CN109234398A - Application of long-chain non-coding RNA MALAT1, HIF1A-AS1 and the XIST in non-small cell lung cancer early diagnosis kit in blood plasma - Google Patents
Application of long-chain non-coding RNA MALAT1, HIF1A-AS1 and the XIST in non-small cell lung cancer early diagnosis kit in blood plasma Download PDFInfo
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Abstract
Application of long-chain non-coding RNA MALAT1, HIF1A-AS1 and the XIST in non-small cell lung cancer early diagnosis kit in blood plasma disclosed by the invention, it is the relative expression quantity that MALAT1, HIF1A-AS1 and XIST in person under test's blood plasma are detected by real-time fluorescence quantitative PCR (GAPDH is internal reference), for non-small cell lung cancer people at highest risk's Early stage NSCLC screening, has many advantages, such as high sensitivity, high specificity, easy to operate and at low cost.
Description
Technical field
The invention belongs to biomedicine fields, in particular in blood plasma long-chain non-coding RNA MALAT1, HIF1A-AS1 and
Application of the XIST in non-small cell lung cancer early diagnosis kit.
Background technique
Lung cancer is the highest malignant tumour of morbidity and mortality in global range.China's lung cancer neopathy number in 2017 reaches
To 800,000, death toll nearly 700,000 accounts for a quarter of all number of cancer deaths, and this data is also with annual 26.9%
Speed increase, it is contemplated that be up to 1,000,000 people by 2025.Clinically about 85% lung cancer is non-small cell lung cancer, is survived within 5 years
Rate is lower than 15%.Due to the limitation of method of early diagnosis, the patient more than 75% misses the best period of operative treatment, thus
Non-small cell lung mortality of carcinoma is caused to remain high.Therefore, tobacco consumption and reduction air are controlled except further taking measures
Pollution is outer, is carried out early using accurate, efficient, quickly repeatable diagnosis marker or technology to non-small cell lung cancer people at highest risk
Phase screening is beneficial to patient and receives treatment in time, improves life quality.
Long-chain non-coding RNA (Long non-coding RNA, lncRNA) is that a kind of transcript length is more than 200nt, no
The RNA of coding protein.These lncRNA are once once not considered biological function, and what is only generated in transcription " makes an uproar
Sound ".But in recent years, more and more researches show that the exception of lncRNA level with including lung cancer including many tumours generation,
Develop related.
(the Metastasis-associated lung adenocarcinoma of lung cancer metastasis associated retroviral sheet 1
Transcript 1, MALAT1) be initially comparison lung cancer metastasis tumor and non-small cell lung cancer transfer process in find, and by
Think closely related with lung cancer Highly invasive and metastasis characteristic and the undesirable prognosis of patient.In addition, in each organ-tissue of human body
In have the expression of MALAT1.Multinomial research confirms that the differential expression of MALAT1 can be used as important tumor marker.
HIF1A-AS1 high expression in tumor of kidney and abdominal aneurvsm shows that its ginseng may participate in the generation of tumour.It passes through cell
Interaction between apoptotic proteins plays a significant role in cell proliferation in vitro and apoptosis.X chromosome inactivation specific transcription
Factor lncRNA (X-inactive specific transcript, XIST) is XIST gene encoding production, and main adjust is fed
Laticiferous cell x chromosome inactivation.Research confirms XIST high expression in certain tumours, such as glioblastoma, breast cancer and son
Palace cancer shows the diagnosis that XIST can be used as biomarker for tumor disease.Inside and outside is the experimental results showed that knock out
It can inhibit tumour growth after XIST gene, XIST is prompted to play a significant role in the occurrence and development of malignant tumour.But due to
Single lncRNA is used to have the defects that specific low, poor sensitivity as Testing index, therefore the present invention is simultaneously to multiple
LncRNA, which carries out joint-detection, becomes the key for improving specificity and sensitivity.Yet there are no MALAT1, HIF1A-AS1 and
Research and application of the XIST in non-small cell lung cancer early diagnosis.
Summary of the invention
The main object of the present invention is to provide MALAT1, HIF1A-AS1 and XIST gene and examines in non-small cell lung cancer early stage
Application in disconnected kit, also resides in and provides a kind of non-small cell lung cancer early diagnosis kit.
To achieve the above object, a kind of non-small cell lung cancer early diagnosis kit proposed by the present invention, including amplification and
Detect long-chain non-coding RNA MALAT1, HIF1A-AS1, XIST and internal reference upstream and downstream primer.
It preferably, further include that real-time fluorescence quantitative PCR reagent, reverse transcription reagents and RNA extract reagent.
Preferably, the internal reference is GAPDH gene.
Preferably, the upstream and downstream primer includes:
MALAT1 upstream primer: 5 '-AAAGCAAGGTCTCCCCACAAG-3 ',
MALAT1 downstream primer: 5 '-GGTCTGTGCTAGATCAAAAGGC-3 '；
HIF1A-AS1 upstream primer: 5 '-TCTTCGGTACTTTACGCACCC-3 ',
HIF1A-AS1 downstream primer: 5 '-TTTTCCTCTGCGTTCTGGAGC-3 '；
XIST upstream primer: 5 '-TCAGCCCATCAGTCCAAGATC-3 ',
XIST downstream primer: 5 '-CCTAGTTCAGGCCTGCTTTTCAT-3 '；
GAPDH upstream primer: 5 '-GCACCGTCAAGGCTGAGAAC-3 ',
GAPDH downstream primer: 5 '-TGGTGAAGACGCCAGTGGA-3 '.
Preferably, the present invention also provides long-chain non-coding RNA MALAT1, HIF1A-AS1 and XIST in blood plasma non-small thin
Application in born of the same parents' lung cancer early diagnosis kit, the kit by real-time fluorescence quantitative PCR detect blood in MALAT1,
HIF1A-AS1 and XIST expression is to determine whether suffer from non-small cell lung cancer.
Long-chain non-coding RNA MALAT1, HIF1A-AS1 and XIST are in non-small cell lung cancer in blood plasma disclosed by the invention
Application in early diagnosis kit, by real-time fluorescence quantitative PCR detect person under test's blood plasma in MALAT1, HIF1A-AS1 and
The relative expression quantity (GAPDH is internal reference) of XIST, if the ratio between MALAT1 relative expression quantity and normal person are higher than in person under test's blood plasma
1.22, and the ratio between HIF1A-AS1, XIST relative expression quantity and normal person are respectively lower than 0.65 and 0.56, then person under test is with non-
A possibility that Small Cell Lung Cancer, is higher than 93.7%.
Non-small cell lung cancer people at highest risk is detected using the present invention, there is high sensitivity, high specificity, operation letter
The advantages that single and at low cost.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is general
Logical technical staff every other embodiment obtained without creative efforts belongs to what the present invention protected
Range.
In addition, the description for being related to " first ", " second " etc. in the present invention is used for description purposes only, and should not be understood as referring to
Show or imply its relative importance or implicitly indicates the quantity of indicated technical characteristic." first ", " are defined as a result,
Two " feature can explicitly or implicitly include at least one of the features.In addition, the technical solution between each embodiment can
It to be combined with each other, but must be based on can be realized by those of ordinary skill in the art, when the combination of technical solution occurs
Conflicting or cannot achieve when, will be understood that the combination of this technical solution is not present, also not the present invention claims protection model
Within enclosing.
In embodiments of the present invention, the non-small cell lung cancer early diagnosis kit, including expand and detect the non-volume of long-chain
Code RNA MALAT1, HIF1A-AS1, XIST and internal reference upstream and downstream primer.
It further, further include that real-time fluorescence quantitative PCR reagent, reverse transcription reagents and RNA extract reagent.
Further, the internal reference is GAPDH gene.
In the present embodiment, in blood plasma long-chain non-coding RNA MALAT1, HIF1A-AS1 and XIST in non-small cell lung cancer
Application in early diagnosis kit, the kit detect MALAT1, HIF1A-AS1 in blood by real-time fluorescence quantitative PCR
With XIST expression to determine whether suffering from non-small cell lung cancer, method is as described below:
1, the RNA (miRNeasy Serum/Plasma Kit) in blood plasma is extracted
(1) plasma sample to be extracted is taken out from -70 DEG C of ultra low temperature freezers, is put on trash ice and is thawed；
(2) sample 12000g is extracted, at 4 DEG C, is centrifuged 10min；
(3) supernatant is transferred in EP pipe, carries out RNA extraction by miRNeasy Serum/Plasma Kit specification.
2, RNA reverse transcription (PrimeScriptTM RT Master Mix)
(1) reaction system is as follows:
| 5x PrimeScript RT Master Mix | 4ul |
| Blood plasma RNA | 14ul |
| RNase-free H2O | 4ul |
| Total | 20ul |
(2) response procedures are as follows:
| 37℃ | 15min |
| 85℃ | 5sec |
| 4℃ | ∞ |
3, real-time fluorescence quantitative PCR (SYBR Premix Ex TaqTM II)
(1) reaction system is as follows:
Wherein, upstream and downstream primer sequence is specific as follows:
MALAT1 upstream primer: 5 '-AAAGCAAGGTCTCCCCACAAG-3 ',
MALAT1 downstream primer: 5 '-GGTCTGTGCTAGATCAAAAGGC-3 '；
HIF1A-AS1 upstream primer: 5 '-TCTTCGGTACTTTACGCACCC-3 ',
HIF1A-AS1 downstream primer: 5 '-TTTTCCTCTGCGTTCTGGAGC-3 '；
XIST upstream primer: 5 '-TCAGCCCATCAGTCCAAGATC-3 ',
XIST downstream primer: 5 '-CCTAGTTCAGGCCTGCTTTTCAT-3 '；
GAPDH upstream primer: 5 '-GCACCGTCAAGGCTGAGAAC-3 ',
GAPDH downstream primer: 5 '-TGGTGAAGACGCCAGTGGA-3 '.
Embodiment 1
(1) 30 Patients with Non-small-cell Lung and 30 normal persons are chosen, are detected according to above-mentioned method, as a result such as
Shown in table 1,2,3:
| Group | MALAT1 relative expression quantity |
| Patients with Non-small-cell Lung | 35.13±2.15 |
| Normal person | 28.63±3.02 |
Table 1: clinically Patients with Non-small-cell Lung and MALAT1 relative expression quantity in normal human blood
| Group | HIF1A-AS1 relative expression quantity |
| Patients with Non-small-cell Lung | 23.85±4.13 |
| Normal person | 36.77±3.76 |
Table 2: clinically Patients with Non-small-cell Lung and HIF1A-AS1 relative expression quantity in normal human blood
Table 3: clinically Patients with Non-small-cell Lung and XIST relative expression quantity in normal human blood
Show through the foregoing embodiment:
If the ratio between MALAT1 relative expression quantity and normal person are higher than 1.22, and HIF1A-AS1, XIST phase in person under test's blood plasma
0.65,0.56 is respectively lower than to the ratio between expression quantity and normal person, then person under test reaches with a possibility that non-small cell lung cancer
93.7%.
Embodiment 2
10 Patients with Non-small-cell Lung and 10 normal persons are randomly selected, the method according to the invention is detected, to
The ratio between the relative expression quantity of MALAT1, HIF1A-AS1 and XIST and normal person meet higher than 1.22, are lower than in survey person's blood plasma
0.65,0.56, show to carry out non-small cell lung cancer using the expression of MALAT1, HIF1A-AS1 and XIST in blood plasma early
Phase diagnosis has very high specificity and sensitivity.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (5)
1. a kind of non-small cell lung cancer early diagnosis kit, it is characterised in that: including expanding and detecting long-chain non-coding RNA
MALAT1, HIF1A-AS1, XIST and internal reference upstream and downstream primer.
2. non-small cell lung cancer early diagnosis kit according to claim 1, it is characterised in that: further include real-time fluorescence
Quantitative PCR reagent, reverse transcription reagents and RNA extract reagent.
3. non-small cell lung cancer early diagnosis kit according to claim 1, it is characterised in that: the internal reference is
GAPDH gene.
4. non-small cell lung cancer early diagnosis kit according to claim 1, it is characterised in that: the non-volume of the long-chain
Code RNA and internal reference upstream and downstream primer include:
MALAT1 upstream primer: 5 '-AAAGCAAGGTCTCCCCACAAG-3 ',
MALAT1 downstream primer: 5 '-GGTCTGTGCTAGATCAAAAGGC-3 '；
HIF1A-AS1 upstream primer: 5 '-TCTTCGGTACTTTACGCACCC-3 ',
HIF1A-AS1 downstream primer: 5 '-TTTTCCTCTGCGTTCTGGAGC-3 '；
XIST upstream primer: 5 '-TCAGCCCATCAGTCCAAGATC-3 ',
XIST downstream primer: 5 '-CCTAGTTCAGGCCTGCTTTTCAT-3 '；
GAPDH upstream primer: 5 '-GCACCGTCAAGGCTGAGAAC-3 ',
GAPDH downstream primer: 5 '-TGGTGAAGACGCCAGTGGA-3 '.
5. long-chain non-coding RNA MALAT1, HIF1A-AS1 and XIST is in non-small cell lung cancer early diagnosis kit in blood plasma
Application, it is characterised in that: the kit by MALAT1, HIF1A-AS1 in plasma by real-time PCR and
XIST expression is to determine whether suffer from non-small cell lung cancer.
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Cited By (4)
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| CN109735570A (en) * | 2019-02-19 | 2019-05-10 | 浙江大学 | The preparation and its application of the adipose-derived mescenchymal stem cell excretion body of XIST modification |
| CN110541027A (en) * | 2019-08-21 | 2019-12-06 | 昆明医科大学第一附属医院 | Application of lncRNA HIF1A-AS1 in resisting deep vein thrombosis |
| CN110699457A (en) * | 2019-10-30 | 2020-01-17 | 深圳瑞科生物科技有限公司 | Primer group and kit for detecting lung cancer |
| CN111893190A (en) * | 2020-09-15 | 2020-11-06 | 中国医科大学附属第一医院 | Application of lncRNA XIST as gastric cancer diagnosis marker |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109735570A (en) * | 2019-02-19 | 2019-05-10 | 浙江大学 | The preparation and its application of the adipose-derived mescenchymal stem cell excretion body of XIST modification |
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| CN110541027A (en) * | 2019-08-21 | 2019-12-06 | 昆明医科大学第一附属医院 | Application of lncRNA HIF1A-AS1 in resisting deep vein thrombosis |
| CN110699457A (en) * | 2019-10-30 | 2020-01-17 | 深圳瑞科生物科技有限公司 | Primer group and kit for detecting lung cancer |
| CN111893190A (en) * | 2020-09-15 | 2020-11-06 | 中国医科大学附属第一医院 | Application of lncRNA XIST as gastric cancer diagnosis marker |
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