| Backgroud:Nowadays the incidence of malignant tumor is bincreasing, which has become one of the main threats to human health and life around the world. Testicular cancer is one of the most common malignant tumors among the young male aged between 15 and 35. In recent years, the trend of testicular cancer’s incidence all over the world is increasing significantly. According to statistics, the incidence of testicular cancer is growing at a rate of 1%-2% per year. In China, the incidence and mortality of testicular cancer are both at a rate of about 1/100 thousand. Compared with other tumors, the degree of malignancy of testicular cancer is higher, although the incidence rate of it is lower.95% of testicular tumors are the testicular germ cell tumors (TGCTs). It is reported that male infertility is a clinical risk factors of TGCTs. According to American research, in the past 25 years, the prevalence rate of testicular cancer has been increasing significantly, which is similar to the rate of the increase of male infertility among the male aged between 15 and 35.A large clinical study with big number of cases and sample data shows that the patients who suffer from the oligospermatism,asthenozoospermia and teratozoospermia are 20 times more likely to have germ cell tumors than normal individuals.After many years of research and analysis, although we have researched on the pathogenesis of cancer in great depth,and had a large number of clinical data and experimental data, we still donot know how the tumor cells originate. The embryonic origin of tumor cells can explain the origin of tumor cells. The hypothesis says that the tumor cells are originated from the embryo. And therefore we can get new ideas and methods or anti-tumor effect. With the development of molecular oncology, cell biology, embryology experiment, experimental oncology and immune embryology,the comparative study of tumor formation and embryonic development is carried on in-depth. After doing a great number of experiments and research,we have found that there is an inherent connection between the tumor formation and the embryonic development.According to the theory of the origin of the tumor, the tumor cells originate from the stem cells, while the primordial germ cells derive from the embryonic germ cells. Like tumor cells,the primordial germ cells also have the migration, proliferation and differentiation activities. Therefore In some certain conditions,Oosperms and tumor cell have some kind of similarity. Experimental results show that the tumor cells, like a fertilized egg,can grow into embryos. If we implant an animal fertilized egg into the testis,it can grow into an teratoma. The tumor cells and the fertilized eggs have a lot of similarities in gene level,protein level and biological behavior. Spata6(spermatogenesis-associated protein-6)is an evolutionarily conserved testis specific genes, and, like Spata 1, and Spata 3,it is also a sperm occurred related gene and protein. Spata6 inactivation may result in male infertility and acephalic spermatozoa.Since Spata 6 is specifically expressed in haploid of germ cells. According to the background above, we hypothesize that Spata 6 is likely to be involved in TGCTs. To prove this hypothesis,we will raise and reduce Spata6 gene expression level to study and understand the effects of Spata6 to the proliferation and apoptosis of human embryonal carcinoma cells derived cell line NTera2 cell and also the changes of the downstream apoptosis regulatory proteins.Our goal is to explore whether Spata6 is involved in the apoptosis of cell tumor of the testis or not.Apoptosis is defined as a stable and orderly cell death which are controlled by genes.Cell apoptosis is different from cellnecrosis. Compared with necrosis, apoptosis is not a passive process, but an active process. It involves the activation, expression and regulation of a series of genes. Under the pathological conditions, it is not a phenomenon of self injury, but an initiative death process to adapt the existing environment better.During the course of the occurrence of cancer, cell apoptosis is involved in the initiation of cancer, and it plays a negative role in the occurrence of cancer. On the other hand, there is a close connection between cell apoptosis and male infertility. There is a very clear connection between sperm apoptosis and male infertility because sperm apoptosis can affect the semen volume, sperm density, sperm motility rate, malformation rate and so on. Sperm apoptosis is a process controlled rigorously by multiple genes. These geneslike C-myc, tumor suppressor gene P53, are very conservative in the species like Bcl-2 family, caspase family. The Bcl-2 family is a class of proteins playing an important role in the progress of cell apoptosis. Bcl-2 is the most representative of inhibiting cell apoptosis gene while Bax is the most representative of promoting cell apoptosis gene.Bcl-2 and Bax play an important role in the regulation of tumor cell apoptosis. In tumor cells, Bax means decrease, while bcl-2 means increase. Downregulation of Bax or up regulation of Bcl-2 can inhibit multiple tumor cell apoptosis induced by various factors. On the contrary, up-regulation of Bax or down-regulation of Bcl-2 promotes apoptosis of many kinds of tumor cells. According to the background above, we hypothesize that transfected with pcDNA3.1 (+)-Spata6 and Spata6 siRNA affect the expression of the genes relating to cell apoptosis, Bcl-2 and Bax, which leads to the changes in testicular tumor cell apoptosis and proliferation activity.Therefore, we hypothesized that Spata6 may be involved in TGCTs. To confirm the hypothesis, our study is aimed to explore the role of SPATA6 in TGCTs. Our results may provide a fundamental research for searching a new target.Research objective:1.By transfecting NTERA-2 cells withthe recombinant expression vector pcDNA3.1 (+)-Spata6 and specific siRNA target sequence for Spata6, we up-regulated and down-regulated the expressions of Spata6 to study the changes of Spata6 protein expression using Western blotanalysis.2. By transfecting NTERA-2 cells withthe recombinant expression vector pcDNA3.1 (+)-Spata6 and specific siRNA target sequence for Spata6, we investigatedthe change of NTERA-2 cell’s viability between different groups by MTT cell proliferation assay.3.Bytransfecting NTERA-2 cells withthe recombinant expression vector pcDNA3.1 (+)-Spata6 and specific siRNA target sequence for Spata6, we studied the effect of Spata6 expression changeon NTERA-2 cells’ apoptosis using flow cytometry.4.Bytransfecting NTERA-2 cells withthe recombinant expression vector pcDNA3.1 (+)-Spata6 and specific siRNA target sequence for Spata6, the expression level of Bax and Bcl-2 proteins were analyzed by Western blottingto study the effect of changing Spata6 gene expression on the Bax and Bcl-2 protein level in NTERA-2 cells and to investigate the effects and mechanism of Spata6 gene expression on NTERA-2 cell.5.Subcutaneous tumor of nude mice was performed with subcutaneo-ustransplantation of NTERA-2 cells. EntransterTM-in vivo transfection method was used to interfere expression of spata6 in the tumors of mice, then growth conditions of the tumors were observed, and volume of the tumors in mice was measured every a week.the tumors were taken out after 21 days and volume and weight of the tumors were measured.then volume and weight variations of the tumors in the mice were observed and effects of down regulation of Spata6 on germ cell tumor of testicle were investigated.Results1.Effects of pcDNA3.1 (+)-Spata6 and Spata6-siRNA transfection on Spata6 gene expression level in NTERA-2 cellsThe expression of Spata6 protein in the Spata6c group was significantly increased in 24h after transfection compare to the control group and Spata6c+si Spata6 group (P< 0.05). However, the expression of Spata6 protein in NTERA-2 cells was markedly diminished after 24h by transfection with Spata6-siRNA.2.Effects of pcDNA3.1 (+)-Spata6 and Spata6-siRNA transfection on NTERA-2 cell viabilityThree groups have no statistical significance in 24h after transfection (P> 0.05). In 48h and 72h after transfection, the results of MTT showed that the cell viability was significantly decreased by transfection with Spata6-siRNA,but was increased clearly by transfection withpcDNA3.1 (+)-Spata6 compared with the control group3.Effects of pcDNA3.1 (+)-Spata6 and Spata6-siRNA transfection on apoptosis in NTERA-2 cellsNo significance change was observed among the control group,Spata6c group, and Spata6c+si Spata6c group in percentages of apoptosis cell (P>0.05). But the percentages of apoptosis cell were significantly higher in the si Spata6 group than thosein the other three groups.4.Effects of pcDNA3.1 (+)-Spata6 and Spata6-siRNA transfection on the expression level of Bax and Bcl-2After transfection of Spata6-siRNA, theBax levels protein increase was statistically significant, but the Bcl-2 protein level was markedly decreased than that in the control group and Spata6c group. But no significant differences were found between the control group and the Spata6c group.5.Effects of Spata6 siRNA transfection on NTERA2 cells tumor of nude miceWeight of the tumors in the Spata6 siRNA group was significant lighter than that in the control siRNA group and physiology buffer group. Difference trend of tumor volume was the same as that of tumor weight. Volume of the tumors in the Spata6 siRNA group was significantly smaller than that in the control siRNA group and physiology buffer group when the nude mice were put to death after 21 days.DiscussionIn an effort to demonstrate more function of Spata6 gene in Testicular germ cell tumors, TGCTs, we generated the eukaryotic expression vectorpcDNA3.1 (+)-Spata6 using gene engineering technology and design and synthesize the specific Spata6-target siRNA. By deliverying the vector and siRNA to NTERA-2 cell-line respectively weup-regulated and down-regulated the expressions level of SPATA6 gene in human EC-derived cell line NTERA-2 The Spata6 protein expression levels were determined by Western blot. The results showed that the levels of Spata6 protein were significantly reduced after transfection of Spata6 siRNA compared with control group, but were statistically increased after transfection of pcDNA3.1 (+)-Spata6, suggesting that the recombinant plasmid was successfully expressed. To explore the effect of SPATA6 after transfection on cell proliferation and apoptosis rate, MTT and FCM assay were performed. We found that the cell viability was significantly reduced after transfection of Spata6 siRNA, but was raised after transfection of pcDNA3.1 (+)-Spata6 compared with the control group. Moreover, the percentages of apoptosis cell were the highest after transfection of Spata6 siRNA than other groups. resultsof nude mice’s experiment were the same as cytology experimental results, tumorigenic ability of NTERA2 cells with silent Spata6 gene was significantly lower than cells in the control groups.The results indicated that down-expression of Spata6 inhibited the cell proliferation and induced cell apoptosis.Our research demonstrate the potential of Spata6 as a specific target in the treatment of TGCTs cancer. The research holds important in vitro experimental evidence that reveal the great necessity for the following animal in vivo tumor model study or clinical investigation. |
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