CN103571955A - Application of DUSP-9 (dual-specificity phosphatase-9) gene to detection of renal clear cell carcinoma - Google Patents
Application of DUSP-9 (dual-specificity phosphatase-9) gene to detection of renal clear cell carcinoma Download PDFInfo
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Abstract
The invention discloses an application of a DUSP-9 (dual-specificity phosphatase-9) gene to the detection of renal clear cell carcinoma, and belongs to the technical field of genetic engineering. The application comprises the following steps of comparing the mRNA (messenger ribonucleic acid) expression quantities of DUSP-9 genes in a renal tissue sample and normal renal tissues in an RT-PCR (reverse transcription-polymerase chain reaction) way, and determining that the sample is a renal clear cell carcinoma sample if the mRNA expression quantity in the sample is lower than the mRNA expression quantity in the normal renal tissues; or performing immunohistochemical measurement on the renal tissue sample, and determining that the DUSP-9 in the renal tissue sample is lowly expressed and that the sample is the renal clear cell carcinoma sample when a staining index of the renal tissue sample is less than or equal to 4. The application has the advantages that (1) experiments show that the DUSP-9 gene in a patient suffering from renal clear cell carcinoma is lowly expressed, and a new diagnostic index is provided for the diagnosis of the patient suffering from renal clear cell carcinoma; (2) the experiments show that the DUSP-9 gene can be used as an independent index for the judgment of DUSP-9 prognosis.
Description
Technical field
The invention belongs to gene engineering technology field, be specifically related to the application of DUSP-9 gene in detecting clear cell carcinoma of kidney.
Background technology
Clear cell carcinoma of kidney (ccRCC) is a kind of common urinary system malignant tumour that global range distributes.Although there is huge lifting in ccRCC treatment in recent years, sickness rate is still raising gradually, and 30% ccRCC patient shows as transfer at first, and the patient experience nephrectomy up to 40% still develops into local recurrence or transfer.Although what have been found that E&H factor and ccRCC is relevant, ccRCC occur and the concrete molecular mechanism of development still unclear.DUSP-9 (dual specificity phosphatase enzyme-9) belongs to dual specificity protein phosphatase subfamily.DUSP-9 is the negative regulator enzyme (as: ERK, JNK, p38) in mitogen-activated protein kinase family, and it is relevant with differentiation to the propagation of cell.Extensive parallel order-checking research is verified in ccRCC patient, and the expression of DUSP-9 is lowered.Yet, the correlation report of not publishing at present.Therefore, the expression level of DUSP-9 and the relation between clinical importance are still needed and are illustrated.Our this research is mainly expression and the clinical application thereof of wanting to probe into DUSP-9 in ccRCC patient.
Summary of the invention
The object of the invention is to disclose the application of DUSP-9 gene in detecting clear cell carcinoma of kidney.
The object of the invention is to be achieved through the following technical solutions:
The application of DUSP-9 gene in detecting clear cell carcinoma of kidney.
The application of DUSP-9 gene described in technique scheme in detecting clear cell carcinoma of kidney, wherein DUSP-9 gene low expression in clear cell carcinoma of kidney.
The application of DUSP-9 gene described in technique scheme in detecting clear cell carcinoma of kidney, comprises the steps:
(1), sample this nephridial tissue, the RNA of extraction nephridial tissue;
(2), the nephridial tissue RNA of sample is carried out to reverse transcription, obtain the cDNA of sample nephridial tissue;
(3), take the nephridial tissue cDNA that step (2) obtains is masterplate, take primer pair P as primer, carry out RT-PCR reaction, the mrna expression amount of the DUSP-9 gene in the mrna expression amount of the DUSP-9 gene in sample nephridial tissue and normal kidney tissue is contrasted, when in sample, the mrna expression amount of DUSP-9 gene is lower than the mrna expression amount of normal DUSP-9 gene, this sample is clear cell carcinoma of kidney sample; Wherein primer pair P is:
DUSP-9 coding strand: 5 '-TATGCCACGCCCTTTGAG-3 ',
DUSP-9 noncoding strand: 5 '-CACAGCAGGATGTAGGAGATGA-3 '.
The application of DUSP-9 gene described in technique scheme in detecting clear cell carcinoma of kidney, wherein, the condition of described RT-PCR is: 50 ° of C 2 minutes and 95 ° of C 2 minutes, 1 circulation; 15 seconds, 55 ° C of 95 ° of C 30 seconds and 72 ° of C 40 seconds, 40 circulations.
The application of DUSP-9 gene described in technique scheme in detecting clear cell carcinoma of kidney, wherein, described application is by nephridial tissue sample is carried out to immunohistochemical methods mensuration, when Di≤4 of nephridial tissue sample, the low expression of DUSP-9 in this nephridial tissue sample, this sample is clear cell carcinoma of kidney sample.
The present invention has following beneficial effect:
1, the present invention has disclosed the low expression in clear cell carcinoma of kidney patient of DUSP-9 gene by experiment, for clear cell carcinoma of kidney patient's diagnosis provides a kind of new diagnostic index;
2, the present invention by experiment (as the analysis P value < 0.005 of table 2) disclosed DUSP-9 gene and can be used as the independence index of judging DUSP-9 prognosis;
3, the present invention has disclosed the relation between DUSP-9 gene and clear cell carcinoma of kidney, in conventional diagnosis of renal carcinoma pathological replacement, the expression analysis of DUSP-9 can be added, as the index of judging patient's prognosis; In the process of section, tumor staining meeting is used to immunohistochemical methods process, and cell dyeing process simple and convenient simultaneously, draws Di in conjunction with positive after dye levels and dyeing (and cell) after dyeing cell proportion, ≤ 4 is prognosis mala, and >=5 is prognosis bona; The complexity of minimizing experiment that can be as much as possible, better judgement patient's histological type and prognosis.
Accompanying drawing explanation:
Fig. 1 is that the RT-PCR of the DUSP-9 genetic expression of 46 routine ccRCC tumor samples and adjacent normal kidney tissue sample analyzes;
Fig. 2 is the immunohistochemical analysis of 107 routine ccRCC specimens and adjacent healthy tissues;
Fig. 3 is the coloured differently situation of adjacent normal kidney tissue sample and ccRCC tumor tissues sample in the immunohistochemical analysis of DUSP-9 protein expression;
Fig. 4 be by Kaplan-Meier analyze and the expression of the resulting DUSP-9 of timing sequence test on the impact of surviving.
Embodiment
For making technical scheme of the present invention be convenient to understand, below in conjunction with concrete test example, to DUSP-9 gene of the present invention, the application in detecting clear cell carcinoma of kidney is further described.
The application of test example 1:DUSP-9 gene in detecting clear cell carcinoma of kidney:
One, case sample and reagent:
1, patient and tissue sample:
(1) 46 examples of, collecting year December in February, 2008 to 2009 complete radical nephrectomy and through ccRCC patient's's (each patient's disease stage is classified and classified according to tumor histology of Cancer center of Zhongshan University and american cancer joint committee in 2002) of proved by pathology cancerous tissue and contiguous healthy tissues sample, male 40 examples wherein, female's 6 examples, 50 years old mean age (scope: 37 – 75 years old), and analyze with RT – PCR.Nephridial tissue after excision is immersed in to RNAlater (test kit immediately; Germany) in, and place as early as possible-4 ℃ of refrigerator overnight, be finally stored in-80 degrees Celsius of cryogenic refrigerators standby.
(2), in addition, we to the embedding sample of 211 routine patient's ccRCCs cancerous tissues of collecting for 1999 to 2007 and wherein the sample of the adjacent healthy tissues of 107 examples (this 107 example is selected from 211 routine patients) carry out immunohistochemical experiment, 211 routine patients' feature is as shown in " clinical pathology variable " in table 1 and " n " hurdle.Data shows that these patients did not accept Radiotherapy chemotherapy in the preoperative.Cancer center of Zhongshan University carries out tumor histology and clinical diagnosis to these patients, and according to < < american cancer joint committee in 2002, the TNM of renal cell carcinoma classification > > classifies and classifies each patient's disease stage.This research obtains the approval of examination board of research institution of Zhongshan University, and all patients signed Informed Consent Form all.
Contacting between the expression of table 1 hyaline cell renal cell carcinoma patient DUSP-9 and clinical pathologic characteristic
2, main agents
(1), RNAlater (QIAGEN, Germany);
(2)、DNase I(RNase Free)；
(3), TRIzol reagent solution (Invitrogen; Carlsbad, CA);
(4), reversed transcriptive enzyme (M-MLV) (Fermentas; American);
(5)、SYBR Green master mix(Invitro-gen；Carlsbad，CA)；
(6), RT-PCR instrument (ABI 7000) Applied biosystems.
Subsequent operations of the present invention is all carried out according to the specification sheets of mentioned reagent and instrument.
Two, method:
(1), RT-PCR:
1, the extracting of sample RNA:
(1), the healthy tissues sample of patient's ccRCCs cancerous tissue sample and vicinity is put into RNAlater and carry out preservation;
(2), according to Trizol reagent (Invitrogen company, the U.S.) specification sheets, carry out following operation steps:
A, homogenized (Homogenization):
By 10-30mg tissue, add 1ml TRIzol, with electric homogenizer or the abundant homogenate of disposable grinding pestle, from homogenate, extract total RNA.
B, layering (Phase Separation): (a), sample adds after TRIzol, room temperature is placed 5min, makes the abundant cracking of sample.
Step: centrifugal 10 minutes of 4 ℃ of 12,000 rpm, gets supernatant.(b), every 1ml TRIzol adds 200 μ l chloroforms, thermal agitation mixes rear room temperature and places 3-5 min and make its natural phase-splitting.C, RNA precipitate (RNA Precipitation):
4 ℃ 12, the centrifugal 10-15min of 000rpm.Sample can be divided into three layers: yellow organic phase, and middle layer and colourless water, RNA mainly, in water, transfers to water (conventionally can draw 550 μ l) in new pipe;
(3), according to the DNA of total RNA in the step removal process (2) in the DNase I working instructions of RNase-free, pollute.
2, cDNA's is synthetic:
Use reversed transcriptive enzyme (M-MLV) (Fermentas; American)
(1), in Microtube pipe, prepare the template ribonucleic acid/primer mixed solution shown in table 2, full dose 6 μ l.
Table 2 template ribonucleic acid/primer mixed solution
| Reagent name | Usage quantity |
| RNA(extracts) | 1ng |
| Specific Primer（10 μM） | 1 μl |
| Sterile purified water | up to 6 μl |
(2), 70 ℃ of insulations after 10 minutes rapidly at chilling on ice more than 2 minutes.
(3), the centrifugal several seconds makes the denaturing soln of template ribonucleic acid/primer be gathered in Microtube pipe bottom.
(4), in above-mentioned Microtube pipe, prepare the inverse transcription reaction liquid shown in table 3.
Table 3 inverse transcription reaction liquid
| Reagent name | Usage quantity |
| Above-mentioned template ribonucleic acid/primer distortion solution | 6ul |
| 5×M-MLV Buffer | 2 μl |
| Each 10 mM of dNTP Mixture() | 0.5 μl |
| RNase Inhibitor（40 U/μl） | 0.25 μl |
| RTase M-MLV（RNase H-）（200 U/μl） | 1 μl |
| Sterile purified water | up to 10 μl |
(5), 42 ℃ are incubated 1 hour.
(6), 70 ℃ of insulations cooled on ice after 15 minutes, the cDNA solution obtaining can be directly used in the synthetic or PCR amplification of 2nd-Strand cDNA etc., during PCR amplification, the usage quantity of cDNA solution is 1 μ l～5 μ l.
3, via the synthetic mRNA step of RT-PCR method:
RT-PCR reaction composition (totally 20 μ l), as shown in table 4:
Table 4 RT-PCR reaction system
Note: reaction solution can at room temperature configure, and all reagent please don't be placed on ice.
RT-PCR reaction conditions: 50 ° of C (2 minutes) and 95 ° of C (2 minutes), 1 circulation; 95 ° of C (15 seconds), 55 ° of C (30 seconds) and 72 ° of C (40 seconds), 40 circulations.
Above-mentioned steps all operates according to the specification sheets of reality and instrument, uses TRIzol reagent solution (Invitrogen in experimental implementation process; Carlsbad, CA) extract whole RNA, use DNase I (RNase Free) to remove DNA and pollute; Press reversed transcriptive enzyme (M-MLV) (Fermentas; American) transcribing the total RNA of system 1ng, to carry out reverse transcription be cDNA.CDNA accepts relative mRNA level and the GAPDH (as an internal control) and corresponding primer pairing that real-time quantitative PCR is evaluated DUSP-9
DUSP-9 coding strand: 5 '-TATGCCACGCCCTTTGAG-3 ',
DUSP-9 noncoding strand: 5 '-CACAGCAGGATGTAGGA-GATGA-3 ';
GAPDH coding strand: 5 '-GCTCTCTGCTCCTCCTGTTC-3 ',
GAPDH noncoding strand: 5 '-GACTCCGACCTTCACCTTCC-3 '.
Fluorescent quantitation amplification (RT-PCR) is by the rt-PCR instrument operation of Applied biosystems (ABI 7000).The reaction system of RT-PCR is totally 20 μ l, comprising the cDNA (step 2cDNA is synthetic) of 1 μ l, the every a pair of Oligonucleolide primers of 10 μ l SYBR Green master mix and 40nm; Reaction conditions is: 50 ° of C 2 minutes and 95 ° of C 2 minutes, 1 circulation; 15 seconds, 55 ° C of 95 ° of C 30 seconds and 72 ° of C 40 seconds, 40 circulations.Calculate the regression curve of each sample, and according to SPSS software (Version 17.0 SPSS Inc.), calculate the cycle threshold of relative populations mRNA.Relative expression's level of target gene is standardized as the geometric mean of reference gene GAPDH.By the data analysis of compare threshold cycle (2-Δ CT) to us.
(2), immunohistochemical methods is measured:
Operation steps
(1), paraffin section is made:
1, fixing: to get tissue, with PBS, rinse, put into fixedly 12h of 4% paraformaldehyde phosphate buffered saline buffer.
2, dehydration: remove stationary liquid, use distilled water flushing 3 times, then rinse 2 times with 50% alcohol, with alcohol, dewater step by step, 70% alcohol 1 day, 80% alcohol spends the night, 95% alcohol 3h, raw spirit (I), (II) each 2h.
3, transparent: 1:1 raw spirit dimethylbenzene 45min, dimethylbenzene (I), (II) each 30min.
4, embedding: (carrying out in thermostat container) immerses paraffin, 1:1 dimethylbenzene paraffin (58 ℃) 45min, paraffin (I), (II), (III) be 2.5h altogether, with paraffin (III) investing tissue.
5, section: the tissue block after embedding through finishing after, with slicing machine, be cut into 5-7 μ m, paraffin band.
6, paster: will organize paraffin mass to open up sheet in 50 ℃ of warm water, and then can stick on slide glass with processing clean slide glass Lao Pian， taeniae telarum.
(develop a film: dry → gluing after 1%HCl soaked overnight, distilled water flushing, 95% alcohol-pickled 2h: anticreep agent poly-lysine)
7, roasting sheet: roasting sheet 2h in 68 ℃ of thermostat containers.
(2), SP three-step approach:
1, dewaxing: before dewaxing, organization chip should be placed to 60min at room temperature, or toast 20min in 60 ℃ of thermostat containers, rear with dimethylbenzene (I), (II) immersion, altogether 25min.
2, aquation: raw spirit (I), (II) each 2min, come downwards to 95%, 80%, 70% each 2min of alcohol (I) (II).
3, PBS rinses 2-3 time, each 5min.
4, blocking-up: 3%H
2o
2deionized water (or 80% methyl alcohol) is hatched 10min, active with deactivating endogenous peroxydase.
5, PBS rinses 2-3 time, each 5min.
6, antigen retrieval: set to 0 in .01M citrate buffer (PH 6.0) with boiling (95 ℃, 15-20min), more than naturally cooling 20min, then use cold water flush mug, quickening is cooled to room temperature.
7, PBS rinses 2-3 time, each 5min.
8, sealing: drip normal goats serum confining liquid, incubated at room 20min, gets rid of unnecessary liquid.
9, drip the anti-50 μ l of I, the standing 1h of room temperature, or 37 ℃ of 1h, or 4 ℃ spend the night (need at 37 ℃ of rewarming 45min).
10, PBS rinses 2-3 time, each 5min.
11, drip anti-40～50 μ l of II of horseradish peroxidase-labeled, the standing 1h of room temperature, or 37 ℃ of 1h.
12, PBS rinses 2-3 time, each 5min
13, drip SP (Streptavidin-peroxidase), room temperature or 37 ℃ are hatched 30min-1h.
14, PBS rinses 2-3 time, each 5min.
15, colour developing: the DAB 5-10min that develops the color, grasps dye levels (endochylema is brown person and is judged to be positive cell) under the microscope; The configuration of developer: add 1 developer A (DAB) in 1ml water, shake up, then add 1 developer B (H
2o
2), shake up, then add 1 developer C (phosphoric acid buffer), shake up;
16, tap water rinses 10 minutes termination reactions.
17, redye: haematoxylin redyeing 2min, hydrochloride alcohol differentiation.
18, tap water rinses 10-15min.
19, conventional dehydration, transparent, mounting (drop in and organize side with neutral gum, then cover with cover glass), microscopy.
(3), interpretation of result:
5 high power lens visuals field (400X) are selected in every section, and applies image analysis system carries out analyzing after quantitative gray scale scanning.
Method by immunohistochemical methods to 211 routine patient's ccRCC cancerous tissue samples and wherein the DUSP-9 of the sample of 107 routine patients' ccRCC adjacent healthy tissues express and measure.In brief, paraffin-embedded sample being cut into 5 μ m puts at 65 ℃ roasting 30 minutes.By dimethylbenzene and aquation row dewaxing treatment, bury in 0.01 M citrate buffer agent (pH value 6.0) antigen recovery buffer zone, then put into microwave oven and carry out antigen recovery.In methyl alcohol, accept 3% hydrogen peroxide and eliminate endogenous peroxidase activity, by 10% bovine serum albumin non-specific binding, hatch subsequently.DUSP-9 (Abcam; Cambrigde, MA, USA) be that monoclonal antibody by mouse detects.Sample and anti-DUSP-9 antibody (1:250) is hatching all night under 4 ° of C.Negative control group is by replacing and obtain with main antibody and antibody diluent.After phosphate-buffered salt PBS washed, put it in (MaxVisionHRP-Polymer anti-Mouse IHC Kit) test kit and place 15-20 minute at 37 ℃.Make tissue infiltration at the amino 9-ethyl carbazole of 3-, use phenodin counterstain, dehydration, is finally fixed in Crystal Mount.
By two, independently observer is to fixing with formalin, and the dye levels of the cancerous tissue of paraffin embedding part is looked back and gives a mark.The ratio of these cell expressings DUSP-9 does not change from 0% to 100% not etc., and staining power also from weak to strong.It is as follows that tumour cell DUSP-9 expresses ratio score: 0 for not having positive cell, <5% Di is that 1,6% ~ 25% Di is that 2,26% ~ 50% Di is 3,51% ~ 75% Di is that 4, >, 75% Di is 5; The ratio from 0% to 100% that percentage ratio is wherein cell expressing DUAP-9 in section is not etc.According to average optical to tinctorial strength classification (referring to Tsuchiya A, Sakamoto M, Yasuda J, Chuma M, Ohta T, Ohki M, Yasugi T, Taketani Y, Hirohashi S:Expression profiling in ovarian clear cell carcinoma:identification of hepatocyte nuclear factor-1 beta as a molecular marker and a possible molecular target for therapy of ovarian clear cell carcinoma. Am J Pathol 2003, 163:2503-2512, Saussez S, Cucu DR, Decaestecker C, Chevalier D, Kaltner H, Andr é S, Wacreniez A, Toubeau G, Camby I, Gabius HJ, Kiss R:Galectin 7 (p53-induced gene 1): a new prognostic predictor of recurrence and survival in stage IV hypopharyngeal cancer. Ann Surg Oncol 2006, 13:999-1009. and Bao S, Ouyang G, Bai X, Huang Z, Ma C, Liu M, Shao R, Anderson RM, Rich JN, Wang XF:Periostin potently promotes metastatic growth of colon cancer by augmenting cell survival via the Akt/PKB pathway.Cancer Cell 2004, 5:329-339.): 0, not dyeing, 1, weak dyeing (faint yellow), 2, appropriateness dyeing (yellowish brown), 3, strong dyeing (brown).Utilize the ratio of staining power mark and DUSP-9 positive tumor cell to multiply each other and obtain Di.We according to Di value assess DUSP-9 optimum and malignant change renal tissue expression, score value is 0,1,2,3,4,5,6,8,9,10,12 and 15.It is by timing sequence test that DUSP-9 expresses obtaining of cutoff value, on the basis in overall survival rate, by measurement variability, obtains.Determining of best cutoff value: Di >=5 are considered to high DUSP-9 expresses, and Di≤4 are considered to the low expression of low DUSP-9.
Three, statistical study:
Statistical study all realizes based on SPSS17.0 software.We check to analyze the importance of difference between ccRCC specimens mRNA and protein expression and adjacent healthy tissues according to the t of rt – PCR, immunohistochemical analysis, matched samples.By the chi square test of comparative example, analyze the expression of DUSP-9 and the relation of clinical importance.By Kaplan-Meier method, paint to obtain survival curve, and compare with timing sequence test.We think that Proportional hazards all can exist in all Cox regression models.By Cox proportional hazard model, the meaning of various existence variablees is carried out to multiplexed analyses, it is meaningful that p<0.05 is considered to be in statistics.
Four, conclusion:
1, real-time quantitative RT-PCR is analyzed the expression of DUSP-9:
The transcriptional level of DUSP-9 is to be detected and determined by the quantitative rt of the pairing of 46 routine ccRCC tumor samples and adjacent normal kidney tissue sample-PCR; Result as shown in Figure 1, the relative expression's level that has 45 tumor samples (T) DUSP-9 mRNA well below pairing adjacent normal kidney tissue sample (N) (N=46, P<0.001); Wherein Fig. 1 is the real-time quantitative RT-PCR analysis (p<0.001, paired sample t check) that DUSP-9 expresses, and in Fig. 1, T is ccRCC specimens sample, and N is adjacent normal kidney tissue sample; The bottom of the rectangular box in N is 25%, and top is 75%, and middle line of delimitation is 50%; Two ends T shape lines represent the 2.5th hundredths and the 97.5th hundredths.
2, paraffin-embedded 107 groups of ccRCC samples (T) and the immunohistochemical analysis expressed with the protein level of the DUSP-9 of the adjacent normal kidney tissue (N) of its pairing:
By the immunohistochemical analysis of carrying out to paraffin-embedded 107 routine ccRCC specimens and adjacent healthy tissues, thereby determine protein expression amount and Subcellular Localization, result as shown in Figure 2, in 102 routine tumour patient tissue samples the expression level of DUSP-9 albumen to be starkly lower than adjacent normal kidney tissue sample (N) (N=102, P<0.001); The expression that wherein Fig. 2 is DUSP-9 is through the result of immunohistochemical analysis, and in Fig. 2, T is ccRCC sample, and N is adjacent normal kidney tissue sample; In ccRCC, DUSP-9 albumen is in low expression as shown in Figure 2, and in tumor tissues, the expression of DUSP-9 albumen will be lower than the adjacent healthy tissues (n=107, p<0.001) of pairing.The bottom of box and top represent minimum quartile and the highest quartile, and what approach the middle band representative of box is median, and the T shape band at two ends represents respectively 2.5% and 97.5%.
In normal renal tissue, special DUSP-9 is positioned in the cytoplasmic yellowish brown particle of nephrocyte (as shown in Figure 3A).In 102 routine tumour patient tissue samples, the expression level of DUSP-9 protein expression will be lower than contiguous healthy tissues sample (p < 0.001, the t experiment of paired sample, Fig. 3 B).Fig. 3 is the immunohistochemical analysis of DUSP-9 protein expression, in figure A, healthy tissues is sample as a comparison, figure B, figure C and D refer to tumor tissues, DUSP-9 albumen is after through dyeing, according to difference, express degree and show dyeing in various degree, this dyed color is darker, illustrates that the expression of DUSP-9 is higher, comparatively speaking, grade malignancy is lower; DUSP-9 derives from nucleus and tenuigenin; Wherein 3A figure is the histochemical stain of adjacent normal kidney tissue sample, and 3B is ccRCC tumor tissues sample, and 3A and 3B show a negative staining infiltration tumor region that forms sharp contrast; Wherein Fig. 3 A is the histochemical stain (400 *) of healthy tissues sample; 3B is passive or weakens DUSP-9 in the dyeing (400 *) of cancerous tissue; 3C is that DUSP-9 dyeing is in the moderate dyeing of cancerous tissue (400 *); 3D is DUSP-9 high strength dyeing (400 *) in most of tumour cells.
3, contacting of 211 routine patients' ccRCC DUSP-9 expression and Clinical symptoms:
For further research DUSP-9 is on ccRCCs patient's impact and prognosis, we use immunohistochemical analysis to assess the expression of DUSP-9 in 211 routine ccRCCs specimens pieces.In 211 routine tumor samples, 117 examples demonstrate the low expression of DUSP-9 (Di≤4), yet 94 routine samples show DUSP-9 high expression level (Di >=5).
The expression of DUSP-9 and the contact of various clinicopathologic features are listed in table 1.Expression degree and the sex (p=0.031) of DUSP-9 in ccRCC sample, pathological staging (p=0.001), Fuhrman classification (p=0.002), tumor size (p=0.001), T is (p=0.001) by stages, and N classifies (p=0.012), shifts (p=0.005), recurrence (p<0.001) is relevant, but there is no great dependency with the age.
As shown in table 1, the low expression of DUSP-9 is respectively T1-46.7%, T2-60.0%, T3/4-80.5% (P=0.001, chi square test) with the corresponding relation of T stage (T by stages).In the ccRCCs patient of tumor size≤7cm, have the 47.1% low expression of not observing DUSP-9, corresponding in the ccRCCs patient of tumor size > 7cm ratio be 70.7% (p=0.001, chi square test).In N stage, in N0, the probability of the low expression of DUSP-9 is that the probability that 52.2%, N+ occurs is 77.8% (p=0.012, chi square test).In having the group of transfer, the low expression probability of DUSP-9 is 82.6% simultaneously, and is 48.6% without the probability shifting.The low expression probability of finding DUSP-9 in recurrence group is 96.6%, and in not recurrence group, has accounted for 48.6% (p<0.001, chi square test).
4, survival analysis:
The expression that we utilize Kaplan-Meier to analyze and timing sequence test is assessed DUSP-9 is on the impact of surviving.5 years survival rates of DUSP-9 high expression level group patient are 97%, but are 62.1% (Fig. 4 A) in low expression group.Timing sequence test shows, these two groups of survivals significantly different (p <0.001).For judgement DUSP-9 expresses the relation with prognosis, we according to pathological staging by 211 routine patients be divided into three subgroups (differentiation of this subgroup according to be kidney case by stages: T1: tumour maximum diameter is less than or equal to 2.5cm, is confined in kidney peplos; T2: tumour maximum diameter is greater than 2 5cm, kidney distortion, tumour is still positioned at coating; T3a: tumors invading and perirenal fat, T3b: tumors invading and vein; T4: tumour has been invaded adjacent organs; T1, T2, T3 and T4 are respectively with respect to I, II, III and IV phase), no matter in I, II group (n=151; Log-rank, p=0.023; Fig. 4 b), III group (n=31; Logrank, p=0.036; Fig. 4 C) or IV group (n=29; Log-rank, p=0.038; Fig. 4 D), the group of all DUSP-9 high expression levels, its survival time is significant prolongation all.Single argument Cox regression analysis shows, tumor size, T are by stages, N classification, transfer, Fuhrman classification, DUSP-9 expresses and Overall survival significant correlation (table 5) all.In addition, polynary Cox regression analysis prompting, for ccRCC patient, only have the expression of DUSP-9 and the independent prognostic factor (p=0.005, table 5) that Fuhrman classification is only its overall survival of judgement, and other factors not independent relevant to ccRCC survival.
Relation between the survival rate that table 5 Cox regression analysis shows and staging
5, discuss:
Clear cell carcinoma of kidney accounts for 2% of all cases of cancers, and in the whole world, is maintaining 1.5～5.9% average annual growth.Initial treatment is radiotherapy or kidney Partial Resection normally, and this is still current topmost therapeutic modality.Unfortunately, ccRCC patient starts radiation and chemotherapy tolerance, and meanwhile, molecular targeted therapy plays good result for the treatment of to ccRCC patient.Therefore, current crucial prerequisite is exactly the biomarker that can filter out the specific molecular of ccRCC.Although there is numerous molecular marked compounds, as p53, vascular endothelial growth factor (VEGF), hypoxia inducible factor, Ki67 (diffusion), as the influence factor of research ccRCCs patient prognosis, but current can not making a return journey from molecular mechanism aspect explained developing of ccRCC.From the extensive parallel sequencing analysis carrying out, show, in ccRCCs patient, DUSP-9 expresses and is suppressed.DUSP-9 is the member of dual specificity protein enzyme family, belongs to Phosphoric acid esterase subfamily, only expresses in placenta, kidney and fetus period.In addition, DUSP-9 and squamous cell carcinoma (SCC) are relevant, and can induce independently the generation of squamous cell carcinoma.DUSP-9, by the phosphoric acid of dephosphorylate serine/threonine and Tyrosine O-phosphate residue, loses activity the target kinase of squamous epithelium cancer cell.The increment of DUSP-9 and cell and break up a kind of negative regulator enzyme (p38 MAPK SAPK) of relevant ，Shi map kinase family.The people such as Molly Kulesz-Martin find that DUSP-9 reconstruct meeting causes the death of G2-M relevant cell and microtubule to interrupt.The loss of DUSP-9 can be SCCs by the innocent tumour induction of mouse epithelial.The DUSP-9 of malignant cell expresses reconstruct, can inducing cell death, form tumor suppression.Yet, as far as we know, the key features of this research has been the on the one hand first report clinical meaning that DUSP-9 expresses in ccRCCs patient.On the other hand, this is also to study to be first intended to assess DUSP-9 as the clinical indices of clinical potential disease progress and as the possibility of the prognostic evaluation index of tumour patient existence.
In this research, we find that DUSP-9 mRNA compares and has significant difference with contiguous normal kidney tissue sample with protein expression in patient ccRCCs.In addition, immunohistochemical analysis shows, DUSP-9 is that moderate is expressed to low in ccRCCs patient, but in contiguous healthy tissues high expression level.Accordingly, the patient's sample that we have studied a large amount of clinical ccRCC finds that the level of DUSP-9 reduces.The expression of DUSP-9 reduces to by stages relevant with prognosis with sex, pathological staging, Fuhrman classification, tumor size, recurrence, TNM.The survival of patients time of the low expression of DUSP-9 is relatively shorter, and the patient's of DUSP-9 high expression level survival time is relatively longer.In addition, we are divided into 3 groups according to pathological staging by patient, obtain the relation of expression and the prognosis of DUSP-9, and we find is all a valuable prognostic indicator at the ill all stage D USP-9 of ccRCC patient.Consistent with the report of previous other cancers, the low expression indication ccRCC patient prognosis mala of DUSP-9.In addition, DUSP-9 expresses relevant to low Fuhrman classification, and this result is not mated with other statisticses.Yet we find in survival analysis, no matter be in Fuhrman classification I level, II level, III level or IV level, the high DUSP-9 of tumour patient expresses and compares low DUSP-9 to express obvious Overall survival longer.We observe, and have the Fuhrman classification of more case all very low.In addition, this is one and independent take hospital as basic retrospective study.Except this is observed, we are also special finds that DUSP-9 expression is relevant to low Fuhrman classification.
The TNM of ccRCC is by stages closely related with Fuhrman classification and its prognosis.In our research, the result of single argument Cox regression analysis show tumor size, T by stages, N classification, shift the expression of Fuhrman classification, DUSP-9 and Overall survival significant correlation.In addition, multivariate Cox regression analysis demonstration, only has DUSP-9 to express and Fuhrman classification is the independently prognostic factor of the overall survival rate of ccRCC patient.Our result shows, DUSP-9 expression level and clinical pathological characteristic are closely related, and can be used as a potential index of ccRCCs patient evaluation prognosis.
Our research is one and simple take hospital as basic retrospective study.It should be pointed out that the experimental result that may have uncertain error and have influence on us.We are necessary extensively to sum up some potential Confounding Factor, and doing a polycentric Huo Yi community is basic perspective study.In addition, the dependency of DUSP-9 and the above-mentioned molecular marked compound of mentioning need to be done further investigation.Obviously, we need to do more research, probe into the expression of DUSP-9 in ccRCC and other gene as the relation between p38.
The present invention, by a large amount of clinical samples, has proved that the low expression of DUSP-9 in ccRCC and poor prognosis thereof are relevant.Result shows, in primary ccRCCs patient, DUSP-9 is playing the part of the role of an evaluate its prognosis and potential inhibition tumour.We can assess primary ccRCCs patient's prognosis according to the expression of results of DUSP-9.In future, DUSP-9 may become treatment ccRCCs patient's a new potential target spot.
The above, it is only preferred embodiment of the present invention, not the present invention is done to any formal and substantial restriction, all those skilled in the art, do not departing within the scope of technical solution of the present invention, when utilizing disclosed above technology contents, and the equivalent variations of a little change of making, modification and differentiation is equivalent embodiment of the present invention; Meanwhile, the change of any equivalent variations that all foundations essence technology of the present invention is done above embodiment, modification and differentiation, all still belong in the scope of technical scheme of the present invention.
SEQUENCE LISTING
<110> Shenzhen City Second People's Hospital
The application of <120> DUSP-9 gene in detecting clear cell carcinoma of kidney
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Claims (5)
- The application of 1.DUSP-9 gene in detecting clear cell carcinoma of kidney.
- 2. the application of DUSP-9 gene according to claim 1 in detecting clear cell carcinoma of kidney, is characterized in that: DUSP-9 gene is low expression in clear cell carcinoma of kidney.
- 3. the application of DUSP-9 gene according to claim 1 and 2 in detecting clear cell carcinoma of kidney, comprises the steps:(1), sample this nephridial tissue, the RNA of extraction nephridial tissue;(2), the nephridial tissue RNA of sample is carried out to reverse transcription, obtain the cDNA of sample nephridial tissue;(3), take the nephridial tissue cDNA that step (2) obtains is masterplate, take primer pair P as primer, carry out RT-PCR reaction, the mrna expression amount of the DUSP-9 gene in the mrna expression amount of the DUSP-9 gene in sample nephridial tissue and normal kidney tissue is contrasted, when in sample, the mrna expression amount of DUSP-9 gene is lower than the mrna expression amount of normal DUSP-9 gene, this sample is clear cell carcinoma of kidney sample; Wherein primer pair P is:DUSP-9 coding strand: 5 '-TATGCCACGCCCTTTGAG-3 ',DUSP-9 noncoding strand: 5 '-CACAGCAGGATGTAGGAGATGA-3 '.
- 4. the application of DUSP-9 gene according to claim 3 in detecting clear cell carcinoma of kidney, is characterized in that, the condition of described RT-PCR is: 50 ° of C 2 minutes and 95 ° of C 2 minutes, 1 circulation; 15 seconds, 55 ° C of 95 ° of C 30 seconds and 72 ° of C 40 seconds, 40 circulations.
- 5. the application of DUSP-9 gene according to claim 1 and 2 in detecting clear cell carcinoma of kidney, it is characterized in that: described application is by nephridial tissue sample is carried out to immunohistochemical methods mensuration, when Di≤4 of nephridial tissue sample, the low expression of DUSP-9 in this nephridial tissue sample, this sample is clear cell carcinoma of kidney sample.
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| CN105148259A (en) * | 2015-09-29 | 2015-12-16 | 武汉大学 | Function and application of dual-specificity phosphatase 12(DUSP12) in treating cardiac hypertrophy |
| CN109022585A (en) * | 2018-09-10 | 2018-12-18 | 路君 | PBLD gene is preparing the application in clear cell carcinoma of kidney diagnosis and prediction prognosis drug |
| CN109055562A (en) * | 2018-10-29 | 2018-12-21 | 深圳市颐康生物科技有限公司 | A kind of biomarker, predict clear-cell carcinoma recurrence and mortality risk method |
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| WO2009062199A1 (en) * | 2007-11-09 | 2009-05-14 | Fox Chase Cancer Center | EGFR/NEDD9/TGF-β LNTERACTOME AND METHODS OF USE THEREOF FOR THE IDENTIFICATION OF AGENTS HAVING EFFICACY IN THE TREATMENT OF HYPERPROLIFERATIVE DISORDERS |
| WO2013036282A2 (en) * | 2011-09-07 | 2013-03-14 | The Trustees Of Columbia University In The City Of New York | Downregulation of inflammatory micrornas by ilt3 |
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| SONG WU ET AL.: "Decreased expression of dual-specificity phosphatase 9 is associated with poor prognosis in clear cell renal cell carcinoma", 《BMC CANCER》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105148259A (en) * | 2015-09-29 | 2015-12-16 | 武汉大学 | Function and application of dual-specificity phosphatase 12(DUSP12) in treating cardiac hypertrophy |
| CN105148259B (en) * | 2015-09-29 | 2018-12-14 | 武汉大学 | Dual specificity phosphatase enzyme 12(DUSP12) treating function and application in myocardial hypertrophy |
| CN109022585A (en) * | 2018-09-10 | 2018-12-18 | 路君 | PBLD gene is preparing the application in clear cell carcinoma of kidney diagnosis and prediction prognosis drug |
| CN109055562A (en) * | 2018-10-29 | 2018-12-21 | 深圳市颐康生物科技有限公司 | A kind of biomarker, predict clear-cell carcinoma recurrence and mortality risk method |
| CN109055562B (en) * | 2018-10-29 | 2022-12-20 | 深圳市颐康生物科技有限公司 | Biomarker and method for predicting recurrence and death risk of renal cell carcinoma |
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