CN106244592B - A kind of long non-coding RNA and its application in diagnosis/treatment non-small cell lung cancer - Google Patents
A kind of long non-coding RNA and its application in diagnosis/treatment non-small cell lung cancer Download PDFInfo
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- CN106244592B CN106244592B CN201610719970.6A CN201610719970A CN106244592B CN 106244592 B CN106244592 B CN 106244592B CN 201610719970 A CN201610719970 A CN 201610719970A CN 106244592 B CN106244592 B CN 106244592B
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Abstract
The invention belongs to genetic engineering field, in particular to application of the LINC00111 in preparation prediction non-small cell lung cancer prognosis and target drug treatment；The LINC00111 expression up-regulation in NSCLC, the expression by changing LINC00111 have an impact invasion, the migration etc. of non-small cell lung cancer cell, strike low LINC00111 expression and are able to suppress cell proliferation of NSCLC and induce cell apoptosis.
Description
Technical field
The invention belongs to genetic engineering field, in particular to a kind of long non-coding RNA and its in diagnosis/treatment non-small cell
Application in lung cancer.
Background technique
Genomics studies have shown that human genome there are about 20000 protein coding genes, about total gene number ratio
2%, remaining most of gene is transcribed into non-coding RNA (non-codingRNA, ncRNA).NcRNA can be divided into according to its length
Transcription initiation RNA, Piwi protein-interacting RNA, Microrna, little nucleolar RNA and long non-coding RNA (longnon-
CodingRNA, lncRNA) etc..LncRNA is that a kind of transcript length is more than 200 nucleotide and itself does not encode albumen
RNA molecule.LncRNA be initially believed to be rna plymerase ii transcription by-product, be subgenomic transcription " noise ", do not have
Biological function.However, it has recently been demonstrated that they can influence base by a variety of different mechanism, in a variety of levels
The expression of cause.There are diversity for the mode of LncRNAs controlling gene expression, show the gene expression that lncRNAs is relied on
The diversity of regulatory mechanism.In different mechanisms, lncRNAs effect is different, can not only be used for main transcription regulatory factor, and can
As one of total regulatory factor, regulating and controlling effect is played jointly with other components.LncRNAs is big for the level of gene expression regulation
It can be divided on body: (1) apparently modify level modulation, by modifying enzyme interacting with various chromatin, chromatin is repaired
Decorations, change its conformation, activate or inhibit the expression of related gene.Especially in Embryonic Stages, lncRNAs participation causes to lose
The maintenance of allele the later stage experssion silencing, Apparent character of biography, extremely for metazoan normal development and cell differentiation
It closes important.(2) transcriptional level control, LncRNAs are formed by the combination and assembly of adjusting transcription factor with regulating and controlling sequence DNA
Three chain cpds, rna regulation polymerase II, the methods of transcription interference are realized.(3) post-transcriptional level regulates and controls, by with it is complementary
MRNA forms dsRNA, the processes such as processing, montage, transhipment, translation and the degradation of mRNA is influenced, to adjust the expression of gene.
LncRNAs becomes the new heat of tumor research because of the latent effect that it is manifested in carcinogenic and tumor suppression approach
Point.In recent years, the certain LncRNAs of many studies have shown thats and corresponding human tumor are closely related.By regulating and controlling a series of biologies
Modes, the LncRNAs such as function or interference normal function, including Transcriptional Silencing, alternative splicing rise in the occurrence and development of tumour
Important function.
Show that the morbidity and mortality of lung cancer are equal in countries in the world according to the data that the World Health Organization (WHO) is announced
In the country of the trend obviously risen, especially industry prosperity.In developed country, lung cancer is one of most common malignant tumour,
Arrange first, the 2nd, 3 of column women common cancer of male's common cancer.Lung cancer has accounted for pernicious swollen at the end of the 20th century
The first place of tumor death.Non-small cell lung cancer accounts for the 80% of all lung cancer, can be divided into gland cancer, squamous carcinoma, maxicell lung cancer etc..Many lungs
Cancer patient has shifted in diagnosis, often loses operative chance, and also not good enough for the chemotherapy regimen effect of lung cancer, and 5 years
Survival rate is lower than 5%, needs to find new, effectively to treat lung cancer method.With going deep into for genetic engineering research, scientist
Keen interest is shown to tumour medicine is developed with genetic engineering.Inventor has found LINC00111 in treatment non-small cell lung
There is good application prospect in terms of cancer.
LINC00111 is the LncRNA that a length is 469bp, is to provide normal lung tissue and cancerous lung tissue by inventor
Sample carries out chip by Boao Biological Co., Ltd and prepares the significant highly expressed RNA in cancerous lung tissue filtered out.
LINC00111 has found and names for the first time have novelty by inventor.
Summary of the invention
The object of the present invention is to provide non-small thin for diagnosing non-small cell lung cancer (NSCLC) prognosis situation or as treating
The long non-coding RNA (LINC00111) of born of the same parents' lung-cancer medicament target spot, nucleotides sequence are classified as SEQ ID NO:1,
SEQ ID NO:1:
TCCATACACTCCGTCTCCTGAAGGGGAAGCGGGCTCTTCTCAGATGCACAGGGACAATGTGAAAATCCT
GTCCTCAGATTGAGAGGCTGTTTCGTGGGCACCGAATTCGGGGTCAGGAAAGCAGCCTGCATCCACGAGTATCCTCG
GGTTACTAAGTGGGGCCAGTGGCTCCAGGTGTAACCCATTTAAGTTTGCCAGACAGCCGGGATGTTCTGGTGAAGGA
TCTGAAGTGTATGGCCACACCAGTCCCAGAAGAGCCTGGGAGAAGGGAAGATGGTGAACACAGTGGAGTTCTGCTGC
AAAGCCGAAGATGGTTCTGGCACGTGGCATGACCCACATGACTCAACATCAGGAGATCTGACTTCATAAAAGTGAAC
TATCACAATGCTGCTTTGCAAGCTGTGTGTGAGTGTAAAAGCGTTGAAACTTCCTCAATAAATGAAAAGATATCTTT
AAAAAAAAAAAAAAA
The invention further relates to the markers of long non-coding RNA to judge examining for Treatment for Non-small Cell Lung prognosis situation in preparation
Application in stopping pregnancy product, the marker includes but is not limited to:
(1) in conjunction with described in the combination of primer/primer sets of the long non-coding RNA or fluorescent marker long non-coding RNA
Primer/primer sets；
(2) in conjunction with the small molecule compound of the long non-coding RNA；
(3) in conjunction with the large biological molecule of the long non-coding RNA, the large biological molecule includes but is not limited to: antibody
Or the antibody or antibody functional fragment of antibody functional fragment, fluorescent marker, rna binding protein or its function fragment, fluorescent marker
Rna binding protein or its function fragment.
The invention further relates to nucleotide sequence such as SEQ ID NO:2 and the SEQ ID of the primer sets for long non-coding RNA
Shown in NO:3:
SEQ ID NO:2:F1:CCTGGGAGAAGGGAAGA；
SEQ ID NO:3:R1:AAGCAGCATTGTGATAGTT.
The invention further relates to include the marker for judge Treatment for Non-small Cell Lung prognosis situation reagent or
Kit.
The invention also includes nucleotide sequence such as the SEQ ID NO:4, SEQ ID of the siRNA for long non-coding RNA
Shown in NO:5 or SEQ ID NO:6:
SEQ ID NO:4 CAGTGGCTCCAGGTGTAACCCATTT
SEQ ID NO:5 GGGATGTTCTGGTGAAGGATCTGAA
SEQ ID NO:6 CAACATCAGGAGATCTGACTTCATA
The invention also includes the markers for judging reagent or the examination of Treatment for Non-small Cell Lung prognosis situation
Agent box.
The invention also includes the inhibitor for treating the drug or pharmaceutical composition of non-small cell lung cancer.
Technical solution
The present invention will be further explained by examples below, but does not limit the present invention.
Generality explanation:
The experimental method of the dated actual conditions in end in embodiment is substantially all and writes according to Sambrook, J et al.
" Molecular Cloning:A Laboratory guide (the 3rd edition) " (MolecularCloning:ALaboratoryManual, 3rded. Huang Peitang etc.
Translate, Science Press .2002.8) described in condition and method or according to condition proposed by material supplier and method into
Row, it is well known standard method that other technologies being not described in, which correspond to for those skilled in the art,.
Material of the invention: cell strain, slow virus interference carrier and the culture medium referred in the application has commodity confession
Answer or with other approach can for obtained by the public, they are only for example, to the present invention be not uniquely, can be respectively with other suitable
Tool and biomaterial replace.
The interference sequence for LINC00111 for designing specificity, to interfere the tract of GAPDH gene as control.
The siRNA of synthesis is transfected into non-small cell lung cancer cell strain A549 cell, plays the role of lowering LINC00111 expression.
Detailed description of the invention
The LINC00111 of Fig. 1 qRT-PCR survey normal lung epithelial cell strain 16HBE and non-small cell lung cancer cell strain A549
Expression, GAPDH are set as internal reference.
Fig. 2, mtt assay, which are surveyed, changes LINC00111 expression front and back to Non-small Cell Lung Cancer A 549 strain progress cisplatin treated
IC50.
Fig. 3, mtt assay, which are surveyed, changes LINC00111 expression front and back to Non-small Cell Lung Cancer A 549 strain progress docetaxel
The IC50 of processing.
Fig. 4, flow cytometry, which are surveyed, changes LINC00111 expression front and back non-small cell lung cancer cell strain A549 for cis-platinum
Changes of cell apoptosis, wherein a is A549/si-NC, and b A549/si-LINC00111, c compare for apoptosis rate.
Fig. 5, flow cytometry survey change LINC00111 expression front and back non-small cell lung cancer cell strain A549 for more west he
The changes of cell apoptosis of match, wherein a is A549/si-NC, and b A549/si-LINC00111, c compare for apoptosis rate.
Specific embodiment
The basic verification test of embodiment 1LINC00111 expression in non-small cell lung cancer
(1): expression of the LINC00111 in normal lung tissue and cancerous lung tissue
1. chip preparation and analysis: preparing normal lung tissue and cancerous lung tissue mark according to the requirement of Boao Biological Co., Ltd
This, transfers to Boao Biological Co., Ltd to carry out chip preparation, and mankind's long-chain non-coding RNA chip V1.0 version completes chip point
Analysis.
2. chip results are analyzed: expression quantity of the LINC00111 in lung cancer is compared with having raised 29.08 times in normal lung tissue；
Prompt LINC00111 that may play a role as an oncogene in lung cancer.
(2): qRT-PCR analyzes LINC00111 in normal lung epithelial cell strain 16HBE and non-small cell lung cancer cell strain
Expression quantity in A549
1. method: Trizol reagent extracts cell total rna, and qRT-PCR uses SYBRGreenPCRMasterMix
(TAKARA, Dalian, China), people GAPDH is as internal reference.
2.qRT-PCR result: as shown in Figure 1.
3. interpretation of result: LINC00111 low expression in 16HBE, the high expression in A549.
Embodiment 2 inhibits LINC00111 to express the therapeutic effect for non-small cell lung cancer
1. flow cytomery Apoptosis:
The nucleotide sequence of the siRNA of LINC00111 such as SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6 institute
Show:
SEQ ID NO:4 CAGTGGCTCCAGGTGTAACCCATTT
SEQ ID NO:5 GGGATGTTCTGGTGAAGGATCTGAA
SEQ ID NO:6 CAACATCAGGAGATCTGACTTCATA
To interfere the tract of GAPDH gene as control, and (the above carrier and segment are inserted into slow virus carrier
It is synthesized by invitrogen company).Packaged slow virus carrier is infected into non-small cell lung cancer cell strain A549 cell, is played
The effect for lowering LINC00111 expression screens stably transfected cell line with G418 after 48h, is named as A549/si-LINC00111
(control is A549/si-NC).It constructs si-LINC00111 slow virus interference carrier and infects A549 cell strain (A549/si-
LINC00111).Empty carrier transfection A549 cell strain (A549/si-NC) is set as control group.By these cell strain kinds in 6 orifice plates
On, 3 × 105 cells/wells.Every group is given cis-platinum or docetaxel processing, and flow cytometry surveys Level of Apoptosis after 48h.
Compared with the control group, it after infecting LINC00111 slow virus interference carrier, gives cis-platinum or docetaxel processing goes out
Existing Apoptosis increases, and non-small cell lung cancer cell can be increased to the sensibility of chemotherapeutics by prompting to reduce LINC00111 expression,
And induce cell apoptosis, as a result referring to Figure of description 4,5.
The detection of 2 cell cycles
The flow cytomery cell cycle: building LINC00111 slow virus interference carrier infects A549 cell strain
(A549/si-LINC00111), empty carrier transfection A549 cell strain (A549/si-NC) is set as control group.By these cell strain kinds
In 6 orifice plates, 3 × 105 cells/wells.Every group is given cis-platinum or docetaxel processing, and rear flow cytometry surveys cell week for 24 hours
Phase.
After A549 infection LINC00111 slow virus interference carrier compared with the control group, cis-platinum or docetaxel processing are given
Phase cell cycle G1 occur blocks increase, and prompting, which reduces LINC00111 expression, can inhibit the proliferation of non-small cell lung cancer cell.
Above-mentioned experimental result prompt, which reduces LINC00111 expression, can increase non-small cell lung cancer cell to chemotherapeutics
Sensibility, and promote the apoptosis of cell, it can be used as the intervention target spot of oncotherapy, as a result referring to Figure of description 4,5.
3 cells resistance related experiment of embodiment
1.MTT surveys cell IC50: building LINC00111 slow virus interference carrier transfects A549 cell strain (A549/si-
LINC00111), empty carrier transfection A549 cell strain (A549/si-NC) is set as control group.By these cell strain kinds in 96 orifice plates
On, 2500 cells/wells.Every group is given cis-platinum or docetaxel processing, and MTT decoration method detects cell viability after administration 3 days, into
And calculate respective drug IC50.
2. result measures: as shown in Figure 2,3.
3. interpretation of result: in A549 cell strain, give the DDP si-LINC00111 experimental group IC50 of processing:
11.01ug/ml, control group IC50 (NC): 23.26ug/ml；Give the DTX A549/si-LINC00111 experimental group of processing
IC50:13.42ug/ml, control group IC50 (NC): 25.69ug/ml.It observes this phenomenon, prompts to reduce LINC00111 expression
It can promote non-small cell lung cancer cell apoptosis, improve cell chemosensitivity.
Claims (2)
1. detecting the primer sets of long non-coding RNA LINC00111 in the kit that preparation is used for diagnosing non-small cell lung cancer
Using, it is characterised in that its nucleotides sequence is classified as shown in SEQ ID NO:2 and SEQ ID NO:3.
2. inhibit application of the siRNA of long non-coding RNA LINC00111 in preparation treatment non-small cell lung cancer drug,
It is characterized by: nucleotide sequence is as shown in SEQ ID NO:4, SEQ ID NO:5 or SEQ ID NO:6.
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