| Background and objectives:Since the people become industralized man,the human tumor become increasely, and Urologic cancer has constantly threatening our life and health.According to the reports,the incidence of Urinary System has always on the rise in Europe from the year 2003 to 2007,among which the incidence of the male’s kidney cancer reached up to 31.4/100000,and it still increased by the speed of 3.7-7.0/1000000 annually on average.Bladder cancer is commonly seen in western and asian countries in urothelial carcinoma. Though the diagnosis and treatment techniques have greatly improved,the incidence and death rate of bladder cancer still increase year by year,Testis cancer has always been the multiple tumors among the male aging from 15 to 40.As the statistical result shows in the United States.among the Hispanic white males aging from 15 to 39 in America,the morbidity of testicular germ cell tumors has increased by 58% from the year 1992 to 2010.However,the morbidity of the urinary system tumor in China is relatively low. According to the statistics in China’s 30 major cities and countries,the attack rate of kidney rate and other urinary tumors reached 6.63/100 000 from the year 1998 to 2002,which is rising all the time,and among which the morbidity of kidney cancer goes up fast.Kidney cancer does not rank among the first few major malignant tumors affecting the health of residents in our country,but its rising trend should be attached more important Over the years,the diagnosis and treatment teachniques have considerably improved,but the attack rate and death rate of the tumors hve still been on the rise.Therefor,to study newer and more effective diagnosis techniques is more beneficial for lowering the hazards of tumors and building human’s health.In recent years, more and more people want to find valuable genes and protein as the tumor markers for dianosis and therapy. The tumor markers are biochemical gene or protein in blood,body fluid and tissue giving hints to the existence of the tumors.when the tissue become tumor, it will existence or its quantitis become more or its become mor activity. It can help us dianosis tumors,even attack the tumor through attack this gene or protein. Because of the heterogeneity of tumors, the morphological staging and grading cannot be used to judge the biological behavior of tumors.So search the new tumor markers with high sensitivity and strong specificity holds significant meaning to diagnosis tumor and improve tumor patients’prognosis. In our previously study, we detect the genes of expression in prostate tissue through transcriptome and proteome technology, Using integrative transcriptome and proteome to detect 4 fresh prostate cancer tissues and paired 4 adjacent benign tissues of prostate. I is found that there are 1207 differently expressed genes,among tumor and benign,which 652 differently expressed genes are up-regulated, and 555 are down-regulated.Through proteomics and comparison,we total check 2566 paired protein spots,there are 89 spots up-regulated in the group of tumors.(the ratio between tumors and non-tumors is more than 2 times,p<0.01),and there are 66 spots down-regulated, (the ratio between tumors and non-tumors is less than 2 times,p<0.01).Based on the the gene chip and proteomics, we can find out 14 common differently expressed molecules(protein).Through retrieve GEO data,we can filter 6 candidate proteins and check its concentrate in blood among the 14 differential proteins,and further confirm that inosine monophosphate dehydrogenase 2 is highly expressed in prostate cancer tissue and it is quantity correlated with the clinical stages,pathological grading and transference of the tumors through the experiment of prostatic cancer tissue.And it also confirms that this gene holds good value to diagnosis prostatic cancer and help us see patients’pronosisIMPDH2 is widely expressed in organs. It not only exists in each organ of human’s and other mammal,but also in microorganism,There are two types of IMPDH, that is, two similar isozymes IMPDH1 and IMPDH2,Both of the subtypes are expressed in many tissues.Every isozyme is made up of 514 amino residues,and it has 84% amino acid sequence homology. IMPDH is the key enzyme in synthesize purine,and it makes IMP oxidized into XMP.XMP, catalyzed by GMP enzyme synthesis GMP and then transfer into GTP and dGTP.GTP and dGTP is necessary nucleotide in synthesize DNA and RNA.In the process of nucleic acid synthesis,compared with ATP,GTP is the major factor to limit the speed.The synthetic way is called the classic synthetic way of guanine nucleotide.The reaction of IMPDH’s catalysis serves to limit the speed in the synthetic way.The guanine nucleotide is the key enzyme substrate fir the synthesis of DNA and RNA,and it plays a very important role in cells’ growth,differentiation and apoptosis,and cellular signal transduction.IMPDH1 activity is constructive expression.Its expression is usually lower than IMPDH2,and it is not influenced by cell proliferation and differentiation and so on.But IMPDH2 shows induced expression.It is the positive regulator in proliferating cell.It’s ability of expression in proliferating cells and cancer cells gradually improves.Therefore,IMPDH2 hols significant meaning to the reaserch on tumors.By reading literature,it is found that IMPDH2 is highly expressed in colorectal cancer,cervical cancer,melanoma,leukemia and other cancers,and it is somewhat related to pathological grading and clinical stages of cancer and shows this gene is valuable for judging the patient’s prognosis.Since Kononen presents the concept of tissue chip for the first time,the teachnology of tissue chip is widely used in the search of cancer genes,the occurrence and growth of cancers, the verification of biological molecular markers related with prognosis,the internal quality control of the diagnosis in clinical laboratory,and the confirmation of the infectious disease and so on. and it solves many knotty problems.The teachnology of tissue chip is to evenly arrange dozens and hundreds of tissue samples on the glass slide to form a miniature tissue slice.And it truly realizes a lot of presentation imformation acquired by high throughput on tissue slice through various dyes,including normal HE dyes, histochemistry dyes,immunohistochemical dyes,in-situ hybridization dyes and so on.The teachnology of tissue chip can upgrade the results of morphologic observation to the level of high standard,and lower the influences of subjective factors and objective conditions on the results.And it has become an important basis of predicting the prognosis of the cancer patients and molecular targeted therapy markers.Based on the research foundation of the predecessors and the outcome of our long-standing research on genomics and proteomics,the experimentalGiven all that.In order to explore the value of,this research would aim to collect prostatic cancer tissue samples, then detect IMPDH2 genes and proteins and compare the differential expression of IMPDH2 in prostatic cancer and benign tissue,and then verify IMPDH2’s expression in prostatic issue in the big sample by the immunohistochemical.The tissue chip and immunohistochemical technology is used to explore and analyze the relativity between the expression level of IMPDH2 and the clinical and pathological characteristica of the prostatic cancer patients.And then detect kidney cancer, bladder cancer,testicular cancer by immunohistochemistry to explore and analyze the expression of IMPDH2 in the 4 most commonly tumors in urinary systems,and explore the gene’s involvement in the transference and recurrence and other unhealthy prognostic process of the tumors in urinary system,and demonstrate whether it will become an independent index to predict the biochemistry,reccurrence,transfering progression and expectation of life,and whether the gene possesses the new value as the tumor markers.Materials and MethodsIMPDH2’s expression in prostatic cancer and its clinical outcomes research1.1MaterialsThis research is authorized by Ethics Committee at the first Peolple’s Hospital in Guangzhou,and the patients sign the informed consent and sampling consent. For quantitative real-time reverse-transcriptase PCR(qRT-PCR) and Western blot analyses, frozen samples of 20 primary PCa tissues and 11 adjacent benign prostate tissues were collected from the tissue bank at Guangzhou (the patients should be diagnosed with prostatic cancer for the first time,and they should not be treated with any radiotheraphy,chemotherapy and endocrinotherapy before operation and 11 benign tissues are both derived from the patients of urinary surgery at No.1 People’s Hospital in Guangzhou during the period from October,2012 to August,2013.(After operation,tissues were taken and fetched to the pathology department at NO.1 People’s Hospital in Guangzhou for freeze and paraffin section,and they are verified to be prostatic cancer by pathology,and there is no limit to staging.)After the samples are taken,they are put into the liquid nitrogen tanks for quick-freeze,and they are preserved at the lower temperature of -80 ℃ until they are used.And 96 cases of prostatic cancer with detailed clinical information and 81 cases of paratumor prostate tissue chip(These patients did not receive androgen ablation treatment and radiotherapy)are purchased at Xinchao Biotechnology company in Shanghai.(The article number is HPro-Ade180PG-01)They are used for the IMPDH2 immunohistochemical experiment. The first antibody reagents of IMPDH2 are purchased from abcam company.(The article number is EPR8364(B)1.2 Methods1.2.1 RT-PCR Quantitative PCR was used to examine the expression status of IMPDH2 mRNA in PCa and non-cancerous prostate tissues. The cDNA templates for qRT-PCR were synthesized from RNA samples. The primers of IMPDH2 genes and b-actin which was used as an internal reference were shown as following.,And we could compare the circulation data of samples and internal reference samples to reach the results of the expression of sample genes.1.2.2 Western blotProteins were extracted from prostate cancer and benign prostate tissue 48 h post-transfection for Western blot analyses. Proteins (40 μ g) were fractioned on SDSPAGE and transferred onto Hybond nitrocellulose membranes (GE Healthcare). The membranes were blocked with 5% skim milk in PBS-Tween 20 and probed with anti-IMPDH2(rabbit monoclonal antibody, ab131158,Abcam Co Ltd, USA) at a dilution of 1:1,000 or anti-bactin antibody (sc-47778, Santa Cruz, USA). The resultswere visualized with the SuperSignal West PICO chemiluminescent detection system (Pierce Biotechnology).b-actin was used as an internal loading control.1.2.3 ImmunohistochemistryThe specimens were fixed in 10% neutral buffered formalin and subsequently embedded in paraffin. The paraffinembedded tissues were cut at 4 lm and then deparaffinized with xylene and rehydrated for further H & E or peroxidase (DAB) immunohistochemistry staining employing DAKO EnVision System (Dako Diagnostics, Switzerland). Briefly,following a brief proteolytic digestion and a peroxidase blocking of tissue, the slides were incubated overnight with the primary antibody against IMPDH2 (rabbit monoclonal antibody, ab131158, Abeam Co Ltd, USA) at a dilution of 1:200, at 4_C. After washing, peroxidaselabeled polymer and substrate-chromogen were then employed to visualize the staining of the interested protein.In each immunohistochemistry run, negative controls were carried out by incubated overnight with PBS instead of the primary antibody.Following a hematoxylin counterstaining, immunostaining was scored by two experienced pathologists, who were blinded to the clinicopathological data of the patients. Every pathologist score independent,finally,the scores of the two pathologists will be compared and any diverging scores were trained through re-examining the stainings by both pathologists to achieve a consensus score. The number of positive-staining cells in ten representative microscopic fields was counted and the percentage of positive cells was calculated. Given the homogenicity of the staining of the target proteins, tumor specimens were scored in a semiquantitative manner. The percentage scoring of immunoreactive cells was as follows:0 (0%),1 (1-10%),2 (11-50%) and 3 ([50%). The staining intensity was visually scored and stratified as follows:0 (negative), 1(weak),2 (moderate) and 3 (strong). A final immunoreactivityscores (IRS) was obtained for each case by adding the percentage and the intensity score.2.IMPDH2’s clinical screening study in urinary system.2.1 MaterialsThe kidney,bladder and testis tissue microarray are bought from Alina company in Xi’an.The serial number of kidney tissue microarray is KD244,there are 12 groups of kidney cancer tissues and paired comparison tissues adjacent to carcinoma.The patients include 9 grown-up male and 3 female ading from 33 to 76.And average age of theirs is 55.The chip data consist of the pathological clinical stages and TNM stages of the patients.The serial number of the bladder tissue microarray is BL481,including 39 cases of bladder cancer tissues and 8 cases of normal control,there are 35 male and 12 female patients aging betwwen 21 and 88.And the average age of theirs is 54.The chip data include the patients’pathological clinical stages and TNM stages.The serial number of the testicular tissue chip is TE281,including 40 cases of testicular cancer tissues and 8 cases of normal control.The age of the patients ranges from 15 to 81,and their average age is 43.The chip data include the patients’TNM stages,non-pathological grading and clinical stages.2.2 ImmunohistochemistryThe immunohistochemical detection is made about the kidney,bladder and testicular tissue microarray,and the method of detection is the same as that of the prostate tissue microarray.3.The methods of statistical analysisThe software package of SPSS18.0(SPSS Inc,USA) is adopted to make the data processing.The two independent-samples T test is made betwwen the expression of IMPDH2mRNA in Statistical Pea and benign tissues and that of western blot’s test in prostate cancer and benign tissues.If there is heterogeneity of variance,t is adopted to make the test.Pearson chi-square test is used to analyze the relative relationship between IMPDH2’s immunohistochemical staining expression in PCa and benign tissues and IMPDH2 protein stain and cancer clinical pathology.Among the multiple sets of mean,one-way ANOVA varience is used to analyze the comparision betwwen prostate cancer expression profile Taylor data mRNA score and prostate cancer clinical pathological parameters.. Differences were considered statistically significant when P<0.05. Pair t test is used to analysis the protein with expression in kidney cancer and para-carcinoma tissues, make the independent sample t test about testicular cancer,and normal testicular tissues,analyze the difference of different pathological grading,clinical stages and TNM stages in tumor tissues When there is heterogeneity of variance,t’ test is adopted.The measurement data of normal distribution is expressed by (?).When p is less than 0.05,there is statistical significance.4.Results4.1 The gene chips are used to screen out the up-regulation expression MCCC2 in prostate cancer tissues in preliminary experiments. Between the Gene TRAP1 and IMPDH2,IMPDH2 has the highest up-regulation multiple,and the rising multiple reaches 1.98 times.(chart one)Chartl:the results of gene chips combined with GEO to analyze IMPDH2’s up-regulation expression in prostate cancer tissues. Figl:IMPDH2 overexpress in Pca4.2 IMPDH2’s expression in prostate cancerThe result of RT-PCR shows:IMPDH2 mRNA’s expression level obviously improves in prostate cancer compared with that in BPH tissues. (9.22 ± 2.49vs 5.06 ± 1.45, P<0.01)。 The result of Western blot shows:MPDH2’s expression level in prostate cancer is higher than that in BPH,(Pca=0.674±0.029 vs Benign=0.418 ± 0.140, P<0.001)。 The result of immunohistochemistry shows:IMPDH2 protein’s expression signal in prostate cancer is claybank,and most of its expression is in cytoplasm and cell membrane,and less is karyon,and its positive rate in prostate cancer tissues is 100%. And the positive expression in benign tissues is in karyon,and its positive expression rate is 80%.IMPDH2 protein presents high expression in prostate cancer tissues in general, obviously higher than benign tissues immunohistochenical tissues. IRS (0-2):benign 17cases VS PCa Ocase,, (3-4): benign45 cases VS Pca21 cases, (5-6):benign 19 cases VS PCa 75 cases(chart 2, P<0.01)。 IMPDH2 protein is between low and moderate expression and high expression,and it has obvious statistical significance in tumor stages,transference or non-transference and clinical progression of the patients.IMPDH2 protein’s expression has no difference with the patents’ age. (P=0.13)。 And its expression level in high-ranking prostate cancer is obviously higher than that in low-ranking prostate cancer.(P=0.003) And its expression level in metastatic prostate cancer is remarkedly higher than that in non-metastatic prostate cancer.(P=0.01), During progressive stage,the expression level of prostate cancer is also remarkedly higher than that in localized prostate cancer (P=0.012),the corresponding data also testify our immnohistochemical result.4.3 IMPDH2’s expression in other tumors in urinary system.The positive rate of IMPDH2 protein expression is 100% in kidney cancer and para-carcinoma tissues.IMPDH2 protein presents high expression in kidney cancer tissues in general,obviously higher than that in para-carcinoma control tissues. (IRS: KDCa=4.67±1.07 vs compare=3.42±0.99 P=0.02)。IMPDH2 protein is between low and moderate expression and high expression in kidney cancer.And it has no obvious statistical meaning in the patients’ age,tumor stages and pathological grading.The positive rate of expression in bladder cancer is 92.31%(36/39),the expression rate is 100% in normal bladder tissues.But it is on mucosal surface of bladder,especially in the tectorial cell.INPDH2 protein generally represents high expression in bladder cancer tissues,obviously higher than normal control tissue. (IRS:BCLCa=4.33±1.88 vs compare=2.88±0.35 P=0.00)o IMPDH2 protein is between moderate expression and high expression in bladder cancer tissue,and it has no obvious statistical meaning in the patients’age,tumor stages and pathological grading.The positive rate of IMPDH2 protein in carcinima cancer and normal testicular tissues is 100%.IMPDH2 protein presents high expression in testicular cancer tissues in general,but it also presents high expression in normal testicular rissues.Both of them have no varience of statistic. (IRS:TECa=4.22±1.31 vs compare=4.25±0.46P=0.96)。 The IMPDH2 protein is between moderate expression and high expression in testicular cancer tissue.And it has no statistical meaning in the patients’ age,tumor stages and pathological grading.Conclusion1.Compared with the benign prostate tissues,IMPDH2’s expression obviously im proves in prostate cancer.Compared with paratumor cancer,IMPDH2’s expression obv iously improves in kidney cancer.Compared with the normal cells,IMPDH2’s expressi on obviously improves.Compared with the normal cells,There is little diversity about IMPDH2’s expression in testicular cancer2.There is no obvious difference between the pathological grading and clinical st ages in the patients of kidney cancer,bladder cancer and testicular cancer.In prostate c ancer,there is positive correlation between this protein’s expression and the patients’c linical stages,tranference or non-transference,and the grading of Gleason.3.IMPDH2 holds a certain value to the diagnosis and treatment of the tumors in u rinary system as the tumor marker,probably as diagnostic sign of kidney cancer,bladd er cancer and prostate cancer and the biological targeted goal.However,it has the highvalue as the tumor marker in prostate cancer. |
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