CN109825596A - The internal reference miRNA of cancer of the esophagus sample and its application - Google Patents
The internal reference miRNA of cancer of the esophagus sample and its application Download PDFInfo
- Publication number
- CN109825596A CN109825596A CN201910317987.2A CN201910317987A CN109825596A CN 109825596 A CN109825596 A CN 109825596A CN 201910317987 A CN201910317987 A CN 201910317987A CN 109825596 A CN109825596 A CN 109825596A
- Authority
- CN
- China
- Prior art keywords
- mirna
- cancer
- sample
- esophagus
- internal reference
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Abstract
The present invention relates to the internal reference miRNA of cancer of the esophagus sample and its applications, wherein, it include hsa-miR-423-5p and/or hsa-miR-16-5p for the internal reference miRNA as cancer of the esophagus sample, wherein, hsa-miR-423-5p, shows that gene expression abundance is high, fluctuating range is small in the sample, without the specific of significant difference between different type sample, it is suitable as the internal reference miRNA of the cancer of the esophagus sample of cancer of the esophagus sample, especially Chinese population.
Description
Technical field
The present invention relates to miRNA detection fields, in particular to the internal reference miRNA of cancer of the esophagus sample and its application.
Background technique
The cancer of the esophagus (esophageal carcinoma, EC) is high-incidence one of the malignant tumor of digestive tract in the whole world, full generation
There are about 400,000 people to die of this disease every year on boundary, seriously endangers common people's health；Incidence of Esophageal Cancer has apparent regional disparity, and China is
One of Esophageal Cancer area in the world, average attack rate are about 22.14/10 ten thousand, and the same period death rate 16.77/10 ten thousand occupies pernicious
The 4th of tumor mortality rate.The cancer of the esophagus has the characteristic for resisting chemicotherapy, focal recurrence or far-end transfer, and five year survival rate is about
For 15-25%.The early symptom of the cancer of the esophagus is not true to type more, and progressive stage is using progressive dysphagia as typical clinical manifestations.
MiRNA is small point of non-coding that one kind is widely present in eucaryote, average length about 22 (19-24) a nucleotide
Sub- RNA, by combining the 3 ' noncoding regions of target mRNA (messenger RNA, mRNA), specificity inhibits said target mrna, in base
Because playing key effect in expression post-transcriptional control, such as various cancers closely related with disease.
For the cancer of the esophagus, current result of study shows miRNA in the generation, development and diagnoses and treatment of the cancer of the esophagus
It plays a significant role, is expected to realize that the early diagnosis of the cancer of the esophagus, prognosis/curative effect monitoring even cancer of the esophagus are controlled by miRNA
It treats and novel targets is provided.
However, miRNA is in the research of the cancer of the esophagus and using still by larger limitation although having obtained certain progress.Each
MiRNA researcher must face and very stubborn problem is when detecting health volunteer and cancer of the esophagus associated patient,
Lack reliable reference gene, the comparison of miRNA between sample is caused to become difficult.In addition, grinding about cancer of the esophagus miRNA at present
Study carefully the white race crowd for focusing primarily upon American-European countries, lacks the miRNA internal reference suitable for subspecies crowd, especially Chinese population.
In view of this, the present invention is specifically proposed.
Summary of the invention
The first object of the present invention is to provide hsa-miR-423-5p answering as the internal reference miRNA of cancer of the esophagus sample
With the internal reference miRNA has the expression height in normal subjects and patient with esophageal carcinoma, is easy to detect, and expression
It is almost the same, it is suitble to the advantages of uniforming, is the high-quality internal reference miRNA of esophagus cancer diagnosis.
The second object of the present invention is to provide a kind of esophagus cancer diagnosis reagent and/or diagnostic kit, the diagnosis examination
Agent and/or diagnostic kit include the detection reagent of cancer of the esophagus miRNA marker and the detection reagent of aforementioned internal reference miRNA, are somebody's turn to do
Diagnostic reagent and/or diagnostic kit include high-quality internal reference miRNA, help to obtain accurate diagnostic result.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
Application of the Hsa-miR-423-5p as the internal reference miRNA of cancer of the esophagus sample.
In some specific embodiments, the subject of the sample is asian population, it is preferable that the asian population
For Chinese population.
In some specific embodiments, the sample is from asymptomatic subject or cancer of the esophagus suspected patient.
In some specific embodiments, patient with esophageal carcinoma of the sample before treatment or the oesophagus after treatment
Cancer patient.
In some specific embodiments, the sample is tissue samples or body fluid sample, and the body fluid sample is preferred
For blood sample, optionally, the blood sample is serum sample or plasma sample.
In some specific embodiments, the internal reference miRNA is used for the standardization of purpose miRNA.
In some specific embodiments, the application includes: purpose miRNA and internal reference miRNA in measurement sample
The absolute concentration of the absolute concentration of purpose miRNA and internal reference miRNA is compared, after being standardized by absolute concentration
MiRNA concentration.
The invention further relates to: a kind of esophagus cancer diagnosis reagent and/or diagnostic kit, the diagnostic reagent and/or diagnosis
Kit includes the detection reagent of cancer of the esophagus miRNA marker and the detection reagent of internal reference miRNA, and the internal reference miRNA includes
hsa-miR-423-5p。
In some specific embodiments, the internal reference miRNA further includes hsa-miR-16-5p.
In some specific embodiments, the detection reagent of the cancer of the esophagus miRNA marker and the inspection of internal reference miRNA
Test agent is reagent suitable for following method: real-time fluorescence quantitative PCR, digital pcr, fluorescent dye determination, Resonance Light Scattering Method,
Sequencing or biomass spectrometry.
In some specific embodiments, the detection reagent and internal reference miRNA of the cancer of the esophagus miRNA marker
Detection reagent be reagent suitable for real-time fluorescence quantitative PCR, including amplimer and Taqman probe.
Compared with prior art, the invention has the benefit that
Hsa-miR-423-5p of the present invention the cancer of the esophagus and cancer beside organism expression without significant difference, expressed in serum
Abundance is high, meanwhile, Chinese population serum sample testing result shows that hsa-miR-423-5p equally maintains high abundance to express, together
When normal subjects it is consistent with the gene expression abundance of patient with esophageal carcinoma, stability is good.It is excellent that Hsa-miR-423-5p can be used as the cancer of the esophagus
Matter internal reference miRNA is especially suitable for Chinese population or even asian population.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the qRT-PCR testing result of hsa-miR-451a in embodiment 2, wherein Normal indicates normal tested
Person, Cancer indicate cancer of the esophagus subject；
Fig. 2 is the qRT-PCR testing result of hsa-miR-320a in embodiment 2, wherein Normal indicates normal tested
Person, Cancer indicate cancer of the esophagus subject；
Fig. 3 is the qRT-PCR testing result of hsa-miR-423-5p in embodiment 2, wherein Normal indicates normal tested
Person, Cancer indicate cancer of the esophagus subject；
Fig. 4 is the qRT-PCR testing result of hsa-miR-423-5p in embodiment 3, wherein Normal indicates normal tested
Person, Cancer indicate cancer of the esophagus subject；
Fig. 5 is the qRT-PCR testing result of hsa-miR-16-5p in embodiment 4, wherein Normal indicates normal tested
Person, Cancer indicate cancer of the esophagus subject；
Fig. 6 is the qRT-PCR testing result of hsa-miR-16-5p in comparative example 1, wherein Normal indicates normal tested
Person, Cancer indicate cancer of the esophagus subject.
Specific embodiment
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by purchase.
Embodiment 1
The present embodiment is intended to preliminary screening and obtains candidate cancer of the esophagus internal reference miRNA, specific screening technique and the following institute of result
Show:
(1) select following 7 miRNA as the candidate cancer of the esophagus internal reference miRNA:hsa-miR-361-5p, hsa-miR-
186-5p, hsa-miR-26a-5p, hsa-miR-191-5p, hsa-miR-451a, hsa-miR-423-5p and hsa-miR-
320a。
(2) by the data source of TCGA (The Cancer Genome Atlas) database, aforementioned 7 miRNA is investigated and are existed
Human esophageal carcinoma and its expression of cancer beside organism, the results showed that, aforementioned 7 miRNA are equal in the cancer of the esophagus and cancer beside organism
It is not significantly different.
(3) by the data source of miRNA-seq database, expression quantity of aforementioned 7 miRNA in serum is investigated, as a result
Show that the abundance of wherein hsa-miR-361-5p and hsa-miR-186-5p in serum is too low, is not suitable as internal reference
miRNA。
(4) consulting literatures find that hsa-miR-26a-5p, hsa-miR-191-5p can inhibit or promote respectively the cancer of the esophagus
The proliferation and transfer of cell are not suitable as cancer of the esophagus internal reference miRNA.
(5) by aforementioned screening step, hsa-miR-423-5p, hsa-miR-451a and hsa-miR-320a is chosen and is used for
Subsequent authentication.
Embodiment 2
The purpose of the present embodiment is intended to investigate hsa-miR-423-5p, hsa-miR-451a and hsa-miR-320a in China
Gene expression abundance in crowd normal subjects and cancer of the esophagus subject's sample, to investigate it if appropriate for as cancer of the esophagus internal reference
miRNA.Wherein, subject is by 32 normal subjects and 32 cancer of the esophagus subject groups at being selected from Chinese population；Detect sample
This is serum, detection method qRT-PCR.Specific detecting step is as follows:
(1) RNA is extracted
Serum RNA is extracted using miRNeasy Serum/Plasma kit (Qiagen), operating procedure is as follows: 250 μ l blood
This centrifugation of final proof (10,000RPM, 5mins) removes cell fragment, and 200 μ l supernatants are transferred to the new centrifuge tube except RNA enzyme；
1,000 μ l of QIAzol lysate is added, according to the operating procedure that kit provides, is finally dissolved in 30 μ l removal RNA enzyme water,
It is frozen in -80 DEG C spare.
(2) miRNA reverse transcription
Using the special loop-stem structure primer of miR, the reverse of miRNAs is carried out using TaqMan MicroRNA Reverse Transcription
Record, reaction system are as shown in table 1.
1 miRNA reverse transcription reaction system of table
| MiRNA reverse transcription reaction system | Dosage (μ l) |
| 10x RT buffer | 0.60 |
| 5x RT primer | 1.2 |
| Multi Scribe TM Reverse | 0.42 |
| 100mM dNTPs | 0.06 |
| Rnase Inhibitor | 0.06 |
| RNA | 1.2 |
| RNase-free H2O | 2.46 |
The reaction condition of miRNA reverse transcription is as shown in table 2, after reaction product in -20 DEG C save.
The reaction condition of 2 miRNA reverse transcription of table
| Temperature | Time |
| 16℃ | 30min |
| 42℃ | 30min |
| 85℃ | 5min |
(3) qRT-PCR detects miRNAs expression
Using AppliedBiosystems (ABI) company QuantStudio DX instrument, qRT-PCR detection is carried out, each
Sample is repeated twice.Reaction system is as shown in table 3.
3 qRT-PCR reaction system of table
| QRT-PCR reaction system | Dosage (μ l) |
| TaqMan small RNA assay(20X) | 0.5 |
| production RT reaction | 3 |
| TaqMan Universal Master Mix II, no UNG | 5 |
| RNase-free H2O | 1.5 |
The reaction condition of qRT-PCR is as shown in table 4.
The amplification condition of 4 qRT-PCR of table
(4) qRT-PCR testing result
QRT-PCR method detects hsa-miR-423-5p, hsa-miR-451a and hsa-miR-320a expression, specific to detect
As a result as shown in Figures 1 to 3.The result according to Fig. 1-2 it is found that hsa-miR-451a and hsa-miR-320a in normal subjects
It is high with abundance in the serum of cancer of the esophagus subject, but its expression quantity shows significant difference (P in two groups of different subjects
=1.19E-7, P=8.99E-5).And as shown in figure 3, hsa-miR-423-5p is in normal subjects and cancer of the esophagus subject
Abundance is high in serum, and in two group data sets of normal subjects and cancer of the esophagus subject, discrete type is small, on gene expression abundance not
Significant difference (P=0.225) is shown, therefore, hsa-miR-423-5p is suitable as the reference gene of cancer of the esophagus miRNA.
Embodiment 3
The present embodiment is intended to enlarged sample quantity, further verifies hsa-miR-423-5p in normal subjects and the cancer of the esophagus
The expression of subject's sample.Wherein, subject is by 100 normal subjects and 100 cancer of the esophagus subject groups at selecting
From Chinese population；Detection sample is serum, detection method qRT-PCR.Specific detecting step is referring to embodiment 2.Specific detection
As a result referring to fig. 4.
Result is it is found that in normal subjects and cancer of the esophagus experimenter's serum according to Fig.4, hsa-miR-423-5p's
Expression is almost the same, no significant difference (P=0.18), and gene expression abundance is higher, is suitable as the internal reference of cancer of the esophagus miRNA
Gene.
Embodiment 4
Hsa-miR-16-5p is often used in American-European crowd as blood internal reference.The present embodiment is intended to verify hsa-miR-
Expression of the 16-5p in American-European normal subjects and cancer of the esophagus subject sample.Wherein, subject is by 20 normal subjects
With 20 cancer of the esophagus subject groups at being selected from American-European crowd；Detection sample is serum, detection method qRT-PCR.Specific inspection
Step is surveyed referring to embodiment 2.Specific testing result is referring to Fig. 5.
Result is it is found that in normal subjects and cancer of the esophagus experimenter's serum according to Fig.5, hsa-miR-16-5p's
Expression is almost the same, no significant difference (P=0.584), and gene expression abundance is higher, is suitable as American-European cancer of the esophagus miRNA
Reference gene.
Comparative example 1
This comparative example is intended to investigate existing cancer of the esophagus internal reference hsa-miR-16-5p in Chinese population normal subjects and oesophagus
Expression in cancer experimenter's serum.Wherein, subject by 100 normal subjects and 100 cancer of the esophagus subject groups at,
It is selected from Chinese population；Detection sample is serum, detection method qRT-PCR.Specific detecting step is referring to embodiment 2.Specifically
Testing result is referring to Fig. 6.
Result is it is found that table of the hsa-miR-16-5p in normal subjects and cancer of the esophagus experimenter's serum according to Fig.6,
There is more significant difference (P=1.05E-13) up to level, be not suitable as the cancer of the esophagus internal reference of Chinese population or even asian population
miRNA。
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
Claims (10)
- Application of the 1.Hsa-miR-423-5p as the internal reference miRNA of cancer of the esophagus sample.
- 2. application according to claim 1, which is characterized in that the subject of the sample is asian population, it is preferable that institute Stating asian population is Chinese population.
- 3. application according to claim 1, which is characterized in that the sample is doubted from asymptomatic subject or the cancer of the esophagus Like patient.
- 4. application according to claim 1, which is characterized in that patient with esophageal carcinoma of the sample before treatment is controlled Patient with esophageal carcinoma after treatment.
- 5. application according to claim 1, which is characterized in that the sample is tissue samples or body fluid sample, the body Liquid sample is preferably blood sample, and optionally, the blood sample is serum sample or plasma sample.
- 6. described in any item applications according to claim 1~5, which is characterized in that the internal reference miRNA is used for purpose miRNA Standardization；Preferably, the standardization includes: the absolute concentration for measuring purpose miRNA and internal reference miRNA in sample, by mesh The absolute concentration of miRNA and the absolute concentration of internal reference miRNA be compared, the miRNA concentration after being standardized.
- 7. a kind of esophagus cancer diagnosis reagent and/or diagnostic kit, which is characterized in that the diagnostic reagent and/or diagnostic reagent Box includes the detection reagent of cancer of the esophagus miRNA marker and the detection reagent of internal reference miRNA, and the internal reference miRNA includes hsa- miR-423-5p。
- 8. diagnostic reagent according to claim 7 and/or diagnostic kit, which is characterized in that the internal reference miRNA is also wrapped Include hsa-miR-16-5p.
- 9. kit according to claim 7 or 8, which is characterized in that the detection reagent of the cancer of the esophagus miRNA marker Detection reagent with internal reference miRNA is the reagent suitable for following method:Real-time fluorescence quantitative PCR, digital pcr, fluorescent dye determination, Resonance Light Scattering Method, sequencing or biomass spectrometry.
- 10. kit according to claim 9, which is characterized in that the detection of the cancer of the esophagus miRNA marker tries The detection reagent of agent and internal reference miRNA are the reagent suitable for real-time fluorescence quantitative PCR, including amplimer and Taqman are visited Needle.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910317987.2A CN109825596B (en) | 2019-04-19 | 2019-04-19 | Internal reference miRNA of esophageal cancer sample and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910317987.2A CN109825596B (en) | 2019-04-19 | 2019-04-19 | Internal reference miRNA of esophageal cancer sample and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109825596A true CN109825596A (en) | 2019-05-31 |
| CN109825596B CN109825596B (en) | 2022-03-15 |
Family
ID=66875749
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910317987.2A Active CN109825596B (en) | 2019-04-19 | 2019-04-19 | Internal reference miRNA of esophageal cancer sample and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109825596B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100285471A1 (en) * | 2007-10-11 | 2010-11-11 | The Ohio State University Research Foundation | Methods and Compositions for the Diagnosis and Treatment of Esphageal Adenocarcinomas |
| CN103602747A (en) * | 2013-11-28 | 2014-02-26 | 山东大学齐鲁医院 | Internal reference substance for detecting bladder cancer serum miRNA and its detection primers and use |
| CN106636309A (en) * | 2015-10-30 | 2017-05-10 | 益善生物技术股份有限公司 | Probe composition for detecting esophagus cancer related markers and kit of probe composition |
| EP3276009A1 (en) * | 2016-07-29 | 2018-01-31 | Gdanski Uniwersytet Medyczny | Microrna expression markers for crc development |
| CN109371137A (en) * | 2018-12-29 | 2019-02-22 | 中山大学孙逸仙纪念医院 | Hsa_circ_0007986 is as new biomarker object space method and application in detection esophagus cancer patient blood serum |
-
2019
- 2019-04-19 CN CN201910317987.2A patent/CN109825596B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100285471A1 (en) * | 2007-10-11 | 2010-11-11 | The Ohio State University Research Foundation | Methods and Compositions for the Diagnosis and Treatment of Esphageal Adenocarcinomas |
| CN103602747A (en) * | 2013-11-28 | 2014-02-26 | 山东大学齐鲁医院 | Internal reference substance for detecting bladder cancer serum miRNA and its detection primers and use |
| CN106636309A (en) * | 2015-10-30 | 2017-05-10 | 益善生物技术股份有限公司 | Probe composition for detecting esophagus cancer related markers and kit of probe composition |
| EP3276009A1 (en) * | 2016-07-29 | 2018-01-31 | Gdanski Uniwersytet Medyczny | Microrna expression markers for crc development |
| CN109371137A (en) * | 2018-12-29 | 2019-02-22 | 中山大学孙逸仙纪念医院 | Hsa_circ_0007986 is as new biomarker object space method and application in detection esophagus cancer patient blood serum |
Non-Patent Citations (2)
| Title |
|---|
| XIAOLEI LIU ET AL.: ""Identification of suitable plasma-based reference genes for miRNAome analysis of major depressive disorder"", 《JOURNAL OF AFFECTIVE DISORDERS》 * |
| 谢子钧等: ""血浆miR-10b对食管癌的诊断价值"", 《广东医学》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109825596B (en) | 2022-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20200370127A1 (en) | Biomarkers in Peripheral Blood Mononuclear Cells for Diagnosing or Detecting Lung Cancers | |
| US11421283B2 (en) | Circulating microRNA as a marker for hepatocellular carcinoma | |
| JP2020141684A (en) | Microrna biomarkers for gastric cancer diagnosis | |
| Bansal et al. | Feasibility of microRNAs as biomarkers for Barrett's esophagus progression: a pilot cross-sectional, phase 2 biomarker study | |
| US11136628B2 (en) | Biomarkers useful for detection of types, grades and stages of human breast cancer | |
| US20200308655A1 (en) | Plasma Microribonucleic Acids as Biomarkers for Endometriosis and Endometriosis-Associated Ovarian Cancer | |
| EP3122905B1 (en) | Circulating micrornas as biomarkers for endometriosis | |
| EP2596136B1 (en) | Marker consisting of plasma microrna and new method for diagnosis of hepatocellular carcinoma | |
| CN108949969B (en) | Application of long-chain non-coding RNA in colorectal cancer | |
| KR102096498B1 (en) | MicroRNA-4732-5p for diagnosing or predicting recurrence of colorectal cancer and use thereof | |
| Takahashi et al. | Current status of molecular diagnostic approaches using liquid biopsy | |
| TW201625797A (en) | Method and biomarkers for accessing the risk of having colorectal cancer | |
| CN107326092B (en) | Application of colorectal cancer-related miRNA as biomarker and colorectal cancer detection kit | |
| CN109825596A (en) | The internal reference miRNA of cancer of the esophagus sample and its application | |
| CN108728439A (en) | The finger-print of tiny RNA composition and its application in Diagnosis of Bladder | |
| KR102096499B1 (en) | MicroRNA-3960 for diagnosing or predicting recurrence of colorectal cancer and use thereof | |
| TWI626314B (en) | Method for accessing the risk of having colorectal cancer | |
| JP7299765B2 (en) | MicroRNA measurement method and kit | |
| EP3545109A1 (en) | In vitro | |
| US20150080229A1 (en) | Plasma microribonucleic acids as biomarkers for endometriosis and endometriosis-associated ovarian cancer | |
| US20230295733A1 (en) | Marker consisting of plasma microrna and a new method for diagnosis of hepatocellular carcinoma |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |