HWAPL Gene Core Promoter Localization And Its Relationship With C-Myc Protein

Cervical cancer is the fourth most common malignancy in women in world.And the incidence rate of cervical cancer is second in female.Every year,528 cases of women were diagnosed with cervical cancer,266000 women died of cervical cancer.Cervical cancer cases in developing countries accounted for 85% of the global total.The incidence of cervical cancer is 7.5/100,000,and the mortality rate is 3.4/100,000 in China.Cervical cancer has become one of the malignant tumors which seriously threaten the health of women.WAPL gene was found in Drosophila melanogaster in 1977.It was located in the Drosophila X chromosome 2D5-2D5.In mitosis,the main function of protein which was encode by WAPL gene,was management the heterochromatin structure and the maintenance of sister chromatid adhesion.The mechanism of action was that the two regulatory subunits of the adhesion protein can form a three element complex,which can promote the separation of adhesion protein,with the WAPL expression product.The mutation of WAPL gene could co-interfere with the normal close and coordinate of sister chromatid.But it did not affect the aggregation and separation of the chromatid.hWAPL gene was the homologous sequence of WAPL gene in human.It was about 30,793 bp and was located in 10q23.2 HWAPL protein is a kind of polymeric anchoring protein,which is highly homologous to the conserved region of WAPL protein.In mitosis prophase,polymerization of chromosome arm could be dissociate in time by h WAPL protein.But excessive expression of h WAPL gene could lead to premature dissociation of sister chromatid.The high expression of h WAPL lead to not only cell chromosome breakage but also non integral or multi nuclear cells appearance,which could cause instability of chromosome,confusion mitosis and tumor.In the study of multiple cancer tissues and cell lines,it was found that the h WAPL gene was highly expressed only in cervical cancer.And it was a specifically over-expressed gene in cervical cancer.In the study of normal cervical epithelium,CIN and cervical cancer,the expression of h WAPL gene was gradually increased along with the progression of cervical lesions.HWAPL gene played an important role in the occurrence and development of cervical cancer.HWAPL gene had the characteristics of oncogene.HWAPL gene pathway had a variety of functions.HWAPL gene may play the role of S phase check point or other apoptotic pathway.Inhibiting the expression of h WAPL gene could cause apoptosis in cervical cancer cell line.And inhibiting the expression of h WAPL gene in animal could inhibit the growth of tumor obviously.At the same time,the polymorphism of h WAPL gene was closely related to the susceptibility of cervical cancer.Myc gene was an oncogene.c-Myc gene was a member of the family.c-Myc gene of human was located in 8q24.The protein encode by c-Myc gene was a nuclear protein with the function of DNA binding.The protein was an important transcription factor and plays an important role in the nuclear information transmission.c-Myc protein,which was synthesized in the cytoplasm,was transfer into the nucleus.c-Myc protein,which was a transcription factor,was bound with specific DNA sequence CACGTG core sequence.They could not only activate or enhance the transcription of target genes and expression,but also promote cell proliferation.It was also involved in the occurrence and development of tumor.The functions of c-Myc gene included inhibiting cell differentiation,promoting cell proliferation,regulating cell cycle,and participating in apoptosis and so on.The expression product of the cell cycle was necessary for the evolution of G0/G1 to Sphase in the cell cycle.It initiated the cell cycle through various cell cycle elements.Under physiological conditions,the proper expression of c-Myc gene maintained normal cell proliferation and apoptosis.If the c-Myc was activated into oncogene,it wouldl cause the cell to break away from the restriction of the normal growth regulation and transform to malignant phenotype,and restrain the apoptosis.In normal cells,the expression of c-Myc was tightly regulated,and was expressed at a low level.c-Myc gene was closely related to the occurrence and development of cervical cancer.The expression of c-Myc was significantly higher in cervical cancer than that in normal cervical tissues.Amplification and over expression of c-Myc gene might be an early event in the development of cervical cancer.The expression of c-Myc gene increased with the degree of malignancy of cervical lesions increased.The expression of c-Myc was significantly enhanced in the cervical cancer and CIN3 tissues compared with CIN1 and CIN2.The expression of c-Myc gene increased with the FIGO staging,pathological grading and the degree of differentiation.The expression of c-Myc gene could be used as an important indicator to judge the prognosis of cervical cancer.As a specifically over-expressed gene in cervical cancer,h WAPL gene has a very important significance in early diagnosis,gene diagnosis and biological treatment of cervical cancer.But it is not clear that the reason,regulation mechanism and regulation of specifically over-expressed gene in cervical cancer.There is no relevant research about h WAPL gene,such as the promoter,transcription factor and signal transduction pathways and so on,at home and abroad.Firstly,the promoter region of the h WAPL gene and its position relative to the h WAPL gene were studied and defined in the study.Secondly,according to the promoter region of h WAPL gene,the transcription factors of h WAPL gene were predicted by the methods of bioinformatics.According to the predicted results,the c-Myc protein was selected as the research object to make sure whether the c-Myc protein has transcriptional activation.Finally,the study focused on whether the c-Myc gene expression product can be combined with the promoter region of h WAPL,whether it is the transcription factor of h WAPL gene.Objects: 1)To identify the promoter region of the h WAPL gene and its position relative to the h WAPL gene.2)To study whether the c-Myc protein has transcriptional activity 3)To study whether the c-Myc protein can be combined with the promoter region of h WAPL gene.To whether it is a transcription factor h WAPL gene.The study was divided into three parts as follows:Part I h WAPL gene core promoter localizationObjects Study and define the location of the core promoter of h WAPL gene.Methods Luciferase assay was used.Steps were as follows: 1)The h WAPL gene upstream 3000 bp was selected;2)The upstream h WAPL 3000 bp was separated into two sections,respectively 2617-1317 bp and 1317-1bp.Steps were as follows: Construction of the vector,the two target gene respectively with pmd18-t vector connection,transformation and detection;construction of fusion expression vector p GL4.17;Plasmid Extraction;Luciferase activity assay after transfection of Lipofectamine 3000 cells;3)By analysis,the h WAPL promoter was located in the-1317—-1bp of the h WAPL gene upstream;4)The h WAPL gene upstream-1317—-1bp was divided into three segments,-900—-1bp,-600—-1bp,-300—-1bp,respectively.The luciferase assay was used respectively.The steps were same as above.Conclusion The core promoter of h WAPL gene was located in-1bp—-300 bp of the h WAPL gene upstream.Part II Study on bioinformatics prediction of h WAPL transcription factors and transcriptional activity of c-Myc proteinObjects According to the promoter region of h WAPL gene,the transcription factors of h WAPL gene were predicted by the methods of bioinformatics.Using the PWM prediction algorithm of FRANSFAC database,ENCODE data prediction algorithm and JASPAR data prediction algorithm,h WAPL gene transcription factors were predicted.c-Myc protein,which is one of the results of three prediction,was choose to verify whether the c-Myc protein is a transcription factor of h WAPL gene.Study on the transcriptional activation of c-Myc protein by yeast two hybrid.Methods 1)Yeast two-hybrid self activation experiment was used.Steps were as follows: construction of c-Myc-p GBKT7 vector;2)Preparation of yeast cell AH109 competent cell;3)PGBKT7 and p GADT7 co-transformed yeast strain AH109;4)Cloned to SD/-Leu/-Trp;5)Positive clones to SD/-Ade/-His/-Leu/-Trp culture medium;X-gal detection experiment.Conclusion 1)c-Myc protein is one of the transcription factors of h WAPL gene in the three prediction results.2)Yeast two hybrid results showed that all of the Y187 strains were cloned in the culture medium.It meant that the success of a total conversion.3)The SD/–Leu/–Trp culture medium was picked to SD/–Ade/–His/–Leu/–Trp culture medium.Except the control group,the other experimental groups were cloned.4)The SD/–Ade/–His/–Leu/–Trp culture medium was picked to SD/–Ade/–His/–Leu/–Trp culture medium which contain X-gal.The clone became blue.c-Myc protein had transcriptional activity.Part III Study on the relationship between c-Myc protein and the core promoter of h WAPL geneObjects To study whether the c-Myc protein could be combined with the h WAPL gene promoter region,whether it is a transcription factor of h WAPL gene.Steps were as follows:Methods 1)Yeast one-hybrid experiment was used.Steps were as follows;2)Construction of C-Myc-p GADT7 and h WAPL-p HIS2 vector;3)Preparation of yeast cell Y187 competent cell;4)c-Myc-p GADT7 and h WAPL-p HIS2 vector co-transformed yeast strain AH109;5)Cloned to SD/-Leu/-Trp;6)Positive clones to SD/-Trp-Leu-His culture medium;7)Yeast one-hybrid experiment.Conclusion 1)Yeast two hybrid results showed that all of the Y187 strains were cloned in the culture medium.It meant that the success of a total conversion.2)The SD/–Leu/–Trp culture medium was picked to SD/–His/–Leu/–Trp culture medium.All the experimental groups were cloned.3)It meant that 3-AT of 20 m M could not inhibit the expression of histidine.4)When 3-AT of 40 m M was used,other experimental groups were not cloned,except positive controls.c-Myc protein could not be combined with the h WAPL gene promoter region.c-Myc protein was not a transcription factor of h WAPL gene.Conclusion The core promoter of h WAPL gene was located in-1bp—-300 bp of the h WAPL gene upstream.c-Myc protein had transcriptional activity c-Myc protein could not be combined with the h WAPL gene promoter region.c-Myc protein was not a transcription factor of hWAPL gene.