MicroRNA Differential Expression Profile In Nephroblastoma Cell Line And Normal Embryonic Kidney Cell Line

Background and objectiveNephroblastoma, also known as Wilms tumor, is the most common pediatric urologic tumors, accounting for 8% of solid tumors and 80% urinary system cancer with children. It comes from precursors of pluripotent renal cells, which could produce undifferentiated embryonic cells, original epithelial and mesenchymal organization and so on. Early accurate diagnosis and timely treatment is critical for improving long-term survival of nephroblastoma patients. Currently, although the 5-year survival has been increased by combining surgery, chemotherapy and radiotherapy, patients with advanced stage often recurrenced. Therefore, early diagnosis and treatment is very important to Wilms tumor patients.MicroRNAs (miRNAs),19-22 nucleotide-long, are small non-protein-coding RNAs that inhibit gene expression at the posttranscriptional level. The target recognition is based on complementarity of the seed sequence of the miRNA (7-8 nt at the 5'end) to a specific sequence motif within the 3'untranslated region of the mRNA target. In recent years, it is a hotspot of the life science research. Several reports showed that miRNAs are oncogenes or tumor suppressor genes. High expression of miRNAs play a role of oncogenes by down-regulating the expression of mRNA in tumors. When genetic or epigenetic changes or miRNA is damaged in processing decreased expression of miRNA, miRNA would not play the role of tumor suppressor genes. The different types of tumors have different types miRNAs expression profile, which suggests tumor miRNAs have tissue-specificity. There exist interactions between miRNAs and mRNA in vivo, such as one miRNA can adjust more than one target and vice versa. These interactions make up complex regulatory network and lead to the formation of tumors finallly. Therefore, understanding total differential expression of miRNAs in tumor is necessary which is the basis studying the pathogenesis of Wilms tumor.MethodsNephroblastoma cell line G401 and normal embryonic kidney cell line CCC-HEK-1 were purchased from cell Resource Center of Concord in Bejing. Three samples from G401 cell line and another 3 samples from CCC-HEK-1 cell line were chosen as experimental group and control group, respectively. Total RNA extraction by Trizol method, identify the quality and concentration of RNA by NanoDrop ND-1000 at 260nm and 280 nm and denaturing Agarose Gel Electrophoresis. It was screening differentially expressed miRNAs between Wilms tumor cell line and normal embryonic kidney cell by miRNA microarray(11.0).To verify the reliability of the results, we selected ten differentially expressed miRNAs in between G401 and CCC-HEK-1 cell lines, two-fold increase (fold change>2) of the miRNAs were 6, ranked as hsa-miR-387,hsa-miR-18b,hsa-miR-183 hsa-miR-199a-5p,hsa-miR-214,hsa-miR-130a, and two-fold decrease (fold change> 2) of themiRNAs were 4,ranked as hsa-miR-143,hsa-let-7f,hsa-miR-886-5p,hsa-miR-125b. Real-time PCR used to detect by U6 as internal and the data used 2-ΔΔCT method of analysis, the final result consistent with the microRNA microarray and Real-time PCR, which suggested microRNA microarray is reliable.ResultsAll of the samples were analyzed by miRNA microarray.130 differential expressed miRNAs were obtained., which included 71 up-regulated and 59 down-regulated miRNAs (fold change>2). Four-fold increase (fold change>4) ofthe miRNAs were 27,10-fold increase (fold change>10) of the miRNAs were 5, ranked as hsa-miR-1255b,hsa-miR-378,hsa-miR-190,hsa-miR-421,hsa-miR-18b, A total of 59 of the miRNAs down-regulate, there were 2o miRNAs are up to 4 times (fold change> 4), there were 9 are up to 10 times (fold change> 10), ranked as hsa-miR-143,hsa-miR-377,hsa-let-7f,hsa-miRPlus-F1158,ebv-miR-BHRF1-2,hsa-miRPlus-F1205,hsa-miRPlus-A1087,hsa-miR-342-5p,hsa-miR-155. Overall, there were significant differences in the expression of microRNA between G401 and CCC-HEK-1 cell lines.We selected ten differentially expressed miRNAs hsa-miR-378,hsa-miR-18b,hsa-miR-183,hsa-miR-199a-5p,hsa-miR-214,hsa-miR-130a,hsa-miR-143,hsa-let-7,hsa-miR-886-5p and hsa-miR-125b, by the real-time PCR method validation,2-ΔΔCT value is 1.98,4.47,1.96,2.69,6.45,2.48,0.53,0.07,0.15 and 0.66 respectively.CouclusionThe results showed there were several differential expressed miRNAs between nephroblastoma cell line G401 and normal embryonic kidney cell line CCC-HEK-1, and some of differential expressed miRNAs have associated with tumor formation and metastasis,including miR-17,miR-18a,miR-19b,miR-20a,miR-190,miR-183,miR-30d,miR-143,miR-377,let-7f,miR-378 and miR-421 and so on. In additon, our study suggest that miRNAs play a key role in tumor formation and metastasis. The consistentence of microRNA microarray analysis and Real-time PCR results maybe indicate miRNA microarray is true and reliable, as well as rapid and effective method of detection of miRNA.