| Testicular germ cell tumors(TGCTs)are a group of diverse tumors composed of different pathological types and clinical manifestations.They originate from dysfunctional embryonic germ cells and can occur in or outside the gonad.TGCTs are the most common type of cancer among young males,with the rising incidence in most parts of the world.TGCTs can be divided into two main histological types,seminoma and non-seminoma,the latter including embryonal carcinoma,teratoma,choriocarcinoma and yolk sac tumor.Among them,embryonic cancer cells(EC)belong to the most malignant type of TGCTs and are considered to be the malignant cell corresponding to embryonic stem cells(ES)because both are pluripotent.TGCTs are sensitive to radiotherapy and chemotherapy.Most TGCTs patients have a good prognosis,but 15-30% of patients with metastasis have poor prognosis or even death,suggesting that there might be some unknown abnormal genetic events contributing to TGCTs malignancy.However,the genetic drivers of malignant transformation of TGCTs have not been fully elucidated.Therefore,it is necessary to find new diagnostic markers and therapeutic targets for TGCTs.So far,more than 50 TGCTs susceptibility loci have been found through genome-wide association studies(GWAS),many of which are located in non-coding regions of the genome,suggesting that non-coding RNAs including miRNAs may influence the development of TGCTs.Therefore,miRNAs are expected to lead to breakthroughs in the diagnosis,prognosis and treatment of TGCTs.miR-196a-5p is a conservative miRNA derived from its precursor miR-196 a.Studies have shown that miR-196a-5p is associated with tumorigenesis,such as acting as an oncogene in gastrointestinal stromal tumors,or acting as a tumor suppressor gene in malignant melanoma,but its role and mechanism in TGCTs remain unclear.In recent years,a series of studies have shown that nerve cells are one of the important components of tumor microenvironment.Cancer cells attract nerve fibers by secreting neurotrophic factors,which stimulate nerve growth.In turn,various neurotrophic growth factors/receptors are upregulated in cancer to promote cancer cell survival,proliferation and invasion.For example,gastric and colorectal cancer stem cells have been found to have the potential to produce neurons.However,the key drivers or regulatory factors in tumor neurogenesis or tumor neuro-dependence have not been fully identified and need further study.In present study,the expression characteristics of miR-196a-5p in TGCTs and influence on TGCTs cell biology behavior were analyzed.The nuclear receptor gene NR6A1 is determined to be a target of miR-196a-5p.It's demonstrated that NR6A1 has the role of promoting TGCTs proliferation,migration and invasion.We also confirmed that the cellular adhesive molecular E-cadherin was a new target gene of NR6A1 in TGCTs.Regulation relationship and mechanism between NR6A1 and Ecadherin has been discussed.In addition,from the perspective of tumor cell neurogenesis,we studied the role of NR6A1 participating in the regulation of EMT process and promoting the neuro-like transformation of testicular tumor cells,and explained the mutual regulation relationship among miR-196a-5p/NR6A1/Ecadherin axis.These funding would provid potential molecular targets for the diagnosis and treatment of TGCTs.The main research contents and results of this study are as follows:(1)miR-196a-5p inhibited the proliferation,migration and invasion of testicular embryonic cancer cells: It was confirmed that miR-196a-5p was down-regulated in testicular tumor tissues by GEO database analysis combined with RT-q PCR detection of clinical samples.Subsequently,the results of MTT assay,scratch assay,Transwell assay and WB assay showed that miR-196a-5p could significantly inhibit the proliferation,migration and invasion of NT-2 and NCCIT testicular embryonic cancer cells,indicating that miR-196a-5p may act as tumor suppressor in TGCTs.(2)Determination and functional verification of the target gene for miR-196a-5p: All predicted target genes of miR-196a-5p were downloaded using Targetscan,then interacted with all proliferation and differentiation related genes in GO database.The nuclear receptor gene NR6A1 was screened out to be a target gene with high score.The expression of miR-196a-5p and NR6A1 in TGCTs was confirmed to be negative correlation by bioinformatics analysis and PCR experiments.Further,the results of Western blot,RT-q PCR and double luciferase reporter gene experiments proved that miR-196a-5p directly targeted NR6A1 and inhibited NR6A1.In addition,the results of functional rescue experiments confirmed that NR6A1 could partially rescue the inhibitory effect of miR-196a-5p on the proliferation,migration and invasion of embryonic cancer cells.(3)Nuclear receptor NR6A1 promote proliferation,migration and invasion of embryonic cancer cells by inhibiting CDH1: RNA-Seq was used to detect the influence of NR6A1 overexpression on NT-2 cells,and GO analysis showed that overexpression of NR6A1 could significantly negatively regulate the cell adhesion behavior of NT-2 cells.The results of RT-q PCR,Western blot,immunofluorescence and other experiments showed that NR6A1 significantly inhibited the expression of adhesion molecule E-cadherin.Further analysis of DR0 sites in CDH1(E-cadherin encoding gene)promoter region revealed that CDH1 gene promoter contained three DR0 sites.CDH1 was identified as a new target gene of NR6A1 in TGCTs by Western blot,chromatin immunoco-precipitation(CHIP)-PCR and dual-luciferase reporter gene assay.Functional rescue experiments confirmed that interference CDH1 could partially rescue the inhibition of NR6A1 on proliferation,migration and invasion of embryonic cancer cells.Mechanistically,the results of Co-IP confirmed that NR6A1 interacts with methylase Dnmt1 to regulate CHD1 methylation level,suggesting that NR6A1 might recruit Dnmt1 to the promoter DR0 site of CDH1 to methylate CDH1 promoter and inhibit its transcription.In addition,the clinical sample analysis based on the Starbase database proved that miR-196a-5p/NR6A1/CDH1 had a mutual regulatory relationship in TGCTs.(4)NR6A1 promoted neuro-like characteristic transformation of embryonic cancer cells: In vitro nude mouse tumor formation experiment showed that the growth rate of xenograft tumor of overexpressing NR6A1 in NT-2 cells was significantly higher than that of the control group,and NR6A1 overexpression promoted the tumor formation of NT-2 cells in vitro.Immunohistochemical assay showed that the expression levels of PCNA and MAP2 in xenograft tumors were significantly increased after NR6A1 overexpression.A neural-like differentiation model of NT-2 embryonic carcinoma cells induced by visual sulfonic acid(RA)was established in vitro,which was used to detect the expression changes of MAP2 under NR6A1 interference.The results confirmed that after interference with NR6A1,MAP2 expression no longer appeared even under the induction of RA,indicating that NR6A1 may play a role in the neuro-like Characteristic transformation of TGCTs cells.(5)miR-196a-5p/NR6A1/E-cadherin axis regulates the occurrence and development of TGCTs: The relationship between miR-196a-5p /NR6A1/E-cadherin in the neuro-like transformation process of embryonic cancer cells was discussed by using the cell model of NT-2 neuro-like differentiation induced by RA in vitro.It was found that NR6A1 and N-cadherin expression levels upregulated with the increase of RA concentration.The expression levels of OCT4 and E-cadherin decreased gradually.The expression of miR-196a-5p and NR6A1 remained reciprocal,further implying the targeting relationship between miR-196a-5p and NR6A1 in TGCTs.In addition,it was found that both RNA interference with NR6A1 and overexpression of miR-196a-5p could affect the transformation between Ecadherin/N-cadherin during neuro-like differentiation of embryonic cancer cells,suggesting that NR6A1 may regulate the EMT process.In order to confirm the correlation of NR6A1,E-cadherin and MAP2 in TGCTs at the tissue level,15 testicular germ cell tumor tissues and 15 normal testicular histopathological specimens were collected for immunohistochemical analysis.The results showed that NR6A1 was highly expressed in TGCTs compared with normal testicular tissue.The expression of E-cadherin was low in TGCTs.MAP2 is highly expressed in TGCTs.Correlation analysis showed that there was no significant correlation between ECadherin and MAP2 in TGCTs.NR6A1 was negatively correlated with e-cadherin expression,while NR6A1 was positively correlated with MAP2,suggesting that NR6A1 plays an important role in the neuro-like transformation of embryonic cancer cells. |
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