CN103361408A - Application of molecule miR-150 in screening preparation of colorectal cancer detecting medicament - Google Patents
Application of molecule miR-150 in screening preparation of colorectal cancer detecting medicament Download PDFInfo
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Abstract
The invention provides application of molecule miR-150 in screening preparation of a colorectal cancer detecting medicament. The molecule miR-150 is screened out by using a muParaflo microfluidic chip by the inventor of the invention; a paraffin section preparation of a clinical colorectal cancer patient is detected by an established digoxigenin labeled probe of miR-150 and an established detection method of qRT-PCR (Quantitative Real-Time-Polymerase Chain Reaction), respectively; results show that miR-150 low expression level in the clinical colorectal cancer patient indicates short lifetime of the patient and poor sensitivity of the patient to postoperative chemotherapy, and miR-150 high expression level indicates long lifetime of the patient and good sensitivity of the patient to postoperative chemotherapy. Compounds having high sensitivity to miR-150 can be screened by the method, and the compounds can be used for developing a kit for detecting the colorectal cancer. The kit is composed of compounds sensitive to the molecule miR-150 as active ingredients and a medicinal carrier. Simple detection of the miR-150 expression level is advantageous for monitoring of the colorectal cancer patient after healing and determination of chemotherapy sensitivity of the patient, and thus provides an effective method for clinical prevention and treatment of the colorectal cancer; high clinical application value and social benefit are created.
Description
Technical field
The present invention relates to pharmaceutical chemistry, be specifically related to the application of biomarker molecule miR-150 in screening preparation detection large bowel cancer medicine.
Background technology
Large bowel cancer is one of common cancer, worldwide its sickness rate occupy the 3rd of malignant tumour, along with improving constantly with dietary structure of standard of living changes, the sickness rate of China's large bowel cancer also increases year by year, particularly then faster in big city amplification, as 1984-2004 two during the decade, the District of Shanghai incidence of colorectal male sex increases to 23.61/10 ten thousand by 16,/10 ten thousand, the women then increases to 20.43/10 ten thousand by 14.26/10 ten thousand.The factor that affects at present the large bowel cancer curative effect is a lot, and the level of signification that lacks prognosis and the monitoring for the treatment of susceptibility still belongs to the major issue that urgent need solves.
MicroRNA (miRNA) molecule (miRNA) is the study hotspot of molecular biology, genetics and oncology in recent years.MiRNA is the newfound non-coding small RNA molecular of a class, usually at the post-transcriptional level regulate gene expression.At present, research for miRNA modulate tumor genesis has become important branch in the tumor research field, the famous laboratory of being engaged in more in the world Tumorigenesis and treatment research turns to this research field, the research of some magazine follow-up story Tumor-assaciated miRNA potential functions and anticarcinogenic effect one after another.See following report: 1.Weidhaas J. utilizes miRNA explaination oncobiology. lancet-tumour magazine .2010; 11:106-7.2.Calin GA, Croce CM.miRNA is as the sign of human tumor. natural cancer summary 2006; 6:857-66. studies show that in a large number that in recent years the progress of miRNA and kinds of tumors exists close relationship, it both can be used as cancer suppressor gene, the activity of downward modulation proto-oncogene; Also can be used as oncogene, the activity of downward modulation cancer suppressor gene, and have conservative property, timing and tissue specificity highly.Therefore, miRNA participates in bringing into play very important regulating effect in the processes such as organism metabolism and tumor development at regulate tumor cell propagation, differentiation and apoptosis.Thereby determined its particular advantages in tumour patient clinical prognosis and treatment are monitored.Borralho PM, Kren BT, Castro RE, et al.miRNA-143 have reduced Human Large Intestine Carcinoma Cells to be the vigor of HCT116 and to have strengthened susceptibility to 5 FU 5 fluorouracil. FEBS's magazine 2009; 276:6689-700. report: the instantaneous expression miR-143 (being a kind of of Microrna) that crosses causes cell viability to reduce about 60% in colon carcinoma cell line HCT116, and the miR-143 overexpressing cell system that uses antibiotic-screening is exposed to 5 FU 5 fluorouracil (5-Fluorouracil, 5-FU), find that the increase of miR-143 stably express is associated with cell viability minimizing and apoptosis increase.Research also confirms above change and nuclear fragmentation and caspase-3, and the increase of-8 and-9 (caspase-3 ,-8 ,-9) activity is relevant.In addition, miR-143 can cause extracellular regulated protein kinase 5, NF-κ B and the downward modulation of Bcl-2 protein expression, and its down-regulated expression is more obvious after being exposed to 5-FU.This has pointed out miR-143 to participate in regulating Growth of Cells as cancer suppressor gene, apoptosis and some chemotherapy side effect key protein, and increase colon carcinoma cell line to the susceptibility of 5-FU.Chen X, Guo X, Zhang H, et al.Role of miR-143 is by the biological action of regulation and control target gene KRAS in large bowel cancer occurs. oncogene 2009; 28:1385-92. report: found in transfection among the colon carcinoma cell line Lovo of miR-143, the expression of KRAS (a kind of proto-oncogene) (action target spot of miR-143) significantly reduces, and transfection miR-143 specific inhibitor then increases KRAS protein expression level in the Lovo clone.The Lovo clone of using the miR-143 inhibitor to process shows stronger multiplication capacity.This explanation miR-143 can suppress the CRC cell line growth by the translation that suppresses KRAS.But, existing these researchs only are at the fundamental aspect screening miRNA relevant with large bowel cancer, and utilize isolated cells experiment and in the body experimentation on animals, confirm biological function and molecular mechanism of action that it is potential, do not screen from clinical extensive sample angle and analyze the large bowel cancer biomarker with potential clinical prognosis and treatment monitoring value, thereby fail to find out effective crucial miRNA molecule yet.The inventor utilizes the miRNA chip directly to screen analysis in the different steps from the large bowel cancer progression (normally, adenoma, the gland cancer) clinical sample, found out the molecule miR-150 (being a kind of of Microrna) that plays a crucial role, clinical extensive sample analysis of later stage finds that expression level and the large bowel cancer of miR-150 is closely connected.Therefore, molecule miR-150 (Chinese) is researched and developed, significant.
Summary of the invention
Technical problem to be solved by this invention is the application of research and design molecule miR-150 in pharmacy.
The invention provides the application of molecule miR-150 in preparation detection large bowel cancer medicine.
The inventor utilizes the miR-150 in micro-fluid chip and pcr chip screening large intestine healthy tissues, adenoma, the adenocarcinoma tissue, and utilizes PCR to carry out the result.The result shows: miR-150 presents notable difference in large intestine healthy tissues, adenoma, adenocarcinoma tissue and expresses (A), and the statistical analysis between each group all has significance (B); Tomour specific miRNA chip analysis has also shown the significant difference (C) of miR-150 expression level between three groups of samples; The qRT-PCR analytical results has also confirmed the differential expression of miR-150.
And further utilize the result of miR-150 expression level in the probe in detecting large bowel cancer of digoxigenin labeled and the healthy tissues sample.Found that: snRNA U6 (micro ribonucleic acid U6) all presents positive expression as positive control in large bowel cancer and pairing tissue samples, without significant difference (A/B); The expression of miR-150 probe in the Colorectal Carcinoma sample of digoxigenin labeled will be starkly lower than the expression level (C/D) in the normal mucosa; And Scramble control is: for a kind of negative control sequence of miR-150 probe design, all negative in two groups of samples.By the probe in detecting miR-150 expression level of qRT-PCR (real-time quantitative polymerase chain reaction) and digoxigenin labeled and the result of PATIENTS WITH LARGE BOWEL prognosis judgement.The result shows: the Cutoff (section value) that utilizes Tumor/Normal (cancer/normal) ratio is divided into whole group that miR-150 is low to express and two groups of high expression levels, and the low miR-150 group of expressing has significance (A) at large bowel cancer and the difference of matching between healthy tissues; The low survival of patients phase of expressing miR-150 will be starkly lower than the patient (B) of high expression level miR-150 in the tissue in the Colorectal Carcinoma; Only utilize Cutoff (section value) in the tumour whole group to be divided into miR-150 is low to express and two groups of high expression levels, the result shows that also the survival of patients phase of the low miR-150 of expression in the Colorectal Carcinoma will be starkly lower than the patient (C) who accuses expression miR-150 in the tissue; And only utilize Cutoff among the Normal whole group to be divided into miR-150 is low to express and two groups of high expression levels, the survival of patients phase that the result shows the low miR-150 of expression in the Colorectal Carcinoma with organize in patient's no significant difference (D) of high expression level miR-150; Utilize simultaneously situ Analysis PATIENTS WITH LARGE BOWEL paraffin section, the result has confirmed that also the survival of patients phase of the low miR-150 of expression will be starkly lower than the patient (E) who accuses expression miR-150 in the tissue in the Colorectal Carcinoma.
In addition, the result who judges by probe in detecting miR-150 expression level and the PATIENTS WITH LARGE BOWEL postoperative chemotherapy susceptibility of qRT-PCR and digoxigenin labeled.The result shows: the low survival of patients phase of expressing of miR-150 obviously shortens in large bowel cancer II and III phase patient, the relation that its susceptibility to chemotherapy obviously reduces (A/D) during analyzing respectively the expression level of miR-150 in large bowel cancer II phase or III phase patient and postoperative chemotherapy, survival of patients, the result also finds: the low survival of patients phase of expressing obviously shortens, and its susceptibility to chemotherapy obviously reduces (B/E, C/F).
By above-mentioned test, illustrate that the expression level of miR-150 and large bowel cancer are closely connected, therefore, molecule miR-150 can be used for screening the medicine that preparation detects large bowel cancer.
Molecule miR-150 of the present invention obtains by following method:
The extraction (Trizol method) of I, total RNA
(1) pulverizes adding 1ml TRIzol in large intestine healthy tissues, adenoma, the adenocarcinoma tissue, mixing on the vortex oscillator;
(2) add the chloroform of 1ml volume, the abundant mixing 1 minute of turning upside down left standstill 5 minutes under 25 ℃ of the room temperatures;
(3) 4 ℃, 12,000rpm changes supernatant liquor over to new 1.5ml centrifuge tube after centrifugal 15 minutes, adds isopyknic Virahol, puts upside down gently mixing, and room temperature left standstill 5 minutes;
(4) 4 ℃, 12000rpm removes supernatant after centrifugal 10 minutes, adds 2ml70% ethanol in precipitation, and 4 ℃, 12000rpm centrifuge washing precipitation 15 minutes;
(5) remove supernatant, after 25 ℃ of room temperatures of precipitation are dried, add the abundant dissolution precipitation of water of RNase-free reagent, measure A260 and A280 value; 1.2 purifying and the quality inspection of total RNA;
II, the total RNA of purifying
(1) gets RNA 70ug and adjust to 100uL with the water of RNase-free reagent, upper rnase centrifugal mini column chromatography;
(2) add 350uL Buffer RLT (RLT damping fluid) (adding first 10uL acetone among the 1mL Buffer RLT), fully mixing;
(3) add the 250uL dehydrated alcohol, fully mixing;
(4) will mix liquid and be added in the rnase centrifugal mini column chromatography, greater than the centrifugal 15s of 8,000rpm;
(5) abandon filtrate, add 500uL Buffer RPE (adding dehydrated alcohol, dehydrated alcohol=1: 4 before using for the first time), greater than the centrifugal 15s of 8,000rpm;
(6) abandon filtrate, add 500uL Buffer RPE, greater than the centrifugal 2min of 8,000rpm;
(7) RNeasy mini spin column is moved into a new collection tube, maximum speed of revolution 16, the centrifugal 1min of 000rpm;
(8) liquid in the rnase centrifugal mini column chromatography is added in an EP (Eppendorf centrifuge tube) pipe, the careful water that adds the 30uL RNase-free reagent in the middle of film, room temperature is placed 1min;
(9) maximum speed of revolution 16,000rpm, and centrifugal 1min obtains total RNA;
III, cDNA microarray miR-150: utilize Microarray Experiments, the total RNA sample of 2-5ug obtains little RNA＜300nt by the little centrifuging post separation of YM-100; Use polysaccharase to add poly (A) (polyadenylic acid A) tail at the little RNA 3 ' end that is separated to, 1 oligonucleotide mark is connected with this polyA tail is used for follow-up fluorescent mark again; In two sample experiments, come two RNA samples of mark with two different markers; Hybridization utilizes the Circulation of micro circulation pump to spend the night at micro-fluid chip to carry out hybridization.On micro-fluid chip, every detection probes all be by a chemically modified nucleoside acid encoding section (with target microRNA (miRNA) complementary (method derives from miRBase kind database), http://microrna.sanger.ac.uk/sequences/) or with other RNA (Quality Control or custom IC sequence) complementation) and a spacer that is formed by polyoxyethylene glycol (spacing of expansion coding section and matrix) form.It is synthetic that detection probes all uses PGR (photogenerated reagent, video picture reagent) chemical method to carry out original position.The hybridization melting temperature(Tm) is to carry out balance by the detection probes of chemically modified.The 100 μ L6xSSPE damping fluids contain 25% methane amide (6mM EDTA, pH 6.8 for 0.90M NaCl, 60mM Na2HPO4) are used in hybridization, and hybridization temperature is 34 ℃.Detect the specific Cy3 of applying marking and Cy5 fluorescence dye after the hybridization.Utilize laser scanner (GenePix4000B, Molecular Device) collection hybridization image and use Array-Pro image analysis software (Media Cybernetics) to carry out the image digitazation conversion.Data analysis at first is the subduction background value, then uses LOWESS to filter 2 (Locally-Weighted Regression) and carries out signal normalization.For two mark experiments, the ratio (log2 conversion, balance) of 2 groups of detection signals and the p-values of t-test will be calculated; When p-value＜0.01, be considered to the significance difference opposite sex and express, therefrom filter out the most significantly miR-150 of difference.
The above-mentioned expression level that shows miR-150 or miR-150 can be used as the independent factor that prediction prognosis and treatment monitoring are judged to the detection of large bowel cancer chemotherapy.Utilize this method can screen the compound strong to miR-150 susceptibility, will be to the compound of molecule miR-150 sensitivity for the preparation of the medicine that detects large bowel cancer.
The serve as reasons test kit of detection large bowel cancer that the compound of molecule miR-150 sensitivity is formed as activeconstituents and pharmaceutical carrier of described medicine.
Expression level by easy detection miR-150 is conducive to the judgement of PATIENTS WITH LARGE BOWEL Prognosis scoveillance and chemosensitivity, for the clinical prevention large bowel cancer provides effective ways, has larger clinical value and social benefit.
Description of drawings
Fig. 1 is for utilizing difference miRNA in micro-fluid chip screening large intestine healthy tissues, adenoma, the adenocarcinoma tissue.Fig. 2 is the relative expression value of miR-150 in micro-fluid chip screening large intestine healthy tissues, adenoma, adenocarcinoma tissue.The result shows: with the carcinogenesis of human (healthy tissues-adenoma-gland cancer) of large bowel cancer, the expression level of miR-150 descends gradually, and the statistical analysis between each group all has significance.
Fig. 3 is the miR-150 in pcr chip screening large intestine healthy tissues, adenoma, the adenocarcinoma tissue.Fig. 4 is that qRT-PCR check analysis miR-150 is at the expression level of large intestine healthy tissues, adenoma, adenocarcinoma tissue.
Fig. 5 is the result who utilizes miR-150 expression level in the probe in detecting large bowel cancer of digoxigenin labeled and the healthy tissues sample.
Fig. 6 is the result who detects miR-150 expression level in 239 routine large bowel cancers and normal pairing tissue by qRT-PCR.
Fig. 7 is miR-150 high expression level group and low expression group in the PATIENTS WITH LARGE BOWEL tissue (Cutoff that utilizes tumour/normal ratio whole group is divided into miR-150 is low to express and two groups of the high expression levels) comparative result of lifetime.
Fig. 8 is miR-150 high expression level group and low expression group in the PATIENTS WITH LARGE BOWEL tissue (utilize Cutoff in the tumor group whole group is divided into miR-150 is low to express and two groups of the high expression levels) comparative result of lifetime.
Fig. 9 is miR-150 high expression level group and low expression group in the PATIENTS WITH LARGE BOWEL tissue (utilize Cutoff in the tumour pairing normal group whole group is divided into miR-150 is low to express and two groups of the high expression levels) comparative result of lifetime.
Figure 10 utilizes the Probe In Situ Hybridization experiment of digoxigenin labeled to detect miR-150 expression level and the relation of survival of patients phase in the 185 routine large bowel cancer paraffin sections.
Figure 11 is for detecting the analytical results of chemosensitivity judgement behind miR-150 expression level in the Colorectal Carcinoma (Cutoff that utilizes tumour/normal ratio whole group is divided into miR-150 is low to express and two groups of high expression levels) and the clinical II of large bowel cancer phase, the III phase operation in patients by qRT-PCR.
The analytical results of Figure 12 for judging by chemosensitivity behind miR-150 expression level and clinical II phase of large bowel cancer, the III phase operation in patients in the Probe In Situ Hybridization experiment detection large bowel cancer paraffin section tissue of digoxigenin labeled.
Embodiment
Extract RNA from large intestine healthy tissues, adenoma, gland cancer:
1.1 the extraction (conventional Trizol method) of total RNA
(1) pulverizes respectively large intestine healthy tissues, adenoma, adenocarcinoma tissue (each 10mg), and add respectively 1ml TRIzol (Shanghai Invitrogen company), mixing on the vortex oscillator.
(2) add the 1ml chloroform, the abundant mixing 1 minute of turning upside down left standstill 5 minutes under 25 ℃ of the room temperatures.
(3) 4 ℃, 12,000rpm changes supernatant liquor over to new 1.5ml centrifuge tube after centrifugal 15 minutes, adds isopyknic Virahol, puts upside down gently mixing, and room temperature left standstill 5 minutes.
(4) 4 ℃, 12000rpm removes supernatant after centrifugal 10 minutes, adds 2ml70% ethanol in precipitation, and 4 ℃, 12000rpm centrifuge washing precipitation 15 minutes.
(5) remove supernatant, after 25 ℃ of room temperatures of precipitation are dried, adding is without the abundant dissolution precipitation of the water 1ml (water of RNase-free reagent) (Shanghai Xi Mei biotech firm) of RNA enzyme, measure A260 and A280 value (752 type UV-light spectrophotometers, Qilin Analytical Instrument Co., Ltd., Nanjing).
1.2 purifying and the quality inspection of total RNA
Use QIAGEN Rneasy Kit purifying (test kit of the purifying RNA of German QIAGEN company) the lower operation of total RNA room temperature (25 ℃))
1.2.1 the total RNA of purifying (QIAGEN RNeasy Mini Kit) (test kit of the purifying RNA of German QIAGEN company)
(1) gets RNA 70ug and adjust to 100uL with the water of RNase-free reagent, (the maximum applied sample amount of RNeasy mini spin column (rnase centrifugal mini column chromatography) is 100ugRNA, so generally get RNA 70ug).
(2) add 350uL Buffer RLT (Shanghai Invitrogen company) (adding first 10uL acetone among the 1mL Buffer RLT), fully mixing.
(3) add the 250uL dehydrated alcohol, fully mixing.
(4) will mix liquid and be added in the rnase centrifugal mini column chromatography, greater than the centrifugal 15s of 8,000rpm.
(5) abandon filtrate, add 500uL Buffer RPE (adding dehydrated alcohol, dehydrated alcohol=1: 4 before using for the first time), greater than the centrifugal 15s of 8,000rpm.
(6) abandon filtrate, add 500uL Buffer RPE, greater than the centrifugal 2min of 8,000rpm.
(7) RNeasy mini spin column is moved into a new collection tube, maximum speed of revolution 16, the centrifugal 1min of 000rpm.
(8) liquid in the rnase centrifugal mini column chromatography is added in an EP (Eppendorf centrifuge tube) pipe, the careful water that adds the 30uL RNase-free reagent in the middle of film, room temperature is placed 1min.
(9) maximum speed of revolution 16,000rpm, centrifugal 1min.
1.2.2 total RNA quality examination (agarose gel electrophoresis method)
(1) the configuration electrophoretic buffer 50TAE (the special-purpose electrophoretic buffer that Ai Jia Bioisystech Co., Ltd in Changsha produces) that DEPC (DEPC) processes is stand-by behind the autoclaving.
(2) use H before the use electrophoresis chamber
2O
2Soak 15min, 1TAE electrophoretic buffer (the special-purpose electrophoretic buffer that Ai Jia Bioisystech Co., Ltd in Changsha produces) 1ml is poured in the water flushing of then processing with DEPC into.
(3) take by weighing the 0.5mg agarose, add 1TAE electrophoretic buffer 1ml, preparation glue is done electrophoretic analysis.
(4) observe, take pictures at the gel imaging instrument, preserve image, the total RNA quality of preliminary assessment.Total RNA does not have decomposition by analysis.
2. cDNA microarray miR-150: utilize Microarray Experiments to need the total RNA sample of 2-5 μ g (microgram).By the little centrifuging post of YM-100 (Millipore) separate obtain little RNA (＜300nt).Use Poly (A) polysaccharase to add poly (A) tail at the little RNA 3 ' end that is separated to, again 1 oligonucleotide mark is connected (ligation) with this poly (A) tail and is used for follow-up fluorescent mark.In two sample experiments, come two RNA samples of mark with two different markers.Hybridization utilizes the Circulation of micro circulation pump (Atactic Technologies) to spend the night at μ Paraflo micro-fluid chip to carry out 1.On micro-fluid chip, every detection probes all be by a chemically modified nucleoside acid encoding section (with target microRNA (miRNA) complementary (method derives from miRBase kind database), http://microrna.sanger.ac.uk/sequences/) or with other RNA (Quality Control or custom IC sequence) complementation) and a spacer that is formed by polyoxyethylene glycol (spacing of expansion coding section and matrix) form.It is synthetic that detection probes all uses PGR (photogenerated reagent, video picture reagent) chemical method to carry out original position.The hybridization melting temperature(Tm) is to carry out balance by the detection probes of chemically modified.The 100 μ L6xSSPE damping fluids (Shanghai Xi Mei biotech firm) contain 25% methane amide (6mM EDTA, pH 6.8 for 0.90M NaCl, 60mM Na2HPO4) are used in hybridization, and hybridization temperature is 34 ℃.Detect the specific Cy3 of applying marking and Cy5 fluorescence dye after the hybridization.Utilize laser scanner (GenePix4000B, Molecular Device) collection hybridization image and use Array-Pro image analysis software (Media Cybernetics) to carry out the image digitazation conversion.Data analysis at first is the subduction background value, then uses LOWESS to filter 2 (Locally-Weighted Regression) and carries out signal normalization.For two mark experiments, the ratio (log2 conversion, balance) of 2 groups of detection signals and the p-values of t-test will be calculated; When p-value＜0.01, be considered to the significance difference opposite sex and express, therefrom filter out the most significantly miR-150 of difference.
1.qRT-PCR detection method:
(1.3RT reverse transcription) reaction: according to the TaqMan MicroRNA Reverse Transcription Kit (test kit of miRNA reverse transcription, Shanghai Xi Mei biotech firm) specification sheets operation adds the total RNA of 5 μ l purifying that embodiment 1 obtains (the RNA concentration after diluting with the water of deoxyribonuclease is 0.5-1 μ g/ μ l) in the reaction.Reaction system is as follows: (volume unit: μ l)
After all reagent add, of short duration centrifugal mixing, 16 ℃ of 30min, 42 ℃ of 30min obtain cDNA behind 85 ℃ of 5min, and-20 ℃ save backup.Be template with the reverse transcription product again, use the ABI7300 quantitative PCR instrument of ABI company to carry out the quantitative fluorescent PCR reaction.
1.4Real time PCR reaction: take above-mentioned eDNA as template, RNU24 is internal reference, detects the relative expression's level of miR-150 in the different experiments group, reaction system is as follows: (volume unit: μ l)
After mixing, add above-mentioned reacted reverse transcription system 10 μ l, so the total reaction system is 20 μ l.Reaction conditions is: 95 ℃ of denaturation 10min; 95 ℃ of 15s, 60 ℃ of 60s, 40 circulations.Use the ABI7300 quantitative PCR instrument of ABI company to carry out the quantitative fluorescent PCR reaction, (C represents cycle (cycle number) to detect the Ct value of each template, T represents threshold (dividing value)), all samples all repeats 3 times, and negative control is take DEPC water (water of deoxyribonuclease) as template.
1.5 data analysis: with RNU24 as confidential reference items, by 2
-Δ Δ CTMethod (international algorithm) is calculated the difference of miRNA expression level.
The Probe In Situ Hybridization detection method of (2.DIG digoxin) mark (peroxidase Color Appearance System):
2.1 paraffin section de-waxing is to water: dimethylbenzene twice, 10 minute/time; Dehydrated alcohol 10 minutes once; 90% ethanol 10 minutes once; 80% ethanol 10 minutes once; 0.1M PBS flushing three times.
2.2 put room temperature 10 minutes in the punching liquid, punch to change histiocytic permeability to cell and make fast permeates cell membranes smoothly of probe, with 0.1M PBS (phosphate buffered saline buffer) flushing three times.
2.3 put hydrogen peroxide confining liquid room temperature 20 minutes, with sealing endogenous catalase.0.1M PBS flushing three times.Drip compound digestion working fluid (purchasing the Science and Technology Ltd. in Shanghai Xi Tang), cover tissue surface, room temperature 10-30 minute.(the condition situation according to experiment tissue and experiment is determined digestion time and temperature) 0.1M PBS flushing three times.0.2xSSC flushing is (room temperature 3 minutes) once.
Cover 37 ℃ of wet boxes of tissue and hatched 1-2 hour 2.4 drip the prehybridization working fluid, after throw off cover glass and wash three times with 25 ℃ of 0.2xSSC room temperatures, washed 5 minutes at every turn.Dripping 37 ℃ of wet boxes of crossing work liquid covering tissue hatched 4 hours.Throwing off cover glass washes three times with 37 ℃ of 2xSSC (0.30mol/l NaAc/0.030mol/L Sodium Citrate liquid), each washing 5 minutes, 0.2xSSC wash three times for 37 ℃, each washing 5 minutes, 0.1mol TBS (Tris-buffered saline, buffer saline) washes 3 times for 37 ℃, washed 5 minutes at every turn.
2.5 dripping 37 ℃ of wet boxes of digoxin biotin labeled probe covering tissue incubated 45-60 minute.0.1M PBS (phosphate buffered saline buffer) flushing three times was washed 5 minutes at every turn.The high quick peroxidase streptavidin mixture working fluid of rear dropping covers 37 ℃ of wet boxes of tissue and hatched 45 minutes.With 0.1M PBS flushing three times, washed 5 minutes more at every turn.
2.6DAB colour developing, termination reaction was washed in distillation when the optical microphotograph Microscopic observation was obvious with extracellular background color contrast gradient contrast to the positive color of cell endoplasm.Cytoplasm shows the positive reaction of blue-purple granule.Hematorylin is redyed, and karyon is blue.
2.7 carry out the expression intensity IOD scoring that in situ hybridization detects clinical PATIENTS WITH LARGE BOWEL Pathologic specimen miR-150 by the digoxin labelled probe for miR-150: the scoring of expression intensity scoring=staining power scoring * positive rate.Staining power represents that with negative (0 minute), the weak positive (1 minute), the medium positive (2 minutes), strong positive (3 minutes) positive rate is divided into 0-25% (1), 26-50% (2 minutes), 51-75% (3 minutes), 76-100% (4 minutes).Sample is quadruplicate, and sheet is seen in two pathologist double blindings, gets mean.
3. interpretation of result
Fig. 1 is for utilizing difference miRNA in micro-fluid chip screening large intestine healthy tissues, adenoma, the adenocarcinoma tissue.Found that: with the carcinogenesis of human (healthy tissues-adenoma-gland cancer) of large bowel cancer, the expression level of miR-150 descends gradually, and the statistical analysis between each group all has significance.
Fig. 2 is the relative expression value of miR-150 in micro-fluid chip screening large intestine healthy tissues, adenoma, adenocarcinoma tissue.The result shows: with the carcinogenesis of human (healthy tissues-adenoma-gland cancer) of large bowel cancer, the expression level of miR-150 descends gradually, and the statistical analysis between each group all has significance.
Fig. 3 is the miR-150 in pcr chip screening large intestine healthy tissues, adenoma, the adenocarcinoma tissue.The result shows: with the carcinogenesis of human (healthy tissues-adenoma-gland cancer) of large bowel cancer, the expression level of miR-150 descends gradually, and the statistical analysis between each group all has significance.
Fig. 4 is that qRT-PCR check analysis miR-150 is at the expression level of large intestine healthy tissues, adenoma, adenocarcinoma tissue.The result shows: with the carcinogenesis of human (healthy tissues-adenoma-gland cancer) of large bowel cancer, the expression level of miR-150 descends gradually, and the statistical analysis between each group all has significance.
Fig. 5 is the result who utilizes miR-150 expression level in the probe in detecting large bowel cancer of digoxigenin labeled and the healthy tissues sample.Found that: snRNA U6 all presents positive expression at large bowel cancer and pairing in the tissue samples as positive control, between large intestine healthy tissues and adenocarcinoma tissue without significant difference; The expression of miR-150 probe in the Colorectal Carcinoma sample of digoxigenin labeled will be starkly lower than the expression level in the normal mucosa; And Scramble control is all negative in two groups of samples.
Fig. 6 is the result who detects miR-150 expression level in 239 routine large bowel cancers and normal pairing tissue by qRT-PCR.The result shows: the Cutoff (section value) that utilizes tumour/normal ratio is divided into whole group that miR-150 is low to express and two groups of high expression levels, the difference of the low miR-150 group of expressing between large bowel cancer and pairing healthy tissues has significance, and the difference not statistically significant of the miR-150 of high expression level group between large bowel cancer and pairing healthy tissues.
Fig. 7 is miR-150 high expression level group and low expression group in the PATIENTS WITH LARGE BOWEL tissue (Cutoff that utilizes tumour/normal ratio whole group is divided into miR-150 is low to express and two groups of the high expression levels) comparative result of lifetime.Found that: the survival of patients phase of the low miR-150 of expression will be starkly lower than the PATIENTS WITH LARGE BOWEL of high expression level miR-150 in the tissue in the Colorectal Carcinoma, and the result has remarkable statistical significance.
Fig. 8 is miR-150 high expression level group and low expression group in the PATIENTS WITH LARGE BOWEL tissue (utilize Cutoff in the tumor group whole group is divided into miR-150 is low to express and two groups of the high expression levels) comparative result of lifetime.The result shows: the survival of patients phase of the low miR-150 of expression will be starkly lower than and accuse the PATIENTS WITH LARGE BOWEL of expressing miR-150 in the tissue in the Colorectal Carcinoma, and the result has remarkable statistical significance.
Fig. 9 is miR-150 high expression level group and low expression group in the PATIENTS WITH LARGE BOWEL tissue (utilize Cutoff in the tumour pairing normal group whole group is divided into miR-150 is low to express and two groups of the high expression levels) comparative result of lifetime.The result shows: patient's no significant difference of high expression level miR-150 in the survival of patients phase of low expression miR-150 and the tissue in the Colorectal Carcinoma, the result does not have remarkable statistical significance.
Figure 10 utilizes the Probe In Situ Hybridization experiment of digoxigenin labeled to detect miR-150 expression level and the relation of survival of patients phase in the 185 routine large bowel cancer paraffin sections.Found that: the survival of patients phase of the low miR-150 of expression will be starkly lower than and accuse the PATIENTS WITH LARGE BOWEL of expressing miR-150 in the tissue in the Colorectal Carcinoma.
Figure 11 is for detecting the analytical results of chemosensitivity judgement behind miR-150 expression level in the Colorectal Carcinoma (Cutoff that utilizes tumour/normal ratio whole group is divided into miR-150 is low to express and two groups of high expression levels) and the clinical II of large bowel cancer phase, the III phase operation in patients by qRT-PCR.The result shows: the clinical II of large bowel cancer, III phase and II the phase+III phase patient in, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously shortens with respect to the PATIENTS WITH LARGE BOWEL of miR-150 high expression level lifetime; The clinical II of the large bowel cancer of not accepting chemotherapy, III phase and II the phase+III phase patient in, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously shortens with respect to the PATIENTS WITH LARGE BOWEL of miR-150 high expression level lifetime; Its susceptibility to chemotherapy of accepting adjuvant chemotherapy obviously reduce (A/D) analyze respectively in large bowel cancer II phase or III phase patient the clinical II of large bowel cancer, III phase and II the phase+III phase patient in, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously shortens with respect to the PATIENTS WITH LARGE BOWEL of miR-150 high expression level lifetime, and namely the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously reduces with respect to the PATIENTS WITH LARGE BOWEL of the miR-150 high expression level susceptibility to adjuvant chemotherapy.
The analytical results of Figure 12 for judging by chemosensitivity behind miR-150 expression level and clinical II phase of large bowel cancer, the III phase operation in patients in the Probe In Situ Hybridization experiment detection large bowel cancer paraffin section tissue of digoxigenin labeled.The result shows: the clinical II of large bowel cancer, III phase and II the phase+III phase patient in, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously shortens with respect to the PATIENTS WITH LARGE BOWEL of miR-150 high expression level lifetime; The clinical II of the large bowel cancer of not accepting chemotherapy, III phase and II the phase+III phase patient in, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously shortens with respect to the PATIENTS WITH LARGE BOWEL of miR-150 high expression level lifetime; Its susceptibility to chemotherapy of accepting adjuvant chemotherapy obviously reduce (A/D) analyze respectively in large bowel cancer II phase or III phase patient the clinical II of large bowel cancer and II the phase+III phase patient in, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously shortens with respect to the PATIENTS WITH LARGE BOWEL of miR-150 high expression level lifetime, being the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 obviously reduces with respect to the PATIENTS WITH LARGE BOWEL of the miR-150 high expression level susceptibility to adjuvant chemotherapy, and in its patient of the clinical III of large bowel cancer, the low PATIENTS WITH LARGE BOWEL of expressing of miR-150 and PATIENTS WITH LARGE BOWEL no significant difference lifetime of miR-150 high expression level do not have statistical significance.
Therefore, according to the expression level that detects miR-150, can be used for analyzing the existence of judging large bowel cancer and development etc.
Claims (5)
1. the application of molecule miR-150 in screening preparation detection large bowel cancer medicine.
2. application according to claim 1 is characterized in that, described molecule miR-150 obtains by following method:
The extraction of I, total RNA
(1) pulverizes adding 1ml TRIzol in large intestine healthy tissues, adenoma or the adenocarcinoma tissue, mixing on the vortex oscillator;
(2) add the chloroform of 1/5 volume, the abundant mixing 1 minute of turning upside down left standstill 5 minutes under 25 ℃ of the room temperatures;
(3) 4 ℃, 12,000rpm changes supernatant liquor over to new 1.5ml centrifuge tube after centrifugal 15 minutes, adds isopyknic Virahol, puts upside down gently mixing, and room temperature left standstill 5 minutes;
(4) 4 ℃, 12000rpm removes supernatant after centrifugal 10 minutes, adds 70% ethanol of 2/5 volume in the precipitation, and 4 ℃, 12000rpm centrifuge washing precipitation 15 minutes;
(5) remove supernatant, after 25 ℃ of room temperatures of precipitation are dried, add the abundant dissolution precipitation of water of RNase-free reagent, measure A260 and A280 value; 1.2 purifying and the quality inspection of total RNA;
II, the total RNA of purifying
(1) gets RNA 70ug and adjust to 100 μ L with the water of RNase-free reagent, upper rnase centrifugal mini column chromatography;
(2) add 350uL Buffer RLT, add first 10 μ L acetone among the 1mL Buffer RLT), abundant mixing;
(3) add 250 μ L dehydrated alcohols, fully mixing;
(4) will mix liquid and be added in the rnase centrifugal mini column chromatography, greater than the centrifugal 15s of 8,000rpm;
(5) abandon filtrate, add 500uL Buffer RPE, add dehydrated alcohol, dehydrated alcohol before using for the first time: Buffer RPE=1: 4, greater than the centrifugal 15s of 8,000rpm;
(6) abandon filtrate, add 500uL Buffer RPE, greater than the centrifugal 2min of 8,000rpm;
(7) RNeasy mini spin column is moved into a new collection tube, maximum speed of revolution 16, the centrifugal 1min of 000rpm;
(8) liquid in the rnase centrifugal mini column chromatography is added in the EP pipe, the careful water that adds 30 μ L RNase-free reagents in the middle of film, room temperature is placed 1min;
(9) maximum speed of revolution 16,000rpm, and centrifugal 1min obtains total RNA;
III, cDNA microarray miR-150: utilize Microarray Experiments, the total RNA sample of 2-5ug obtains little RNA＜300nt by the little centrifuging post separation of YM-100; Use polysaccharase to add poly A tail at the little RNA 3 ' end that is separated to, 1 oligonucleotide mark is connected with this polyA tail is used for follow-up fluorescent mark again; In two sample experiments, come two RNA samples of mark with two different markers; Hybridization utilizes the Circulation of micro circulation pump to spend the night at micro-fluid chip to carry out hybridization, on micro-fluid chip, every detection probes is all complementary by a chemically modified nucleoside acid encoding section or complementary and spacer that is comprised of polyoxyethylene glycol forms with other RNA, and detection probes is all used PGR video picture reagent chemical method to carry out original position to synthesize; The hybridization melting temperature(Tm) is to carry out balance by the detection probes of chemically modified, the 100 μ L 6xSSPE damping fluid 0.90M NaCl that contain 25% methane amide are used in hybridization, 60mM Na2HPO4,6mM EDTA, pH 6.8, and hybridization temperature is 34 ℃, detect the specific Cy3 of applying marking and Cy5 fluorescence dye after the hybridization, utilize laser scanner collection hybridization image and use the Array-Pro image analysis software to carry out the image digitazation conversion, therefrom filter out the most significantly miR-150 of difference.
3. application according to claim 1 is characterized in that, the described compound that screens molecule miR-150 sensitivity that is applied as.
4. application according to claim 3 is characterized in that, described be applied as with to the compound of molecule miR-150 sensitivity for the preparation of the medicine that detects large bowel cancer.
5. application according to claim 4 is characterized in that, the serve as reasons test kit of detection large bowel cancer that the compound of molecule miR-150 sensitivity is formed as activeconstituents and pharmaceutical carrier of described medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105363042A (en) * | 2015-10-21 | 2016-03-02 | 苏州圣诺生物医药技术有限公司 | Medicine composition and application thereof |
| CN112553334A (en) * | 2014-06-13 | 2021-03-26 | 东丽株式会社 | Colorectal cancer detection kit or device and detection method |
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2012
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Non-Patent Citations (3)
| Title |
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| MA Y等: "miR-150 as a potential biomarker associated with prognosis and therapeutic outcome in colorectal cancer", 《GUT.》 * |
| 杨长成等: "大肠癌相关microRNA 研究进展", 《中国肿瘤临床》 * |
| 邹健等: "MicroRNA在人结肠癌干细胞中的表达谱", 《世界华人消化杂志》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112553334A (en) * | 2014-06-13 | 2021-03-26 | 东丽株式会社 | Colorectal cancer detection kit or device and detection method |
| CN105363042A (en) * | 2015-10-21 | 2016-03-02 | 苏州圣诺生物医药技术有限公司 | Medicine composition and application thereof |
| CN105363042B (en) * | 2015-10-21 | 2022-05-06 | 圣诺生物医药技术(苏州)有限公司 | Pharmaceutical composition and application thereof |
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