell renal cell carcinoma and verify the low expression of miR-9-5p and its clinical value.2.Explore the effect of miR-9-5p on the biological characteristics of renal cancer cells in vitro.3.Predict the potential targets of miR-9-5p and explore its related mechanisms.4.Explore the expression of miR-9-5p in serum exosomes of patients with clear cell renal cell carcinoma.Method:1.We checked out previous studies and found the differentially expressed miRNAs in clear cell renal cell carcinoma,then detected miR-9-5p in 44 pairs of clear cell renal cell carcinoma tissue with its adjacent tissue,clear cell renal cell carcinoma cell line 786-O and SN12-PM6 by qRT-PCR.2.We constructed the control plasmid(pFlag-cmv2)and miR-9-5p expression plasmid(phsa-miR-9),then transfected into 786-O and SN12-PM6 cells,and analyzed the expression of miR-9-5p in these cells by qRT-PCR.3.We detected the changes in proliferation,invasion and metastasis of transfected 786-O cells by CCK8 and transwell assays.4.We predicted the target genes of miR-9-5p by Targetscan and other target prediction softwares,then detected the expression of these potential target genes in clear cell renal cell carcinoma tissues and their adjacent tissues as well as 786-O and SN12-PM6 cells by qRT-PCR and Western blot.5.The serum exosomes of 4 patients with clear cell renal cell carcinoma and 4 healthy controls were separated by ultracentrifugation and observed by transmission electron microscopy.The miRNA expression profiles in exosomes were detected and analyzed by HiSeq deep sequencing and analyzed by bioinformatics techniques.6.We measured the expression of miR-9-5p in serum exosomes in 14 patients with clear cell renal cell carcinoma and 6 healthy controls by qRT-PCR.Result :1.Previous researches revealed that the expression of miR-9-5p was downregulated 7.6-fold in clear cell renal cell carcinoma cancer tissues compared with its adjacent tissues by high-throughput microarray analysis of miRNA expression profiles and it also was down-regulated in other tumors as a tumor suppressor.The result of qRT-PCR showed that miR-9-5p was significantly down-regulated(p = 0.0001<0.05)in 93.2%(41/44)patients with clear cell renal cell carcinoma with an average of 3.8 times lower than their adjacent normal tissue;the expression of miR-9-5p was down-regulated by 836.3-fold in 786-O and 24.1-fold in SN12-PM6 compared with the clear cell renal cell carcinoma tissue;the expression of miR-9-5p was not correlated with the gender,age,tumor stage,grade,and size of patients with clear cell renal cell carcinoma.2.The expression of miR-9-5p in 786-O and SN12-PM6 cells transfected with phsa-miR-9 was respectively upregulated by 21610.3-fold and 4599.5-fold.3.After upregulation of miR-9-5p,the proliferation,invasion and metastasis of 786-O cells were significantly inhibited.4.Target prediction softwares showed that there were miR-9-5p seed binding sites on the 3’UTR of CXCR4,NFκB1,FN1,and Bcl-2,and the mRNA expression levels of these four genes were 71-fold,5.8-fold,19.3-fold,and 9.7-fold in clear cell renal cell carcinoma tissues,and the protein levels were also increased.CXCR4 was down-regulated in 786-O and SN12-PM6 cells with high expression of miR-9-5p.5.Sequencing results of serum exosomes in 4 patients with clear cell renal cell carcinoma and 4 healthy controls showed that miR-9-5p were upregulated in serum exosomes in patients with clear cell renal cell carcinoma.6.The expression of miR-9-5p was upregulated in serum exosomes in patients with clear cell renal cell carcinoma.Conclusion:miR-9-5p is down-regulated in clear cell renal cell carcinoma and plays a role in anti-oncogene by inhibiting the proliferation and metastasis of clear cell renal cell carcinoma;CXCR4 may be a direct target of miR-9-5p,and its high expression in clear cell renal cell carcinoma may be due to the down-regulation of miR-9-5p;clear cell renal cell carcinoma may discharge the tumor suppressor miR-9-5p by exosomes. |
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