The Initial Study Of F10 Gene Up-regulated In The Hydatidiform Mole

Hydatidiform mole(HM) is a type of gestational trophoblastic disease with unpredictable malignant potential and disease incidence is 1/100—1/500.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 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 cellF10 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.MethodsDDPCR 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.Results1.The expression levels of F10 were different among the cells.The first one was Bel7402,HIC and HepG2 cells.The following cells were PC,293 and MGC.The lowest cells were 16HBE and A549.2.The differently expressed strips were amplicated and then analysised by sequenceing.The annexinI,BASP1,STAT1 gene were Higher level expression while IPLA2,DATF1 gene were lower level expression in F10 tranfected group. The result was justified by the fluorescent quantitation PCR technology.Summary1.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.Part two-Effect of F10 gene on the activities of transcription factorWe cloned F10 gene which up-regulated in HM by suppression subtractive hybridization(SSH),F10 EST cDNA(Genbank code:AB196290).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 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.Methods1.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- 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.Results1.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-CMV2/F10~+ 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. 1.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene AP1-Luc were cotransfected A549 cell respectively.After being transfected for 24h,48h,72h we measured and compared the lucifrease activity of different groups to evaluate the effect of the F10 gene on the important signal transduction pathways.We found that after transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously lower than the other groups.We can conclude that F10 gene can be up-regulated the transcription activity of AP1.2.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene NFκB-Luc were cotransfected A549 cell respectively.After being transfected for 24h,48h and 72h we measured and compared the lucifrease activity of different groups to evaluate the effect of F10 gene on the important signal transduction pathways.We found that after transfecting for 48 hours the lucifrease of pRc-CMV2/F10~+ group has been obviously higher than the other groups.We can conclude that F10 gene can down-regulated the transcription activity of NFκB.3.Plasmids pRc-CMV2/F10~+,pRc-CMV2,pRc-CMV2/F10~- and reporter gene pHSE-luc,pCRE-luc,pSRE-luc,pGRE-luc cotransfected A549 cell respectively. Lucifrease activity of different groups has no obvious difference.Part three Effect of F10 Gene on the activities of cell growthAccording to the first two parts results F10 gene possibly can promote cell promation and inhibit cell apoptosis and lead to cell development abnormally.The malignant tumors are related to the cell growth and development abnormally.So we detected the PCNA and cyclinD1 two types of protein which play important role in the cell cycle.The cell proliferation in vitro and flow cytometry technology were used to observe F10 gene's function.Methods1.The influence of F10 gene on the cell multiplication and cell life cycle,plasmids pRc-CMV2/F10~+ and pRc-CMV2 transfected A549 cell respectively.The influence of F10 on cell multiplication and cell life cycle were detected by MTT and flow cytometry.2.The expression of PCNA and cyclin D1 were detected by immunohisochemistry.Results1.According to the A549 cell's 6 days growth curve the two groups growth tendency has obvious difference.The pRc-CMV2/F10 group's cell proliferation was higher than the control.2.The pRc-CMV2/F10 group's the ratio of G2/M cell was 1.2 times higher and the ratio S cell was 2.1 times higher than the control after transfecting 48 hours.The result of two groups was obviously different.3.The result of immunohisochemistry showed that the expression of PCNA in the A549/F10 group was higher than the control.The expression of cyclin D1 was the same as the PCNA's among the groups.Summary1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.According to the A549 cell's 6 days growth curve the two groups growth tendency was obvious difference.The pRc-CMV2/F10 group's cell proliferation was higher than the control.The results reflected that F10 gene can promote cell multiplication.2.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.We detected the influence of F10 on the cell cycle by flow cytometry technology. The pRc-CMV2/F10 group's the ratio of G2/M cell is 1.2 times higher and the ratio S cell is 2.1 times higher than the control after transfecting 48 hours.The results reflect cue that F10 gene can promote cell multiplication by influence the cell cycle.3.The expression of PCNA and cyclin D1 were detected by immunohisochemistry.The expression of PCNA in the A549/F10 group is higher than the control.The same as the expression of cyclin D1.The results cue that F10 gene can promote cell multiplication by up-regulating the expression of PCNA and cyclin D1.Part four Construction and identification the stable expression system of F10 geneThe gene transduction is a technology widely used when studying the gene function.Constructing the stable expression system of gene is necessary step.So constructing the cell line which stable express F10 gene has significant role on study the F10 gene function.Methods1.The A549 cell was transfected with plasmids pRc-CMV2/F10~+,pRc-CMV2 respectively in order to Construct and identify the stable expression system of F10 gene in eukaryocytes.The positive cell clones were screened by G418.2.The insertion and expression of F10 gene in the A549 cells were analyzed by fluorescent quantitation PCR technology.Results1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.The positive cell clones were screened by G418 for three months.We obtain two cell lines of stable express pRc-CMV2/F10~+ and one cell line of stable express pRc-CMV2.2.The result of fluorescent quantitation PCR shows that the level of express F10 gene in pRc-CMV2/F10~+ group is higher than the pRc-CMV2 group.The result is obvious difference.Summary1.The plasmids pRc-CMV2/F10~+,pRc-CMV2 transfected A549 cell respectively.The positive cell clones were screened by G418 for three months.We obtain two cell lines of stable express pRc-CMV2/F10~+ and one cell line of stable express pRc-CMV2.2.The result of fluorescent quantitation PCR reflects that the level of express F10 gene in pRc-CMV2/F10~+ group is higher than the pRc-CMV2 group's.3.The pulmonary carcinoma cell line A549 with stable expression of F10 gene was established,which will be a good model for further study on biological functions of F10 gene.