CN112999238B - Application of LINC00167 in preparation of medicine for inhibiting tumor angiogenesis - Google Patents

Abstract

The invention discloses an application of LINC00167 in preparing a medicine for inhibiting tumor angiogenesis, and belongs to the technical field of biological medicines. LINC00167 is a lncRNA closely related to ionizing radiation stimulation, and its expression level increases significantly after X-ray irradiation, and increases with time after irradiation. At the same time, the expression level of LINC00167 is positively correlated with the level of tumor angiogenesis, which means that it may play a critical role in the response of the tumor to radiation stimuli; by utilizing the point, the high expression of the LINC00167 after irradiation can be artificially inhibited, so that the effect same as that of a clinical common vascular inhibitor is achieved, the prognosis of a tumor patient after radiotherapy is improved, and the recurrence probability of the tumor is reduced.

Description

Application of LINC00167 in preparation of medicine for inhibiting tumor angiogenesis

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of LINC00167 in preparing a medicine for inhibiting tumor angiogenesis.

Background

Long non-coding RNA (Long no-coding RNA, lncRNA) is an RNA fragment without protein coding function, and the length of the RNA fragment is generally greater than 200 nucleotides, and the RNA fragment widely exists in human genome, and accounts for 98% of the total human genome, that is, only about 2% of RNA in human genome has protein coding function; therefore, lncRNA was once thought by scientists to be a "garbage gene". In recent years, with the progress of research, scientists have found that lncRNA can not encode protein, but can exert biological functions by combining with DNA or RNA, even with protein, and form a fine and complex biological regulation network. At present, research in the field of biology proves that lncRNA participates in biological processes such as programmed cell apoptosis, epigenetics, metastasis and generation of cancer cells, stem cell transformation and the like. In addition, the expression of lncRNA in cancer and normal paracancer tissues is different, which provides a potential biological target for treating cancer, and scientists can up-regulate or down-regulate the lncRNA by using medicines or biotechnology, thereby achieving the aim of treatment.

At present, the situation of tumor angiogenesis aggravated after radiotherapy occurs in the process of conventional photon radiotherapy (XRT), and the situation has a promotion effect on the distant metastasis and recurrence of tumors, which has a negative effect on the prognosis of patients with radiotherapy. Whether lncRNA participates in the human stress process caused by ionizing radiation and plays an important role is yet to be proved.

Therefore, the problem to be solved by the technical personnel in the field is to provide the application of LINC00167 in preparing the medicine for inhibiting tumor angiogenesis.

Disclosure of Invention

In view of the above, the invention provides an application of LINC00167 in preparation of a drug for inhibiting tumor angiogenesis, wherein the expression of LINC00167 is up-regulated after radiotherapy, and a target is provided for inhibiting the level of tumor angiogenesis after radiotherapy by inhibiting LINC00167, so that the radiotherapy effect is improved, and the prognosis of a tumor patient is improved.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of LINC00167 as a vascular inhibitor in preparing a tumor radiation adjuvant therapy medicament, wherein the nucleotide sequence of LINC00167 is shown as SEQ ID NO: 1 is shown.

Further, the application of the knock-down long-chain non-coding RNA LINC00167 in preparing a medicament for inhibiting tumor angiogenesis is provided.

Further, the application of the LINC00167 knockdown cell strain in preparing a medicament for inhibiting tumor angiogenesis.

The up-regulation of LINC00167 plays an important role in the tumor angiogenesis process, and has the potential of becoming a vascular inhibitor and being applied to clinical tumor radiation-assisted therapy.

According to the technical scheme, compared with the prior art, the invention discloses and provides the application of LINC00167 in preparing the medicine for inhibiting tumor angiogenesis, LINC00167 is lncRNA closely related to ionizing radiation stimulation, the expression level of LINC00167 is obviously increased after X-ray irradiation, and the expression level is higher and higher along with the prolonging of the time after the irradiation. At the same time, the expression level of LINC00167 is positively correlated with the level of tumor angiogenesis, which means that it may play a critical role in the response of the tumor to radiation stimuli; by utilizing the point, the high expression of the LINC00167 after irradiation can be artificially inhibited, so that the effect same as that of a clinical common vascular inhibitor is achieved, the prognosis of a tumor patient after radiotherapy is improved, and the recurrence probability of the tumor is reduced.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph showing the expression of LINC00167 according to the present invention in lung cancer tissues and normal lung tissues;

wherein, red represents the expression level in tumor tissue, and num (t) number (tumor); gray represents the expression level in normal lung tissue, num (n) ═ number (normal), lucc: lung Squamous Cell Carcinoma, for Lung Squamous Cell Carcinoma, Lung Adenoccancomoma, for Lung Adenocarcinoma;

FIG. 2 is a graph showing the expression levels of LINC00167 according to the present invention in different lung cancer stages;

wherein, stageI, II, III and IV respectively represent stage I, stage II, stage III and stage IV of lung cancer;

FIG. 3 is a graph showing the correlation between LINC00167 expression levels and survival rates of lung cancer patients according to the present invention;

FIG. 4 is a graph showing the expression change of LINC00167 in irradiated lung cancer cells A549 in accordance with the present invention;

wherein, p < 0.05;

FIG. 5 is a pictorial representation of the effect of conditioned medium after irradiation of A549 on tumor angiogenesis in accordance with the present invention; the scale bar is 200 μm;

FIG. 6 is a graph showing the quantitative results of the effect of conditioned medium after irradiation of A549 on tumor angiogenesis in accordance with the present invention, and Nb-mes, the number of mes, indicates the number of HUVEC cells interconnected to form vascular luminal-like meshes; wherein, p < 0.05;

in fig. 5 to 6, Control: a non-illuminated group; IR (2 Gy): an irradiation group;

FIG. 7 is a graph showing the overexpression or knock-down of the level of LINC00167 in accordance with the present invention; wherein, p < 0.001;

FIG. 8 is a schematic representation of the effect of conditioned medium of the present invention on tumor angiogenesis after overexpression or knock-down of LINC00167 in A549 cells; the scale bar is 200 μm;

FIG. 9 is a graph showing the quantification of the effect of LINC00167 on tumor angiogenesis in conditioned media after overexpression or knock-down of LINC00167 in A549 cells according to the present invention; wherein, p < 0.01;

in fig. 7 to 9, Control: a549 cells; pcDNA3.1-LINC 00167: a549 cells transfected with pcDNA3.1-LINC 00167; shLINC 00167: a549 cells transfected with shLINC 00167.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

(1) The invention screens out a long-chain non-coding RNA with up-regulated expression after irradiation by a biological experiment technology: LINC00167, the nucleotide sequence of which is as follows:

attttgggtgggactaagcaaagacgaaacaccctcccagtctatggggtagtctggaccctggcttatctggttcccttaatcctttaggtacttacacgacgttgggtgaggggagctggaagatcagcgggtccccgatcctctctccctaggggtgatagaaatcaaccccccccgccacctccaggtggttcttcccgccgcagagtgggagcagcataaaaggcggggtcttattagcataatctgcctggcaacctgatctcgacgttatatgattggttaaactctgtaagccccatgccgtgcctagagacgagaaacgctggggcgaactgacaggcccagatttaggagtcatgacgaagatcctgagacgcttattcagtctgcagagaagccaaaaggagattcacataatggcccaatgactgaaggcctctttatttcgtgctctgctgttcgtgtaaaacccaatcgcagagcgggtctcaggcggcgctcgcctgcgtttctactctcagccaatcagaaaacccgactcttcgcattggggtcaagcccccgctgcggcccgcgtgctaacggggaggaagcttccagctgtgcctgggtgtctcgggcgccgagggcagcctgcgcccgtgctaagcccgcctcccgcgcgcccgagggcccggtgtcccggaagacgcgcgggggtgaggcggccctcgcgtccgcccgccccgccacagattgcctccgaagtggcctggccgtggagcgccggcgaaaaccgaactcccgcccggctcccggagtcgggagcctgccggggtctcgccctcaggacccccagggagcggccgggcgccgcctatcaccctgacccaccgcaccttgagagcgctttcactgcgcctcttttttctcctctaccaatctggaaaggaagtgagtgtcatttcagacattgagaaacattattcaacactggttacccagtgactcagctcactccttaaccgcaggcttccacatcctgctgtcccttaaaattgccttcacaaagggcagccaagagcccagctttggagagatacccagctccaactacccactggcaggcaatacgcagggcagctagatggcagagagaccttggaccttcggaccttcggggagcggttttcccttcggtgatatctaccttgctatgttcgtgatatctaccttgctacgttattttgaagattaagtgagctcttactttaccgagtaaagtgctttgcttagcacactgtctggaacttaaatgagaagtactgacaatataacttacaaaaatcctaaactattgcctgagaaaaagagggagacaggacagctaagcaaaagacgagaatagaaaacacccacagcgtgaaaaactgtaaaattaactcaatagtattctctttactgttttttgttttgttttgttgttttgttttttgatacggagtctcgctctgttgcccaggctggagtgcagtggtgagatctgggctcattgcaacctctgcctcccgggttcaagcgattctcctgcctcagcctctctagtagctgagattacaggcatgcaccaccacgcccggctaatttttgtattttcagtagagatggggtttccccatgttggccaggctggtctcaaactctaacgtcaagtgattctcccgccttggccttccaaattgctgggattacagatgtgagccactgcgtctagtctaccattttgtttctttaaatagctggaaacaacgtggaaagaaaagagagcttttttttttttttaaacttcgtgacagggtgcggtggctcacgcctgtaatcccagaattttgggaggccgaggcgggtggatcgcttgagcacaggcgttcgagaccagcctgggcaacatagtgagactctgtacaaaaaacaaaaagattagccaggcatggcagtacacatctgtggttccagttaccggcaggctgtgcaggagggttacttgagcccaggaagtcaaggctgcagtgagctgtgatcgtgccactgcactacaacctgggtgatagagcaagtctctgtctttaaaaaaacaaaacaacaacaagaagaaacttcagacagttaactgctttgtaaacaacttcacatctaggccgtgtcttgttcttccttaaaccactgtacaggtggcactggacttagatgaggccgaccttaacatagctagtgagagacactagtagccctactctggcaacacagacatggaaatcagttgcctaaggaaagatcatattggctaagagagtcccttgaatgctcccttttagtgttacgagtagatgaacaatgccgcaggttggacaggtccttctgtgtccttgcaaaagctgccactccacatacccctggcctcagtaagaacggctcacttttagatttaacatcattacatccttgctgatgaactgagatggtcccaactgaggtccactgaccagaaacaacaccacagaacccaaaagagaacgcaccatggggaatggggctattcaaactgaagagcctgagttactctcatggctttagagaaatgttctaaaacttctaagtacataaatatgtgcagggagattcctccccacagagtttttattcagagggtctggagtgaacctcaggaatttgctttttatgtggatttttaaatgatttttaaagtttacactgtaaaattcactcactggggtatacaagaagtatttattgaatatgtactgtgtgccattcatttttctaaacaataaagatagaatagggaacaacagcaacc；SEQ ID NO：1。

(2) the differences in the Expression levels of LINC00167 in lung cancer tissue and normal lung tissue, as well as in the different lung cancer stages, were analyzed in the database gepia (gene Expression Profiling Interactive analysis), and the results are shown in fig. 1 and fig. 2.

The results in FIG. 1 show that LINC00167 is expressed in lung cancer tissues at a higher level than normal lung tissues, but not much higher. The results in FIG. 2 show that, in the lung cancer stage, the expression of LINC00167 is higher in the lung cancer tissues with higher stage and poorer prognosis than in the lung cancer tissues with lower stage. The results show that LINC00167 is related to the development of lung cancer and may have the effect of promoting the development of lung cancer.

(3) Survival curve data is derived from the database OncoLnc, see fig. 3; the results in FIG. 3 show that the LINC00167 high expression group is the data of the website based on the TCGA database, a COX regression risk assessment model is used for selecting the median of the LINC00167 expression levels of all patients as a threshold, the patients with the LINC00167 expression level higher than the threshold are defined as high expression, the prognosis is poor, and the overall survival rate at the same time point is lower than that of the patients with the low expression group; patients below this threshold are defined as under-expressing and have a better prognosis and a higher overall survival rate at the same time than patients in the high expression group; it is suggested that LINC00167 is a negative factor in the prognosis of lung cancer patients, and it may play a role in promoting tumor development.

Example 2 Effect of irradiation on LINC00167 expression

(1) Cell culture

Selecting stable lung cancer cell line A549 cell, culturing with RPMI-1640 complete culture medium, and standing at 37 deg.C with 5% CO₂Culturing in a cell culture box; washing cells with sterile PBS for 2-3 times during passage, digesting with 0.25% pancreatin for 1-2min, and suspending with RPMI-1640 complete culture medium containing 10% fetal calf serum after cells are digested from culture dish; and (4) dividing into new culture dishes, and adding RPMI-1640 complete culture medium for amplification culture.

(2) Irradiation treatment

The cultured A549 cells were divided into control group and experimental group, each group having approximately1×10⁸(ii) individual cells; an electric energy excitation X-ray irradiator (model RAD. SOURCE, RS-2000) is used for carrying out ionization irradiation experiment in the biological experiment, the voltage is 50KV, the dose rate is 1.224Gy/min, the experimental group receives 2Gy X-ray irradiation, and the contrast group does not irradiate; after irradiation, the experimental group and the control group are put into a cell culture box again for culture.

(3) Extraction of RNA

Taking out two groups of cells from the cell culture box, sucking out the culture medium, washing twice by using sterile PBS, and sucking out all PBS; trizol reagent is added to the cell culture dish approximately every 1X 10⁷Adding 1mL of reagent into each cell, and blowing and beating the cells for several times by using a micropipette to completely lyse the cells; cells lysed with Trizol reagent were transferred to RNase-free EP tubes and labeled on the tube wall. Adding 200 mu L of chloroform into each 1mL of Trizol lysate, violently shaking for 30s, standing for 3min, rotating at 12000 r/min, and centrifuging at the low temperature of 4 ℃ for 15 min; sucking 400 μ L of the supernatant, transferring into a new EP tube, adding equal amount of isopropanol, and standing on ice for 10 min; centrifuging at 12000 r/min at 4 deg.C for 15 min; discarding the supernatant to obtain a precipitate, namely RNA, and washing the precipitate by 0.8mL of prepared 75% DEPC alcohol; centrifuging at 12000 r/min at 4 deg.C for 10 min; discarding the supernatant, naturally drying the precipitate, and dissolving with DEPC water at 56-57 deg.C; the concentration of total RNA extracted was determined.

(4) Real-time fluorescent quantitative PCR experiment

Performing total RNA reverse transcription by adopting an mRNA reverse transcription kit of GeneCopoeia company, and adding 2 mu g of total RNA into each 25 mu L system; the reverse transcription was performed at 45 ℃ for 1h, 85 ℃ for 5min, and 4 ℃. Diluting cDNA obtained by reverse transcription by 5 times to be used as a template of quantitative PCR; primer (LINC00167-F/R, GAPDH-F/R) diluted to a concentration of 2. mu.M, with GAPDH as internal control; preparing a system and a set program according to an instruction by adopting a mRNA fluorescent quantitative PCR kit of GeneCopoeia company; and calculating the expression change multiple of the LINC00167 transcript between the experimental group and the control group according to the Ct value of the fluorescent quantitative PCR experiment, and drawing according to the results of three independent experiments.

Wherein, the primer sequences are as follows:

LINC00167-F：5’-CGAACTGACAGGCCCAGATT-3’；SEQ ID NO：2；

LINC00167-R：5’-GAGAGTAGAAACGCAGGCGA-3’；SEQ ID NO：3；

GAPDH-F：5’-AGCCACATCGCTCAGACAC-3’；SEQ ID NO：4；

GAPDH-R：5’-GCCCAATACGACCAAATCC-3’；SEQ ID NO：5。

the results are shown in fig. 4, and the results in fig. 4 show that after lung cancer cell a549 is irradiated with 2Gy X-rays, the expression level of LINC00167 is gradually increased along with the time after irradiation, indicating that the cell can increase the self survival rate by up-regulating the expression of LINC00167 after being irradiated and stimulated.

Example 3 angiogenesis experiment

Culturing A549 cells in 6-well plate, irradiating with 2Gy of X-ray when the cells grow to 60-70% fusion, irradiating at 37 deg.C and 5% CO₂Culturing for 24h under the condition, taking a culture medium of A549 cells, culturing HUVEC cells, and observing the influence of the conditioned medium of an irradiation group and a non-irradiation group on the vascularization level of the HUVEC cells.

The day before the experiment, Matrigel (corning 354234-9148009) was thawed at 4 ℃. Sterilized 200 μ L tips, 1mL tips, and 96-well plates were refrigerated for use. To start the experiment, Matrigel was kept on ice or in an ice box to ensure that the Matrigel concentration was 10mg/mL or more, and was spread evenly in pre-cooled 96-well plates at 100. mu.L per well. The whole culture dish was placed in an incubator and left for 30min to allow the gel to solidify. While waiting for the gel to solidify, HUVEC cells in logarithmic growth phase were taken, digested with 0.25% pancreatin for about 2min, resuspended and adjusted for cell concentration, and then seeded into 96-well plates at about 1X 10/well⁴And (4) cells. At 37 deg.C, 5% CO₂Culturing for 24h under the condition, removing culture solution from each well after 24h, washing twice in PBS (phosphate buffer solution) each well, fixing cells for 10min in 4% paraformaldehyde solution at room temperature, and observing the formation state and number of microtubules under a microscope. Quantitative Image analysis was performed using Image-J software.

By collecting a conditioned medium of A549 cells after being irradiated by X rays, and culturing HUVEC cells by using the conditioned medium, the change of the capability of the HUVEC cells for forming a blood vessel cavity is observed, so that the capability change of lung cancer cells in lung cancer tissues for promoting angiogenesis after irradiation is simulated, and the change is a more key influencing factor for the generation of tumor metastasis after radiotherapy.

The results are shown in fig. 5 and 6, and the results of angiogenesis experiments show that the culture medium of a549 cells can promote the ability of HUVEC angiogenesis after X-ray irradiation.

Example 4 overexpression and knockdown of LINC00167 in A549 cells

(1) The LINC00167 in the A549 cell is overexpressed by constructing a plasmid vector pcDNA3.1-LINC00167, enzyme cutting sites are BamHI and Xho I, and the LINC00167 is amplified by utilizing an upstream primer/a downstream primer through PCR; the primer sequences used were as follows:

an upstream primer: 5'-CGGGATCCATTTTGGGTGGGACTAAGCAA-3', respectively; SEQ ID NO: 6;

a downstream primer: 5'-CCCTCGAGGGTTGCTGTTGTTCCCTATTCTAT-3', respectively; SEQ ID NO: 7;

the PCR reaction system is as follows: mu.g cDNA template, 0.5. mu.L Pyrobest DNA polymerase, 2. mu.L 10 XPyrobest Buffer II, 4. mu.L dNTP mix (2.5 mM each), 1. mu.L upstream and downstream primers (final concentration 0.2. mu.M), and 50. mu.L double distilled water.

The PCR reaction program is: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 180s, 40 cycles.

(2) Knockdown of LINC00167 was designed using the Sigma-Aldrich online software session, sequence as follows:

5’-CCGGCCGAGTAAAGTGCTTTGCTTACTCGAGTAAGCAAAGCACTTTACTCGGTTTTTTG-3’；SEQ ID NO：8；

SEQ ID NO: 8 to pLKO.1-TRC vector (Addgene Plasmid 10878) to obtain shLINC 00167.

(3) Plasmid transfection Lipofectamine 3000Reagent transfection Reagent from Thermo Fisher was used. A549 cells were plated in 6-well plates, 2X 10 cells per well⁵When the cell confluence reaches 60-70%, plasmid transfection is started, a serum-free and double-antibody-free RPMI-1640 culture medium is used for transfection, and the complete culture is carried out by replacing with fresh RPMI-1640 after 6h of transfectionCulturing the cells, culturing the cells continuously, carrying out normal passage when the cell fusion degree reaches 90%, collecting partial cells, extracting total RNA, and detecting the over-expression or the reduction level of LINC00167 by using primers (LINC00167-F/R, GAPDH-F/R), wherein the result is shown in figure 7. FIG. 7 shows that the expression level of LINC00167 is significantly improved by the A549 cell transfected with pcDNA3.1-LINC 00167; the expression level of LINC00167 is obviously reduced by transfecting the A549 cells of shLINC 00167; indicating that the overexpression and knockdown of LINC00167 was successful.

Example 5 Effect of over-expression or knock-down of LINC00167 on HUVEC angiogenesis

The experimental results of example 3 show that the A549 conditioned medium after irradiation has the effect of promoting angiogenesis, and the expression of LINC00167 is up-regulated after irradiation, so that whether the phenomenon is the same as that after irradiation after the LINC00167 is over-expressed in A549 cells or not can be realized.

Respectively culturing A549 cells, A549 cells (A549-LINC00167) for over-expressing LINC00167 and A549 cells for knocking down LINC00167 in 6-well plate at 37 deg.C and 5% CO₂Culturing for 24h under the condition, taking corresponding conditioned medium, respectively culturing HUVEC cells, and observing the influence of the conditioned medium on the blood vessel formation level of the HUVEC cells.

The results, shown in fig. 8 and 9, show that conditioned media overexpressing LINC00167 also have the ability to promote angiogenesis; this ability to promote angiogenesis is also diminished after inhibition of LINC00167 expression. It is shown that LINC00167 plays an important role in signal transmission in radiation-promoted angiogenesis of tumors. In the future, the effect of reducing the tumor metastasis recurrence probability after radiotherapy can be realized by inhibiting the expression of LINC00167 in tumor cells after irradiation.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Suzhou university

Application of LINC00167 in preparation of medicine for inhibiting tumor angiogenesis

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2863

<212> DNA

<213> Artificial Sequence

<400> 1

attttgggtg ggactaagca aagacgaaac accctcccag tctatggggt agtctggacc 60

ctggcttatc tggttccctt aatcctttag gtacttacac gacgttgggt gaggggagct 120

ggaagatcag cgggtccccg atcctctctc cctaggggtg atagaaatca accccccccg 180

ccacctccag gtggttcttc ccgccgcaga gtgggagcag cataaaaggc ggggtcttat 240

tagcataatc tgcctggcaa cctgatctcg acgttatatg attggttaaa ctctgtaagc 300

cccatgccgt gcctagagac gagaaacgct ggggcgaact gacaggccca gatttaggag 360

tcatgacgaa gatcctgaga cgcttattca gtctgcagag aagccaaaag gagattcaca 420

taatggccca atgactgaag gcctctttat ttcgtgctct gctgttcgtg taaaacccaa 480

tcgcagagcg ggtctcaggc ggcgctcgcc tgcgtttcta ctctcagcca atcagaaaac 540

ccgactcttc gcattggggt caagcccccg ctgcggcccg cgtgctaacg gggaggaagc 600

ttccagctgt gcctgggtgt ctcgggcgcc gagggcagcc tgcgcccgtg ctaagcccgc 660

ctcccgcgcg cccgagggcc cggtgtcccg gaagacgcgc gggggtgagg cggccctcgc 720

gtccgcccgc cccgccacag attgcctccg aagtggcctg gccgtggagc gccggcgaaa 780

accgaactcc cgcccggctc ccggagtcgg gagcctgccg gggtctcgcc ctcaggaccc 840

ccagggagcg gccgggcgcc gcctatcacc ctgacccacc gcaccttgag agcgctttca 900

ctgcgcctct tttttctcct ctaccaatct ggaaaggaag tgagtgtcat ttcagacatt 960

gagaaacatt attcaacact ggttacccag tgactcagct cactccttaa ccgcaggctt 1020

ccacatcctg ctgtccctta aaattgcctt cacaaagggc agccaagagc ccagctttgg 1080

agagataccc agctccaact acccactggc aggcaatacg cagggcagct agatggcaga 1140

gagaccttgg accttcggac cttcggggag cggttttccc ttcggtgata tctaccttgc 1200

tatgttcgtg atatctacct tgctacgtta ttttgaagat taagtgagct cttactttac 1260

cgagtaaagt gctttgctta gcacactgtc tggaacttaa atgagaagta ctgacaatat 1320

aacttacaaa aatcctaaac tattgcctga gaaaaagagg gagacaggac agctaagcaa 1380

aagacgagaa tagaaaacac ccacagcgtg aaaaactgta aaattaactc aatagtattc 1440

tctttactgt tttttgtttt gttttgttgt tttgtttttt gatacggagt ctcgctctgt 1500

tgcccaggct ggagtgcagt ggtgagatct gggctcattg caacctctgc ctcccgggtt 1560

caagcgattc tcctgcctca gcctctctag tagctgagat tacaggcatg caccaccacg 1620

cccggctaat ttttgtattt tcagtagaga tggggtttcc ccatgttggc caggctggtc 1680

tcaaactcta acgtcaagtg attctcccgc cttggccttc caaattgctg ggattacaga 1740

tgtgagccac tgcgtctagt ctaccatttt gtttctttaa atagctggaa acaacgtgga 1800

aagaaaagag agcttttttt tttttttaaa cttcgtgaca gggtgcggtg gctcacgcct 1860

gtaatcccag aattttggga ggccgaggcg ggtggatcgc ttgagcacag gcgttcgaga 1920

ccagcctggg caacatagtg agactctgta caaaaaacaa aaagattagc caggcatggc 1980

agtacacatc tgtggttcca gttaccggca ggctgtgcag gagggttact tgagcccagg 2040

aagtcaaggc tgcagtgagc tgtgatcgtg ccactgcact acaacctggg tgatagagca 2100

agtctctgtc tttaaaaaaa caaaacaaca acaagaagaa acttcagaca gttaactgct 2160

ttgtaaacaa cttcacatct aggccgtgtc ttgttcttcc ttaaaccact gtacaggtgg 2220

cactggactt agatgaggcc gaccttaaca tagctagtga gagacactag tagccctact 2280

ctggcaacac agacatggaa atcagttgcc taaggaaaga tcatattggc taagagagtc 2340

ccttgaatgc tcccttttag tgttacgagt agatgaacaa tgccgcaggt tggacaggtc 2400

cttctgtgtc cttgcaaaag ctgccactcc acatacccct ggcctcagta agaacggctc 2460

acttttagat ttaacatcat tacatccttg ctgatgaact gagatggtcc caactgaggt 2520

ccactgacca gaaacaacac cacagaaccc aaaagagaac gcaccatggg gaatggggct 2580

attcaaactg aagagcctga gttactctca tggctttaga gaaatgttct aaaacttcta 2640

agtacataaa tatgtgcagg gagattcctc cccacagagt ttttattcag agggtctgga 2700

gtgaacctca ggaatttgct ttttatgtgg atttttaaat gatttttaaa gtttacactg 2760

taaaattcac tcactggggt atacaagaag tatttattga atatgtactg tgtgccattc 2820

atttttctaa acaataaaga tagaataggg aacaacagca acc 2863

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 2

cgaactgaca ggcccagatt 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence

<400> 3

gagagtagaa acgcaggcga 20

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 4

agccacatcg ctcagacac 19

<210> 5

<211> 19

<212> DNA

<213> Artificial Sequence

<400> 5

gcccaatacg accaaatcc 19

<210> 6

<211> 29

<212> DNA

<213> Artificial Sequence

<400> 6

cgggatccat tttgggtggg actaagcaa 29

<210> 7

<211> 32

<212> DNA

<213> Artificial Sequence

<400> 7

ccctcgaggg ttgctgttgt tccctattct at 32

<210> 8

<211> 59

<212> DNA

<213> Artificial Sequence

<400> 8

ccggccgagt aaagtgcttt gcttactcga gtaagcaaag cactttactc ggttttttg 59

Claims (1)

1. The application of the LINC00167 shRNA in preparing the medicine for inhibiting the angiogenesis of the lung cancer is characterized in that the sequence of the LINC00167 shRNA is shown as SEQ ID NO: shown in fig. 8.

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