Related Study Of The Gene Expression Of P27 And CyclinE In Bladder Carcinoma

From biology angle malignant tumor multiplication shows in two aspects; one hand is tumor cell immortality , the other hand is the dropout of cell division . The regulatory molecule of participated cell cycle includes cyclin , cyclin dependence kinase phosphorylase and cyclin dependence kinase inhibited protein . CKI is discovered recently it is a group of important cell cycle modulin , it can combine with CDKs, cyclins and cyclin - CDKs compound to inhibit CDK activity . CKIs are divided into two families: CIP/KIP and ink4 . CyclinE is CDK2 s regulatory subunit adjusting CDK2 activity. In many tumor tissues cyclinE generates gene amplification and hyperexpression , it has been already confirmed that its a cancer gene. P27 is a important role of CIP/KIP family, the effect in cell cycle regulation caused extensive attention . p27 can combine with cyclinE/ CDK compound to inhibit substrate s phosphorylation , while cyclin E is major adjusted object. This test applies RT - PCR , Western blot and immunohisto-chemistry method ( S - P) to detect both cyclinE gene mRNA and protein expression in 71 pou carcinoma of bladder and 21 pou normal control so as to discuss the relationship between p27 and cyclinE gene.The first part: the research between p27 gene and protein expression in carcinoma of bladderThis test applies RT - PCR , Western blot and immunohistochenmistry method(S -P) to detect the situation both p27 gene mRNA and protein expression in 71 pou carcinoma of bladder and 20 pou normal control so as to discuss the relationship between p27 gene and carcinoma of bladder .Experimental methods1. Test materials; This study applies the mucous tissues of 71 pou carcinoma and 20 pou normal control conduct as study material , samples came from operated patients in hospital .2. Main agent:1) RT - PCR trial: RNA extract, reverse transcription agent , Dnase 1 is GIBCOBRL company product, PCR agent is TaKaRa company product , RNA LA PCR? Kit( AMV) is TaKaRa corporation product.2 ) Western blot trial: Mouse monoclonal antibody p27 , ECLTest Kit is Santa Cruze corporation product3)The immunohistochemistry (S-P) experiments: 10% caprine blood serum, S - P, biotin mark the second antibody is American Zymed Corporation product,p27 mouse monoclonal antibody is Santa Cruze corporation product3. experiment method:1) RT - PCR amplification :total RNA withdraws, after Dnasel digestion, reversal transcripts and synthesizes the first chain of cDNA, PCR amplification, p27 product length is 238bp, inter - contrast use β - actin (318bp) , caraphore-sis detect, with autocaraphoresis - gelatin image analyltical machine analysising the result. The software spsslO. 0 is applied to do statistic analysis.2) Western blot; Protein sample preparation,protein density determination . Use Bio - Rad Corporation DC Protein the assay agent box. Attracts the luminosity value on the ultraviolet spectrophotometer in the 570nm determination . And according to standard curve we obtains the actual protein content . SDS -PAGE electrophoresis , suck the waiting analytical sample , on the basis of protein concentration and adding sample pore volume to determine the adding sample quantity. Trarsmembrane, hybridization, chemiluminescencereaction( ECL). In the ECL Test Kit the detection reagent 1 and detection reagent 2 are mixed with the same volume ,after that isodribbling them on the PVDF membrane,ex-posuring and washing sheet. Obtaining the carcinoma of urinary bladder and normal contrast constitution gel gray degree tite with gelatum scanning analysis sys-tem. Software spsslO. 0 is applied to do statistic analysis.3) The immunnohistochemistry ( S - P) experiments: paraffin section routine HE dyeing, phytomycinphile bioepiderm peroxidized agent enzymic method dyeing ( S - P). PBS displace the first antibody acting as blank , using the knowed breast cancer tissue P27 act as positive control, Observating ten piece perim count under high power microscope,x~2 test.Experimental results1 p27 genie RT - PCR analytic resultThis test applied RT - PCR reaction to screen 71 pou carcinoma of bladder and 21 pou normal control p27mRNA expression, amplification fragment magnitude are coincide with primer contrivance ,gel analytic result progress statistical treatment though software SPSS 10. 0, the test result indicated that the difference of p27 gene expression between anisopathoclass carcinoma of bladder and normal control have statistical significance ( F = 9. 623 , p < 0. 0001) , its expression is obviously decreased in carcinoma of bladder tissue, via Q test , it indicated that the difference among normal control and each carcinoma of bladder class existed statistical significance but among each carcinoma of bladder class dose not exist statistical significance.2. p27 protein Western blot analytic resultThough Western blot reaction to screen 71 pou carcinoma of bladder and 21 pou normal control p27 protein expression , protein magnitude is 27 KD , gel analytic result progress statistical treatment through software SPSS 10.0, the test result indicated that the difference of p27 gene expression between anisopathoclass carcinoma of bladder and normal control have statistical significance ( F = 20. 281, p < 0. 0001 ) , its expression is obviously decreased in carcinoma of bladder tissue, via Q test, , it indicated that the difference among normal control and each carcinoma of bladder class carcinoma of class 1, class 2 and class3 existed statistical significance , but the difference between carcinoma of bladder class2 and class 3 dose not exist statistical significance.3. p27 protein S - P immunanohistochemistry analytic result.P27 protein expression masc location located in cell nucleus . Image analytic result progress statistical treatment though software SPSS1O.O, the test result indicated that the difference of p27 gene expression between anisopathoclass carcinoma of bladder and normal control have statistical significance (F = 12.315, p < 0. 0001) its expression is obviously decreased in carcinoma of bladder architecture , via Q test, , it indicated that the difference among normal control and each carcinoma of bladder class carcinoma of class 1, class 2 and class3 existed statistical significance , but the difference between carcinoma of bladder class2 and class 3 dose not exist statistical significance. In normal control p27 protein expression masc rate is 90.00% ( 18/20) , among it 4 pous are weakly positive , in carcinoma of bladder class 1, class 2 , class 3, p27 protein expression masc rate is 55.00% (11/20) ,30.43% (7/23) and 14.29% (4/28) weakly positive is three pous , one pou , one pou . x2 test result hinted the difference between normal control and carcinoma of bladder class 1 exist statistical significance (x =4.90, p <0.05) ,p27 protein in carcinoma of bladder is less than normal control ,the difference among carcinoma of bladder classl class2(x2 =2. 65, p > 0. 05 ) ,013883 ( x2 - 1- 95, p > 0. 05 ) dose not exist statistical significance.Discussion and conclutionPolyak found and cloned p27K1P1 in 1994, in the meantime Toyoshima and other people also did it . p27 gene lies in chromosome 12p. Human p27 is a heat - resisting cell cyclesuppression protein which is composed by 198 amino acids. , its molecular weight is 27kD. p27 regards as a kind of broad spectral CKI molecule, through with CDK and cyclin mutually interacting to regulate the cellcycle. It may suppress the many kinds of cyclical element - CDK compound. It is found that p27 is major in inhibiting kinase complex of Gl period, and it can specially inhibit the activity of CDK2 - cyclinE kinase . After cyclinE - CDK2 complex formed , its CDK2 -Thrl60 must be phospalated ,then it has the activity of the kinase. P27 inhibits CDK2 -Thrl60 phosphalated through its C terminus, so that it inhibit the kinase % activity, meanwhile it may inhibit the kinase sactibity through binding with cyclinE -CDK2.P27 can inhibit the Gl/S transform . p27 can inhibit the activity of cy-clineE/CDK2 complex through combining with it. p27 plays an important role of Gl/S transform . While the inhibition of p27 and cyclinE/CDK2 complex is mutual. Sheaff found that cyclinE - CDK2 complex can make p27 decrease in cell synthesis, lose inhibition action , make cell cycle entering S period from Gl period. Xu thought p27 completes this process through combining with cyclinE -CDK2 complex. When the p27 expression regulation mechanism has the disorder, which causes p27 the expression to drop or the flaw,then causes the tumor the occurrence. As candidate tumor inhibiting factor p27 ,it prevents the cell to pass Gl/S transforms "the checkpoint" function,thus suppression cell cycle,a-chieving the suppression cell multiplication and making cells have the chance to repair the injured DNA or the mistakes in DNA multiplication .P27 gene seldom mutates or loses in tumor cells. Now we found the change of p27 in very few tumor tissues. Morisetti found nonsense mutation of code 76 in a pou T cell lymphoma which caused the production of short disfunction p27 ; In another example T cell lymphoma discovering the pure lost of p27. Spirin also found nonsense mutation of code 104 in a pou primary breast tumor,and no mutation in control group. As for the consequence of gene 27 targeting, we found p27 - - / - - mice often form pituitary tumors . Li Jinghe applied RT - PCR to test mRNA change of p27 in gastric mucous carcinoma. It showed p27 expressed decreasing in gastric mucous carcinoma .This test applied RT -PCR to identified p27mRNA expression of 71 carcinomas of bladder and 20 normal control tissues, the sizes of amplified fragments are consistent with those of primer designs. Gelatin analytical results showed p27 expression had statistic significance in varied pathological classifications and normal control groups of carcinomas of bladder via the statistical proposals of software SPSS10. 0. This kind of expression obvious decreased in carcinoma of bladder tissues. Via Q test, it showed the differences between the normal control groups and varied groups of carcinoma of bladder have statistic significances, but the differences of every group of carcinoma of bladder have no statistic significances. p27 mutation is rare in most tumor cells,but it can be detected in someindividual tumors. p27 perhaps plays a role in these tumors. Our results are likely to Ii Jinghes ,but the differences of varied pathetic classifications of carcinoma of bladder arent marked . So we conferred that mRNA of p27 may play an important role in the occurrence of carcinoma of bladder.P27 regulates cell cycles mostly in protein expression levels,its protein expression avtivitys change is associated with the formings of tumors. Esposito investigated 108 patients of small cell lung cancers, finding p27 of tumor cells is very low . In another studies about cancers of colon and the breast. We also can find no change of p27, but its protein level is decreased. Facts certificated that the changes p27 protein level have the key role in tumor growing.In this study Western blot and immunohistochemistry results both showed p27 protein expression has statistical significance in varied pathetic classifications and normal control tissues, its expression markly decreased in carcinoma of bladder . Via Q test , it showed between normal control groups and varied carcinomas of bladder , the difference of the first grade and the second grade or the first grade and the third grade have the statistic significance , whereas no statistic significance between the second grade and the third grade. All above show p27 protein gradually decreased with the advancement of carcinoma of bladder. The lost of p27 protein in carcinoma of bladder suggests p27 combines with cyclinE/ CDK2 complex so that p27 inhibits its activity , and inhibition of p27 to the transformation of Gl/S can be restricted. p27 may be a tumor suppression gene associated with carcinoma of bladder .The conclution is:1. This study applied RT—PCR to find mRNA expression of p27 decreased in 71 carcinoma of bladder and normal control groups. It showed p27 may be a tumor suppression gene associated with carcinoma of bladder .2. Western blot and immunohistochemistry detected 71 carcinoma of bladder and normal control groups , finding that p27 of tumor tissues lies in cell nuleus, and p27 protein expression markly decreased in carcinoma of bladder. This result is coincided with that of RT - PCR , so further confirmed p27 may be associated with tumor suppression gene .The second Part : CyclinE Gene and Protein Expression Research hiCarcinoma of BladderThe investigation implies RT - PCR. Western blot and S - P methods , which investigate the protein expression in the tissue of 71 bladder cancer cases patients and 20 control team cases in order to discuss the relation between cy-clinE gene and bladder cancer.Methods1. material: as test 12. the prime reagent; RT-PCR test; as test 1Western blot test; mouse monoclonal antibody cyclinE. ECL kit ( Santa Cruze)Immunohistochemistry( S - P) test; 10% goat serum. S - P. the second antibody (Zymed ,America) ,cyclinE mouse monoclonal antibody(Santa Cruze)3. methods1) RT - PCR amplification ;as test 1 ,the length of cyclinE outcome is 257 - acti (318 bp) bp, control uses2) Western blot; as test 13) S—P reaction : routine HE stain in paraffin section ; strepotin biotin— hyperoxide enzyme stain (S—P). It is as the blank control that using permutated the first antibody with PBS , while using known cyclinE of carcinoma of the breast as the positive control. Under the high time of microscope we observed 10 fields and accounted . via x2 test.Experimental result1. RT - PCR analytical results of cyclinE geneThis test repeatedly screen mRNA expression of cyclinE gene in 71 pou carcinoma of bladder and 20 pou normal control tissue, the conclusion is that the size of amplified segments is coincided with the primer. Gel analytical results are statistical managed via spsslO. O,the results show cyclinE gene expression hasstatistical significance in varied pathetic classification of carcinoma of bladder and normal control tissues ( F = 10. 964, p < 0. 0001) , and its expression markly increased in carcinoma of bladder tissues. Via Q test ,it showed between normal control groups and varied carcinomas of bladder , the difference of the first grade and the second grade or the first grade and the third grade have the statistic significance , whereas no statistic significance between the second grade and the third grade.2. Western bolt analytical results of cyclinE proteinThough repeatedly screening 71 pou carcinoma of bladder and 20 pou normal cyclinE protein expression of control tissues, the size of protein is definited 42KD. Gel analytical results are statistical managed via spsslO. 0,the results show cyclinE gene expression has statistical significance in varied pathetic classification of carcinoma of bladder and normal control tissues ( F = 13.415,p<0. 0001) , its expression markly increased in carcinoma of bladder tissues. Via Q test ,it showed that the difference had the statistical significance between normal control groups and varied carcinomas of bladder , or among the first grade , the second grade, the third grade of the carcinoma of bladder.3. S - P immunohistochemical analytical results of cyclinE proteinsThe positive portions of cyclinE protein expression lies in cell nuleus. Picture analytical results are statistical managed via soft spss 10.0. The results show cyclinE gene expression has statistical significance in varied pathetic classification of carcinoma of bladder and normal control tissues (F = 13. 415, p <0. 0001) , its expression markly increased in carcinoma of bladder tissues. Via Q test ,it showed that the difference had the statistical significance between normal control groups and varied carcinomas of bladder , or among the fir st grade , the second grade, the third grade of the carcinoma of bladder.In normal control tissues , the positive rate of cyclinE protein expression is 15.00% (3/20) ,no weak positive; CyclinE protein expression positive rate of the first grade of carcinoma of bladder is 50. 00% (10/20) , the second grade 65. 22% (15/23) ,the third grade 82. 14% (5/28). There is a pou weak positive in every grade . X2 test results present the difference between the normal control tissue and the first group of the carcinoma of bladder have the statistical signifi-cance (X2 =4. 90,p <0.05) ,cyclinE protein expression increased in carcinoma of bladder than that in normal tissues. The differences between the first grade and the second grade have no statistical significance(X = 1.02,p >0.05) ,and the first grade and the third grade has also no statistical significance ( X = 1. 90, p>0.05).Discussion and ConclutionTwo American investigation teams discovered cyclin E gene when screening the human cDNA library, which lies in 19ql2 - 13 ,transcripting 2. 2kb mRNA, coding the protein of 395 amino acid whose MW is about 50 KD. As it is known that it has a highly conservative central area which is commonly called cyclin box, and it can combine CDK . The C segment of cyclin E is longer and rich in P,E,S,T residues. There are two degradation forms of cyclin E gene, one is hy-drolyzed by calcium proteinase type adjustment protein in associaion with PEST segment, the other is degraded by the ubiquitin form in with C segment .CyslinE acts mostly in the Gl advanced stage. The mechanism has been confirmed : After cyclins combines CDKs, , it phosphorylate its substrate, for instance pRB and release the TF E2F, which makes DNA systhesize go on and the Gl stage in the cell cycle enters the S stage. cyclinE is the primary regulating factor, if we make cyclin E antibody inject the culture mammary animal cell, which makes the cell cycle stops in the Gl stage.CDK is a kind of protein activating enzyme family. Every CDKs and several cyclins combine and form dimer , which acts in the different stage in the cell cycle. Cyclin E combines CDK2 and its activeness hasnt achieved the peak in the end of Gl. CKIs has two kinds, and one includes p21 and p27,the other includes pl6,pl5. The prime function of CKIS is restraining the activation of CDK. The investigation indicates the activation of cyclinE - CDK2, A - CDk2 depend on P53, which are restrained in the ionization radiation , it is supposed that it is caused by the transcript activation. p27 is another member of the p21 family, it also binds with cyclin E/CDK complex and inhibits the substrate phos-phorylation , while cyclin E - CDK2 is the major adjusted object of p27 .The more investigation indicates that tumorous occurrence is associated with cyclin and other gene which is related with cell life abnormal expression ,many cancer such as breast cancer , lcolon carcinoma gastric cancer , leukemia , all discover gene amplification and protein expression imbalance , using carcinogenic agent induces mouse into esophageal carcinoma and sikn cancer , investigate the change of cyclin E , among the induced - animal esophageal carcinoma , the mRNA of cyclin E increased obviously during normal tissue , precancerous change, cancer process . cyclin E increased gradually ,it is indicated that cell cyclin E occurrence in esophageal carcinoma is early event , among cyclinE transgenic mice , female mouse breast cancer occurrence is 10% . indicate the effect of cyclin E in breast cancer occurrence . the model feature is that a couple of lacteal duct specific promoter locates in cyclin E gene , the homoexpression during duration of preganancy and feeding time may induce visible breast cancer occurrence, Keyomarsi first observated the gene change of cyclinE in cancer , indicate the role of oncogene cyclin may contact with activated ras gene to make mouse embryo fibroblast malignant transformated ,so that it can confirm cyclin is oncogene.Although many disease dose not exist gene amplification , mRNA expression also increase , or neither gene amplification nor mRNA expression increased , but many isomerism formal protein exists in tumor. From 35KD to 50KD , accordingly considered that it exists post - transcriptional control , breast cancer solid tumor cyclin E progressing Western test discovered that 90% tumor tissure expression increased , with the increasing of breast cancer staging and hierarchica-tion cyclin E expression increased gradually, estrogen receptor negative group and poorly differentiated group both exist cyclin E homoexpression , accordingly it can conduct a prognostic index, deeped investigation indicated that cyclin E correlated kinase activity is obviously higher than normal tissue . but in majority recurrence leukemia the expression of cyclin E is higher than the frist occurrence ,the level of expression reflect the advancement of disease , control p27mRNA expressio This test applied RT - PCR reaction to screen 71 pou carcinoma of bladder and 21 pou normal n P27 protein expression masc location located in cell nucleus , gel analytic result progress statistical treatment though software SPSS...