Screening And Functional Studies On Differential Expression Genes Of Caski Cell Lines Induced By Exposing To The Space Environment

AIM:This study will use the suppression subtractive hybridization library in the ground and space cervical cancer CaSki cells to select the differentially expressed genes and investigate the functions of the genes.METHODS:The hybridization membrane was made by the library, and 48A9Caski and ground CaSki were labed P32 before the reverse Northern hybridization. The differentially expressed genes were screened by autoradiography. The gene expression was inhibited or enhanced by the method of RNA interference or constructing eukaryotic expression vector. MTT, flow cytometry and in vivo tumor growth assay were used to investigate the biologic change of CaSki cells. The sensitivity of CaSki cells to anti-tumor drugs was checked. The mechanism of the gene was further studied by RT-PCR and Western blot.RESULTS:1.We found 33 differentially expressed genes which were associated with cytoskeleton, cell differentiation, apoptosis, and gene transcription and so on. The differentially expressed genes associated with tumor established the foundation for tumorigenesis and development. STC1 was one of most significant genes. 2. The expression of STC1 in cervical cancer tissues was low compared with normal tissues. MTT assay showed that the speed of CaSki/siSTC1 cells was quick compared to negative control CaSki/siNC. The colony conforming assay showed the colony numbers of CaSki/siRNA cells were elevated. Flow cytometry demonstrated the G1 phase cells of CaSki/siRNA cells reduced. Transwell assay showed the invasion ability of CaSki/siRNA cells enhanced. In vivo tumor growth assay showed that the tumor growth of CaSki/siRNA cells enhanced.3. MTT assay showed that the speed of CaSki/STC1 cells transfected with pcDNA3.1-STC1 vector was slow compared to negative control CaSki/NC. The colony conforming assay showed the colony numbers of CaSki/STC1 cells were reduced. Flow cytometry demonstrated the G1 phase cells of CaSki/STC1 cells enhanced. Transwell assay showed the invasion ability of CaSki/STC1 cells decreased. In vivo tumor growth assay showed that the tumor growth of CaSki/STC1 cells reduced. STC1 might be used to treat the tumor.4. Cisplatin, thapsin, and rapamycin inhibited the proliferation and induced the apoptosis of CaSki cells. When the expression of STC1 gene enhanced, these drugs readily suppressed the cell proliferation and induced the cell apoptosis. These demonstrated that STC1 sensitized CaSki cells to cisplatin, thapsin, and rapamycin, and provided a new way of curing tumor.5. In mRNA and protein level, when the expression of STC1 decreased and elevated, the expression level of NF-κB accordingly changed. The expression between STC1 and NF-κB was positive correlation. When cisplatin or thapsin was added into CaSki or HeLa cells, the mRNA level of Caspase-3 increased and the expression of STC1 and NF-κB also increased. Chromatin immunoprecipitation (CHIP) showed that NF-κB combined with the promoter of STC1 gene. These results demonstrated that NF-κB combined with promoter of STC1 and evoke the gene expression, which induced the apoptosis of cervical cancer cells.CONCLUSION:The way that constructing suppression subtractive hybridization library combining with reverse Northern blot selected the differentially expressed genes is a high throughput and efficient screening. The expression in CaSki cells can inhibit the proliferation and induce the apoptosis of cells by nuclear factor NF-κB. These results establish the foundation for carcinogenesis and development and might be a new road to cure tumor.