CN107557470A - A kind of detection method using people MUC13 as hepatocellular carcinoma specific markers - Google Patents
A kind of detection method using people MUC13 as hepatocellular carcinoma specific markers Download PDFInfo
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- CN107557470A CN107557470A CN201710947982.9A CN201710947982A CN107557470A CN 107557470 A CN107557470 A CN 107557470A CN 201710947982 A CN201710947982 A CN 201710947982A CN 107557470 A CN107557470 A CN 107557470A
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Abstract
The present invention relates to biological field.Purpose is to provide a kind of detection method using people MUC13 as hepatocellular carcinoma specific markers, this method passes through the quantitative detection to people MUC13, the relevant information of hepatocellular carcinoma is obtained, and the survival of patients time prediction based on liver cancer MUC13 expressions is carried out according to the PCR amplifications used in detection.Technical scheme is：A kind of detection method using MUC13 as hepatocellular carcinoma specific markers, comprise the following steps：(1) human tissue sample is obtained from tested crowd, then carries out RNA extractions and reverse transcription；(2) MUC13 in human tissue sample is detected；(3) if MUC13 Δs CT prompts have hepatocellular carcinoma in tested tissue samples less than normal structure sample MUC13 Δ CT average values in tested tissue samples；Detection in the step 2 is carried out by fluorescence real-time quantitative nucleic acid amplification detection (QPCR)；Used MUC13 fluorescence real-time quantitative PCRs (QPCR) primer is：5 ' CCTTCGGTGTGATTATTATGGC 3 ' of forward direction；Reverse 5 ' GCATCTGGCTGTCTCTGGAG 3 '.
Description
Technical field
The present invention relates to biological field, is specifically used as hepatocellular carcinoma specific markers using people's Mucin gene MUC13
Detection method.
Background technology
Hepatocellular carcinoma (Hepatocellular carcinoma, HCC, abbreviation liver cancer), it is the 5th evil occurred frequently in the world
Property tumour, fatal rate rank the 3rd.The liver cancer patient number in China remains high at present, accounts for the 55% of global hepatocarcinoma patient,
Have a strong impact on the health of people.
For diagnosing cancer of liver in addition to traditional pathological diagnosis and iconography auxiliary diagnosis, molecule diagnosis is in the ascendant at present,
Judge that patient disease hypotype and progression of disease situation provide strong evidence for individuation.Common liver cancer molecular diagnostic markers thing
Have：Alpha-fetoprotein, heat shock protein 70, phosphatidylinositols, gamma glutamyltransferase, TGF (TGF-β 1) and pancreas
Island element like growth factor II etc..
Traditional pathological diagnosis and iconography auxiliary diagnosis has reliable diagnosis effect to liver cancer diseases, but lacks
Individuation suggesting effect, there is no any suggesting effect to the gene-correlation information of tumour yet, be therefore helpless to follow-up gene yet
Targeted therapy.And existing liver cancer molecular diagnostic markers thing includes conventional alpha-fetoprotein etc., its false positive rate and false negative rate
It is higher, easily cause mistaken diagnosis or fail to pinpoint a disease in diagnosis.
Judge that liver cancer patient prognostic indicator has many, it is conventional for pathological staging, clinical stages, Cure outcome etc..In addition
Numerous genes are found have suggesting effect to liver cancer patient prognosis, such as serum alpha-fetoprotein, tumor tissues p53, cell angle egg
White 10/19, osteopontin, Dickkopf-1 etc., but not yet find optimal parameter or combination at present.Therefore it is special further to find it
Opposite molecule mark has important clinical meaning.
The content of the invention
The technical problems to be solved by the invention are to overcome the shortcomings of above-mentioned background technology, there is provided a kind of to be made with people MUC13
For the detection method of hepatocellular carcinoma specific markers, this method obtains the phase of hepatocellular carcinoma by the quantitative detection to people MUC13
Information is closed, and the survival of patients time based on liver cancer MUC13 expressions can be carried out according to the PCR amplifications used in detection
Prediction.
Technical scheme provided by the invention is：
A kind of detection method using MUC13 as hepatocellular carcinoma specific markers, comprise the following steps：
1) human tissue sample is obtained from tested crowd, then carries out RNA extractions and reverse transcription；
2) MUC13 in human tissue sample is detected；
3) if being detected MUC13 Δs CT in tissue samples, less than normal structure sample MUC13 Δ CT average values, prompts quilt
There is hepatocellular carcinoma in inspection tissue samples；
Detection in the step 2 is carried out by fluorescence real-time quantitative nucleic acid amplification detection (QPCR)；Used MUC13
Fluorescence real-time quantitative PCR (QPCR) primer is：
Forward direction 5 '-CCTTCGGTGTGATTATTATGGC-3 '
Reverse 5 '-GCATCTGGCTGTCTCTGGAG-3 '.
QPCR in the step 2, the control primer used for：
Forward direction 5 '-CTCTTAGCTGAGTGTCCCGC-3 '
Reverse 5 '-CTGATCGTCTTCGAACCTCC-3 '.
As recommendation, in the step 2, the MUC13 Δ CT average values of normal structure sample are 15.5.
RNA extractions and reverse transcription in the step 1), are carried out RNA extractions using Trizol and are reversed using RNA
Record kit is reversed into complementary DNA (cDNA).
It is pure using denaturing polyacrylamide gel electrophoresis method (PAGE) when the MUC13PCR primers and control primer synthesis
Change.
The MUC13PCR primers and control primer using tri-distilled water in use, be diluted to 10 μM of working solution concentration.
The beneficial effects of the invention are as follows：
1) molecular marked compound of liver cancer has many, but specificity is bad.Present invention discover that liver cancer of the MUC13 44.0%
Significantly raised in patient, and the clinical parameter such as related to the formation of patient tumors coating, tumor size, Tumor thrombus and tumor grade
Correlation, therefore inventor thinks that MUC13 can be used as liver cancer-specific molecular marked compound.
2) serum alpha-fetoprotein, tumor tissues p53, CK10/19, osteopontin, Dickkopf-1 etc.
As the molecular indexes of the existence anticipation of liver cancer patient, but anticipation value is all preferable not to the utmost, finds the index that disease existence prejudges
It is significant.Present invention discover that it is 64.1 ± 5.8 months that MUC13, which expresses high patient's mean survival time, it is substantially less than
MUC13 expresses 89.2 ± 5.2 months mean survival times of low patient.As a result it is that liver cancer existence can be used as pre- to prove MUC13
The good molecular marked compound sentenced.
Brief description of the drawings
Fig. 1 is that in liver cancer tissue, (each point represents one to figure to MUC13 albumen compared with the expression in liver normal structure
Sample).
Fig. 2 is microphoto ratio of the MUC13 albumen in liver cancer tissue (Non-tumor) and liver normal structure (Normal)
Compared with figure.
Fig. 3 is MUC13 albumen figure compared with liver cancer tissue (T) detects with the Western in liver normal structure (N).
Fig. 4 is the MUC13 expressions for analyzing to obtain based on QPCR to the total life span of liver cancer patient (A) and disease-free survival
The Kaplan-Meier analysis charts of time (B).
Fig. 5 is to the total life span of liver cancer patient (A) and disease-free based on the MUC13 expressions that immunohistochemistry obtains
The Kaplan-Meier analysis charts of life span (B).
Fig. 6 is that the predictive value of the total life span of liver cancer patient is analyzed based on the MUC13 Δ CT values that QPCR analyzes to obtain
Figure.
Embodiment
Further illustrated below in conjunction with embodiment shown in the drawings.
Detection method provided by the invention using MUC13 as hepatocellular carcinoma specific markers, comprise the following steps：
1) human tissue sample is obtained from tested crowd, then carries out RNA extractions, and reverse transcription is into complementary DNA (cDNA)；
2) MUC13 in human tissue sample is detected；Detection is carried out by QPCR, and used MUC13PCR primers are：
Forward direction 5 '-CCTTCGGTGTGATTATTATGGC-3 '
Reverse 5 '-GCATCTGGCTGTCTCTGGAG-3 '.
Compareing primer is：
Forward direction 5 '-CTCTTAGCTGAGTGTCCCGC-3 '
Reverse 5 '-CTGATCGTCTTCGAACCTCC-3 '
Above primer sequence is synthesized by Shenzhen Huada Genetic Technology Co., Ltd；
Purified during synthesis using PAGE methods (denaturing polyacrylamide gel electrophoresis)；During using primer, tri-distilled water need to be used dilute
It is 10 μM to release to working solution concentration.
If 3) it is detected (the MUC13 tables that MUC13 Δs CT in tissue samples is less than normal structure sample MUC13 Δ CT average values
It is in inverse ratio up to horizontal and MUC13 Δs CT levels), then prompt that there is hepatocellular carcinoma in tested tissue samples.
1.MUC13 quantitative detecting step and key reagents and instrument
Key reagents and instrument
1)Trizol(Invitrogen,Carlsbad,CA)
2) RNA Reverse Transcriptase kits (Roche, Basel, Switzerland)
3) SYBR Green PCR kits (Roche, Basel, Switzerland)
4) regular-PCR instrument
5) quantitative real time PCR Instrument
Detect committed step
1) flesh tissue sample is obtained and handled
Fresh specimens are typically derived from living body puncture or operation gross specimen.The tissue of acquirement should be as early as possible as 4 degree of ice chests
In, and be transferred within 5-10 minutes in liquid nitrogen.Sample is no less than 1 hour in the immersing in liquid nitrogen time.Sample can be in liquid nitrogen
It is long-term to preserve or preserved in -80 degree refrigerators no more than 6 months.
1) RNA is extracted
A) take 100mg tissues to be put into mortar, add a small amount of liquid nitrogen, rapid grind into powder；
B) 1ml Trizol cracking tissues are added；
C) lysate is transferred to 1.5ml centrifuge tubes, adds 0.2ml chloroforms, and 12,000g is centrifuged 15 minutes after shaken well；
D) supernatant is drawn into another centrifuge tube, adds 0.5ml isopropanols, 12,000g is centrifuged 10 minutes after mixing；
E) supernatant discarding, the ethanol of 1ml 75% is added to sediment, 8,000g centrifugation 5min, abandons supernatant；
F) sediment is air-dried, adds tri-distilled water dissolving RNA；
G) OD values are surveyed with ultraviolet specrophotometer, calculates RNA concentration and purity.- 80 DEG C of preservation RNA after packing.
2) RNA is reversed using RNA Reverse Transcriptase kits
A) following reagent is added in PCR pipe：
B) 65 DEG C of regular-PCR instrument is incubated 5 minutes, 0 DEG C of ice-water bath 1 minute；
C) following reagent is added in pipe after of short duration centrifugation：
D) of short duration centrifugation again after mixing；
E) 50 DEG C of regular-PCR instrument is incubated 60 minutes；
F) 70 DEG C of regular-PCR instrument is incubated 15 minutes inactivation RT activities, -80 DEG C of preservation products.
3) QPCR (fluorescence real-time quantitative nucleic acid amplification detecting system) is detected
Carried out using FastStart Universal SYBR Green Master (Roche fluorescence quantitative kits)
QPCR.Amplified reaction is carried out using quantitative real time PCR Instrument：50 DEG C are reacted 2 minutes, and 95 DEG C are reacted 10 minutes, and 95 DEG C are reacted 15 seconds,
60 DEG C are reacted 1 minute, and 95 DEG C are reacted 15 seconds, and 60 DEG C are reacted 15 seconds, totally 40 circular responses.Reaction product is quantitative determined
Analyzed with melt curve analysis, carry out parallel laboratory test three times respectively.MUC13 relative expression levels' computational methods are in tissue：2-ΔCT
Each Δ CT=CT (MUC13)-CT (18S).
QPCR expands 20ul system compositions：
(MUC13 control primers carry out QPCR detections in another same system, and two systems are only primer differences).
2.MUC13 is detected in 168 liver cancer patients
Inventor have collected the fresh specimens (comprising normal liver tissue by cancer and cancerous tissue) of 168 hepatectomies,
With reference to above operating procedure, using above primer and reagent to all carry out real-time quantitative PCRs, to the mRNA water of MUC13 genes
It is flat to be detected.As a result show that expressions of the MUC13 in liver cancer tissue is significantly higher than normal structure (Fig. 1).
Table1.Clinicopath ological correlation of Muc13 expression in Hcc
3.MUC13 abnormal level defines in liver cancer tissue
As shown in table 1：MUC13 Δs CT average values are 15.5 in 168 Carcinoma side normal tissues.In liver cancer tissue
There are 74 (accounting for 44.0%) higher than the value, less than having 94 (accounting for 56.0%) for the value.The clinical parameter of corresponding case is entered
Row analysis, it is found that the high expression of Muc13 is related to the formation of patient tumors coating, tumor size, Tumor thrombus and tumor grade
(Table 1, statistical check P<0.05, Chi-square Test, χ test).
4. immunohistochemistry verifies expression of the MUC13 in liver cancer tissue
Based on immunohistochemical method, inventor is verified to above QPCR results, have detected 119 liver normal groups
It is abcam companies to knit with the expression of liver cancer tissue MUC13 albumen (micro- enlarged photograph shown in Figure 2), antibody
ab65109.Statistical result showed MUC13 abnormal expressions in liver cancer are elevated to be had 51 (accounting for 42.9%), uninflated to have 68
Example (accounting for 57.1%).Prove the MUC13QPCR detection primers and its detection method and the immune group of MUC13 albumen of inventor's design
Weave chemistry detection method has similar recall rate.
5. immune protein trace verifies expression of the MUC13 in liver cancer tissue
In addition, inventor detects to the immune protein trace of 16 fresh liver normal structures and liver cancer tissue protein extract
As a result, it was also found that the number of cases that expression (T) of the MUC13 in tumor tissues is significantly higher than the expression (N) of normal structure (accounts for for 8
50%), have no notable elevated number of cases for 8 (referring to Fig. 3；Internal reference when house-keeping gene GAPDH is Protein Detection, two samples
When GAPDH is horizontal consistent, it is believed that sample applied sample amount is equal).Be also demonstrated that inventor design MUC13QPCR detection primers and its
Detection method, there is similar recall rate with the immune protein trace detection method of MUC13 albumen.
6.MUC13 to the predictive value of survival of patients time
Inventor detects to obtain expressions of the MUC13 in liver cancer tissue according to QPCR, to total life span of patient
Kaplan-Meier analyses are carried out, it is found that 5 years survival rates of the high patient of MUC13 expression are substantially less than MUC13 and express low trouble
Person, MUC13 expressed low patient's mean survival time as 89.2 ± 5.2 months, and MUC13 expresses high patient's mean survival time
For 64.1 ± 5.8 months (Fig. 4-A).Kaplan-Meier analyses are carried out to the disease-free survival time of patient, find MUC13 expression
5 years survival rates of high patient express low patient (Fig. 4-B) less than MUC13.
Inventor is also further verified to the conclusion.Based on 119 liver normal structures and liver cancer tissue MUC13 eggs
The testing result of the immunohistochemical method of white expression, Kaplan-Meier analyses show that MUC13 expresses 5 years of high patient always
Survival rate and disease-free survival rate are substantially less than MUC13 and express low patient (Fig. 5-A and Fig. 5-B).Prove autonomous based on inventor
The survival analysis result and be based on MUC13 immunoreaction scorings that the MUC13QPCR detection primers and its detection method of exploitation are drawn
The survival analysis result that chemical detection method is drawn has similar predictive value.
As a result primer and the detection means designed based on the present invention is proved, can effectively detect new proto-oncogene
Whether MUC13 is expressed in liver cancer tissue in the presence of abnormal.
The primer and detection means designed based on the present invention, inventor are had found：As the Δ CT of QPCR results>When 15.5, suffer from
The mean survival time of person is contemplated to 89.2 ± 5.2 months；When 10.2<During Δ CT≤15.5, the mean survival time of patient is pre-
Phase is 63.4 ± 6.3 months；As Δ CT≤10.2, the mean survival time of patient is contemplated to 54.8 ± 8.0 months (see figure
6).As a result primer and the detection means designed based on the present invention is proved, the value with the good prediction survival of patients time.
Sequence table
<110>Zhejiang University
<120>A kind of detection method using people MUC13 as hepatocellular carcinoma specific markers
<160>2
<210>1
<211> 20
<212>DNA
<213>Artificial sequence
<220>
<222>DNA（Or DNA）Sequence（1）…（20）
<223>MUC13 PCR forward primers
<400>1
CCTTCGGTGT GATTATTATGGC 20
<110>Zhejiang University
<120>A kind of detection method using people MUC13 as hepatocellular carcinoma specific markers
<160>2
<210>2
<211>20
<212>DNA
<213>Artificial sequence
<220>
<222> DNA（Or DNA）Sequence（1）…（20）
<223>MUC13 PCR reverse primers
<400>2
GCATCTGGCT GTCTCTGGAG 20
Claims (6)
1. a kind of detection method using MUC13 as hepatocellular carcinoma specific markers, comprise the following steps：
1) human tissue sample is obtained from tested crowd, then carries out RNA extractions and reverse transcription；
2) MUC13 in human tissue sample is detected；
If 3) it is detected MUC13 Δs CT in tissue samples, less than normal structure sample MUC13 Δ CT average values, to prompt tested
There is hepatocellular carcinoma in tissue samples；
Detection in the step 2 is carried out by QPCR；Used MUC13 fluorescence real-time quantitative PCRs primer is：
Forward direction 5 '-CCTTCGGTGTGATTATTATGGC-3 '
Reverse 5 '-GCATCTGGCTGTCTCTGGAG-3 '.
2. the detection method according to claim 1 using MUC13 as hepatocellular carcinoma specific markers, it is characterised in that：
QPCR in the step 2, the control primer used for：
Forward direction 5 '-CTCTTAGCTGAGTGTCCCGC-3 '
Reverse 5 '-CTGATCGTCTTCGAACCTCC-3 '.
3. the detection method according to claim 1 or 2 using MUC13 as hepatocellular carcinoma specific markers, its feature exists
In：In the step 2, the MUC13 Δ CT average values of normal structure sample are 15.5.
4. the detection method according to claim 3 using MUC13 as hepatocellular carcinoma specific markers, it is characterised in that：
RNA extractions and reverse transcription in the step 1), carry out RNA extractions using Trizol and utilize RNA Reverse Transcriptase kits
It is reversed into complementary DNA.
5. the detection method according to claim 4 using MUC13 as hepatocellular carcinoma specific markers, it is characterised in that：
When the MUC13 PCR primers and control primer synthesis, purified using denaturing polyacrylamide gel electrophoresis method.
6. the detection method according to claim 5 using MUC13 as hepatocellular carcinoma specific markers, it is characterised in that：
The MUC13 PCR primers and control primer using tri-distilled water in use, be diluted to 10 μM of working solution concentration.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114502189A (en) * | 2019-07-19 | 2022-05-13 | 安特卫普大学 | Mucin isoforms in diseases characterized by barrier dysfunction |
Citations (2)
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| JP2004159600A (en) * | 2002-11-14 | 2004-06-10 | Center For Advanced Science & Technology Incubation Ltd | Method for diagnosing cancer and kit for diagnosing cancer |
| CN105420195A (en) * | 2013-12-27 | 2016-03-23 | 中山大学附属肿瘤医院 | Cancer suppressor gene ATOH8 and application of encoding protein of ATOH8 to iPSC preparation |
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2017
- 2017-10-12 CN CN201710947982.9A patent/CN107557470A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2004159600A (en) * | 2002-11-14 | 2004-06-10 | Center For Advanced Science & Technology Incubation Ltd | Method for diagnosing cancer and kit for diagnosing cancer |
| CN105420195A (en) * | 2013-12-27 | 2016-03-23 | 中山大学附属肿瘤医院 | Cancer suppressor gene ATOH8 and application of encoding protein of ATOH8 to iPSC preparation |
Non-Patent Citations (1)
| Title |
|---|
| WILLIAMS,S.J.等: "Homo sapiens transmembrane mucin MUC13 (MUC13) mRNA, complete cds", 《GENBANK DATABASE》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114502189A (en) * | 2019-07-19 | 2022-05-13 | 安特卫普大学 | Mucin isoforms in diseases characterized by barrier dysfunction |
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