CN110016507A - Application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis - Google Patents
Application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis Download PDFInfo
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Abstract
The present invention discloses a kind of application of SYT8 gene expression variation in gastric cancer prognosis and diagnosis, and the expression by detecting SYT8 gene carries out prognosis for gastric cancer, has preferable prediction effect, preliminary guidance is provided for later period further treatment.Simultaneously the present invention provides a kind of siRNA for blocking SYT8 gene expression, have the effect of preferably inhibiting SYT8 gene expression.The present invention has preferable inhibitory effect and application prospect.
Description
Technical field
The present invention relates to technical field of pharmaceutical biotechnology, especially a kind of SYT8 gene expression variation is in gastric cancer prognosis and diagnosis
In application.
Background technique
Gastric cancer is one of most common malignant tumour in the whole world, and morbidity and mortality worldwide occupy the 4th respectively
And second.China is the hotspot of gastric cancer, and morbidity and mortality occupy second, and there are about 450,000 people to die of stomach every year
Cancer.The major reason for causing gastric cancer mortality high is the Preventive and drug resistance of tumour.In early carcinoma of stomach patient into
Row scope or operation excision, can get good prognosis, therapeutic modality traditional at present, such as operation, chemicotherapy treatment means
Poor to the treatment curative effect of Metastasis of Gastric Cancer, 5 years survival rates nearly 30 years of gastric cancer are hovered always 20%~30%.Therefore, it to mention
5 years survival rates of high gastric cancer have to look for the new more effective therapeutic strategy of research.
Peritonaeum is a common Malignant Tumor Recurrence and DISTANT METASTASES IN position, and peritonaeum transfer is the master of late gastric cancer patient
Extremely because.Although being substantially a kind of disease that can not be cured, in iconography research, discovery can change entire therapeutic strategy
Small peritonaeum deposit is usually highly difficult, and celioscopy by stages and biopsy is needed to be confirmed.It is therefore, clinical that there is an urgent need to one kind
The biomarker that energy Accurate Prediction gastric cancer peritoneum shifts risk, helps clinic to make strategy appropriate.
SYT8 gene is a kind of transmembrane protein relevant to growth factor and transferring anti-cancer medicine, the height in stomach organization
Expression has maximum Hazard ratio, has high specific to gastric cancer peritoneum transfer, is confirmed as the only of peritonaeum recurrence-free survival rate
Vertical Prognostic Factors.Public data relevant to SYT8 expression has not been reported in gastric cancer, especially examines in gastric cancer peritoneum transfer prognosis
Application in disconnected is related to not yet.
Summary of the invention
The purpose of the present invention is to provide a kind of application of SYT8 gene expression variation in gastric cancer prognosis and diagnosis, pass through
Research confirms that SYT8 can be applied to the index prediction prognosis of diagnosis of gastric cancer peritonaeum transfer therapeutic effect；SYT8 can be used as gastric cancer peritoneum
The novel targets of transfer treatment new drug and biological immune treatment.
The technical solution of the present invention is as follows: a kind of application of SYT8 gene expression variation in gastric cancer prognosis and diagnosis, this is answered
With to prepare diagnosing gastric cancer and/or prognosis kit with Syt8 gene, include the primer for qRT-PCR in the kit
Pair and corresponding reagent.
Contain following primer sequence: Syt8 detection primer pair: upstream primer: 5 '-in the kit
GCTTCTCTCTCCGGTACGTG-3'；Downstream primer: 5 '-AGGAAGGTGAAGGCCTCATT-3 '；Internal control primer pair: GAPDH
Upstream primer: 5 '-CAGCTGAGAGGGAAATCGTG-3 ', downstream primer: 5 '-CGTTGCCAATAGTGATGACC-3 '.
Syt8 gene expression blocking agent preparation treatment gastric cancer medicament, Syt8 gene expression blocking agent are the interference of Syt8 gene
RNA。
RNA interfering is siRNA, shRNA, RNAi or chemistry disruption inhibitor.
RNA interfering includes justice RNA segment and antisense RNA fragment, the justice RNA segment and the antisense RNA fragment
It is complementary.
The justice RNA segment and the length of antisense RNA fragment are 15-27 nucleotide；Preferably, length is
19-23 nucleotide；Optimal, length is 19,20 or 21 nucleotide.
The small molecules interference RNA is hair clip type single stranded RNA, including just RNA segment, stem ring segment and antisense RNA piece
Section, separated among just RNA segment and antisense RNA fragment by stem ring segment；Wherein, just RNA segment and antisense RNA fragment are mutual
It mends.
Interference RNA sequence is configured to gene interference slow virus carrier, the carrier is that will encode specificity inhibition
The gene fragment clone of the interference microRNA of Syt8 gene expression obtains after entering slow virus carrier, can generate people's Syt8 gene
Small molecules interference RNA.
The slow virus carrier can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-
tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、
pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、
pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-
puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-
GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or
Any in pLenti6.2/N-Lumio/V5-GW/lacZ.
The gastric cancer is primary gastric cancer.
The present invention has the advantages that prognosis is carried out for curing gastric cancer patient by the expression of detection SYT8 gene,
With preferable prediction effect, preliminary guidance is provided for later period further treatment.The present invention provides a kind of blockings simultaneously
The siRNA of SYT8 gene expression has the effect of preferably inhibiting SYT8 gene expression.The present invention has preferable inhibitory effect
And application prospect.
Detailed description of the invention
Fig. 1 is that qRT-PCR compares normal gastric mucosa, stomach organization, and gastric cancer peritoneum shifts the expression water of SYT8 in oncocyte
Flat variation comparison diagram；
Fig. 2 is the SYT8 expression and Prognostic significance figure of Patients with Gastric Cancer；
Fig. 3 is that siRNA interferes SYT8 gene expression effect picture.
Specific embodiment
SYT8 gene expression dose predicts the marker purposes of gastric cancer peritoneum transfer patient's prognosis, is based on SYT8 gene expression
Level can predict the prognosis of gastric cancer peritoneum transfer patient, the low patient's good prognosis of SYT8 expression, the high trouble of expression
Person's poor prognosis.
Detection and application method, a. based on the prediction gastric cancer peritoneum transfer patient's prognosis of SYT8 gene expression dose are extracted swollen
The cell total rna of tumor patient's primary carcinoma tissue specimen；CDNA is synthesized by template reverse transcription of RNA；Using cDNA as template, SYT8 is used
The specific primer and fluorescence labeling probe of gene and house-keeping gene carry out real-time quantitative PCR amplification, calculate SYT8 gene mRNA
Relative expression quantity；B. the SYT8 of Patients on Recurrence transfer positive case and relapse and metastasis negative case is shifted according to gastric cancer peritoneum
Mrna expression amount determines the critical value of Index for diagnosis.SYT8 mrna expression amount is lower than patient's good prognosis of critical value, and is higher than
Patient's poor prognosis of critical value.
The detection method, with SYT8 mrna expression amount in primary cancerous tissue to tumor patient Index for diagnosis, expression quantity
Low patient is then judged as good prognosis, expression quantity it is high be then judged as poor prognosis.
The present invention confirms that SYT8 can be applied to the index prediction prognosis that diagnosis of gastric cancer peritonaeum shifts therapeutic effect by research；
SYT8 can be used as the novel targets of gastric cancer peritoneum transfer treatment new drug and biological immune treatment.SYT8 expression is blocked to can inhibit gastric cancer swollen
Tumor growth, inhibits gastric cancer tumor cell, can be used as the novel targets of curing gastric cancer new drug and biological immune treatment.
In addition, the present invention provides the siRNA, sequence such as SEQ ID NO:1 that a species specificity inhibits SYT8 gene expression
Shown in 2.
In addition, the present invention provides a kind of gene interference slow virus carrier, inhibit SYT8 gene expression for specificity will be encoded
Interference microRNA gene fragment clone enter slow virus carrier after obtain, can generate people's SYT8 gene small molecule interference
RNA。
The slow virus carrier can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-
tGFP、pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、
pLKO.1-puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、
pLKO.1-puro-UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-
puro-IPTG-3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-
GW/U6-laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or
Any in pLenti6.2/N-Lumio/V5-GW/lacZ.
Examinations SYT8 shifts expression (Fig. 1) in oncocyte and normal gastric cells in stomach cancer cell, gastric cancer peritoneum.
Clinical samples inclusion criteria: receiving surgical resection case, and postoperative pathological is diagnosed as gastric cancer；Percutaneous biopsy obtains
Complete tissue sample is obtained, pathological diagnosis is gastric cancer.Participation originally grind before the patients surgery or biopsy to make internal disorder or usurp receiving, radiotherapy with
And immunization therapy, patient endorsed informed consent form, and audit by Ethics Committee.
Method: postoperative and biopsy normal gastric mucosa, stomach organization are obtained.QRT-PCR, qRT-PCR behaviour are carried out after extract RNA
Make the operating method that step is this field routine, required primer is respectively as follows: SYT8 upstream primer: 5 '-
GCTTCTCTCTCCGGTACGTG-3 ', downstream primer: 5 '-AGGAAGGTGAAGGCCTCATT-3 '；GAPDH upstream primer: 5 '-
CAGCTGAGAGGGAAATCGTG-3 ', downstream primer: 5 '-CGTTGCCAATAGTGATGACC-3 '.
The expression water of SYT8 in qRT-PCR comparison normal gastric mucosa, gastric cancer tumor tissue and gastric cancer peritoneal metastases cell
Flat variation.
As a result: as shown in Figure 1, the expression of quantitative PCR data discovery SYT8 is lower in normal gastric mucosa, relatively just
It often organizes, increases about 4.1 times in stomach organization, increase nearly 8.1 times in gastric cancer peritoneum transfer tissue.The every group analysis of quantitative PCR
10 samples, GAPDH is accurate as internal reference instruction detection, meets the requirements.
Immunohistochemical staining uses U.S. Dako company EnVisionTMTwo step method detection system detects SYT8 albumen table
It reaches.The positive percentage statistics of sample is carried out according to IRS commonly used in the art.Staining power and sun can be taken into account using IRS
Property cell percentage.
As shown in table 1, immunohistochemistry inspection discovery normal gastric mucosa only has 3.2% for the SYT8 positive, and stomach organization is
86.9%, gastric cancer peritoneum transfer group is woven to 91.6% (table 1).Immunohistochemistry checks that every group is 10 samples.In order to avoid subjectivity
Property, sample is randomly assigned, the Independent Observers for not knowing about sample properties by 2 before analysis encode.When given
Between interval in, each observer at least assesses all samples (SYT8 positive or negative) twice.
Table 1
| Normal gastric cells | Stomach organization | Gastric cancer peritoneum shifts tissue |
| 3.2% | 86.9% | 91.6% |
As can be seen from the above results, SYT8 quantitative PCR and immunohistochemistry inspection can be used to the transfer of diagnosis of gastric cancer peritonaeum, have
Help predict peritonaeum recurrence.
The relationship (Fig. 2) of examinations stomach cancer cell SYT8 Positive Level and prognosis.
There is complete follow-up to record 36 Patients with Gastric Cancer for selection, obtains pathological section and carries out SYT8 immunohistochemistry inspection (behaviour
It is same as above as step).The SYT8 immunohistochemistry inspection of 36 Patients with Gastric Cancer is divided into three groups, and (weakly positive 1.0, the positive are 3.0, Qiang Yang
Property be 5.0) every group 12, carry out prognosis tracking, obtain the life span of patient.
As a result: as shown in Fig. 2, prognostic data shows that gastric tumor cells Positive Level and prognosis life span are closed in inverse ratio
System, strong positive patient's poor prognosis, life cycle is short, and weakly positive patient's prognosis is preferable, and life cycle is longer.Conclusion, detection SYT8 exempt from
Epidemic disease group level can be used to predict patients with gastric cancer prognosis, and can be used as the index of predicted treatment effect.
Implement the influence (Fig. 3) of SYT8 interference tumour.
1, the preparation of people SYT8 gene RNAi slow virus
(1) screening is directed to the effective siRNA target spot of people SYT8 gene
People's SYT8 gene information is transferred from Genbank；The effective siRNA target of SYT8 gene is directed to using DNAMAN design
Point.In region coded sequence (CDS) of SYT8 gene, 2 effective siRNA target sequences for being directed to SYT8 gene are devised.
It is respectively as follows: SYT8-1:cccagaagcccctgaggagctg；SYT8-2:tactgcaagcgggaggcggagg.
For the double-stranded DNA Oligo sequence of siRNA target spot synthesis both ends I containing Age and EcoR I restriction enzyme site cohesive end；With
Age I and EcoR I restriction enzyme acts on pGCSIL-GFP carrier, makes its linearisation, and agarose gel electrophoresis identifies enzyme
It is sliced section.And the RNAi carrier containing SYT8 is constructed, it is named as pGCSIL-GFP-SYT8-1-shRNA and pGCSIL-GFP-
SYT8-2-shRNA, while pGCSIL-GFP-control negative control matter is constructed according further to the test method of this field routine
Grain.It is simultaneously that the vector introduction is spare into slow virus.
2, the silence efficiency of real-time fluorescence quantitative RT-PCR method detection SYT8 gene
Pancreatin digestion is carried out using the human gastric cancer MKN45 cell of logarithmic growth phase and normal human oral epithelial cells, is made
(cell number is about 5.0 × 10 to cell suspension4/ ml), culture culture 72 hours in 24 orifice plates, culture to cell fusion degree reaches
About 40%.According to plural number (MOI of MKN45 is 20) value is infected, the virus of appropriate amount is added, culture medium is replaced in culture afterwards for 24 hours,
After time of infection reaches 3 days, cell is collected.According to the Trizol operational manual of Invitrogen company, extracted total RNA.
According to the M-MLV operational manual of Promega company, by RNA reverse transcription obtain cDNA (2 DEG C of reaction 1h of reverse transcription reaction system,
Then water-bath 10min inactivates reverse transcriptase in 70 DEG C of water-baths).
Real_time quantitative detection is carried out using ABI7500 type Real time PCR instrument (Life).For example above-mentioned reality of the primer of gene
It applies described in example.Experimental result shows that the SYT8 mRNA expression of human gastric cancer MKN45 cell has dropped respectively in experimental group
98.5% and 99.2%, there is preferable inhibition expression effect.
3: the proliferative capacity of the tumour cell of SYT8-shRNA slow virus is infected in detection
Cell prepared by above-mentioned experiment 2 collects each experiment for being in logarithmic growth phase after time of infection reaches 3 days
Group cell.Complete medium is resuspended into cell suspension (5.0 × 104/ ml), it is about 2000/hole with cell density, is inoculated with 96 holes
Plate.Every group of 5 multiple holes, every 200 μ l of hole.After completing plate, 37 DEG C, 5%CO2 incubator culture are set.Since after bed board second day,
It is primary with the 1st, 3 and 5 day measurement read plate of Cellomics instrument (Thermo Fisher).By adjusting Cellomics
The quantity of the cell with green fluorescence in scanning orifice plate every time is accurately calculated, to data in the input parameter of arrayscan
Statistics drawing is carried out, cell Proliferation curve is drawn.As a result, it has been found that the human gastric cancer MKN45 that two SYT8shRNA slow virus are infected is thin
After born of the same parents cultivate 5 days in vitro, vigor cell number has dropped 81.9% and 82.8% respectively, shows that SYT8 gene silencing causes to swell
Tumor cell proliferation ability is suppressed.
The present invention is explained detailedly by specific implementation above.It will be apparent to one skilled in the art that the present invention and unlimited
In embodiment listed herein, protection scope is defined by the appended claims, and the following examples are only to show
The mode of example illustrates the present invention, so that the present invention is more readily understood.
Claims (11)
1. a kind of application of SYT8 gene expression variation in gastric cancer prognosis and diagnosis, it is characterised in that: the application is utilization
Syt8 gene prepares diagnosing gastric cancer and/or prognosis kit, includes the primer pair and phase for qRT-PCR in the kit
The reagent answered.
2. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 1, it is characterised in that: institute
It states and contains following primer sequence: Syt8 detection primer pair: upstream primer: 5 '-GCTTCTCTCTCCGGTACGTG- in kit
3'；Downstream primer: 5 '-AGGAAGGTGAAGGCCTCATT-3 '；Internal control primer pair: GAPDH upstream primer: 5 '-
CAGCTGAGAGGGAAATCGTG-3 ', downstream primer: 5 '-CGTTGCCAATAGTGATGACC-3 '.
3. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 1, it is characterised in that:
Syt8 gene expression blocking agent preparation treatment gastric cancer medicament, Syt8 gene expression blocking agent is Syt8 gene RNA interfering.
4. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 3, it is characterised in that: dry
Disturbing RNA is siRNA, shRNA, RNAi or chemistry disruption inhibitor.
5. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 4, it is characterised in that: dry
Disturbing RNA includes justice RNA segment and antisense RNA fragment, and the justice RNA segment and the antisense RNA fragment are complementary.
6. application of the SYT8 gene expression variation according to claim 4 or 5 in gastric cancer prognosis and diagnosis, feature exist
In: the justice RNA segment and the length of antisense RNA fragment be 15-27 nucleotide；Preferably, length is 19-23
Nucleotide；Optimal, length is 19,20 or 21 nucleotide.
7. application of the SYT8 gene expression variation according to claim 4 or 5 in gastric cancer prognosis and diagnosis, feature exist
In: the small molecules interference RNA be hair clip type single stranded RNA, including just RNA segment, stem ring segment and antisense RNA fragment, just
Separated among adopted RNA segment and antisense RNA fragment by stem ring segment；Wherein, just RNA segment and antisense RNA fragment are complementary.
8. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 7, it is characterised in that: dry
The sequence for disturbing RNA is shown in SEQ ID NO:1-2 is any.
9. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 8, it is characterised in that: will
Interference RNA sequence is configured to gene interference slow virus carrier, and the carrier is that will encode specificity to inhibit Syt8 gene expression
Interference microRNA gene fragment clone enter slow virus carrier after obtain, can generate people's Syt8 gene small molecule interference
RNA。
10. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 9, it is characterised in that:
The slow virus carrier can be selected from: pLKO.1-puro, pLKO.1-CMV-tGFP, pLKO.1-puro-CMV-tGFP,
pLKO.1-CMV-Neo、pLKO.1-Neo、pLKO.1-Neo-CMV-tGFP、pLKO.1-puro-CMV-TagCFP、pLKO.1-
puro-CMV-TagYFP、pLKO.1-puro-CMV-TagRFP、pLKO.1-puro-CMV-TagFP635、pLKO.1-puro-
UbC-TurboGFP、pLKO.1-puro-UbC-TagFP635、pLKO-puro-IPTG-1xLacO、pLKO-puro-IPTG-
3xLacO、pLP1、pLP2、pLP/VSV-G、pENTR/U6、pLenti6/BLOCK-iT-DEST、pLenti6-GW/U6-
Laminshrna, pcDNA1.2/V5-GW/lacZ, pLenti6.2/N-Lumio/V5-DEST, pGCSIL-GFP or
Any in pLenti6.2/N-Lumio/V5-GW/lacZ.
11. application of the SYT8 gene expression variation in gastric cancer prognosis and diagnosis according to claim 1, it is characterised in that:
The gastric cancer is primary gastric cancer.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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2019
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|---|---|---|---|---|
| WO2016143697A1 (en) * | 2015-03-06 | 2016-09-15 | 国立大学法人名古屋大学 | Method for testing peritoneal dissemination of gastric cancer by syt13, syt8, and anos1 expression levels, test kit, molecularly targeted therapeutic screening method, and therapeutic agent |
| US20190024179A1 (en) * | 2015-03-06 | 2019-01-24 | National University Corporation Nagoya University | Method for testing peritoneal dissemination of gastric cancer by expression level of syt13, syt8, or anos1, test kit, method for screening molecularly targeted therapeutic agent, and therapeutic agent |
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| MITSURO KANDA等: "Integrated multigene expression panel to prognosticate patients with gastric cancer", 《ONCOTARGET》 * |
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