The initial study of F10 gene up-regulated in the hydatidiform mole 510515

The initial study F10 gene up-regulated in the hydatidiform mole 3047065830672234

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The initial study of F10 gene up-regulated in the hydatidiform mole Name: Cao Xiao Min Supervisor: Professor Xing Fu Qi ABSTRACT Hydatidiform mole (HM) is a type of gestational trophoblastic disease with unpredictable malignant potential and disease incidence is 11001500. The mechanism of generation and malignant transformation of Hydatidiform mole is not definite. There are several theories about HM at present as listed below: 1. Epidemic investigation reflects that the Hydatidiform mole is related to the community culture. The higher level the culture is, the lower possibility Hydatidiform mole occurs. 2. The theory of heredity concludes that Hydatidiform mole is most likely caused by abnormal fertilizations. 3. The number of individual s pregnancies is a critical factor of Hydatidiform mole generation. 4. The excessive number of uterine aspiration, dilatation and curettage is an important factor of HM. 5. The oncogene, tumor-suppressing genes and apoptosis regulator genes are involved in HM generation. However a considerable number of researches point out that inherit predisposing genes which were not reported at present possibly exist in the genome of hydatidiform mole. Suppression subtractive hybridization (SSH) was used to assess molecular pathogenesis of HM. According to comparing the gene expression pattern of the similar gestational ages of HM with normal first-trimester trophonema, we cloned F10 gene form the subtracted cdna libraries which up-regulated in HM and its function is unknown at present. In the i

ABSTRACT previous researches, F10 gene was considered to be associated with trophoblastic tumor and adenocarcinoma, but the relationship is unclear. Therefore, it is important to do further research on biological functions of F10 gene in order to reveal the generation mechanism of HM and noval treatment and precaution for HM. Part one The expression spectrum analysis of F10 gene transfected cell F10 gene is a noval gene and its function is unknown. According to the results which we have obtained, we concluded that F10 gene may participate in the malignant change of HM and tumor s invasion behavior. Our research also shows that F10 gene is related to the cancer generation. In order to identify the function of F10 gene, we obtain the different downstream express genes from the genetic transcription level by DDPCR method and indirectly predict the function of F10 gene. Methods DDPCR technology was used when we analysised F10 gene expression spectrum and screened the F10 gene function-related genes. The cell line with lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402, HIC, HepG2, PC, A549, MGC, 16HBE and 293 cell lines. The coarsely screening experiment was performed after the prc-cmv2/f10, prc-cmv2, prc-cmv2/f10 plasmids transient transfecting A549 cells respectively. The mrna of cell was extracted after transfecting for 24 hours. The differently expressed genes were screened by DDPCR among 4 groups. The different expression strips were amplicated and the products were connected to T-Vector and then analyzed by sequenceing. The result was confirmed by fluorescent quantitated PCR. Results 1. The expression levels of F10 were different among the cells. The first one was ii

Bel7402, HIC and HepG2 cells. The following cells were PC293 and MGC. The lowest cells were 16HBE and A549. 2. The differently expressed strips were amplicated and then analysised by sequenceing. The annexinibasp1 STAT1 gene were Higher level expression while IPLA2DATF1 gene were lower level expression in F10 tranfected group. The result was justified by the fluorescent quantitation PCR technology. Summary 1. The cell which lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402, HIC, HepG2, PC, A549, MGC, 16HBE, 293 cell lines. The first were Bel7402, HIC and HepG2 cells. The following cells were PC, 293 and MGC. The lowest cells were 16HBE and A549. 2. According to the results of F10 gene expression spectrum analysis and screening the F10 gene function-related genes, we found that annexini, STAT1,BASP1 gene were higher level expression while IPLA2, DATF1 gene were lower level expression in F10 tranfected group. We initially conclude that F10 is related to cell proliferation and apoptosis. Key words: Different display PCR; Cell Proliferation; Fluorescent quantitated PCR Part two - Effect of F10 gene on the activities of transcription factor We cloned F10 gene which up-regulated in HM by suppression subtractive hybridization (SSH), F10 EST cdna(genbank codeab196290).the cell which lower expression of F10 gene was screened by immunohistochemistry and fluorescent quantitated PCR among the Bel7402, HIC, HepG2, PC, A549, MGC, 16HBE and 293 cell lines. According to F10 gene expression spectrum analysis and screening the F10 gene function-related genes by DDPCR we initially conclude that F10 is related to cell proliferation and apoptosis. Genetic transcription is a significant regulation of life iii

ABSTRACT and a hot spot research at present. NF-κB and AP1 are core factors about cell proliferation, apoptosis, differentiation and transcriptional control. The transcription factor phse, pcre, psre and prce participate in many signal transmission regulation. Therefore we observe the effect of F10 gene on the activities of transcription factor to further study the F10 gene biological fuction. Methods 1. Plasmids prc-cmv2f10, prc-cmv2, prc-cmv2f10 and reporter gene AP1-Luc, NFκB-Luc, phse-luc, pcre-luc, psre-luc, pgre-luc were cotransfected A549 cell respectively. After transfecting for 24h, 48h and 72h measuring and compareing the lucifrease activity of different groups were used to evaluate the effect of the F10 gene on the important signal transduction pathways. 2. The activity of DNA bingding AP1 and NF-κB were detected by EMSA. Results 1. The lucifrease activity can be detected after F10 and AP1-Luc plasmids are transfected to four groups for 24 hours and increased gradually. After transfecting for 48 hours the lucifrease of prc-cmv2f10 group has been obviously higher than the other groups. After transfecting for 72 hours the lucifrease of three groups has no difference and the results were confirmed by the EMSA. 2. The lucifrease activity can be detected after F10 and NFκB-Luc plasmids transfecting for 24 hours and increased gradually among three groups. After transfecting for 48 hours the lucifrease of prc-cmv2 F10 group has obviously lower than the other groups. After transfecting for 72 hours the lucifrease of three groups has no distinction and the results were confirmed by the EMSA. 3. The lucifrease activity has no visible difference among the three groups transfected with the lucifrease report plasmid of phse-luc, pcre-luc, psre-luc, prc-luc. iv