CN106075445B - The new application of tRF-Leu-CAG - Google Patents
The new application of tRF-Leu-CAG Download PDFInfo
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- CN106075445B CN106075445B CN201610295963.8A CN201610295963A CN106075445B CN 106075445 B CN106075445 B CN 106075445B CN 201610295963 A CN201610295963 A CN 201610295963A CN 106075445 B CN106075445 B CN 106075445B
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
Abstract
The present invention relates to the new applications of tRF-Leu-CAG a kind of.NSCLC is one of highest cancer of lethality, and effective method of early diagnosis has important influence to the treatment and prognosis of disease.Present invention demonstrates that tRF-Leu-CAG is broken from the anticodon loop of tRNA-Leu-CAG-1 or tRNA-Leu-CAG-2, length is 34 nt.Using qRT-PCR method validation tRF-Leu-CAG, the expression in NSCLC cell line obviously raises the present invention.Cell Proliferation and the variation of cell cycle discovery are detected by CCK8 and fluidic cell cycle analysis means, tRF-Leu-CAG inhibitor is able to suppress the cell Proliferation of H1299, while G1/S being inhibited to convert, cell block in the G1 phase.For the first time report of the tRF-Leu-CAG inhibitor as preparation treatment NSCLC drug disclosed in it, the invention clinically provide certain application value for the preparation of NSCLC therapeutic agent.
Description
Technical field
The present invention relates to a kind of application of the inhibitor of tRF, especially a kind of inhibitor of tRF-Leu-CAG is controlled in preparation
Treat the application in non-small cell lung cancer drug.
Background technique
Lung cancer is most commonly seen, death rate highest in world wide, maximum to population health and life threat pernicious swollen
One of tumor.Lung cancer can be divided into Small Cell Lung Cancer (small cell lung cancer, SCLC) and non-small cell lung cancer (non
small cell lung cancer,NSCLC).In China, about 80% lung cancer is diagnosed as NSCLC, it mainly includes gland
Cancer, squamous cell carcinoma, maxicell undifferentiated carcinoma three categories.Most of patients with lung cancer illness early stage and non-evident sympton, thus
Lead to make a definite diagnosis that the time is later, and therapeutic effect is poor, the lower feature of disease prognosis rate.Even if treatment method achieves huge hair
Exhibition, the five year survival rate of NSCLC are still not higher than 15%.
Critical elements of the tRNA with classical cloverleaf structure as cell protein synthesis machine, have possessed tens
Year thoroughgoing and painstaking research history.But be far from end for the understanding of its function, and it is as potential gene expression tune
The function of control molecular precursor is just gradually being recognized at present.Newest multinomial result of study shows to lead in various kinds of cell system
It crosses high-flux sequence and finds that certain derives from the small fragment RNA (tRNA-derived small RNA, tRF) of tRNA, these are cut
Cutting product is considered having phase interaction with a variety of miRNA process systems key molecules (protein in such as Dicer, Ago family)
Ability.Meanwhile the result of study of reporter gene detection system also implies, these small fragment RNAs have diving for similar miRNA
In adjusting function, important adjustment effect may be played when cell copes with external environment stimulation.
TRF is a kind of non-coding single stranded RNA, and length is 14-35 (nucleotide, nt), in specific environment can be from
The end 3' 5' of tRNA is broken to obtain.Some tRF are broken under the action of anticodon loop shears enzyme from mature tRNA
It arrives, length 30-35nt, therefore referred to as tRNA half molecule.Remaining tRF is then divided into 3'U tRF, 3'CCA tRF and 5'
tRF.3'U tRF is usually to shear to obtain by tRNA enzyme and RNA enzyme P in maturation in core in tRNA, and be transported to thin
In cytoplasm.And 3'CCA tRF and 5'tRF are then to be sheared to obtain from mature tRNA by Dicer enzyme and angiogenin.But
Different from miRNA and piRNA, the research at present about tRF biological function is also very limited.
Summary of the invention
One of the objects of the present invention is to provide the inhibitor of tRF-Leu-CAG a kind of to hinder in H1299 cell in preparation
Application in cell proliferation.The tRF-Leu-CAG is tRNA half molecule, and the fragment length of tRF-Leu-CAG is 34nt,
The sequence of tRF-Leu-CAG is GTCAG GATGG CCGAG CGGTC TAAGG CGCTG CGTT.
The second object of the present invention is to provide the inhibitor of tRF-Leu-CAG a kind of in preparation retardance H1299 cell
G1/S converts the application in drug.The tRF-Leu-CAG is tRNA half molecule, and the fragment length of tRF-Leu-CAG is 34nt,
The sequence of tRF-Leu-CAG is GTCAG GATGG CCGAG CGGTC TAAGG CGCTG CGTT.
The third object of the present invention is that the inhibitor for providing a kind of tRF-Leu-CAG treats non-small cell lung cancer in preparation
Application in drug.TRF-Leu-CAG specificity overexpression, inhibition of tRF-Leu-CAG in the common NSCLC cell line in part
Agent can inhibit cell Proliferation and the cell cycle of NSCLC, can be used as the application of preparation treatment non-small cell lung cancer drug.Institute
Stating tRF-Leu-CAG is tRNA half molecule, and the fragment length of tRF-Leu-CAG is 34nt, and the sequence of tRF-Leu-CAG is
GTCAG GATGG CCGAG CGGTC TAAGG CGCTG CGTT。
Implementation steps of the invention are as described below:
First, reads number of the tRFs in lung cancer sample is detected by database sample collection and high-flux sequence, and
Screening obtains the tRFs of abnormal expression.
Second, the present invention has detected expression of the tRF-Leu-CAG in common NSCLC cell line by qRT-PCR technology
Situation, as the result is shown its significantly high expression.
Third, the present invention detect cell Proliferation by CCK8, and the flow cytomery cell cycle confirms tRF-Leu-
CAG inhibitor can significantly inhibit NSCLC cell Proliferation and cell cycle progress.
To implement as procedure described above, all parts of the invention will be described later in detail below, it need to be pointed out that: it is above-mentioned
And the content combination of each specific features of ensuing disclosure is also within the scope of the present invention.
Present invention demonstrates that tRF-Leu-CAG is the anticodon from tRNA-Leu-CAG-1 or tRNA-Leu-CAG-2
It is broken at ring, length 34nt.The present invention is using qRT-PCR method validation tRF-Leu-CAG in NSCLC cell line
Middle expression obviously raises.The variation of cell Proliferation and cell cycle is detected by CCK8 and fluidic cell cycle analysis means
It was found that the inhibitor of tRF-Leu-CAG is able to suppress the cell Proliferation of H1299, while G1/S being inhibited to convert, cell block is existed
The G1 phase.For the first time report of the inhibitor of tRF-Leu-CAG disclosed in it as preparation treatment NSCLC drug, the invention exist
Clinically certain application value is provided for the preparation of NSCLC therapeutic agent.
Detailed description of the invention
The reads number distribution situation of Fig. 1 tRNA-Leu, wherein (A) tRNA-Leu-CAG-1 is in 30 pairs of database lung cancer samples
Reads number in product obviously rises.(B) tRNA-Leu-CAG-2 is on the reads number in 30 pairs of database lung cancer samples is obvious
It rises.
Fig. 2 tRF-Leu-CAG is a kind of tRNA half molecule, wherein the fragment length of (A) tRF-Leu-CAG is 34nt.
(B) & (C) tRF-Leu-CAG degrades to obtain from the anticodon loop of tRNA-Leu-CAG-1 or tRNA-Leu-CAG-2, is
TRNA half molecule.
Up-regulation trend is presented in Fig. 3 tRF-Leu-CAG in NSCLC cell line.
Fig. 4 tRF-Leu-CAG inhibitor is able to suppress cell Proliferation and the cell cycle of NSCLC, wherein (A)
MiR-1251-5p inhibitor can be used as tRF-Leu-CAG inhibitor, lower tRF-Leu-CAG expression.(B)
TRF-Leu-CAG inhibitor is able to suppress the cell Proliferation of H1299.(C) tRF-Leu-CAG inhibitor is able to suppress
The cell cycle of H1299.
Specific embodiment
Ensuing disclosure will the present invention is further explained in conjunction with specific example.These examples are used merely to explain the present invention, without
For limiting the scope of the invention.Specific experiment condition or experimental method are not specified in following Examples, usually according to conventional strip
Part --- i.e. item described in " molecular cloning " (Molecular Cloning:A Laboratory Manual, 3rd ed.)
Part, or implement according to the normal condition proposed by manufacturer.
Embodiment one: high-flux sequence detects expression number of the tRF in NSCLC sample and screens
We have collected 30 pairs of NSCLC sample messages by SRA database, and are counted by the means of high-flux sequence
According to analysis.
According to data analysis it is found that in 30 pairs of database samples, the Reads number distributed area of tRF is 17-34nt,
In have the tendency that the Reads number presentation of 27 kinds of tRNA obviously rises (FC>1.3, P<0.05), and tRNA-Leu and tRNA-Val are accounted for
The ratio highest arrived, respectively 37% and 26%.
TRF-Leu-CAG is determined as research object, and in database sample by screening, and tRNA-Leu-CAG-1 (schemes
1A) and the trend obviously risen is presented in the reads number of tRNA-Leu-CAG-2 (Figure 1B) in cancer.
By qRT-PCR product is detected can determine tRF-Leu-CAG length be 34nt (Fig. 2A), be from
What the anticodon loop of tRNA-Leu-CAG-1 (Fig. 2 B) and tRNA-Leu-CAG-2 (Fig. 2 C) were degraded, therefore be one kind
TRNA half molecule.
Expression variation of the embodiment two: tRF-Leu-CAG in mankind's NSCLC cell line
We detect expression of the tRF-Leu-CAG in human lung cancer cell line by the method for qRT-PCR.
The cell line that the present invention uses includes: that H23 is p53+/-Lines, A549 Kras+/+It is non-small
Cell lung carcinoma lines, H1299 p53-/-Lines, SPCA-1 are Asian's non-small cell lung cancer cell
System, PC-9 and H1650 are lung adenocarcinoma cell system, and 95-D is people's migration maxicell lung cancer cell line, and 16HBE is on pulmonary branches tracheae
Chrotoplast system.
This example cracks using Trizol reagent and extracts the total serum IgE in sample, uses the One Step of TaKaRa company
PrimeScript miRNA cDNA Synthesis Kit (Code No.D350A) reverse transcription obtains cDNA template.The reagent
Box can carry out Poly (A) tailings reactions to the small non-coding RNA in sample, while introduce Uni-miR qPCR Primer knot
Coincidence point, in sample any sncRNA and cDNA carry out quantitative PCR reaction.Such as 1 institute of table of primer used in the present embodiment
Show.
1 qRT-PCR detection primer sequence of table
According to experimental result it is found that with the internal reference of U6, pulmonary branches tracheal epithelial cell 16HBE is compared, tRF-Leu-CAG is six
Relative expression levels in kind Lines are risen (Fig. 3).
Three: tRF-Leu-CAG inhibitor of embodiment is able to suppress cell Proliferation and the cell cycle of NSCLC
It is compared by the BLAST of ncbi database, it is found that it is certain the sequence of miR-1251-5p and tRF-Leu-CAG have
Similitude.It is found after miR-1251-5p inhibitor is transfected into H1299, the expression quantity of tRF-Leu-CAG presents obvious
It lowers, therefore, it is considered that miR-1251-5p inhibitor can be used as tRF-Leu-CAG inhibitor (Fig. 4 A).Meanwhile it is logical
Cross CCK8 and fluidic cell cycle analysis means detection cell Proliferation and the variation of cell cycle discovery, tRF-Leu-CAG
Inhibitor is able to suppress the cell Proliferation (Fig. 4 B) of H1299, while G1/S being inhibited to convert, cell block in G1 phase (figure
4C)。
Although the present invention describes specific example, there is any to be apparent to practitioners skilled in the art,
The present invention can be made various changes and be changed under the premise without departing from the spirit and scope of the present invention.Therefore, appended right
It is required that covering all these variations within the scope of the present invention.
Claims (3)
1. a kind of inhibitor of tRF-Leu-CAG hinders the application in H1299 cell proliferation in preparation, it is characterised in that:
The inhibitor of tRF-Leu-CAG is able to suppress the cell Proliferation of H1299；The tRF-Leu-CAG is tRNA half molecule, tRF-
The fragment length of Leu-CAG is 34nt, and the sequence of tRF-Leu-CAG is GTCAG GATGG CCGAG CGGTC TAAGG
CGCTG CGTT。
2. a kind of application of inhibitor of tRF-Leu-CAG in preparation retardance H1299 cell in G1/S conversion drug, feature
Be: the inhibitor of tRF-Leu-CAG is able to suppress the cell Proliferation of H1299, while G1/S being inhibited to convert, and cell block is existed
The G1 phase；The tRF-Leu-CAG is tRNA half molecule, and the fragment length of tRF-Leu-CAG is 34nt, the sequence of tRF-Leu-CAG
It is classified as GTCAG GATGG CCGAG CGGTC TAAGG CGCTG CGTT.
3. a kind of application of inhibitor of tRF-Leu-CAG in preparation treatment non-small cell lung cancer drug, it is characterised in that:
The inhibitor of tRF-Leu-CAG is able to suppress cell Proliferation and the cell cycle of NSCLC；The tRF-Leu-CAG is tRNA half
Molecule, the fragment length of tRF-Leu-CAG are 34nt, and the sequence of tRF-Leu-CAG is GTCAG GATGG CCGAG CGGTC
TAAGG CGCTG CGTT。
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| CN109136381A (en) * | 2018-11-04 | 2019-01-04 | 华东医院 | A kind of tRNA molecular marker of diagnosis and treatment adenocarcinoma of lung and its application |
| CN113981044A (en) * | 2021-07-08 | 2022-01-28 | 南京鼓楼医院 | TsrA detection method based on rolling circle amplification and molecular beacon |
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| Endogenous tRNA-Derived Fragments Suppress Breast Cancer Progression via YBX1 Displacement;Hani Goodarzi等;《Cell》;20150507;第161卷;第790–802页 |
| The Hansenula polymorpha (strain CBS4732) genome sequencing and analysis;Massoud Ramezani-Rad等;《FEMS Yeast Research》;20030430;第4卷;第207-215页 |
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