CN108085385A - A kind of colorectal cancer biomarker and its application - Google Patents
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Abstract
The present invention relates to a kind of colorectal cancer biomarker, the biomarker is miR 92a；The present invention also provides a kind of for diagnosing the kit of colorectal cancer, it includes above-mentioned colorectal cancer biomarkers, further include PCR and react common enzyme and reagent；The present invention still further provides above-mentioned colorectal cancer biomarker and is preparing for the application in diagnosis of colorectal carcinoma drug and inhibiting the application in colorectal cancer neonate tumour blood vessel drug preparing.Colorectal cancer biomarker provided by the invention high expression in colorectal cancer cell and tissue, it is closely related with neonate tumour blood vessel increase, its biological function for promoting colorectal cancer angiogenesis can be played by inhibiting the expression of PTEN.
Description
Technical field
The present invention relates to be related to field of biomedicine technology more particularly to a kind of colorectal cancer biomarker and its to knot
The carcinoma of the rectum diagnoses and inhibits the application in colorectal cancer neonate tumour blood vessel.
Background technology
Colorectal cancer (colorectal cancer, CRC) is the common alimentary system malignant tumour of the mankind, and incidence occupies
The 3rd of malignant tumour, onset concealment, the poor river of prognosis.Research in recent years finds that microRNAs (miRNAs) is a kind of unique
Non-coding tiny RNA, high expression or low expression play the role of oncogene or tumor suppressor gene in tumour cell, make cell Proliferation
It is out of hand with breaking up, lead oncogenic occurrence and development.Research confirms that miR-92a is in the kinds of tumors tissue such as colorectal cancer
Overexpression, and it is related to the lymphatic metastasis of colon cancer and prognosis, and therefore, miR-92a is considered as a kind of oncogene, can
Key regulatory effect can be played in CRC occurrence and development.It is the substantially raw of malignant tumour with non-controllable angiogenesis ability
One of object feature, growth, invasion and attack and the transfer of tumour are closely related with angiogenesis.The study found that miR-92a and blood vessel
Endothelial cell is formed, neonate tumour blood vessel is related.However, at present in relation to miR-92a to the shadow of colorectal cancer angiogenesis function
It rings and its regulatory mechanism is unclear.
The present invention analyzes itself and neonate tumour blood vessel by detecting expressions of the miR-92a in Colorectal Carcinoma
Correlation；By transfecting miR-92a-mimic, inhibitor up-regulation or inhibiting in colorectal cancer cell HCT116, SW620
The expression of miR-92a, influences of the observation miR-92a to HUVEC vascular endothelial cell new life functions and to potential downstream
The regulating and controlling effect of target spot PTEN expression, inquiring into miR-92a influences effect and the mechanism of colorectal cancer angiogenesis, is colorectal cancer
Molecular targeted therapy provide experimental basis.
The content of the invention
In view of problems of the prior art, it is an object of the invention to provide a kind of colorectal cancer biomarker and
It is to diagnosis of colorectal carcinoma and inhibits the application in colorectal cancer neonate tumour blood vessel.
For this purpose, the present invention uses following technical scheme：
In a first aspect, the present invention provides a kind of colorectal cancer biomarker, the biomarker is miR-92a.
The present invention has detected attached Shenzhen South Mountain hospital of Guangdong Medical College in June, 2014 to 2015 using qRT-PCR methods
Year December 25 Colorectal Carcinomas and corresponding cancer beside organism through surgery excision and 4 kinds of colorectal cancer cells (HCT116,
SW620, SW480, HT29) in miR-92a expression；Immunohistochemical method detects CD31 sun in colorectal cancer and cancer beside organism
Property expression microvessel density (microvessel density, MVD), Pearson correlation analysis inquire into miR-92a expression
With the correlation of neonate tumour blood vessel MVD.By transfecting miR-92a-mimic, inhihitor up-regulations or inhibition colorectal cancer are thin
The expression of miR-92a in born of the same parents HCT116, SW620, using the different expression of small tube formation assay detection miR-92a to HUVEC
The influence that tubule is formed, Western blot detect the influence to the protein expression of potential target spot PTEN downstream.The result shows that：Knot
The expression of rectum cancer tissue miR-92a is significantly higher than corresponding cancer beside organism (P<0.01)；4 kinds of human colon cancer cell line miR-
The expression of 92a is all remarkably higher than normal intestinal epithelial tissue (P<0.05)；Colorectal Carcinoma CD31 positive microvessel densitys
It is significantly higher than cancer beside organism (P<0.01), miR-92a expressions and colorectal cancer angiogenesis MVD are in notable positive correlation (r=
0.580, P=0.01)；It is small tubular that the HCT116 cell culture supernatants of up-regulation miR-92a expression can remarkably promote HLJVEC
Into (P<0.05)；Up-regulation miR-92a expression can significantly inhibit pten protein expression (P in HCT116 cells<0.01).Cause
This draws：MiR-92a high expression in colorectal cancer cell and tissue, increases closely related with neonate tumour blood vessel；miR-92a
The biological function of promotion colorectal cancer angiogenesis can be played by inhibiting the expression of PTEN.
According to the present invention, the colorectal cancer biomarker miR-92a, can reflect in colorectal cancer patients and
Differential expression in healthy person embodies high sensitive and high specific to diagnosis of colorectal carcinoma.
Second aspect, the present invention also provides a kind of for diagnosing the kit of colorectal cancer, which includes and knot
The relevant miRNA marker of the carcinoma of the rectum；The marker is the combination of miR-16, miR-21 and miR-92a.
In kit provided by the present invention for diagnosing colorectal cancer, except including above-mentioned colorectal cancer biomarker
Outside, further include PCR and react common enzyme and reagent.
Specifically, also containing PCR enzymes, lysate, TE buffer solutions, ultra-pure water and Marker etc. in kit, wherein splitting
Solution liquid for example can be by 50mM Tris-base, and EDTA 2mM, pH are the triton X- of 8.0 and lysate total volume 5%
The mixed solution of 100 compositions；PCR enzymes for example can the Tris-base containing 10mM, the MgCl of the KCl of 50mM, 3mM2、25mM
DNTP Mixture and 0.5U Taq DNApolymerase/ μ l.
The present invention does not do particular determination for other reagents in kit in addition to colorectal cancer biomarker, this
Field technology personnel can make choice according to actual needs.
The third aspect, the present invention also provides the colorectal cancer biomarkers according to first aspect to be used in preparation
Application in diagnosis of colorectal carcinoma drug.
Fourth aspect, the present invention also provides the colorectal cancer biomarkers according to first aspect to be used in preparation
Inhibit the application in colorectal cancer neonate tumour blood vessel drug.
According to the present invention, expression of the colorectal cancer biomarker miR-92a in Colorectal Carcinoma and cell
Level is significantly higher than corresponding cancer beside organism (P<And normal intestinal epithelial tissue (P 0.01)<0.05)；Colorectal Carcinoma CD31 is positive
Microvessel density is significantly higher than cancer beside organism (P<0.01), miR-92a expressions are in notable with colorectal cancer angiogenesis MVD
Positive correlation (r=0.580, P=0.01)；Therefore miR-92a high can be expressed in colorectal cancer cell and tissue, and and tumour
Angiogenesis increase is closely related, can be as colorectal cancer new molecular marker and therapy target.
Compared with prior art, the present invention at least has the advantages that：
Colorectal cancer biomarker miR-92a provided by the invention not only has height in Colorectal Carcinoma and cell
Expression, and increase molecular marker and therapeutic target closely related, therefore that colorectal cancer can be become with neonate tumour blood vessel
Point is so as to fulfill the diagnose and treat to colorectal cancer.
Description of the drawings
Fig. 1 is expressions of the miR-92a in Colorectal Carcinoma and cell, and wherein Fig. 1-A are miR-92a straight in knot
Intestinal cancer tissue is compared with the expression in cancer beside organism, and Fig. 1-B are miR-92a in colorectal cancer cell and normal intestinal epithelial tissue
Expression comparison；
Fig. 2 is Colorectal Carcinoma miR-92a expression and the correlation of neonate tumour blood vessel, and wherein Fig. 2-A are CRC and neighbour
Capilary in nearly tissue is (left：CRC is organized；It is right：The adjacent tissue of tumour；Arrow：The CD31 positive tables of capilary and mark
It reaches, IHC × 400)；Fig. 2-B are CD31 positives microvessel density (MVD) and the comparison in adjacent tissue in CRC；**P<0.01；
Fig. 2-C are the expression correlation of miR-92a expression and MVD in CRC tissues；
Fig. 3 is transfection miR-92a mimic, inhibitor up-regulations or inhibits colorectal cancer cell miR-92a expression water
It is flat；
Fig. 4 is the influence of colorectal cancer cell miR-92a high expression or inhibition expression to HUVEC tubule Forming abilities,
Middle Fig. 4-A are human umbilical vein endothelial cells (HUVEC) in the condition training from the HCT116 cells being overexpressed with miR-92a
The pipe supported in base is formed；Fig. 4-B are human umbilical vein endothelial cells (HUVEC) from the SW620 being overexpressed with miR-92a
Pipe in the conditioned medium of cell is formed；Fig. 4-C are the sealing blood vessels numbers in Fig. 4-A；Fig. 4-D are the closings in Fig. 4-C
Number of blood vessel；*P<0.05；
Fig. 5 is the influence of miR-92a high expression or inhibition expression to the expression of colorectal cancer cell pten protein, wherein, figure
5-A is the combination by miR-92a and phosphatase and tensin homologue (PTEN) 3'UTR of TargetScan software predictions
Site；Fig. 5-B are that the opposite miR-92a in the CRC cells transfected with miR-92a analogies is expressed and by quantitative real time aggregation
The inhibitor that enzyme chain reaction measures；Fig. 5-C are to measure pten protein by western blot to express；Fig. 5-D are to use miR-92a
The relative expression of pten protein in analogies and the CRC cells of inhibitor transfection；*P<0.05, * * P<0.01.
The present invention is described in more detail below.But following examples is only the simple example of the present invention, not generation
Table or limitation the scope of the present invention, protection scope of the present invention are subject to claims.
Specific embodiment
Technical solution to further illustrate the present invention below with reference to the accompanying drawings and specific embodiments.
For the present invention is better described, technical scheme is readily appreciated, of the invention is typical but non-limiting
Embodiment is as follows：
Embodiment 1
1 materials and methods
1.1 material
1.1.1 tissue specimen, which is collected, passes through attached Shenzhen South Mountain hospital of Guangdong Medical College in June, 2014 in December, 2015
The colorectal cancer of surgery excision and corresponding cancer beside organism are (away from borderline tumor>4cm) sample 25.Wherein male 12, women 13
Example；Age 32-84 Sui, average age (59.2 scholar 13.1) year.It is selected in the equal informed consent of patient and by the court's Ethic review
Committee's examination and approval, preoperative to be treated without chemicotherapy, Operated Specimens are preserved respectively in liquid nitrogen or neutral formalin.
1.1.2 cell line Human colorectal cancer cells HCT116, SW620, HT29, SW480 are gifted by Hong Kong Chinese University,
Human umbilical vein endothelial cells HUVEC is gifted by Zhongshan University.
1.1.3 main agents TRIzoI Reagent, Lipofectamine2000, RNA reverse transcriptions and amplification kit
It is purchased from Life Technologies companies of the U.S.；Reverse transcriptase primer, miR-92a sense primers and anti-sense primer, miR-92a-
3p mimic and miR-92a-3p inhibi-for and corresponding negative control are closed by Guangzhou Ribo Bio Co., Ltd.
Into；DMEM in high glucose culture medium is purchased from HyClone companies of the U.S., Human umbilical vein endothelial cells Human Endothelial SFM trainings
Base and the addition factor are supported purchased from LifeTechnologies companies of the U.S., Matrigel matrigels are purchased from U.S. company BD；Rabbit-anti
People PTEN monoclonal antibodies (ab154814, clone number：EPR7495 Abcam companies of Britain) are purchased from；Goat-anti rabbit secondary antibody (111-
035-003) it is purchased from Jackson companies of the U.S.；Rabbit-anti people GAPDH monoclonal antibodies (10494-1-AP) are public purchased from U.S. Proteintech
Department；The anti-human CD31 monoclonal antibodies of mouse (ZM-0044, clone number：1A10) it is purchased from Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge；ABC exempts from
Epidemic disease High iron-diamine method box (PIE-6102) is purchased from Vector companies of the U.S..
1.2 method
1.2.1 it is complete to be incubated at DMEM in high glucose by cell culture colorectal cancer cell HCT116, SW620, HT29 and SW480
Culture medium, Human umbilical vein endothelial cells HUVEC are formed complete using Endothelial SFM basal mediums addition growth factor
Full medium culture, 37 DEG C, 5%CO2Culture is stablized 2~3 generations of passage, and growth period cell of taking the logarithm carries out subsequent experimental.
1.2.2 qRT-PCR methods detection miR-92a expression by TRIzoI reagents specification extraction Colorectal Carcinoma and
Total serum IgE in cell sets reverse transcription condition according to RNA Reverse Transcriptase kits specification.It is inverse that each sample takes 1 μ g total serum IgEs to carry out
Responsive transcription, reaction condition are 42 DEG C of 60min, 70 DEG C of 10min.1 μ L cDNA is taken to carry out PCR amplification, reaction condition is 50 DEG C
2min, 95 DEG C of 10min, 95 DEG C of denaturation 15s, 60 DEG C of annealing extension 1min, 40 cycle.MiR-92a expression is using exhausted in tissue
To quantitative approach, standard curve and regression equation are established according to standard items, calculates the concentration of miR-92a.MiRNAs is quantitatively carried out
Standardization, as a result in total serum IgE/μ g miRNA, content represents (fmol/ μ g total serum IgEs).The RNA extracted in cell using U6 as
Internal reference calculates the relative expression quantity of each group miR-92a, and experiment is repeated 3 times, and calculates average value.
1.2.3 the dehydration of immunohistochemical method detection Colorectal Carcinoma angiogenesis situation tissue specimen routine, stone
Wax embeds, and makes 4 μm of sections.Hot high pressure reparation is carried out using citrate buffer, primary antibody is the anti-human CD31 monoclonal antibodies (1 of mouse:
100), using PBS primary antibody is replaced, using known positive piece as positive control, endothelial cell to be detected according to ABC methods as negative control
CD31 is expressed.Angiogenesis situation is represented with microvessel density (mierovessel density, MVD), with CD31 positive expressions
Brown color vascular endothelial cell or cell cluster represent 1 individual capilary, first select blood vessel high under low power lens (× 100)
Density region randomly selects 5 visuals field under high power lens (× 400), calculates its capilary number average value.
1.2.4 cell transfecting growth period Human colorectal cancer cells HCT116 and SW620 cell of taking the logarithm instantaneously is turned
Dye.Cell inoculation is in 6 orifice plates, and culture cell to degrees of fusion is up to 60% or so.Experimental group is separately added into 50nM miR-92a
The miR-92a inhibitor of mimic and 100nM and 5 μ L transfection reagents；Control group is separately added into corresponding negative control
(negative control, NC).L × Opti-MEM culture mediums adjust final volume and cultivate 6h to 2mL, replace culture medium and continue to train
It supports.Each group cell is collected after cell transfecting 48h for extracting RNA, cell protein and free serum culture supernatant are collected after transfecting 72h
Liquid (conditioned medium) is used for subsequent experimental.
1.2.5 small 96 orifice plate of tube formation assay spreads 50 μ L Matrigel matrigels, inoculation 1 × 10 per hole4A HUVEC,
For 24 hours, experimental group, control group are separately added into corresponding transfection miR-92a mimic, the HCT116 of inhibitor collected for culture
With SW620 cell conditioned mediums 100 μ L, 37 DEG C, 5%CO2It is cultivated in environment, 6h is after micro- Microscopic observation, HUVEC growths
The newborn tubule of conduct 1 for forming closed hoop tubule like structure is counted.Every group sets 3 multiple holes, and 3 visual field meters are taken per hole
Calculate average value.
1.2.6 Western blot detection PTEN expression prepares 10%SDS-PAGE gels, and 30 μ g albumen samples are added in per hole
Product, through SDS electrophoresis 1h protein isolates, electricity of wet process turns pvdf membrane 3h, and 5% skim milk closing 1h adds in rabbit-anti people's PTEN primary antibodies
(1:2000), in 4 DEG C of overnight incubations, TBST is rinsed 3 times, 10min/ times.Add in goat-anti rabbit secondary antibody (1:4000), room temperature is incubated
1h, TBST are rinsed 6 times, 5min/ times.ECL developer solutions are added dropwise, are imaged using chemiluminescence imaging instrument, measure purpose band gray scale
Value, analysis protein expression, pten protein relative expression quantity is calculated by internal reference of GAPDH.
1.3 statistical method
Statistical analysis is carried out using 19.0 softwares of SPSS, measurement data result is represented with x scholar s, is used
Kolmogorov-Smirnov methods of inspection carry out data test of normality, for meeting normal distribution data, equal value difference between two groups
Different comparison is examined using t, and Colorectal Carcinoma miR-92a is expressed uses Pearson correlations with the correlation of angiogenesis MVD
Analysis, with P<0.05 has statistical significance for difference.
2 results
Expression of 2.1 miR-92a in Colorectal Carcinoma and cell
The expression of the cancerous tissue (T) of colorectal cancer patients and cancer beside organism (TAT) miR-92a corresponding thereto
Shown in (Figure 1A).The miR-92a expressions of 25 CRC tissues are (1.102 scholar 0.735) (fmol/ μ g total serum IgEs), significantly high
In cancer beside organism ((0.037 scholar 0.031) (fmol/ μ g total serum IgEs), difference have statistical significance (t=7.405, P<
0.01).With the conspicuous membrane tissue (normal tissue, NT) of normal Colon and rectum epithelium to compare, miR-92a in 4 kinds of CRC cell lines
Expression be all remarkably higher than normal control, difference is respectively provided with statistical significance (P<0.05).Wherein HCT116 cells miR-
92a expression is minimum, SW620 cells miR-92a expression highests (Figure 1B).
2.2 miR-92a express the correlation with colorectal cancer angiogenesis MVD
Immunohistochemical method dyeing detect 25 Colorectal Carcinomas and corresponding blood vessel in tissue adjacent new life situation (Fig. 2-
A, Fig. 2-B and Fig. 2-C).The results show that the MVD of CRC cancerous tissue CD31 positive expressions is (27.400 scholar 7.560), it is significantly higher than
Corresponding cancer beside organism's MVD quantity (10.760 scholar 3.140), difference have statistical significance (t=9.829, P<0.01).
Pearson Correlation analysis showeds, the expression of miR-92a and the microvessel density of CD31 positive are in significantly in Colorectal Carcinoma
Positive correlation (r=0.580, P=0.01).
The cell model that 2.3 miR-92a high expressed and inhibited expression is established
Respectively miR-92a is selected to express relatively minimum, highest HCT116, SW620 cell, by transiently transfecting miR-
92a-mimic, inhibitor raise or inhibit the expression of its miR-92a, as a result shown (Fig. 3) and negative control group ratio
Compared with, after HCT116 cell transfecting miR-92a mimic 48h, level significantly rise (t=7.439, the P of miR-92a<
0.05), after SW620 cell transfectings miR-92a inhibitor 48h, the level of miR-92a significantly reduces (t=-
45.916 P<0.01).
2.4 colorectal cancer cell miR-92a high express or inhibit influence of the expression to HUVEC tubule Forming abilities
Tubule forms experimental result and shows, using transfection miR-92a mimic up-regulation HCT116 cells, niR-92a expression
Conditioned medium effect 6h, HUVEC form the complete quantity for being closed tubule apparently higher than control group (t=5.563, P<
0.05)；On the contrary, application transfection miR-92a inhibitor inhibit SW620 cells, the conditioned medium effect of miR-92a expression
6h, HUVEC form the quantity for being closed tubule significantly lower than control group (t=-6.107, P<0.05, Fig. 4-A, Fig. 4-B, Fig. 4-C and
Fig. 4-D).
2.5 miR-92a high express or inhibit influence of the expression to the expression of colorectal cancer cell pten protein
The target gene of miR-92a is predicted through Targetsean softwares, the Seed Sequences of the results show miR-92a
There is targeting binding site (Fig. 5-A) between PTEN genes 3'UTR, prompt the regulation and control target gene that PTEN may be miR-92a.
Transfection miR-92a mimic make HCT116 cells miR-92a significantly raise, and pten protein band gray value declines 26.40%
(t=-15.176, P<0.01)；Transfection miR-92a inhibitor make miR-92a expressions in SW620 cells significantly drop
Low, pten protein band gray value raises 21.98% (t=10.911, P<0.01, Fig. 5-B, 5-C, 5-D).As a result prompt
The expression of miR-92a is expressed negatively correlated with pten protein.
It can thus be seen that the present invention is further predicted by analysis of biological information finds that PTEN may be miR-92a's
Downstream regulatory target gene.Known PTEN is inhibition molecule important on PI3K/AKT signal paths, can be by mediating VEGF's
Expression participates in the adjusting process of angiogenesis, and the angiogenesis that can promote tumour is lowered in the missing of PTEN or expression；The present invention
It draws, expression and the pten protein expression of colorectal cancer cell miR-92a is negatively correlated, prompts miR-92a that may pass through
The expression for inhibiting PTEN plays regulating and controlling effect, promotes the angiogenesis of colorectal cancer.
In conclusion miR-92a high expression in Colorectal Carcinoma, increases closely related with cancerous tissue angiogenesis.
MiR-92a can play the biological function of promotion colorectal cancer angiogenesis by inhibiting the expression of PTEN.Therefore, miR-92a
Colorectal cancer new molecular marker and therapy target can be become, so as to fulfill the diagnose and treat to colorectal cancer.
Applicant states that the present invention illustrates the detailed construction feature of the present invention by above-described embodiment, but the present invention is simultaneously
Above-mentioned detailed construction feature is not limited to, that is, does not mean that the present invention has to rely on above-mentioned detailed construction feature and could implement.Institute
Belong to those skilled in the art it will be clearly understood that any improvement in the present invention, to the equivalence replacement of component selected by the present invention
And the increase of accessory, selection of concrete mode etc., it all falls within protection scope of the present invention and the open scope.
The preferred embodiment of the present invention has been described above in detail, still, during present invention is not limited to the embodiments described above
Detail, within the scope of the technical concept of the present invention, a variety of simple variants can be carried out to technical scheme, this
A little simple variants all belong to the scope of protection of the present invention.
It is further to note that the specific technical features described in the above specific embodiments, in not lance
In the case of shield, can be combined by any suitable means, in order to avoid unnecessary repetition, the present invention to it is various can
The combination of energy no longer separately illustrates.
In addition, various embodiments of the present invention can be combined randomly, as long as it is without prejudice to originally
The thought of invention, it should also be regarded as the disclosure of the present invention.
Claims (6)
1. a kind of colorectal cancer biomarker, which is characterized in that the biomarker is miR-92a.
2. colorectal cancer biomarker according to claim 1, which is characterized in that the biomarker is in Colon and rectum
High expression, related to neonate tumour blood vessel increase in cancer cell and tissue.
3. a kind of kit for being used to diagnose colorectal cancer, which is characterized in that the kit includes colorectal cancer biological marker
Object；
The biomarker is miR-92a.
4. kit according to claim 3, which is characterized in that the kit further include for PCR reaction enzyme and
Reagent.
5. colorectal cancer biomarker according to claim 1 or 2 is in preparing for diagnosis of colorectal carcinoma drug
Using.
6. colorectal cancer biomarker according to claim 1 or 2 is being prepared for inhibiting colorectal cancer tumor vessel
Application in rebirth medicine.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110007090A (en) * | 2019-03-28 | 2019-07-12 | 清华大学深圳研究生院 | One kind novel molecular diagnosis marker relevant to colon cancer and application thereof |
| CN114107514A (en) * | 2022-01-27 | 2022-03-01 | 天津欧德莱生物医药科技有限公司 | miRNA molecular marker for colorectal cancer diagnosis and kit thereof |
-
2016
- 2016-11-22 CN CN201611046191.0A patent/CN108085385A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110007090A (en) * | 2019-03-28 | 2019-07-12 | 清华大学深圳研究生院 | One kind novel molecular diagnosis marker relevant to colon cancer and application thereof |
| CN114107514A (en) * | 2022-01-27 | 2022-03-01 | 天津欧德莱生物医药科技有限公司 | miRNA molecular marker for colorectal cancer diagnosis and kit thereof |
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