The Expression Of P19^ARF And P18^Ink4c Gene In Cervical Carcinoma And Their Clinical Significance

Cervival cancer is one of the most common therioma of the female procreational system.The morbidity of cervical cancer is the first malignant tumor of female therioma.Though it is one of highest percentage in female pernicious neoplasm causa mortis. The morbidity of cervical cancer presents a growing trend among young women.This probably because younger women’ sex happen early, they have more sex partner, they don’t pay attention to clean sanitation when they have sex. Persistent infection of high risk human papilloma virus is a necessary factor among attacks and progression of uterine neck pernicious neoplasm.In the known15high risk HPV, HPV16and HPV18are two of the most common.The new research demonstrates that they have relation with70%of cervical cancer.P19ARF is a newfound potential tumor suppressor gene.It roots in Ink4a family which is from cyclin dependent kinase inhibitor family. P19ARF gene coding P19ARF protein whose molecular weight is14KD. P19ARF and P16Ink4a have the same coding gene,but their open reading frame are different.Meantime, P19ARF is the upstream gene of P53gene,it can arrest the cell cycle through P53-MDM2pathway.In this way RB protein can be non-phosphorylation.Phosphorylated RB protein can not bind with E2F,as a result signal pathway is interrupted,then P19ARF-MDM2-P53signal pathway is interrupted.Cell cycle can be stoped in G1/S,in order to inhibit attacks and progression of uterine neck pernicious neoplasm.P18Ink4c is a cell cycle control gene and it can inhibit attacks and progression of uterine neck pernicious neoplasm.The transcription product P18Ink4c protein is a cyclin dependent kinase inhibitor.cThe transcription factor E2F activated transcription of genetic material,and it ia able to escape the cell cycle checkpoint,activation of cyclin dependent kinase related.It comes into paly through binding with CDK4/6,this makes cell still in G1phase,preventing cancer cells continue to proliferation.ObjectiveThis study use RT-PCR methods to research the expression of P19ARF and P18Ink4c mRNA in three different condition uterine neck tissue,use immunohistochemistry technique S-P to study the positive expression of P19ARF and P18Ink4c, in order to disuss the expression and clinical ananlysis of P19ARF and P18Ink4c in uterine nack neoplasm.Materials and Methods1MaterialsChoosing47cervical cancer and20CIN samples from third Affiliated Hospital of Zhengzhou University outpatient cervival biopsy and inpatient department patients during junary2011and junary2012,and20normal cervical tissue samples from resectional uterus because of ademomyosis of uterus or uterine myoma.According to FIGO standard,cervical cancer sample including21Ⅰ phase samples,20Ⅱ pahase samples,3Ⅲ phase samples,3Ⅳ phase samples.According pathology classification,19G1samples,15G2samples,13G3samples.Among them,17patients have lymphatic metastasis, and30patients have not lymphatic metastasis.All the patients do not have chemotherapy and radiational and immunology treatment history.All the specimen was drew within5min after downcut.Following close to aseptique principe.One part of materials were put in-80℃liquid nitrogen,another part were put in paraffin embedding,serial section per44um. 2Methods2.1Experimental method and main reagentImmunohistochemisty and RT-PCR,the SPim-sty kit and DAB colouring agent and primary antibodies P18Ink4c polyclonal antibody are bought from Beijing biological bo orson limited company. P19ARF polyclonal antibody is bought from santa cruz biological reagent limited company.2.2ImmunohistochemistyThe tissue is fabricated by10%specific drug solvent and solid chemical substances,serial section per4um.In strict accordance with the kit instructions for operation.The evalution standard of P18and P19protein positive results,to prefer5random field vision in400times microscope.Giving a mark according to the dyeing strength and positive cell percentae of total cell number.According to the dyeing strength rating:zero means no coloring,1means weak positive coloring,3means strong positive coloring,2is between1and3.According to the positive cell rating:≤5%defines0,6%-25%defines1,26%-50%defines2,>50%defines3.The two scores determine the product,0-2is devided into negative,3-9is devided into positive.2.3RT-PCRDesigning primers,extracting tissue total RNA,PCR amplification product using AGAR gel electrophoresis,observing electrophoresis band under ultraviolet rays.Analysis by D-140picture record analysis system.Quqantity of purpose gene expression is computed by the ratio of objective DNA banding and grave value of ACTINA banding.3Statistical methodsThe data is dealed by SPSS17.0.Using K-W test, X2test,Fisher’s exact propability method.Inspection atandard:a=0.05. Results1The expression of P19ARF mRNA in cervical tissueIn progression of uterine neck, lesioning to serious analysis tendency,P19ARF mRNA has gradually lower expression(P<0.05). Its expression are0.098±0.005,0.502±0.009,0.915±0.027respectively and is seated in568bp.2The expression of P19ARF protein in cervival tissueIn progression of uterine neck morbidity,lesioning to serious analysis tendency,P19ARF has gradually lower expression. Its expression are80%、45%、21.28%respectively(P<0.05).3. P19ARF and cervical cancer clinical and pathological characteristicsP19ARF is related with lymph node metastasis(P<0.05).The positive expression of P19ARF in cancer has not relationship with clinical stages and histological grading(P>0.05).4. The expression of P18Ink4c mRNA protein in different cervival tissueThe rate of positive expression of P18Ink4c mRNAprotein in cervical cancer,CIN,normal cervical tissue is gradually died down(P<0.05). Its expression are0.616±0.009,0.331±0.006,0.187±0.002respectively and is seated in479bp.5. The expression of P18Ink4c protein in cervical tissueIn progression of uterine neck morbidity,lesioning to serious analysis tendency, The gradually higher expression of P18Ink4c are10%、45%、65.96%(P<0.05).6. P18Ink4c and cervical cancer clinical and pathological characteristicsIt is related with cervival cancer stages.The expressional rate of P18Ink4c is gradually higher along with lymphatic metastasis and cervical cancer stages increased.And it has not relationship with histological grading. 7. Correlation analysis of P19ARF and P18Ink4c in cervical cancerRT-PCR showed that they do not have linear ralationship.The results showed that there is no obvious correlationship of P19ARF and P18Ink4c in cervical cancer.(P>0.05).Conclusion1. The low expression of P19ARF and high expression of P18Ink4c may with the relevant to the attack and progression of uterine neck neoplasm.2. In cervical cancer P19ARF and P18Ink4c has abnornal expression in gene level and protein level.