CN110438229A - A kind of gastric cancer Prognosis biomarker and its diagnostic kit - Google Patents
A kind of gastric cancer Prognosis biomarker and its diagnostic kit Download PDFInfo
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Abstract
The invention discloses a kind of gastric cancer Prognosis biomarker and its detection kit, the biomarker is tumor microenvironment correlation factor RGS1.The application that the present invention passes through prognostic markers object and detection marker RGS1, it can rapidly, accurately, easily predict gastric cancer prognosis, the hierarchical classification of gastric cancer danger height is realized from molecular level, the patient classification of different prognosis degree is treated significantly, to instruct individuation precisely to treat, medical treatment cost, clinical value with higher have been saved simultaneously.
Description
Technical field
The invention belongs to fields of biomedicine, and in particular to a kind of gastric cancer Prognosis biomarker and its diagnostic reagent
Box, and using the method for marker prediction gastric cancer prognosis and the application of diagnostic kit, more particularly to RGS1 as stomach
The application of cancer prognosis evaluation biomarker and diagnostic kit.
Background technique
Gastric cancer is malignant tumour common in a kind of global range, and prognosis is relatively poor, seriously threatens human health.In
State is the High Risk For Gastric Cancer country, and the morbidity number of cases of gastric cancer and dead number of cases account for global incidence gastric cancer and 42.6% dead He respectively
45.0%, it is the principal disease for seriously endangering Chinese residents health.In recent years, gastric cancer clinical diagnosis and treatment aspect obtain very big
Progress, but due to local recurrence easy after Primary Gastric carcinectomy or transfer, lead to whole 5 annual survival rates of patient relatively
Low, this is also the significant challenge that gastric cancer clinical treatment faces.Studies have shown that stomach organization has very high heterogeneity, it is in addition a
The influence of body difference, the prognosis between different patients have very big difference.In order to judge the state of an illness of patients with gastric cancer in time, according to each
The therapeutic scheme of the prognosis individuation of patient, find simple and effective biomarker for patients with gastric cancer Prognosis for
Clinical treatment has highly important directive significance.
RGS1 encoding histone one kind G-protein signal family regulatory factor, passes through the G α subunit knot in conjunction with the GTP of activation
It closes, serves as GTPase activated protein (GAP), to reduce the signal activity of G-protein, improve conversion ratio of the GTP to GDP.Research
Show that RGS1 may adjust the expression of PD-L1 molecule by MAPK access and PI3K/Akt access in human melanoma cell.
PD1 (programmed death protein 1, apoptosis receptor 1) is the negative immune detection on T cell surface
PD-L1 (Programmed death-ligand 1, the apoptosis ligand of point, specificity factor and tumor cell surface
1) it can inhibit T cell activity after combining, make tumour cell that immunologic escape occur.Research confirmation, what immune and inflammatory mediator was dominated
Microenvironment plays a crucial role in the generation and progress of gastric cancer.In tumor tissues, CD3+T cell quantity is fewer to imply existence
Rate is lower and prognosis has stronger changeability, or even lymphatic metastasis quantity can be made to increase；In addition, about 15% tumour is related
Dead closely related with chronic inflammation or uncontrollable infection, Chronic inflammation can lead to gastric cancer, hepatocellular carcinoma, colorectal cancer etc.
The generation of malignant tumour and related to prognosis.Effect and its expression of the RGS1 in gastric cancer are to patients with gastric cancer prognosis quality
There is not been reported for influence.
It include: at present that application No. is 201210337178.6 granted patents to disclose about the Prognosis marker of gastric cancer
Provide the inhibition expression by detection BCL6B gene diagnose the gastric cancer of individual and determine individual gastric cancer prognosis side
Method.Application No. is 201510092010.7 granted patents to disclose Netrin-4 in preparation detection gastric cancer and prognostic marker
Preparation in purposes, etc..But RGS1 is as gastric cancer Prognosis marker using there is not been reported.It is more smart in order to realize
Quasi- and individuation clinical treatment, needing discovery more can be used for the biomarker of gastric cancer Prognosis and assessment, find more
More high specifics and the molecular marker of sensibility are of great significance to gastric cancer auxiliary diagnosis and prognostic analysis.
Summary of the invention
In view of the drawbacks of the prior art, it the object of the present invention is to provide a kind of gastric cancer Prognosis biomarker and its examines
Disconnected kit.RGS1 is provided as gastric cancer Prognosis marker and its application, is presented in people's clinic stomach organization special
Property height express phenomenon, and the highly expressed patient's prognosis of RGS1 is significantly worse than patient's prognosis of RGS1 low expression in stomach organization, and
With statistical difference, show that RGS1 can characterize the prognosis of patients with gastric cancer with being expressly understood that.
The present invention by prognostic markers object and detect marker RGS1 application, can rapidly, accurately, easily predict stomach
Cancer prognosis realizes the hierarchical classification of gastric cancer danger height from molecular level, distinguishes the patient of different prognosis degree significantly, thus
It instructs individuation precisely to treat, while having saved medical treatment cost, clinical value with higher.
The purpose of the present invention is what is be achieved through the following technical solutions:
In a first aspect, the biomarker is tumour the present invention provides a kind of gastric cancer Prognosis biomarker
Microenvironment correlation factor RGS1.
Second aspect, the present invention provides a kind of tumor microenvironment correlation factor RGS1 as gastric cancer prognostic markers object and inspection
Survey the application of marker.
The third aspect, the present invention provides the primer pair of species-specific amplification biomarker above-mentioned, the primer
To as shown in SEQ ID No.1 and 2.
Fourth aspect, the present invention provides a kind of gastric cancer Prognosis kit, the kit includes reverse transcription reaction
System and qPCR reaction system.
Preferably, in the qPCR reaction system, including primer pair above-mentioned and reference gene primer pair.
Preferably, the reference gene primer pair is as shown in SEQ ID No.3 and 4.
5th aspect detects gastric cancer prognosis biology mark according to the non-diagnostic purpose of kit above-mentioned the present invention provides a kind of
The method of will object, comprising the following steps:
Step 1: extracting the cancerous tissue sample from patients with gastric cancer；
Step 2: using the above-mentioned diagnostic kit for gastric cancer prognosis evaluation, determine the cancer group of patients with gastric cancer in step 1
Knit the expression of RGS1 in sample；
Step 3: the relative expression quantity of gastric cancer Prognosis biomarker RGS1 is obtained by calculation.
The expression of the RGS1 high obtained in the step 3, then prognosis patients with gastric cancer is high mortality；Obtained in step 3
RGS1 low expression, then prognosis patients with gastric cancer is low actual；
Using Kaplan-Meier survivorship curve method analytic approach, variable is expressed as with RGS1, with RGS1 gene relative expression
The median of value is as threshold value.If the expression of RGS1 obtained in step 3 is less than or equal to threshold value, the postoperative life of patients with gastric cancer
The time is deposited greater than 5 years；If the expression of RGS1 obtained in step 3 is higher than threshold value, the patients with gastric cancer post-operative survival rates time is small
In 5 years.
Compared with prior art, the present invention have it is following the utility model has the advantages that
1, discovery tumor microenvironment correlation factor RGS1 can be used as gastric cancer Prognosis marker for the first time, relative to normal group
It knits, is in specificity overexpression phenomenon in gastric cancer tumor, expression quantity level indicates patient's prognosis life cycle；
It 2, can quick, accurate and clear judgement gastric cancer by detecting the expression of the RGS1 of patients with gastric cancer biopsy samples
The prognosis situation of patient provides guidance for the determination of patient treatment protocol.
3, present invention firstly discloses RGS1 as gastric cancer prognostic markers object and detects the application of marker, the marker energy
Enough rapid, accurately, easily prediction gastric cancer prognosis, the Classification and stage of gastric cancer danger level is realized from molecular level, greatly improves stomach
The sensibility and specificity of cancer prognosis detection and assessment.The patient of different prognosis degree is subjected to Classification treatment, is not only directed
Individuation is precisely treated, and has saved medical treatment cost, clinical value with higher.
Detailed description of the invention
Upon reading the detailed description of non-limiting embodiments with reference to the following drawings, other feature of the invention,
Objects and advantages will become more apparent upon:
Fig. 1 is that qPCR method analyzes result to the RGS1 expression difference in gastric cancer tissue sample and control group normal sample；
Fig. 2 is that Kaplan-Meier survivorship curve method analyzes patients with gastric cancer RGS1 expression quantity and the relationship between life cycle
Figure.
Specific embodiment
The following describes the present invention in detail with reference to examples.Following embodiment will be helpful to those skilled in the art
The present invention is further understood, but the invention is not limited in any way.It should be pointed out that those skilled in the art
For, without departing from the inventive concept of the premise, it can also make certain adjustments and improvements.These belong to guarantor of the invention
Protect range.
Embodiment 1:
Fluorescent quantitation qPCR method detects expression quantity of the RGS1 by the Human Stomach Tissue and cancer in sample:
1, RNA is extracted
(1) gastric tissue is ground
The mortar used needed for tissue grinder, grinding pestle, scissors, scalpel, tweezers, spoon are cleaned up, in 60 DEG C
Masking foil is wrapped after baking oven baking 3h, is toasted overnight in 180 DEG C of baking ovens；Carry out tissue grinder before above-mentioned articles be required to by
Liquid nitrogen precooler；The clinical sample for being stored in liquid nitrogen is taken out, the tissue block for cutting soya bean size is put into the mortar being pre-chilled, grinds
Mill, while constantly liquid nitrogen is supplemented in multiple times on a small quantity, until being ground to powdery, this process generally requires 10min-15min；It grinds
After being milled to powdery, suitable pre-cooling TRIzoI is added and continues to grind, so that tissue powder and TRIzol are mixed well；To
TRIzol is transferred in the centrifuge tube of the 1.7mL DNase/RNase Free marked after melting completely, starts to carry out RNA
Extracting is frozen if not extracting immediately in -80 DEG C of ultra low temperature freezers.
(2) extracting and quality inspection of total serum IgE:
The extraction of total serum IgE is carried out using the method for separating RNA in TRIzol specification, the specific steps are as follows:
Centrifuge tube equipped with the tissue by TRIzol cracking is placed in equilibrium at room temperature 5min；Add by every milliliter of TRIzol
Chloroform is added in the ratio of 0.2mL chloroform, covers pipe lid, up and down violent concussion 15sec, stands 5min later, under the conditions of 4 DEG C
12000 × g is centrifuged 15min；Centrifugation can be seen that apparent lamination later, and the careful colourless aqueous phase for drawing supernatant is transferred to
In new 1.7mL DNase/RNase Free centrifuge tube, isopropanol is added in 0.5mL isopropanol/mL TRIzol ratio, on
Under be mixed by inversion, stand 10min at room temperature, 12000 × g is centrifuged 10min under the conditions of 4 DEG C, it is seen that tube bottom has white total
RNA precipitate；It carefully discards supernatant, adds the ratio of 75% ethyl alcohol of 1mL that 75% ethanol washing is added in every milliliter of TRIzol and precipitate, 4
7 500 × g is centrifuged 5min under the conditions of DEG C；Supernatant is sucked, 5min-10min is air-dried, suitable no RNA enzyme water dissolution is added
RNA precipitate, if later not being stored in -80 DEG C using immediately.
After the completion of extraction, prestige amount light splitting luminance meter 2000 is surpassed using Nanodrop, total serum IgE is quantified, use fine jade later
The integrality of sepharose electrophoresis detection RNA, the specific steps are as follows:
The total serum IgE for taking 2 μ L to extract is quantitative with Nanodrop 2000 after dilution appropriate, is made using no RNA enzyme water
Solution when to calibrate.The ratio of the preferable total serum IgE A260/280 of quality should be between 2.0-2.2.Prepare 0.8% fine jade
Sepharose, electrophoretic buffer are 0.5 × TBE, and 120V constant pressure electrophoresis 15min is observed in gel imager later.Completely
RNA sample should show 3 clearly bands in electrophoretogram, from top to bottom respectively represent 28S rRNA, 18S rRNA
With 5S rRNA.
2, the reverse transcription of total serum IgE
Use the PrimeScript of Takara companyTMReverse transcription reagent box carries out reverse transcription, including removal genomic DNA
With two processes of reverse transcription, it is shown that specific step is as follows:
1) genomic DNA in total serum IgE sample is removed, system formulation is as follows:
| 5 × genomic DNA removes buffer | 2μL |
| Genomic DNA remover | 1μL |
| Total serum IgE | 1μg |
| Without RNA enzyme water | Complement to 10 μ L |
| Total volume | 10μL |
It is placed in PCR instrument and is reacted, 42 DEG C of reaction 2min.
2) reverse transcription reaction
Total serum IgE sample after genomic DNA (gDNA) will be removed is divided equally into two pcr pipes, and it is anti-to configure mRNA on ice
Transcription system, system formulation are as follows:
Above-mentioned Oligonucleolide primers and nucleotide random primer are PrimeScriptTMIt is had in reverse transcription reagent box.
It is immediately placed in PCR instrument after soft mixing and carries out reverse transcription reaction, reaction condition are as follows:
45℃ 15min
85℃ 5sec
4℃ 5min
3, fluorescent quantitation qPCR reacts
0.5 μ L reverse transcription product is taken, 19.5 μ L is added to without in RNA enzyme water, dilutes 40 times.Configure qPCR reaction system, body
System is as follows:
| 2 × SsoAdvanced SYBR Green mixed liquor | 5μL |
| CDNA (after dilution) | 1μL |
| Forward primer (10 μM) | 1μL |
| Reverse primer (10 μM) | 1μL |
| Total volume | 10μL |
Rear wink is mixed well from gently attack tube wall is centrifuged again to remove bubble.Be placed on StepOne Plus
In Real-Time PCR System, setting program simultaneously carries out qPCR reaction, and response procedures are as follows:
Gastric cancer sample is each two groups of qPCR of RGS1 and reference gene GAPDH with control group normal sample and (is respectively adopted
Primer pair as shown in SEQ ID No.1 and 2, SEQ ID No.3 and 4), every group 3 parallel to repeat.
4, qPCR data are analyzed
QPCR reaction carries out relative expression quantity analysis, specific calculating process to the initial data (Ct value) of acquisition after completing
It is as follows:
Δ Ct (gastric cancer sample)=Ct (RGS1 in gastric cancer sample)-Ct (the reference gene GAPDH in gastric cancer sample)
Δ Ct (control group normal sample)=Ct (RGS1 in control group normal sample)-Ct is (in control group normal sample
Reference gene GAPDH)
Δ Δ Ct=Δ Ct (gastric cancer sample)-Δ Ct (control group normal sample)
(- Δ Δ Ct) power of relative expression quantity=2 of RGS1
5, result
As a result as shown in Figure 1, the expression quantity (Δ Ct (gastric cancer sample)) of RGS1 is significant in gastric cancer sample is being higher than control group just
Normal sample (Δ Ct (control group normal sample)), difference have statistical difference (p < 0.01).It is raw in conjunction with patients with gastric cancer clinic
Deposit the highly expressed clinical meaning of RGS1 in information analysis cancerous tissue.
The highly expressed clinical meaning of embodiment 2:RGS1
Using R lingware (more new edition 3.5.2), relative expression's magnitude of the RGS1 obtained in above-mentioned qPCR experiment is made
For reference standard, according to the median of RGS1 gene relative expression's magnitude, by 407 patients with gastric cancer be divided into RGS1 high expression group and
RGS1 low expression group.It is expression value median or more when RGS1 expresses data in testing result, then is RGS1 high expression；Detection knot
It is expression value median hereinafter, being then RGS1 low expression that RGS1, which expresses data, in fruit.Using 7.0 software of GraphPad Prism,
In conjunction with its life cycle data, draws Kaplan-Meier survivorship curve analysis result and see Fig. 2.
If the expression of RGS1 obtained in embodiment 1 is less than or equal to threshold value, i.e., in RGS1 gene Relative Expression values
Digit, then the patients with gastric cancer post-operative survival rates time is greater than 5 years；If the expression of RGS1 obtained in embodiment 1 is higher than threshold value,
The patients with gastric cancer post-operative survival rates time was less than 5 years.
As shown in Figure 2, the life cycle that RGS1 high expresses patients with gastric cancer is significantly lower than the existence of RGS1 low expression patients with gastric cancer
Phase, difference have statistical significance (p < 0.01).RGS1 high expression is significant related to the reduction of patients with gastric cancer overall survival,
Prognosis is high mortality, and postoperative recurrence, transfer probability are high, it is proposed that patient is subsequent actively to be checked and treated.RGS1 low expression
Significant related to the raising of patients with gastric cancer overall survival, prognosis is low actual, and postoperative recurrence, transfer probability are low, and prognosis is more
It is optimistic, it is proposed that the subsequent progress regular return visit of patient is checked when necessary and treated.As it can be seen that RGS1 gene phase in stomach organization
It can be used as the foundation for determining poorer prognosis for the expression of normal tissue conspicuousness height.This shows that RGS1 has in gastric cancer prognosis evaluation
It is significant, it can be used as patients with gastric cancer prognostic marker.
Embodiment 3: the preparation of gastric cancer Prognosis kit
According to the correlation of RGS1 and gastric cancer prognosis, gastric cancer prognosis can be predicted by detecting the expression of RGS1,
The present invention provides a kind of diagnostic kits for gastric cancer prognosis evaluation accordingly.It not only include embodiment 1 in the kit
Middle reaction system and the reagent used further include the forward and reverse primer and internal reference of the mrna expression amount of fluorogenic quantitative detection RGS1
GAPDH primer sequence.
Following table is the qPCR primer sequence used in experiment:
Specific embodiments of the present invention are described above.It is to be appreciated that the invention is not limited to above-mentioned
Embodiment, those skilled in the art can make various deformations or amendments within the scope of the claims, this has no effect on this
The substantive content of invention.
Sequence table
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Claims (7)
1. a kind of gastric cancer Prognosis biomarker, which is characterized in that the biomarker be tumor microenvironment correlation because
Sub- RGS1.
2. a kind of application of tumor microenvironment correlation factor RGS1 as gastric cancer prognostic markers object and detection marker.
3. the primer pair of species-specific amplification biomarker described in claim 1, which is characterized in that the primer pair is such as
Shown in SEQ ID No.1 and 2.
4. a kind of gastric cancer Prognosis kit, which is characterized in that the kit includes reverse transcription reaction system and qPCR anti-
Answer system.
5. gastric cancer Prognosis kit according to claim 4, which is characterized in that in the qPCR reaction system, packet
Include primer pair as claimed in claim 3 and reference gene primer pair.
6. gastric cancer Prognosis kit according to claim 5, which is characterized in that the reference gene primer pair is such as
Shown in SEQ ID No.3 and 4.
7. a kind of method of the non-diagnostic purpose detection gastric cancer prognosis biomarker of kit according to claim 4,
It is characterized in that, comprising the following steps:
Step 1: extracting the cancerous tissue sample from patients with gastric cancer；
Step 2: using the above-mentioned diagnostic kit for gastric cancer prognosis evaluation, determine the cancerous tissue sample of patients with gastric cancer in step 1
The expression of RGS1 in this；
Step 3: the relative expression quantity of gastric cancer Prognosis biomarker RGS1 is obtained by calculation.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102037361A (en) * | 2008-03-05 | 2011-04-27 | 加利福尼亚大学董事会 | Molecular diagnosis and classification of malignant melanoma |
| CN106086029A (en) * | 2016-08-24 | 2016-11-09 | 江苏省人民医院 | A kind of long non-coding RNA and the application in diagnosis/treatment gastric cancer thereof |
-
2019
- 2019-08-01 CN CN201910707728.0A patent/CN110438229A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102037361A (en) * | 2008-03-05 | 2011-04-27 | 加利福尼亚大学董事会 | Molecular diagnosis and classification of malignant melanoma |
| CN106086029A (en) * | 2016-08-24 | 2016-11-09 | 江苏省人民医院 | A kind of long non-coding RNA and the application in diagnosis/treatment gastric cancer thereof |
Non-Patent Citations (1)
| Title |
|---|
| SHIHORI TANABE等: "Regulated genes in mesenchymal stem cells and gastric cancer", 《WORLD J STEM CELLS》 * |
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