CN103710451A - Application of PIK3C2G in evaluation and detection kit for curative effect of colorectal cancer chemotherapy - Google Patents
Application of PIK3C2G in evaluation and detection kit for curative effect of colorectal cancer chemotherapy Download PDFInfo
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Abstract
The invention relates to the fields of molecular biology and phymatology, and discloses an application of a biomarker associated with a colorectal cancer. The marker is PIK3C2G, and particularly, deoxyribonucleic acid (DNA) of the marker is inversely related to survival time after colorectal cancer occurrence and chemotherapy of a sufferer relative to copy number variation. The invention also relates to a primer pair, a kit and a method for detecting the PIK3C2G in a colorectal cancer, the primer pair for detecting the PIK3C2G and a primer pair for detecting a reference gene reduced glyceraldehyde-phosphate dehydrogenase(GAPDH) are contained in the kit of the PIK3C2G. An upstream primer base sequence is shown in SEQ ID NO:1. The primer can be applied to detection of the copy number variation of the PIK3C2G through polymerase chain reaction (PCR) amplification, so that detection of the colorectal cancer and evaluation of the curative effect of oxaliplatin (FOLFOX) chemotherapy of the colorectal cancer through the PIK3C2G become possible.
Description
[technical field]
The invention belongs to molecular biology and oncobiology field, relate to a kind of colorectal cancer gene marker and application thereof, meanwhile, relate to the primer pair, test kit and the method that in colorectal cancer, detect PIK3C2G.
[background technology]
Colorectal cancer is one of the most common malignant tumour, and the sickness rate ascendant trend of CHINESE REGION is fairly obvious, occupies the 3rd of whole malignant tumour, serious threat human health.
Along with the development of molecular analysis methods, various new diagnoses and treatments and new medicine continue to bring out, and the result for the treatment of of colorectal cancer has clear improvement.Yet although traditional clinical pathological factors can provide certain curative effect information for colorectal cancer patients, these risk factors can not be quantized well and integrate the degree of colorectal cancer influence prognosis.For individual patients, still seriously rely on traditional histopathology and clinical examination existing neoplasm staging, not high to the evaluation accuracy of curative effect.For this reason, clinical study is badly in need of can easy method of carrying out exactly prognosis and outcome prediction, for the postoperative medication of colorectal cancer patients provides important references.
Phosphatidylinositol 3-kinase (phosphatidylinositol3-kinase, PI3K) family is a proteinoid kinases, participate in regulatory mechanism (the Franke TF in various kinds of cell, Kaplan DR, Cantley LC (1997) PI3K:downstream AKTion blocks apoptosis.Cell88:435-437).PI3K is catalysis phosphatidylinositols (phosphatidylinositol specifically, PI) 3 hydroxyl phosphorylations on ring, the corresponding inositol fat material producing, as 3, [PI (3 for 4-bisphosphate phosphatidylinositols, 4) P2] and 3, 4, [PI (3 for 5-triphosphoric acid phosphatidylinositols, 4, 5) P3] as second messenger in conjunction with and activate multiple target protein, form a signal cascade mixture, thereby regulate the propagation of cell, differentiation, (the Katso R such as survival and migration, Okkenhaug K, Ahmadi K, White S, Timms J, et al. (2001) Cellular function of phosphoinositide3-kinases:implications for development, homeostasis, and cancer.Annu Rev Cell Dev Biol17:615-675).PI3K family is according to substrate be divided into I type, II type and III types different from catalytic subunit.Wherein, I type is with PI (phosphatidylinositol), PIP (Phosphatidylinositol4-phosphate) and PIP2 (Phosphatidylinositol4,5-bis-phosphate) be substrate, it is substrate that II type be take PI and PIP, III type PI3K be take PI as substrate (Djordjevic S, Driscoll PC (2002) Structural insight into substrate specificity and regulatory mechanisms of phosphoinositide3-kinases.Trends Biochem Sci27:426-432).I type PI3K can be further divided into two subclass, by the heterodimer that regulates one of subunit p85 and catalytic subunit p110 α/β/δ to form, it is IA type, the heterodimer that regulates subunit p101 and catalytic subunit p110 γ to form is IB type, and the two has different activate mechanisms and effect in cell.The catalytic subunit of I type and III type forms heterodimer with regulating subunit, and II type does not form heterodimer, has unique C2 structural domain.
Much research in human tumor shows, overexpression often occurs the PI3K of I type, strengthens PI3K catalytic activity, increases the synthetic amount of its albumen simultaneously, promotes cell carcinogenesis, with to become cancer to transform relevant.In colorectal cancer, PIK3CA gene expression amount over 25% increases (Samuels Y, Wang Z, Bardelli A, Silliman N, Ptak J, et al. (2004) High frequency of mutations of the PIK3CA gene in human cancers.Science304:554), the PIK3CA that shows IA type play an important role in tumor development (Vivanco I, Sawyers CL (2002) The phosphatidylinositol3-Kinase AKT pathway in human cancer.Nat Rev Cancer2:489-501).The PI3K of II type is structurally similar to I type, it lacks p85/p101 and additionally has PX and C2 structural domain, mainly be distributed in tenuigenin differently from I type, the PI3K of II type is mainly distributed in (Fry MJ (2001) Phosphoinositide3-kinase signalling in breast cancer:how big a role might it play Breast Cancer Res3:304-312) in membrane structure.
II type PI3K family member comprises PIK3CG and PIK3C2G, studies have found that PIK3CG lacks in colorectal cancer sample and clone, cross and express this gene and can suppress tumour (Sasaki T occurs, Irie-Sasaki J, Horie Y, Bachmaier K, Fata JE, et al. (2000) Colorectal carcinomas in mice lacking the catalytic subunit of PI (3) Kgamma.Nature406:897-902), prompting PIK3CG plays restraining effect in colorectal cancer generating process.PIK3C2G studies have found that it is at ovarian cancer (Lambros MB at present, Fiegler H, Jones A, Gorman P, Roylance RR, et al. (2005) Analysis of ovarian cancer cell lines using array-based comparative genomic hybridization.J Pathol205:29-40) and carcinoma of the pancreas (Harada T, Chelala C, Bhakta V, Chaplin T, Caulee K, et al. (2008) Genome-wide DNA copy number analysis in pancreatic cancer using high-density single nucleotide polymorphism arrays.Oncogene27:1951-1960) significantly amplification in.What is interesting is, PIK3C2G is suppressed by Bcr-Abl in leukemia cell's middle expression of going back to the nest, point out its expression inhibiting (Yu W relevant to cell migration, Sun X, Tang H, Tao Y, Dai Z (2010) Inhibition of class II phosphoinositide3-kinase gamma expression by p185 (Bcr-Abl) contributes to impaired chemotaxis and aberrant homing of leukemic cells.Leuk Lymphoma51:1098-1107), prompting PIK3C2G has the possibility that becomes potential tumor markers, but, what whether PIK3C2G had a biomarker in colorectal cancer may be not studied with concrete application.
[summary of the invention]
Primary and foremost purpose of the present invention is to confirm whether the relative copy number of PIK3C2G changes relevant to colorectal cancer FOLFOX chemotherapeutic efficacy, secondly, invents primer pair and the test kit of a kind of PIK3C2G of detection, then by PIK3C2G, made and detected human colorectal test kit.
To achieve these goals, first invent the primer pair of a kind of PIK3C2G of detection: upstream primer base sequence is as shown in SEQ ID NO:1; Downstream primer base sequence is as shown in SEQ ID NO:2.
The present invention also comprises the test kit of a kind of PIK3C2G of detection, contains above-mentioned primer pair.
The present invention also comprises a kind of test kit that detects colorectal cancer, the primer pair that contains above-mentioned detection PIK3C2G, and for detection of the primer pair of reference gene GAPDH.
The primer pair of described reference gene GAPDH is: upstream primer, as shown in base sequence SEQ ID NO:3; Downstream primer, as shown in base sequence SEQ ID NO:4.
The present invention also comprises the method for PIK3C2G of detection a kind of, comprises the extraction step of DNA; With detecting the primer of the primer pair of PIK3C2G as pcr amplification, carry out pcr amplification; Finally obtain amplified production, and detect judgement.
The PIK3C2G that the present invention finds by research and the relation between colorectal cancer (referring to embodiment), be that the relative copy number of PIK3C2G gene is higher than non-tumour contrast intestinal tissue in Colorectal Carcinoma, therefore, the relative copy number of the PIK3C2G in tumor sample can be used as a mark of colorectal cancer.It is relevant that the present invention also finds that the relative copy number of PIK3C2G increases the lifetime that degree and patient take after FOLFOX chemotherapeutics.With the test kit that this primer pair is designed, can be used in and detect colorectal cancer tumour, make to detect colorectal cancer and judge that FOLFOX curative effect becomes possibility by PIK3C2G.
Because play main antineoplastic active substance in FOLFOX chemotherapy regimen, be 5-FU (5FU) and oxaliplatin, thereby their mode of action is inducing DNA, damage causes apoptosis, therefore, the present invention relates to the index of PIK3C2G as lifetime after the chemotherapeutics of evaluation colorectal cancer patients use inducing DNA damage.
Because DNA copy number reduces, directly cause genetic expression to reduce, therefore, to the present invention also relates to utilize PIK3C2G to express (comprising mRNA and protein level) as evaluating that colorectal cancer occurs and colorectal cancer patients the take chemotherapeutics index of lifetime afterwards.
[accompanying drawing explanation]
Fig. 1 PIK3C2G is as the ROC graphic representation of colorectal cancer mark;
The relative copy number of Fig. 2 PIK3C2G and colorectal cancer patients after FOLFOX chemotherapy lifetime be related to schematic diagram;
The relative copy number of Fig. 3 PIK3C2G is evaluated colorectal cancer patients through FOLFOX chemotherapeutic efficacy schematic diagram.
[embodiment]
The generation essence of tumour is gene amplification, disappearance and the sudden change that chromosome instability causes surely.Therefore, these genes that increase, lack and suddenly change have the potential possibility that becomes tumor markers.Because these genes play an important role simultaneously in tumour generation, resistance and transfer process, therefore, they have the effect that becomes potential drug target spot.
There are some researches show that (Sasaki T appears obviously lacking in PIK3CG in colorectal cancer, Irie-Sasaki J, Horie Y, Bachmaier K, Fata JE, et al. (2000) Colorectal carcinomas in mice lacking the catalytic subunit of PI (3) Kgamma.Nature406:897-902), prompting PIK3CG family plays an important role in colorectal cancer family, there is the possibility that becomes colorectal cancer mark, therefore, the present invention detects the relative copy number difference of colorectal cancer sample and PIK3C2G in check sample by fluorescent quantitative PCR technique, and whether there is significant difference with PIK3C2G copy number in student t test and judge check sample and colorectal cancer sample, then, colorectal cancer patients is carried out to statistics lifetime, by patient be divided into 5 years following and 5 years above two groups, with the relative copy number of two groups of crowd PIK3C2G of student t test and judge, whether have significant difference.Afterwards by the relation of the relative copy number of SPSS software analysis survival of patients phase and PIK3C2G and by logistic regression analysis specificity and susceptibility.
Be below specific embodiments of the invention:
Sample and follow up a case by regular visits to the collection of information
Contriver has collected PATIENTS WITH LARGE BOWEL sample since 2006 from Ruijin Hospital, passes through data compilation and follows up a case by regular visits to, and has therefrom chosen 92 parts of (lifetime below 35 examples 5 years, more than 57 examples 5 years lifetime) III phase colorectal cancer tumour paraffin-embedded tissue samples.
The extracting of paraffin sample DNA
From paraffin-embedded tissue, cut 5 milligrams of samples, put into 2 milliliters of centrifuge tubes, add 1 milliliter of dimethylbenzene, vortex concussion 10 seconds.Under 14000 revs/min of rotating speeds, normal temperature is centrifugal 2 minutes.Abandon supernatant.Add 1 milliliter of absolute ethanol washing precipitation, vortex concussion, under 14000 revs/min of rotating speeds, normal temperature is centrifugal 2 minutes, abandons supernatant.Centrifuge tube is placed in to super clean bench and removes unnecessary alcohol.Add 180 microlitre RTL damping fluids (Qiagen), 20 microlitre Proteinase Ks, mix, and hatch 1 hour for 56 ℃, hatch 1 hour for 90 ℃.Add 200 microlitre AL damping fluids, mix, then add 200 microlitre dehydrated alcohols, mix.Mixed solution is added to QIAamp pillar (Qiagen), centrifugal 1 minute of 8000 revs/min of normal temperature.Open pillar lid, add 500 microlitre AW1 damping fluids (Qiagen), centrifugal 1 minute of 8000 revs/min of normal temperature.Add 500 microlitre AW2 damping fluids (Qiagen), centrifugal 1 minute of 8000 revs/min of normal temperature.Centrifugal 3 minutes desciccator diaphragms of normal temperature under 14000 revs/min of rotating speeds.Add 50 microlitre ATE damping fluids, under 14000 revs/min of rotating speeds, normal temperature is collected DNA for centrifugal 1 minute.
Quantitative fluorescent PCR
Design PCR primer is used for the PIK3C2G that increases, and sequence is shown in SEQ ID NO:1, SEQ ID NO:2.Use 2 * SYBR Green fluorescence dye preparation PCR reaction mixture, sample number and the repeat number of going up as required machine, calculate and prepare PCR reaction mixture, and system is as follows:
| Composition | Volume |
| 2*SYBR?Green | 10μl |
| Primer Mix | 4μM（2.5μl） |
| Template | 20ng（2.5μl） |
| Ultrapure water | 5μl |
| Cumulative volume | 20ul |
Divide and be filled to AXYGEN PCR8 connecting leg, the instantaneous centrifugal PCR system that mixes of microcentrifuge.
Above-mentioned sample is put into IQ5(BioRad) quantitative real time PCR Instrument, SYBR Green method quantitative fluorescent PCR is to analyze the expression of each gene, and PCR programming is as follows:
Denaturation Cycle1:(1X)
Step1:95℃for02:00
PCR circulation Cycle2:(40X)
Step1:95℃for00:15
Step2:60℃for00:20
Step3:72℃for00:20
60 ℃-95 ℃ increase by 0.5 degree per second of solubility curve, gather fluorescence, collect data.
Two couples of qPCR detect primer:
First pair, the primer pair of detection PIK3C2G: upstream primer base sequence is as shown in SEQ ID NO:1; Downstream primer base sequence is as shown in SEQ ID NO:2.
Second pair, the primer pair of reference gene GAPDH is: upstream primer, as shown in base sequence SEQ ID NO:3; Downstream primer, as shown in base sequence SEQ ID NO:4.
Table 1PIK3CG gene is with respect to copy number changing value (the △ Ct=Ct of reference gene GAPDH
pIK3C2G-Ct
gAPDH) and sample survival time (n/d represents that survival time is not definite):
PIK3C2G is as the statistical analysis of colorectal cancer mark
(1) 100 routine samples are divided into two groups: the contrast of inflammatory bowel tissue (93-100) and colorectal cancer sample (1-92).By t, check the relative copy number of PIK3C2G between two groups of samples of comparison whether to have significant difference, result shows between them significant difference.Simultaneously, we originally carry out logistic regression (Logistic regression) to 100 increments, to build the possibility model of the trouble colorectal cancer of each sample of assessment, each sample being obtained by Logic Regression Models is suffered from colorectal cancer probability for generating experimenter's performance curve (ROC curve), as shown in Figure 1.Under curve, AUC area is 1, illustrates that PIK3C2G can become colorectal cancer mark completely.
The relation of existence after the relative copy number of PIK3C2G and colorectal cancer patients FOLFOX chemotherapy
According to the relative copy number of 92 routine colorectal cancer patients PIK3C2G, classify, sub-average patient is first group (Group1), higher than the patient of mean value, is second group (Group2).Utilize KaplanMeier survival curve and Cox Proportional hazards to return the difference of assessment existence.Use IBM SPSS Statistics19 software to carry out above all statistical study.Find that two groups of crowd's survival curves have significant difference (Fig. 2)
KaplanMeier analyzes as shown in Figure 2, and in single factor Cox proportional hazards regression models, PIK3C2G is relative, and copy number is lower, and the survival of patients phase is longer, (95%CI:0.284-0.731, P=0.001).
The relative copy number of PIK3C2G is evaluated colorectal cancer patients through FOLFOX chemotherapeutic efficacy
First 1-92 example sample evidence is divided into two classes lifetime: a class is 5 years more than lifetime; Another kind of is 5 years below lifetime.By the relative copy number of PIK3C2G gene of each sample with carry out experimenter's performance curve (ROC) lifetime and analyze, as shown in Fig. 3 and table 2, area (AUC) value that is experimenter's performance curve below through the performance perameter of the relative copy number of PIK3C2G gene of FOLFOX chemotherapeutic efficacy analysis for assessment of colorectal cancer patients is 0.731, P<0.001, 95% fiducial interval is 0.622-0.841, be greater than 0.5, illustrate that the relative copy number of PIK3C2G gene has good diagnostic value for assessment of colorectal cancer patients through FOLFOX chemotherapeutic efficacy.From ROC curve, select probability threshold value to produce at least 90% specificity.As table 3, estimation range is: the relative copy number of PIK3C2G gene lower than 1.79 for survival the phase higher than the patient of 5 years.
Table 2ROC analytical results
The coordinate of table 3 curve
Assay variable: gene
Claims (9)
- The application of 1.PIK3C2G gene in FOLFOX chemotherapeutical medicine curative effect judgement medicine or test kit.
- 2. application as claimed in claim 1, is characterized in that described chemotherapeutics is the chemotherapeutics of inducing DNA damage.
- 3. application as claimed in claim 2, the chemotherapeutics that it is characterized in that described inducing DNA damage is selected from one or more in the chemotherapeutics of platiniferous class and 5-FU and derivative thereof.
- 4. application as claimed in claim 1, is characterized in that PIK3C2G gene is the biomarker of colorectal cancer, and the relative copy number of its DNA of described mark increases with colorectal cancer and is proportionate; Be negative correlation the lifetime that the relative copy number reduction of DNA and the colorectal cancer patients of described mark carried out after FOLFOX chemotherapy, and described negative correlation is that the relative copy number of PIK3C2G is lower, and lifetime is longer; Copy number is higher relatively, and lifetime is shorter, utilizes described negative correlation to prepare described judgement medicine or test kit.
- 5. application as claimed in claim 4, is characterized in that the relative copy number of described mark reduces the mRNA of PIK3C2G and the reduction of protein level that comprises that relative copy number reduction causes.
- 6. a test kit that detects PIK3C2G, is characterized in that, contains and detects PIK3C2G and reference gene GAPDH primer pair.
- 7. the test kit of detection colorectal cancer as claimed in claim 6, is characterized in that the primer pair of described detection PIK3C2G is:Upstream primer, as shown in base sequence SEQ ID NO:1;Downstream primer, as shown in base sequence SEQ ID NO:2.
- 8. the test kit of detection colorectal cancer as claimed in claim 6, is characterized in that the primer pair of described reference gene GAPDH is:Upstream primer, as shown in base sequence SEQ ID NO:3;Downstream primer, as shown in base sequence SEQ ID NO:4.
- 9. detect a method of PIK3C2G,The extraction step that comprises DNA;It is characterized in that, also comprise:With test kit claimed in claim 6, described DNA is carried out to pcr amplification;Obtain amplified production, and detect judgement.
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| CN104878084A (en) * | 2015-03-04 | 2015-09-02 | 上海市杨浦区中心医院 | Kit for detecting effectiveness of oxaliplatin to colorectal cancer |
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| WO2016112488A1 (en) * | 2015-01-13 | 2016-07-21 | BGI Shenzhen Co.,Limited | Biomarkers for colorectal cancer related diseases |
| CN104878084A (en) * | 2015-03-04 | 2015-09-02 | 上海市杨浦区中心医院 | Kit for detecting effectiveness of oxaliplatin to colorectal cancer |
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| CN105349665A (en) * | 2015-11-27 | 2016-02-24 | 上海锐赛生物技术有限公司 | Kit for evaluating colorectal cancer relapse risk after Oxaliplatin chemotherapy and application thereof |
| CN105349665B (en) * | 2015-11-27 | 2018-06-19 | 上海锐赛生物技术有限公司 | Kit of colorectal cancer risk of recurrence and application thereof after assessment oxaliplatin chemotherapeutic |
| CN111020024A (en) * | 2019-12-05 | 2020-04-17 | 复旦大学附属眼耳鼻喉科医院 | Application of TLR9 |
| CN113774130A (en) * | 2020-06-09 | 2021-12-10 | 碳逻辑生物科技(香港)有限公司 | Biomarkers and methods for selecting a group of chemotherapy responsive patients and uses thereof |
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