| Background and objectiveNephroblastoma,also known as Wilms tumor,was first pathologically described by Dr.Max Wilms in 1899.It is the most common childhood renal malignant tumor,accounting for more than 90%of all cases.The incidence of nephroblastoma in children under 15 years old is about 7/1000000,varying by race and ethnicity,with the highest incidence among black people and the lowest among East Asian people.Nephroblastoma is usually diagnosed under 5 years old,with an average onset age of 38 months.It can be associated with iris absence,developmental delay,hypospadias,unilateral hypertrophy and other developmental malformation.Typical nephroblastoma tissue is composed of blastomal,stromal,and epithelial components.The tumor cells detected with anaplastic cells are called anaplasia type.According to histological type and prognosis,nephroblastoma can be divided into favorable histology(FH)type and unfavorable histology(UH)type.For now,the treatment regimen for nephroblastoma is a combination of surgery,chemotherapy and radiotherapy.The application of treatment usually refer to the regimes made by north American Children's cancer group(COG)or International Society of Pediatric Oncology,SIOP)according to the application of preoperative chemotherapy or not.In recent years,the 5-year survival rate of nephroblastoma has significantly improved,which can exceed 90%in developed countries,and more than 80%in China according to domestic studies.However,surgical treatment combined with radiotherapy and chemotherapy often has serious side effects,and 25%of the survivors may suffer from subsequent complications such as renal failure,cardiac insufficiency,impaired fertility and so on.Moreover,the combined treatment has limited therapeutic effect on children with distant metastasis and anaplastic nephroblastoma.Therefore,it is still of great significance to further explore the molecular characteristics of nephroblastoma for strengthening the understanding of tumor genesis and searching for new therapeutic targets.MicroRNA(miRNA)is a class of endogenous single-stranded noncoding RNAs with 18-25 nucleotides in length,which degrades or inhibits the translation of target mRNA through complete or incomplete complementary pairing with target mRNA,and is a kind of posttranscriptional regulation of gene expression.MiRNA plays an important role in many biological processes such as development,growth and metabolism.In tumors,miRNA can regulate the proliferation,differentiation and apoptosis of tumor cells,such as kidney cancer,lung cancer,stomach cancer,colon cancer,liver cancer and osteosarcoma.Studies have shown that miRNA is abnormal in the regulation of nephroblastoma,and miRNA may play an important role in the occurrence and development of nephroblastoma.At present,a variety of miRNAs have been confirmed to be associated with nephroblastoma,such as Oncomir-1,miR-483-3p/5p,miR-204 and mir-185.It is a common method to identify differentially expressed miRNAs by using miRNAs microarray to screen miRNAs expressed in the tissues,serum or cell lines of nephroblastoma and then perform fluorescence quantitative PCR for verification.However,miRNAs chips also have disadvantages such as lack of recently annotated miRNAs,inability to predict new miRNAs,and low validation rate.Small RNA sequencing(sRNA-Seq)method which has the advantages of small sample size,high throughput and high accuracy,uses second-generation Illumina high-throughput sequencing technology to obtain millions of small RNA sequences at a time.Bioinformatics analysis can quickly identify already-known small RNAs expressed in specific tissues or at specific periods and predict new small RNAs.Meanwhile,the expression,structure and target genes of small RNAs can be further deeply analyzed.The application of small RNA sequencing technology in the study of nephroblastoma can provide a more in-depth understanding of the miRNA expression profile of nephroblastoma and screening of miRNA with differential expression,providing new ideas for the diagnosis and treatment of nephroblastoma.MiRNA-3 63-3p is a newly discovered miRNA in recent years.Many studies have shown that miR-363-3p has anti-tumor effects in a variety of cancers,thus acting as a tumor suppressor.For example,the expression of mirna-363-3p is down regulated in gastric cancer tissues and gastric cancer cell lines,and overexpression of mir-363-3p can inhibit the growth and migration of gastric cancer cells;Compared with para tumor tissues,the expression of miR-363-3p was downregulated in bladder cancer tissues.Overexpression of miR-363-3p inhibited the migration of bladder cancer cells,but overexpression of the target gene BTG2 could reverse the inhibitory effect of miR-363-3p on the migration of bladder cancer cells.In non-small cell lung cancer,overexpression of mir-363-3p can inhibit the proliferation of tumor cells,inhibit the migration and invasion of tumor cells by inhibiting epithelial mesenchymal transformation,and improve the sensitivity to gemcitabine chemotherapy.However,the roles of miR-363-3p in the occurrence and development of nephroblastoma are still unclear.In this study,the differentially expressed miRNAs in Wilms tumor cells were screened by high-throughput assay,and their target genes were analyzed.MiRNA-363-3p was selected as the research object,and the regulation of miR-363-3p and its target genes on the biological characteristics of Wilms tumor were studied.MethodTwenty-one cases of nephroblastoma tissues and matched adjacent normal tissues from patients with nephroblastoma who received surgery in Shandong provincial hospital were collected from April 2016 to April 2019.All patients had not received preoperative chemoradiation,and all specimens had been diagnosed nephroblastoma by two pathology expert separately.Small RNA sequencing was performed on nephroblastoma tissues and matched adjacent normal tissue of 3 children(1 case in stage I,1 case in stage II and 1 case in stage III)to detect the differentially expressed small RNA in the tumor tissues and adjacent normal tissues.The miRNA target genes were predicted by miRanda and TargetScan target prediction software.GO enrichment analysis and KEGG Pathway analysis were performed on the target genes of miRNA with significant differential expression to analyze the ontological annotations and important signaling pathways involved.Differentially expressed miR-363-3p were selected and verified by RT-qPCR in tissue samples of 21 children in this group.It was confirmed that miR-363-3p expression was low in tumor tissues compared with normal tissues.SK-NEP-1 cells with stable up-regulated miR-363-3P expression were established by lentivirus transfection,and the transfection efficiency was verified by RT-qPCR.Cell proliferation ability was detected by CCK-8 assay,and cell migration and invasion ability was detected by Transwell assay.Apoptosis and autophagic death were detected by AO/EB.The target gene of miR-363-3p was predicted by bioinformatics software,and nephroblastoma overexpressed gene(NOV/CCN3)was selected as the target gene for verification.The double luciferase reporter gene method verified that miR-363-3p could target the 3'noncoding region binding to NOV.After miR-363-3p lentivirus was transfected into SK-NEP1 cells,the expression level of NOV protein was detected by Western Blotting to verify its targeted regulation relationship.The expression of NOV at the gene level was verified by RT-qPCR in nephroblastoma and adjacent normal renal tissues by Western Blotting.A total of 57 paraffin samples of children with nephroblastoma who underwent surgery in Shandong Provincial Hospital from August 2009 to June 2016 were selected to analyze the expression of NOV protein in tumor and peritumoral tissues,as well as its relationship with clinicopathological staging and typing.SK-NEP-1 cell line with stable and low expression of NOV was constructed by lentivirus transfection.Cell viability of different groups was detected by CCK-8 experiment.Cell migration ability was detected by Transwell experiment.Apoptosis and autophagic death were detected by AO/EB,and the expression of apoptotic proteins c-Parpl,Bcl2,Bax and c-caspase-3 were detected by Western Blotting.Results1.Analysis of high throughput sequencing results of tumor tissues and adjacent tissues of nephroblastoma1.1 Through high-throughput sequencing detection,it is found that there are 234 differentially expressed miRNAs in nephroblastoma tumor tissues and adjacent normal tissues,including 107 up-regulated and 127 down regulated miRNAs.Through GO analysis,it is found that the target genes of differentially expressed miRNAs are mainly enriched in cell process,physiological regulation and metabolic process in biological process,while cell location is mainly enriched in cell components,organelles and cell membrane,and molecular functions are mainly enriched in binding,catalysis and structural molecular activities.KEGG enrichment analysis showed that the target genes of differentially expressed miRNAs were mainly concentrated in RAS signal pathway,circadian rhythm and cAMP pathway.1.2 After screening,miRNA-363-3p was selected for further verification.It was confirmed that miR-363-3p was low expressed in the tissues of nephroblastoma by RT-qPCR.2.Regulation of miR-363-3p/NOV on biological characteristics of nephroblastoma2.1 SK-NEP-1 cells were transfected with miR-363-3p up lentivirus.After overexpression of miR-363-3p,SK-NEP-1 cells were found to have decreased proliferation and migration ability and increased cell death using CCK8 experiment,Transwell experiment and AO/EB fluorescence double staining.2.2 The double luciferase experiment verified that miR-363-3p could target on the 3' noncoding region of NOV,and the luciferase activity of the wild-type NOV 3' UTR region was significantly inhibited(P<0.05).After transfection with miR-363-3p up lentivirus,the expression level of NOV protein was decreased using Western Blotting,showing that miR-363-3p can bind to the 3'UTR region of NOV and regulate its expression.2.3 Western Blotting showed that the expression of NOV protein was higher in nephroblastoma tissues than normal tissues.Immunohistochemical staining showed that the high expression of NOV protein had no significant correlation with children's age,gender,stage and pathological classification,and was closely related to lymph node metastasis.Through establishing SKNEP-1 cells that stably expressing downregulated NOV protein by NOV shRNA lentivirus transfection,it has been confirmed that NOV knockdown could inhibit the proliferation and migration abilities of SK-NEP-1 cells and promote cell apoptosis by the detection of apoptotic proteins such as cParp1,Bax,Bcl-2 and c-caspase-3 using CCK8 experiment,Transwell experiment,AO/EB double staining experiment and Western Blotting.Conclusion1.High-throughput sequencing results suggested that 234 miRNAs with abnormal expression were present in the tumor tissues of nephrobalstoma,which participate in a variety of biological processes of tumor cells by regulating target genes.2.The expression of miRNA-363-3p was downregulated in nephroblastoma and the overexpression of miR-363-3p could inhibit the proliferation,migration and promote cell death of SK-NEP-1 cells.3.Mir-363-3p can target the 3'UTR region of NOV and regulate its expression.4.NOV was highly expressed in nephroblastoma.Downregulating the expression of NOV can inhibit the proliferation,migration and promote the apoptosis of nephroblastoma cells. |
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