| Part I:Screening and preliminary functional studies of species-specific genesThe reproductive health is currently one of the most widespread concerns. Reproductive diseases had miserably affected our life and the development of next generation. Moreover, the worldwide incidence of reproductive diseases is on the rise year by year. Although a lot of studies had been done for the understanding and treatment of reproductive diseases, it is still insufficient to meet the needs of the prevention and treatment of these diseases. The study is aimed to provide molecular theory for the prevention and control of reproductive diseases througth studying the species specific genes. From an evolutionary perspective, primates and rodents belong to euarchontoglires, but their reproductive strategies are completely different. Human and mice serve as the representatives of the higher primates and rodents, respectively. Human has hemomonochorial, spontaneously decidualization and menstruation, while mouse has hemotrichorial, no spontaneously decidualizatio and no menstruation. The traits of the organism are controlled by genes. Because of the evolutionary conservation of homologous genes in different species, the homologous genes were selected and studied further. Firstly, we downloaded the build67 from the homologous genes database of the NCBI website, which contains 18915 human orthologue genes and 20747 mouse homologous genes. We obtained 248 human specific genes and 680 mouse specific genes througth comparison of Excel software and filtering of the NCBI filters tool. We investigated the tissue specific expression of the specific genes of human and mouse in the UniGene database and analyzed the function of the special genes expressing in the uterus and placenta using the David online software. Furthermore, we investigated the expression levels of these genes protein in the female reproductive system through the human protein atlas. As a result, the protein expression level oî–ˆTN3A1, IL-32, ZNF222, ZNF649 and ZNF773 genes is very high in the placenta, and is weak or not detected in other tissues. Then, according to the tissue specificity of these genes expression and the research reports in previous studies, we selected IL-32, ZNF222 and LAIR2 as the candidate genes for further research. According to the result of PCR experiment, these genes may be human specific genes. Using immunohistochemistry, we found the expression of IL-32 and ZNF222 is high in human chorionic trophoblast cells. Moreover, it had been reported that LAIR2 localised to human chorionic trophoblast cells. Therefore we chose HTR8/SVneo, a human chorionic trophoblast cell line, to make further research into the function of these genes. The knockdown of IL-32 significantly promoted the proliferation, migration and invasion in HTR8/SVneo. The interference of ZNF222 expression evidently inhibited the migration and invasion in HTR8/SVneo, but not proliferation. After the knockdown of LAIR2 of HTR8/SVneo, the invasion was obviously inhibited, but the proliferation was significantly promoted. These results suggested the three human specific genes may play an important role in the pregnancy. We speculated that the specific genes may play an important part in the process of human pregnancy, which may have important meaning for the successful reproduction. This study also provided theoretical foundation in a deeper level for the prevention and therapy of reproductive diseases.Part â…¡:The preliminary study of the role of miR-205 and CHN1 in human cervical cancerCervical cancer is the leading cause of cancer-related death in women worldwide. However, the mechanisms mediating the development and progression of cervical cancer are not clear. In this study, we aimed to elucidate the roles of microRNAs and CHN1 protein in cervical cancer progression. We found that miR-205 and CHN1 protein were highly expressed in human cervical cancer tissue compared with paired normal cervical tissues. The CHN1 gene was shown to be targeted by miR-205 in HeLa cells, a human cervical cancer cell line, using software prediction and dual luciferase assays. Interestingly, transfection with an miR-205 mimic upregulated CHN1 mRNA and protein, while miR-205 inhibitor downregulated CHN1 in high-risk and human papilloma virus (HPV)-negative human cervical cancer cells in vitro, as demonstrated by real-time polymerase chain reaction and western blotting. These data suggested that miR-205 positively regulated the expression ofCHNl. Furthermore, the miR-205 mimic promoted cell growth, apoptosis, migration, and invasion in high-risk and HPV-negative cervical cancer cells, while the miR-205 inhibitor blocked these biological processes. Knockdown of CHN1 obviously reduced the aggressive cellular behaviours induced by upregulation of miR-205, suggesting that miR-205 positively regulated CHN1 to mediate these cell behaviours during the development of cervical cancer. Furthermore, CHN1 was correlated with lymph node metastasis in clinical specimens, as shown by immunohistochemistry. Taken together, our findings showed that miR-205 positively regulated CHN1 to mediate cell growth, apoptosis, migration, and invasion during cervix cancer development, particularly for high-risk HPV-type cervical cancer. These findings suggested that dysregulation of miR-205 and subsequent abnormalities in CHN1 expression promoted the oncogenic potential of human cervical cancer.Part â…¢:The expression of HIF1A and VEGF in the process of menstruationMenstruation is a physiological phenomenon of endometrium periodic shedding and excreting the shedding with bleeding, which is mainly in higher primates including human and a few placental mammals. Abnormal menstruation is related with many gynecological diseases. Studying the mechanism on the endometrium periodic breakdown and bleeding is the key to understand and find new treatment strategy for abnormal bleeding. In this study, we try to establish the mouse menstrual-like model, and test the change of the related genes expression in the model. Firstly, we successfully established the mouse menstrual-like model by physiological progesterone withdrawal in vivo. After the withdrawal of P4 in the model, the level of HIF1A mRNA and protein were raised, and then falling back to the original level, suggesting that HIF1A is related with the endometrial disintegration. In addition, in the mouse menstrual-like model, after the withdrawal of the P4, the expression of VEGF were also raised. These results suggested that HIF1A and VEGF may play an important role in the process of menstruation. |
PDF Full Text Request