CN108676884A

CN108676884A - Tumor marker for predicting bladder cancer

Info

Publication number: CN108676884A
Application number: CN201810565627.XA
Authority: CN
Inventors: 张淑芳; 文小红; 刘熹; 王顺兰; 黄邓高
Original assignee: Haikou Peoples Hospital
Current assignee: Haikou Peoples Hospital
Priority date: 2018-06-04
Filing date: 2018-06-04
Publication date: 2018-10-19

Abstract

The invention discloses a tumor marker for predicting bladder cancer, which is DDX46, wherein a gene target sequence of a DDX46 gene amplification primer is 5'-GATTGTGATTGAAGAAGAA-3', DDX46, a gene upstream primer sequence is 5'-AAAATGGCGAGAAGAGCAACG-3', DDX46, and a gene downstream primer sequence is 5'-CATCATCGTCCTCTAAACTCCAC-3'. The invention applies high-throughput sequencing technology to detect bladder cancer tissues and normal tissues of 8 patients with early preliminary diagnosis of bladder cancer, finds that the DDX46 gene is highly expressed in the bladder cancer tissues, further verifies the DDX46mRNA expression in the bladder cancer tissues and normal tissues of 9 patients by an RT-PCR method, and verifies the DDX46 protein expression in paraffin sections of 56 bladder cancer and 10 normal tissues by an IHC method.

Description

A kind of tumor markers for predicting carcinoma of urinary bladder

Technical field

The invention belongs to gene engineering technology field, it is related to a kind of gene and its amplimer more particularly to a kind of DDX46 Gene magnification primer and its as the application in carcinoma of urinary bladder molecular marker.

Background technology

Carcinoma of urinary bladder occupies the first place of Chinese urological cancer, in west as one of most common malignant tumour of urinary system It is only second to prostate cancer, and the trend risen year by year with rejuvenation is presented in the morbidity of carcinoma of urinary bladder.It is investigated according to Cancer in China in 2015 Data are shown, in most common ten kinds of tumours of male, the incidence of carcinoma of urinary bladder has the tendency that rising during 2000~2011 years, But the death rate keeps relative stability.For carcinoma of urinary bladder in initial diagnosis, about 70%~75% is non-muscle invasive bladder cancer, however Local relapse is up to 70%, wherein the about 20% non-Myometrial involvement carcinoma of urinary bladder of first visit can progress to Myometrial involvement bladder Cancer has the characteristics that easily to recur.Since the carcinoma of urinary bladder cause of disease is complicated, occurrence and development are multifactor a, stage constructions, multi-path Complex process, pathogenesis is simultaneously indefinite, clinically urgently finds molecular marker and the targeting that can early diagnose, recur detection The action target spot for the treatment of.

DDX46 genes belong to DEAD-box family members, are located on chromosome 5q31.1.Some researches show that DDX46 to lead to Overregulate mRNA precursor variable sheer influence digestive organs and brain development, also with the close phase of the differentiation and development of hematopoietic cell It closes.There is scholar to point out recently, DDX46 gene high expressions and the occurrence and development of esophageal squamous cell carcinoma, colon cancer and osteosarcoma are close Cut phase is closed.But effect of the DDX46 genes in carcinoma of urinary bladder is unclear, and there are no document report.

Invention content

The object of the present invention is to provide a kind of tumor markers for predicting carcinoma of urinary bladder, by finding DDX46 genes Target sequence, design DDX46 genes primer sequence, construct interference DDX46 gene expressions slow virus carrier, by striking DDX46 gene expressions find to inhibit proliferation, invasion and the migration of transitional cell bladder carcinoma cell line in low bladder cancer cell lines (5637, T24), Inducing cell apoptosis；Zoopery illustrates the ability that DDX46 genes inhibit urinary bladder carcinoma T24 cell line tumor formation.

To achieve the goals above, the technical scheme is that：A kind of tumor-marker for predicting carcinoma of urinary bladder is provided Object, the tumor markers are DDX46, and the gene target sequence of DDX46 gene magnification primers is 5 '- GATTGTGATTGAAGAAGAA-3 ', DDX46 upstream region of gene primer sequence are 5 '-AAAATGGCGAGAAGAGCAACG-3 ', DDX46 downstream of gene primer sequences are 5 '-CATCATCGTCCTCTAAACTCCAC-3 '.

Application of the DDX46 gene magnifications primer in identifying carcinoma of urinary bladder.

The Bladder Cancer and normal structure of 8 early stage first visit bladder cancer patients of present invention application high throughput sequencing technologies pair It is detected, as a result, it has been found that the high expression in Bladder Cancer of DDX46 genes, further passes through the method validation of RT-PCR 9 DDX46mRNA is expressed in the Bladder Cancer and normal structure of example patient, and 56 carcinomas of urinary bladder of the method validation of IHC and 10 are just The expression of DDX46 albumen in normal tissue paraffin section de.

Description of the drawings

Fig. 1 is that slow virus strikes low carcinoma of urinary bladder T24 and 5637 cell DDX46 gene expressions；

Fig. 2 is that Celigo cell counts detect cell Proliferation figure；

Fig. 3 is that MTT experiment detects cell viability figure；

Fig. 4 is that colony formation detects cell Proliferation figure；

Fig. 5 is Transwell Matrigels and scratch experiment figure；

Fig. 6 is Apoptosis by Flow Cytometry figure.

Fig. 7 is tumor formation in nude mice animal and knurl figure.

Specific implementation mode

The present invention is used to predict the tumor markers DDX46 effect genes proliferation of human bladder cancer cells of carcinoma of urinary bladder, invades and move It moves, it is apoptosis-induced to be as follows：

1, the slow virus carrier of structure interference DDX46 gene expressions, strikes low carcinoma of urinary bladder T24 and 5637 cell DDX46 genes Expression.

It is prepared by 1.1DDX46 gene RNAi slow virus

1) DDX46 genes number is NM_014829, and DDX46 genes is used to lead to as template according to the design principle of RNAi It crosses software evaluation and has selected the target sequences of DDX46 genes and be：5’-GATTGTGATTGAAGAAGAA-3’.Its control sequence For generally acknowledged negative sequence：5’-TTCTCCGAACGTGTCACGT-3’；

2) the suitable restriction enzyme sites of two sections of DDX46 gene shRNA interference sequences addition (such as：The enzyme of AgeI enzymes The restriction enzyme site AATTC and site complementary series G of enzyme site CCGG, EcoRI enzyme), and in 5 ' end addition TTTTT of antisense strand Complementary series, and the end of positive-sense strand 3 ' addition termination signal TTTTT, are prepared into single-stranded DNA oligo segments, then carry out double-strand The synthesis of DNA Oligo.

3) it applies AgeI enzymes and EcoRI enzymes to carry out double digestion to pGC-LV carriers, makes its linearisation；

4) use T4DNA ligases by linear pGC-LV carriers and the DDX46 gene shRNA double-strands of above-mentioned preparation same It is attached in reaction system, is placed in 16 DEG C of reactions overnight；

5) by above-mentioned connection product and the competent escherichia coli cell prepared, while the LB of a 500 μ L is transferred to Antibiotic in fluid nutrient medium and is not added, with the speed shake culture 1h of 200rpm under the conditions of 37 DEG C；It takes containing ammonia benzyl mould The LB solid mediums of element uniformly smear 150 μ L of above-mentioned bacterium solution, and culture medium is placed on 37 DEG C, contain 5%CO₂Incubator in cultivate Overnight.Screening positive bacteria drops into the analysis of row PCR amplification, and the plasmid for finally extracting quality inspection qualification carries out subsequent experimental.

The packaging of 1.2 slow virus

1) by the DDX46 gene recombination plasmids comprising 20 μ g, the pHelper1.0 plasmids of 15 μ g and 10 μ g The mixed solution cotransfection 293T cells of pHelper2.0 plasmids discard all liq in culture dish after 6h, are washed 3 times with PBS, Ensure that remaining transfection mixture is washed away；

2) 293T cells are collected, and the culture solution containing 10% fetal calf serum is added and continues to cultivate 48h, collect cell supernatant Slow virus is obtained, is concentrated by centrifugal purification and obtains high-purity slow virus；

3) slow virus titre detects.

1.3DDX46 genes interfere slow-virus transfection transitional cell bladder carcinoma cell line T24 and 5637

1) T24 and 5637 cells of the exponential phase digested through 0.25% pancreatin are resuspended with complete medium, makes cell A concentration of 3-5 × 104 of suspension/mL are inoculated in 6 orifice plates and continue to cultivate；

2) to plating cells amount up to 15-30% or so appropriate virus is added according to actual conditions, infection carcinoma of urinary bladder is thin in culture Born of the same parents are that the number ratio of virus and cell is indicated with infection multiplicity (multiplicity of infection, MOI) when infection： Urinary bladder carcinoma T24 cell line and the MOI of 5637 cell virus infection are 10, and disease is added in experimental group LV-DDX46-RNAi (3640-1) The amount of poison is 6.67 μ L, and virus titer is 3 × 108TU/mL.The amount that virus is added in negative control group CON053 is 4 μ L, disease Malicious titre is 5 × 108TU/mL.

3) liquid in culture bottle is discarded after 16h after addition virus, conventional medium is added and continues to cultivate；

4) monitoring cell state is paid attention to during cultivating, ensures that experimental group and cellular control unit are in good condition, is not occurred thin Born of the same parents' mortality phenomenon.After replacing conventional medium 56h, culture medium is placed under fluorescence microscope, observes green fluorescence egg The expression of (green fluorescence protein, GFP) in vain, it is that positive cells ratio is felt to calculate luciferase expression Contaminate fluorescence rate；Continue subsequent experimental if infection fluorescence rate reaches 70% or more.

1.4qRT-PCR verifies the expression of DDX46 genes in T24 and 5637 cells

1) it is extracted always according to Shanghai Pu Fei Bioisystech Co., Ltd TRIzol RNA extracts kit operational manuals RNA；

1. cell scraper scrapes the carcinoma of urinary bladder T24 of logarithmic growth phase and 5637 cells are placed in centrifuge tube, be put into centrifuge with The rotating speed of 2000rpm centrifuges 5min, and supernatant is abandoned in suction；

2. 1mL TRIzol are added into centrifuge tube, gently blows and beats cell mass, it is made to mix well, be then allowed to stand for room temperature 5min；

3. centrifugation liquid in pipe is transferred to the new EP of 1.5mL to manage, 200 μ L chloroforms and mixing is added, places 10min；

4. with low-temperature and high-speed centrifuge under the conditions of 4 DEG C, 12800rpm high speed centrifugations 15min；

5. drawing supernatant liquor slowly, it is added in new 1.5mL EP pipes, then measures the isopropyl of isometric 4 DEG C of precoolings Alcohol is simultaneously added in EP pipes, and turn upside down mixing, is placed in 4 DEG C of mixture of ice and water and is stood 10min；

6. with the pelleted by centrifugation 12min of step 4., supernatant is abandoned in suction；

7. a concentration of 75% ethyl alcohol 1mL is added into EP pipes, cleaning 2 times (ethyl alcohol is that DEPC water is now with the current)；

8. 11800rpm high speed centrifugations 5min under the conditions of 4 DEG C, supernatant is abandoned in suction；

9. the placement of EP pipes is spontaneously dried at ambient temperature, RNase-free water dissolutions RNA is added into pipe；

10. detecting the concentration and quality of RNA with Nanodrop2000/2000C spectrophotometers.

2) it is analyzed by software, obtains DDX46 genes and internal reference GAPDH primer sequences are respectively：

DDX46 genes (110bp) upstream primer sequence：5-AAAATGGCGAGAAGAGCAACG-3 ', downstream primer sequence： 5 '-CATCATCGTCCTCTAAACTCCAC-3 ', GAPDH internal reference

(121bp) upstream primer sequence：5 '-TGACTTCAACAGCGACACCCA-3, downstream primer sequence：5’- CACCCTGTTGCTGTAGCCAAA-3’

3) RNA reverse transcriptions are carried out according to the operational manual of reverse transcription M-MLV kits and obtains cDNA；

Mixture is configured according to following table, 30s is centrifuged after vibrating mixing, so that mixture is denaturalized 10min in 70 DEG C of water-baths, so Mixture is placed in 4 DEG C of ice-water baths afterwards, makes Oligo dT and template annealing；

After obtaining above-mentioned mixture, reverse transcription reaction system is prepared on ice, and feed the mixture into reverse transcription reaction In system, after vibrating mixing, 30s is centrifuged；It is placed on after taking-up in 42 DEG C of water baths and is incubated 1h, then 70 DEG C of incubation 10min again, Reverse transcriptase is set to inactivate.The cDNA products of acquisition are finally stored in -20 DEG C of refrigerators.

Note：DNTPs is the mixture of dATP, dCTP, dGTP and dTTP, a concentration of 10mM

4) pcr gene amplification is carried out according to TAKARA SYBR Master Mixture kit operational manuals；It presses Table configures amplification system, and reaction condition is：95 DEG C of 15s, 95 DEG C of 5s, 60 DEG C of 30s, three processes recycle 45 times altogether, every time 60 The annealing of DEG C 30s measures OD values at the end of extending.Make melting curve：95 DEG C of 1min, 95 DEG C → 55 DEG C → 95 DEG C, and often increase 0.5 DEG C, 4s is maintained, and read light absorption value simultaneously；Data analysis is carried out using 2- Δ Δ Ct methods to react DDX46 genes in sample Relative expression levels.Wherein, Δ Ct, which represents DDX46 gene C t values and GAPDH reference genes Ct, is worth difference.

2, Celigo cell counts

1) each group aim cell that exponential phase is digested with pancreatin is resuspended cell with complete medium and carries out cytometer Number, is finally adjusted to 4x10 by the cell concentration that cell liquid is resuspended⁶A/ml；

2) 96 orifice plate of this experimental selection, per hole, cell liquid is resuspended in 100 μ l of addition, is 3 multiple holes of every group of setting, by each group hole Plate is put into the incubator under certain condition (37 DEG C, 5%CO2, humidity suitable) overnight incubation；

3) start within the 2nd day, read plate is carried out with Celigo cell counters, 1 time a day, continuous 5 days, as much as possible same A time carries out read plate；

4) parameter for suitably adjusting analysis software as the case may be, calculates and collects taking in the different each orifice plates of group Number with green fluorescence aim cell, it is (the different continuous 5 days growth curve charts of group) and poor to be charted after finally summarizing Different comparative analysis；

5) it analyzes：Draw the cell growth curve figure based on cell counts and cell Proliferation multiple, both the time be Abscissa, but ordinate is different, and the former ordinate is cell counts, and the ordinate of the latter is cell Proliferation multiple (the ratio between each time point cell counts and first day cell count value).

3, MTT cell viabilities are tested

1) T24 and 5637 cells for collecting exponential phase, it is single cell suspension to be resuspended, and carries out cell count；

2) according to 1500cells/well, every group of 3 multiple holes spread 96 orifice plates 5 and open.After the completion of bed board, 5min is stood, then Plate is placed in each experimental group of microscopically observation and whether cellular control unit is evenly distributed.If being unevenly distributed, bed board again is needed. Culture bottle is finally placed on 37 DEG C, overnight incubation in the incubator of CO2 concentration 5%；

3) 1 to 5 days after bed board, daily same time point, 20 a concentration of 5mg/mL of μ L of MTT were added into each hole, Period without culture medium is needed to change, then sets and continues to be incubated in incubator；

4) after being incubated 4h, the culture solution for abandoning upper layer is gently inhaled, but be unable to the first a ceremonial jade-ladle, used in libation particle in contact hole board bottom portion, PBS is added After soft flushing 2 times, dimethyl sub-maple (DMSO) 100 μ L are added, and are placed on oscillator oscillation, keep first a ceremonial jade-ladle, used in libation particle fully molten Solution is detected with microplate reader per hole OD values at 490nm；

5) data preparation is analyzed.

4, colony formation

1) each group aim cell of pancreatin digestion exponential phase is resuspended cell with complete medium and carries out cytometer Number, is finally adjusted to 5x10 by the cell concentration that cell liquid is resuspended⁶A/ml；

2) this experimental selection 6 orifice plates, per hole, cell liquid, 3 multiple holes of every group of setting, by each group orifice plate are resuspended in 100 μ l of addition (37 DEG C, 5%CO are put into the incubator under certain condition₂, humidity it is suitable) culture, after the 1st time is changed liquid, liquid was changed every 2-3 days Once, while under inverted microscope the upgrowth situation of cell is observed；

3) after 14-16 days, confirm that the counting for forming cell in cell mass is all higher than 50 substantially, while aobvious being inverted fluorescence It takes pictures to it under micro mirror；

4) add appropriate PBS liquid into every hole, mix well, after waste liquid is outwelled；

5) 4% paraformaldehydes of 1mL are added to the purpose that arrived fixed cell in each orifice plate, after 0.5h-1h again Add appropriate PBS liquid into every hole, mix well, after waste liquid is outwelled；

6) 500 μ l GIEMSA dye liquors are added in each orifice plate after, cell is made to catch color, maintain 15-25min；

7) appropriate distilled water is finally added into every hole again, mixed well, after waste liquid is outwelled, totally 3 times, wait for that it is naturally dry It is taken pictures with camera after dry, and collects data and carry out statistical analysis.

5, Transwell Matrigels

1) cell that required number is taken out according to experimental design, is placed in 24 new orifice plates, upwards 100 μ L of interior addition The culture medium of serum-free sets 37 DEG C of incubator 1h；

2) cell suspension is prepared with serum free medium, it is 105/hole (24 orifice plate) to count and adjust cell number；

3) culture medium in upper chamber is sucked, 100 μ L of cell suspension, the culture medium of lower interior addition FBS a concentration of 30% is added 600μL；At the same time, it uses one piece of 96 orifice plates of above-mentioned cell suspension paving MTS as transfer reference, is inoculated with the holes 5000cell/, i.e., It carves and measures OD570；

4) plate being inoculated with is set into 37 DEG C of incubators for 24 hours, small indoor culture medium is abandoned in suction, and sterile cotton balls is gripped gently with tweezers Non-diverting cell is wiped, the metastatic cells 3-5min of Giemsa dyeing liquors dip dyeing film lower surface is then used, cleans cell, dry simultaneously It takes pictures；

5) 96 orifice plates are added in configured in advance 10% 200 μ L of acetic acid, remove counterdie with scissors and tweezers and is placed in vinegar It is completely dissolved to cell in acid, takes 100 μ L to set in new hole and detect OD570；

6) data analysis：The rate of transform：The rate of transform=A transfers/AMTS；It calculates each group and shifts mean value, and calculate standard deviation, lead to It crosses T-Test to analyze to obtain corresponding p value, judges to whether there is significant difference (p between each group<0.05, significant difference is no Then there was no significant difference).

6, scratch experiment

1) by institute's equipment sterilization treatment in need, scratching instrument ultraviolet irradiation 30min before operation；

2) experimental group (LV-DDX46-RNAi) and control group (Control-LV) are added about 3 × 104 in 96 orifice plates respectively The metainfective cell in a/hole, next day cell of being subject to reach 90% or more degree of converging；

3) it replaces low concentration blood serum medium within second day and uses scratching instrument using 96 orifice plate bottom lower end centers as reference standard It gently pushes upwards and forms cut；Then the cell of floating is washed away with serum free medium, and the culture of the serum containing low concentration is added Base, and take pictures；

4) culture medium pulled is placed in 37 DEG C, 5%CO₂Incubator, it is aobvious with fluorescence according to the time point of preliminary experiment selection Micro mirror using 96 hole center shadow regions as reference, take pictures (0h, 3h, 6h or 0h, 4h, 8h) by the center that cut is placed in picture；

5) the cut picture for comparing culture different time, measures the width of scored area in the picture of each hole different time points Degree calculates each group cell migration rate, the difference of final comparison of tumor cell migration ability.

7, Apoptosis by Flow Cytometry

1) each group aim cell of exponential phase is digested with pancreatin, complete medium is resuspended cell and carries out cytometer Number, is finally adjusted to 1x10 by the cell concentration that cell liquid is resuspended⁶A/ml；

2) this experimental selection 6 orifice plates, per hole, cell liquid, 3 multiple holes of every group of setting, by each group orifice plate are resuspended in 100 μ l of addition (37 DEG C, 5%CO2, humidity suitable) overnight incubation is put into the incubator under certain condition 5 days；

3) the above cell is collected in the centrifuge tube of 15ml；

4) 10min at normal temperatures, is centrifuged with the rotating speed of 1300rmp/min, supernatant waste liquid is outwelled, is added into centrifuge tube 2mlD-Hanks liquid (is pre-chilled) at 4 DEG C, shakes 1min, it is made fully to shake up washing cell；

5) upper layer waste liquid is abandoned, then adds 200 μ l1 × binding buffer into centrifuge tube, at normal temperatures, with The rotating speed of 1300rmp/min centrifuges 3min, outwells supernatant waste liquid, totally 2 times；

6) 10 μ l Annexin V-APC dyeing are added into the centrifuge tube, are protected from light, are reacted 15-20min at normal temperatures；

7) 400-800 μ 1 × binding of l buffer (depending on specific visual cell's quantity), 1h are added into centrifuge tube again It is interior to be detected with flow cytometer, and collect data and carry out statistical analysis.

8, tumor formation in nude mice

1) urinary bladder carcinoma T24 cell line for collecting exponential phase, makes a concentration of the 2 × 10 of cell suspension⁷A cell/mL is used Disposable syringe is subcutaneously injected according to every 0.5mL to animal right fore oxter.

2) each group mouse is raised under the same conditions, and the continuous tumor formation situation for monitoring nude mice is opened on the 16th day in inoculation Begin, knurl major diameter and minor axis (unit are accurately measured with vernier caliper：Mm), tumor volume=π/6 × L × W × W, i.e. ,=3.14/ 6×L×W×W.Wherein L represents major diameter, and W represents minor axis.2 times a week, continuous 3 weeks；

3) after being subcutaneously injected 35 days, overdose of sodium pentobarbital is injected to experimental group and all nude mices of control group, is carried out peaceful and comfortable Extremely, and by the method for cervical dislocation confirm its death.With preprepared medical scissors and tweezers excised tumor.With scale As reference, scale is placed on the blank left side and top, animal and knurl are arranged at intermediate blank, records and observes mark Ruler scale shoots animal and knurl photo with digital camera；

4) then each group knurl is put in paraformaldehyde and fixes room temperature preservation by the weight for each knurl that weighs with scale respectively And -80 DEG C of freezen protectives, it arranges and analyzes data.

After the interference slow virus of structure DDX46 genes shown in Fig. 1, low carcinoma of urinary bladder T24 and 5637 cell DDX46 gene tables are struck It reaches, carries out PCR verifications, as a result DDX46 genes express downward.

Low expression DDX46 gene cells proliferation is struck shown in Fig. 2 to be suppressed.

The reduction of low expression DDX46 gene cell vigor is struck shown in Fig. 3.

The clonality that low expression DDX46 gene cells are struck shown in Fig. 4 weakens.

The invasion and transfer ability that low expression DDX46 gene cells are struck shown in Fig. 5 are suppressed.

The apoptosis that low expression DDX46 gene cells are struck shown in Fig. 6 increases.

One-tenth knurl ability of the low expression DDX46 gene cells in nude mouse is struck shown in Fig. 7 to weaken.

Therefore, the primer of DDX46 genes using the present invention can successfully build DDX46 genes interference slow virus, inhibit wing Proliferation, invasion and the migration of Guang cancer, it is apoptosis-induced, and weaken its one-tenth knurl ability in nude mouse.

Above disclosed is only presently preferred embodiments of the present invention, cannot limit the right of the present invention with this certainly Range, therefore equivalent changes made in accordance with the claims of the present invention still fall within the range that the present invention is covered.

Claims

1. a kind of tumor markers for predicting carcinoma of urinary bladder, it is characterised in that：The tumor markers are DDX46, DDX46 genes Target sequence is 5 '-GATTGTGATTGAAGAAGAA-3 ', and the upstream primer sequence in DDX46 gene magnification primers is 5 '- Downstream primer sequence in AAAATGGCGAGAAGAGCAACG-3 ', DDX46 gene magnification primer is 5 '- CATCATCGTCCTCTAAACTCCAC-3’。

2. the tumor markers as described in claim 1 for predicting carcinoma of urinary bladder, it is characterised in that：The DDX46 genes expand Increase application of the primer in identifying carcinoma of urinary bladder.