CN109371136B - Lung adenocarcinoma-related lncRNA and application thereof - Google Patents

Abstract

The invention discloses application of a gene related to lung adenocarcinoma, wherein the gene is LINC02014, the invention discovers that differential expression of LINC02014 is related to generation and development of lung adenocarcinoma for the first time, and verifies that LINC02014 is remarkably up-regulated in lung adenocarcinoma patients through QPCR, and the gene can be used as an index for diagnosing lung adenocarcinoma and applied to clinic. The invention also discloses that by reducing the expression of LINC02014, the proliferation of lung adenocarcinoma cells can be effectively inhibited, the migration and invasion rate of the lung adenocarcinoma cells can be reduced, and the LINC02014 can be used as a drug target for treating lung adenocarcinoma and metastasis thereof.

Description

Lung adenocarcinoma-related lncRNA and application thereof

Technical Field

The invention belongs to the field of biological medicines, relates to lncRNA related to lung adenocarcinoma and application thereof, and particularly relates to application of LINC02014 in diagnosis and treatment of lung adenocarcinoma.

Background

Since the 80 s of the 20 th century, lung cancer has gradually become one of the highest incidence and mortality cancers worldwide, and the incidence and mortality rates show a trend of increasing year by year, and according to the statistical data of the investigation of the american cancer society of 2016, the average mortality rates of lung cancer in male and female patients are 27% and 26%, respectively, which is the first of the mortality rates of malignant tumors. In China, the incidence and fatality rate of lung cancer are rising year by year, and the lung cancer becomes a malignant tumor which has the greatest threat to the health and life of people. In recent years, with the continuous and deep research of lung cancer mutant genes and the research and application of a large number of targeted drugs, the treatment of lung cancer gradually shifts to gene-oriented individualized treatment. In the past, people put the emphasis on coding genes for the research of the individualized treatment of lung cancer, and with the continuous and intensive research, people find that long-chain non-coding RNA (lncRNA) plays a considerable important role in the occurrence and development process of malignant tumors, and meanwhile, the remarkable tumor tissue specificity and the possibility of applying the long-chain non-coding RNA to tumor diagnosis make the long-chain non-coding RNA increasingly become a new hotspot in tumor research. With the continuous development of sequencing technology, more and more researchers adopt high-throughput genome screening technology to identify novel lung cancer according to gene functions. Attempts have been made to develop more accurate diagnostic tools based on the effective typing of lung cancer, and thus, to provide better treatment of lung cancer.

The pathophysiological processes of the disease are regulated by genes, and the genome of the eukaryotic cell is composed of huge and complex DNA sequences and expresses and transmits biological genetic information by transcribing into RNA. As a result of human whole genome sequencing, only less than 2% of gene sequences participate in protein coding, while most of non-coding sequences are transcribed into long-chain non-coding RNA with the length of more than 200 bases, participate in regulation and control of gene expression, and promote or inhibit the occurrence and development of various tumors. The focus of molecular biology research make internal disorder or usurp has been on messenger RNA (mRNA) coding for proteins, and a large number of non-coding RNAs (ncrnas) without protein coding function have been ignored for a long time as "garbage sequences" without biological function. ncRNA does not have the characteristics of typical initiation codon, open reading frame, promoter conserved region, stop codon and the like. Depending on the length of ncRNA, ncRNA is divided into small non-coding RNA (snRNA) and long non-coding RNA, wherein the lnRNA accounts for 80%. The lncRNA is transcribed by RNA polymerase II, participates in the processes of epigenetics, alternative splicing, nuclear entry transport and the like, and the transcription and dysfunction of the lncRNA can function as a tumor suppressor or a cancer-promoting factor. Therefore, the deep research on the biological functions of lncRNA and the relationship between lncRNA and diseases can provide a new idea for clinical diagnosis and treatment.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a molecular marker related to the occurrence and development of lung adenocarcinoma.

The invention also aims to provide a product and a means for diagnosing lung adenocarcinoma, which can judge whether a patient has lung adenocarcinoma or is at risk of lung adenocarcinoma by detecting the expression level of a molecular marker.

The third object of the present invention is to provide a pharmaceutical composition or a method for treating lung adenocarcinoma, which can treat lung adenocarcinoma by specific down-regulation of molecular markers.

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

the invention provides application of a reagent for detecting LINC02014 gene expression in preparation of a product for diagnosing early lung adenocarcinoma.

Further, the agent is selected from:

a probe that specifically recognizes LINC 02014; or

And (3) a primer for specifically amplifying LINC 02014.

Preferably, the primer sequence for specifically amplifying the LINC02014 gene is shown as SEQ ID NO.2 and SEQ ID NO. 3.

The invention provides a product for diagnosing lung adenocarcinoma, which comprises the step of detecting the expression level of LINC02014 gene in a sample by a sequencing technology, a nucleic acid hybridization technology and a nucleic acid amplification technology. Wherein the product includes, but is not limited to, a chip, a formulation or a kit.

Further, the nucleic acid amplification technique is selected from the group consisting of polymerase chain reaction, reverse transcription polymerase chain reaction, transcription mediated amplification, ligase chain reaction, strand displacement amplification and nucleic acid sequence based amplification.

The invention provides application of LINC02014 gene in preparing a pharmaceutical composition for preventing or treating lung adenocarcinoma.

Further, the pharmaceutical composition comprises a down-regulator of LINC 02014. The down-regulating agent is selected from: an interfering molecule targeting LINC02014 or its transcript and capable of inhibiting LINC02014 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the down regulator is siRNA. The preferable siRNA sequence is shown in SEQ ID NO.8 and SEQ ID NO. 9.

Furthermore, the pharmaceutical composition also comprises other medicines compatible with the down regulator and pharmaceutically acceptable carriers and/or auxiliary materials.

The invention provides application of LINC02014 gene in screening potential substances for preventing or treating lung adenocarcinoma.

The invention provides a method for screening potential substances for preventing or treating lung adenocarcinoma, which comprises the following steps:

treating the system expressing or containing the LINC02014 gene with a candidate substance; and

detecting the expression of the LINC02014 gene in the system;

wherein, if the candidate substance can reduce the expression or activity of the LINC02014 gene, (preferably significantly reduced, such as more than 20% lower, preferably more than 50% lower, more preferably more than 80% lower), it is indicative that the candidate substance is a potential substance for preventing or treating lung adenocarcinoma. The system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.

Such potential substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed aiming at the LINC02014 gene or genes upstream or downstream of the LINC02014 gene.

Drawings

FIG. 1 is a graph showing the detection of the expression of LINC02014 gene in lung adenocarcinoma tissue by QPCR;

FIG. 2 is a graph of detection of transfection of LINC02014 in lung adenocarcinoma cells using QPCR;

FIG. 3 is a graph showing the effect of LINC02014 gene on lung adenocarcinoma cell proliferation measured by CCK-8 method;

FIG. 4 is a graph showing the effect of LINC02014 gene on apoptosis of lung adenocarcinoma cells measured by flow cytometry;

FIG. 5-1 is a graph of the effect of LINC02014 on lung adenocarcinoma cell migration using a Transwell chamber;

FIG. 5-2 is a graph of the effect of LINC02014 on lung adenocarcinoma cell invasion using a Transwell chamber.

Detailed Description

According to the invention, through extensive and intensive research, the expression of lncRNA in a lung adenocarcinoma specimen in a tumor tissue and a tissue beside the tumor is detected by adopting an lncRNA chip which covers a database most widely at present through a high-throughput method, an lncRNA fragment with obvious expression difference is found, and the relation between the lncRNA fragment and the occurrence of lung adenocarcinoma is discussed, so that a better way and method are found for the early detection and the targeted treatment of the lung adenocarcinoma. Through screening, the invention discovers the significant up-regulation of LINC02014 in lung adenocarcinoma for the first time. Experiments prove that siRNA interference silences LINC02014, can effectively inhibit proliferation and invasion of lung adenocarcinoma cells, and provides a new way for personalized treatment of lung adenocarcinoma.

Molecular marker

A "molecular marker" is any gene whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, the marker gene may have the nucleotide sequence specified in SEQ ID NO. 1. The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level.

LINC02014 gene

LINC02014 is located on human chromosome 3, NC-000003.12 (130088863..130094304, complement), and has the nucleotide sequence shown in SEQ ID NO. 1. LINC02014 in the present invention includes wild type, mutant or fragments thereof.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the target gene of the present invention. Two sequences are "substantially homologous" (or substantially similar) if, when the nucleic acid or fragment thereof is optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 60% of the nucleotide bases, usually at least about 70%, more usually at least about 80%, preferably at least about 90%, and more preferably at least about 95-98% of the nucleotide bases.

Alternatively, substantial homology or identity exists between nucleic acids or fragments thereof when the nucleic acids or fragments thereof hybridize to another nucleic acid (or the complementary strand thereof), one strand, or the complementary sequence thereof under selective hybridization conditions. Hybridization selectivity exists when hybridization is more selective than the overall loss of specificity. Typically, selective hybridization occurs when there is at least about 55% identity, preferably at least about 65%, more preferably at least about 75% and most preferably at least about 90% identity over a stretch of at least about 14 nucleotides. As described herein, the length of the homology alignments can be a longer sequence segment, in certain embodiments generally at least about 20 nucleotides, more generally at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides.

Thus, the polynucleotide of the invention preferably has at least 75%, more preferably at least 85%, more preferably at least 90% homology with SEQ ID NO. 1. More preferably, there is at least 95%, more preferably at least 98% homology.

The present invention may utilize any method known in the art for determining gene expression. It will be appreciated by those skilled in the art that the means by which gene expression is determined is not an important aspect of the present invention. The expression level of the biomarker can be detected at the transcriptional level.

Detection techniques

The lncrnas of the invention are detected using a variety of nucleic acid techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing, nucleic acid hybridization, and nucleic acid amplification techniques.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to, chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments.

The present invention can amplify nucleic acids (e.g., ncRNA) prior to or simultaneously with detection. Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

The polymerase chain reaction, commonly referred to as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of a target nucleic acid sequence under conditions of substantially constant temperature, ionic strength and pH, wherein multiple RNA copies of the target sequence autocatalytically generate additional copies; ligase chain reaction of LCR uses two sets of complementary DNA oligonucleotides that hybridize to adjacent regions of the target nucleic acid; other amplification methods include, for example, nucleic acid sequence-based amplification commonly known as NASBA; amplification of the probe molecule itself using RNA replicase (commonly known as Q.beta.replicase), transcription-based amplification methods, and self-sustained sequence amplification.

Non-amplified or amplified nucleic acids of the invention can be detected by any conventional means.

Chip and kit

The invention provides products including (but not limited to) formulations, chips or kits for detecting the expression level of the LINC02014 gene in a subject. Wherein the chip includes: a solid support; and oligonucleotide probes orderly fixed on the solid phase carrier, wherein the oligonucleotide probes specifically correspond to part or all of the sequence shown in LINC 02014.

The solid phase carrier comprises an inorganic carrier and an organic carrier, wherein the inorganic carrier comprises but is not limited to a silicon carrier, a glass carrier, a ceramic carrier and the like; the organic vehicle includes a polypropylene film, a nylon film, and the like.

The term "probe" refers to a molecule that binds to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including, but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

Exemplary probes in the present invention include PCR primers as well as gene-specific DNA oligonucleotide probes, such as microarray probes immobilized on a microarray substrate, quantitative nuclease protection test probes, probes attached to molecular barcodes, and probes immobilized on beads.

These probes have a base sequence complementary to a specific base sequence of a target gene. Here, the term "complementary" may or may not be completely complementary as long as it is a hybrid. These polynucleotides usually have a homology of 80% or more, preferably 90% or more, more preferably 95% or more, particularly preferably 100% with respect to the specific nucleotide sequence. These probes may be DNA or RNA, and may be polynucleotides obtained by replacing nucleotides in a part or all of them with artificial Nucleic acids such as PNA (Polyamide Nucleic Acid), LNA (registered trademark, locked Nucleic Acid, bridge Nucleic Acid, crosslinked Nucleic Acid), ENA (registered trademark, 2 '-O, 4' -C-Ethylene-Bridged Nucleic acids), GNA (Glycerol Nucleic Acid), and TNA (Threose Nucleic Acid).

The invention provides a kit which can be used for detecting the expression of LINC 02014. Preferably, the preparation or the kit further comprises a marker for marking the RNA sample, and a substrate corresponding to the marker. In addition, the kit may further include various reagents required for RNA extraction, PCR, hybridization, color development, and the like, including but not limited to: an extraction solution, an amplification solution, a hybridization solution, an enzyme, a control solution, a color development solution, a washing solution, and the like. In addition, the kit also comprises an instruction manual and/or chip image analysis software.

Down-regulating agent and pharmaceutical composition

Based on the discovery of the inventor, the invention provides application of a LINC02014 down-regulator in preparing a pharmaceutical composition for inhibiting lung adenocarcinoma. As used herein, said down-regulator of LINC02014 includes, but is not limited to, inhibitors, antagonists, blockers, nucleic acid inhibitors, and the like.

The LINC02014 down-regulator refers to any substance which can down-regulate the expression of LINC02014 gene or inhibit the transcription of LINC02014 gene, and the substances can be used for preventing or treating lung adenocarcinoma as substances which are useful for down-regulating LINC 02014.

As a preferred mode of the invention, the LINC02014 down-regulator is a LINC02014 specific small interfering RNA molecule. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, which is the RNA interference (RNA interference) process. Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

When screening effective siRNA sequences, the inventor finds out the optimal effective fragment by a large amount of alignment analysis. The inventor designs and synthesizes a plurality of siRNA sequences, and verifies the siRNA sequences by transfecting a lung adenocarcinoma cell line with a transfection reagent respectively, selects siRNA with the best interference effect, and further performs experiments at a cell level, and the result proves that the siRNA can effectively inhibit the expression level of the LINC02014 gene in cells and the proliferation of the lung adenocarcinoma cells.

The nucleic acid inhibitor of the present invention, such as siRNA, can be chemically synthesized or can be prepared by transcribing an expression cassette in a recombinant nucleic acid construct into single-stranded RNA. Nucleic acid inhibitors, such as siRNA, can be delivered into cells by using appropriate transfection reagents, or can also be delivered into cells using a variety of techniques known in the art.

Pharmaceutical composition

The invention also provides a composition comprising an effective amount of said downregulator of LINC02014, and a pharmaceutically acceptable carrier. The composition can be used for inhibiting lung adenocarcinoma. Any of the foregoing downregulators of LINC02014 can be used in the preparation of the composition.

As used herein, the "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. The "pharmaceutically acceptable carrier" refers to a carrier for administration of the therapeutic agent, including various excipients and diluents. The term refers to such pharmaceutical carriers: they are not essential active ingredients per se and are not unduly toxic after administration. Suitable carriers are well known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may comprise liquids such as water, saline, buffers. In addition, auxiliary substances, such as fillers, lubricants, glidants, wetting or emulsifying agents, pH buffering substances and the like may also be present in these carriers. The vector may also contain a cell (host cell) transfection reagent.

The present invention may employ various methods well known in the art for administering the down-regulator or its encoding gene, or its pharmaceutical composition to a mammal. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

Preferably, it can be carried out by means of gene therapy. For example, a downregulator of LINC02014 can be administered directly to a subject by a method such as injection; alternatively, an expression unit (e.g., an expression vector or virus, etc., or an siRNA or shRNA) carrying a LINC02014 down-regulator may be delivered to a target site in a manner that allows expression of the active LINC02014 down-regulator, depending on the type of down-regulator, as will be appreciated by those skilled in the art.

The pharmaceutical composition of the present invention may further comprise one or more anticancer agents. In a specific embodiment, the pharmaceutical composition comprises at least one compound inhibiting the expression of the LINC02014 gene and at least one chemotherapeutic agent. Chemotherapeutic agents useful in the methods of the invention include, but are not limited to, DNA-alkylating agents, anti-tumor antibiotic agents, anti-metabolic agents, tubulin stabilizing agents, tubulin destabilizing agents, hormone antagonists, topoisomerase inhibitors, protein kinase inhibitors, HMG-COA inhibitors, CDK inhibitors, cyclin inhibitors, caspase inhibitors, metalloproteinase inhibitors, antisense nucleic acids, triple helix DNA, nucleic acid aptamers, and molecularly modified viral, bacterial and exotoxin agents.

Pharmaceutically acceptable carriers can include, but are not limited to: viruses, liposomes, nanoparticles, or polymers, and any combination thereof. Relevant delivery vehicles can include, but are not limited to: liposomes, biocompatible polymers (including natural and synthetic polymers), lipoproteins, polypeptides, polysaccharides, lipopolysaccharides, artificial viral envelopes, inorganic (including metal) particles, and bacterial or viral (e.g., baculovirus, adenovirus, and retrovirus), phage, cosmid, or plasmid vectors.

The pharmaceutical compositions of the invention can also be used in combination with other drugs for the treatment of lung adenocarcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, even in the same composition.

The pharmaceutical compositions of the present invention may also be administered separately with other therapeutic compounds, either as separate compositions or in different dosage forms than the primary active ingredient. Some of the doses of the main ingredient may be administered simultaneously with other therapeutic compounds, while other doses may be administered separately. The dosage of the pharmaceutical composition of the present invention can be adjusted during the course of treatment depending on the severity of symptoms, the frequency of relapse, and the physiological response of the treatment regimen.

Drug screening

The invention provides a method for screening a medicament for preventing or treating lung adenocarcinoma, which comprises the following steps:

in the experimental group, a compound to be detected is added into a cell culture system, and the expression level of LINC02014 is measured; in a control group, no test compound is added into the same culture system, and the expression level of LINC02014 is determined; wherein if the expression level of LINC02014 in the experimental group is less than that in the control group, the candidate compound is a down-regulator of LINC 02014.

In the present invention, the method further comprises: the candidate compound obtained in the above step is further tested for its effect of inhibiting lung adenocarcinoma, and if the test compound has a significant inhibitory effect on lung adenocarcinoma, the compound is a potential substance for preventing or treating lung adenocarcinoma.

In the present invention, the term "sample" is used in its broadest sense. In one sense, specimens or cultures obtained from any source, as well as biological and environmental samples, are meant to be included. Biological samples can be obtained from animals (including humans) and encompass liquids, solids, tissues, and gases. Biological samples include blood products such as plasma, serum, and the like. However, such samples should not be construed as limiting the type of sample that is suitable for use in the present invention.

The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the examples, generally following conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations.

Example 1 screening of Gene markers associated with Lung adenocarcinoma

1. Sample collection

Samples of 5 tissues adjacent to and from lung adenocarcinoma were collected. The specimen material-taking part of the lung adenocarcinoma tumor tissue specimen is a main tumor area which is positioned at the junction of 1/3 outside the tumor mass and normal tissue, and obvious necrotic and calcified parts in the center of the tumor and normal lung tissue outside the tumor are excluded; the paracancer normal lung tissue specimen is taken from a part above 5cm of the tumor edge, and no obvious change is observed by naked eyes. All the specimens were obtained with the consent of the tissue ethics committee.

2. Preparation of RNA samples (Using

miRNA kit for operation)

Introducing liquid nitrogen into a mortar, putting the obtained tissue into the mortar, shearing the tissue in the liquid nitrogen and grinding the tissue into powder, putting the tissue into the liquid nitrogen after shearing the tissue into the powder, grinding the tissue into powder, and then transferring the powder into a glass homogenizer; tissue homogenization Trizol reagent was added to a glass homogenizer and the tissue was ground on ice. The homogenized tissue homogenate was transferred to an EP tube without RNase and allowed to stand at room temperature for 5 min. RNA was isolated by extraction according to the instructions in the kit.

The method comprises the following specific steps:

1) and (3) RNA isolation:

0.2m1 chloroform was added to the EP tube, the cap of the EP tube was closed, and shaken vigorously by hand for 15s to mix well. Incubating at room temperature for 5 min. Then centrifuged at 14000g for 15min at 4 ℃. After centrifugation the sample was divided into three layers, with RNA present in the upper aqueous phase.

2) RNA precipitation

Transferring 450 μ l of the separated water phase into a new RNase-free EP tube, adding 450 μ l of isopropanol at a ratio of 1:1, reversing the mixture from top to bottom, mixing the mixture uniformly, incubating the mixture at room temperature for 10min, and centrifuging the mixture at 14000g at 4 ℃ for 10 min.

3) RNA elution

After centrifugation the supernatant was carefully removed and the RNA washed by addition of 1ml of 75% ethanol (enzyme-killed, ready-to-use and pre-cooled on ice) followed by centrifugation at 7500g for 5min at 4 ℃.

4) RNA resolubilization

Carefully remove the washed supernatant, open the EP vial cap in the clean bench, place the RNA sample at room temperature for 5-10min, and air dry. Adding 20-50 μ l of non-RNase treated water, and carrying out water bath in a water bath tank at 55-60 ℃ for 10 min.

5) Mass analysis of RNA samples

And (3) detection by a spectrophotometer:

detecting an RNA sample by a NanoDrop1000 spectrophotometer, wherein the sample for RNA-seq sequencing requires: OD260/OD280 was 1.8-2.2.

And (3) agarose gel electrophoresis detection:

the extracted RNA is subjected to agarose gel electrophoresis, Agilent Technologies 2100Bioanalyzer detects the quality of an RNA sample, and the RNA sample is observed to have obvious main bands of 28S rRNA and 18S rRNA, no degradation, qualified RNA integrity index and concentration meeting the requirements, so that the RNA sample can be used for lncRNA expression profile and screening experiment of a chip.

3. Reverse transcription and labelling

mRNA was reverse-transcribed into cDNA using the Low RNA Input Linear Amplification Kit, and the experimental group and the control group were labeled with Cy3, respectively.

4. Hybridization of

The gene chip adopts Kangcheng organism-Human lncRNA Array, and hybridization is carried out according to the steps of the chip use instruction.

5. Data analysis

Chip results were analyzed using Agilent GeneSpring software, lncRNA with significant differences in expression levels (p <0.05) was screened.

6. Results

The results show that the expression level of LINC02014 in lung adenocarcinoma tissue is significantly higher than that in para-carcinoma tissue.

Example 2 QPCR sequencing verification of differential expression of LINC02014 Gene

1. Large sample QPCR validation was performed on differential LINC02014 gene expression. 35 cases of lung adenocarcinoma paracancerous tissue and lung adenocarcinoma tissue were selected according to the sample collection method in example 1.

2. The RNA extraction procedure was as described in example 1.

3. Reverse transcription

1) Reaction system:

1 mul of RNA template, 1 mul of random primer and 12 mul of double distilled water are added, mixed evenly, centrifuged at low speed, and cooled on ice at 65 ℃ for 5 min.

The following ingredients were added successively to 12. mu.l of the reaction:

5 × 4. mu.l of reaction buffer, 1. mu.l of RNase inhibitor (20U/. mu.l), 2. mu.l of 10mM dNTP mixture, 1. mu.l of AMV reverse transcriptase (200U/. mu.l); fully and uniformly mixing and carrying out centrifugal treatment;

2) conditions for reverse transcription

25℃5min，42℃60min，70℃5min。

3) Polymerase chain reaction

Designing a primer:

QPCR amplification primers were designed based on the coding sequences of LINC02014 gene and GAPDH gene in Genebank and synthesized by Bomader Biotech. The specific primer sequences are as follows:

LINC02014 gene:

the forward primer is 5'-AACAGGACAGATAAGACA-3' (SEQ ID NO. 2);

the reverse primer was 5'-GCAACAGACTAAGACATT-3' (SEQ ID NO. 3).

GAPDH gene:

the forward primer is 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO. 4);

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 5).

Preparing a PCR reaction system:

2 XqqPCR mixture 12.5. mu.l, gene primer 2.0. mu.l, reverse transcription product 2.5. mu.l, ddH₂O 8.0μl。

And (3) PCR reaction conditions: extension reaction at 95 deg.C for 10min, (95 deg.C for 15s, 60 deg.C for 60 s). times.40 cycles, and 60 deg.C for 5 min. The temperature is raised to 1 ℃ every 20s at 75 ℃ to 95 ℃, and a dissolution curve is drawn. SYBR Green is used as a fluorescent marker, PCR reaction is carried out on a Light Cycler fluorescent quantitative PCR instrument, a target band is determined through melting curve analysis and electrophoresis, and relative quantification is carried out through a delta CT method.

5. Statistical method

The experiments were performed in 3 replicates, the data were presented as mean ± sd, statistically analyzed using SPSS18.0 statistical software, and the paired comparison of cancer to paracancerous tissue was performed using t-test, which was considered statistically significant when P < 0.05.

6. Results

The results are shown in fig. 1, LINC02014 is up-regulated in lung adenocarcinoma tissue compared to lung adenocarcinoma para-tissue, with statistical significance (P <0.05), consistent with the results of the chip assay.

Example 3 silencing of LINC02014 Gene

1. Cell culture

Human lung adenocarcinoma cell strain A549 prepared from 10% fetal bovineSerum and 1% P/S RPMI1640 medium at 37 deg.C, 5% CO₂And culturing in an incubator with relative humidity of 90%. The solution was changed 1 time 2-3 days and passaged by conventional digestion with 0.25% EDTA-containing trypsin.

The cells in the culture flask were digested with pancreatin and seeded in 6-well plates to ensure that the number of cells was 2-8X 10⁵Per well, cell culture medium was added. The cell density was observed overnight the next day, and transfection was possible at cell densities above 70%.

2. Design of siRNA

Negative control siRNA sequence (siRNA-NC):

the sense strand is 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.6)

The antisense strand is 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.7)

siRNA-1：

The sense strand is 5'-UAAAAAGCCCCUUUCAUUCGG-3' (SEQ ID NO.8)

The antisense strand is 5'-GAAUGAAAGGGGCUUUUUAC-3' (SEQ ID NO.9)

siRNA-2：

The sense strand is 5'-UGUCUAACUGCUUUCUAAGGA-3' (SEQ ID NO.10)

The antisense strand is 5'-CUUAGAAAGCAGUUAGACACC-3' (SEQ ID NO.11)

siRNA-3：

The sense strand is 5'-UCUUUGAAGACAAAUGGUGGA-3' (SEQ ID NO.12)

The antisense strand is 5'-CACCAUUUGUCUUCAAAGAGA-3' (SEQ ID NO.13)

3. Transfection

The experiment was divided into three groups: a control group (A549), a negative control group (siRNA-NC) and an experimental group (siRNA-1, siRNA-2 and siRNA-3), wherein the negative control group siRNA-NC has no homology with the sequence of the LINC02014 gene and has the concentration of 20 nM/hole, and the transfection is carried out respectively.

4. QPCR detection of transcription level of LINC02014 gene

4.1 extraction of Total RNA from cells

1) The cell culture medium in the 6-well plate was poured off, washed twice with PBS, and 1ml of Trizol reagent was added to each well, and left at room temperature for 5 min.

2) 0.2m of 1 g of chloroform was added and centrifuged for 15min at 12000g with vigorous shaking for 15s at 4 ℃.

3) Transferring the water phase into a new tube, adding 4.5m1 isopropanol, and standing at room temperature for 10 min; centrifuging at 4 deg.C and 10000g for 10 min.

4) The liquid was decanted and the EP tube walls were washed with l ml of 75% ethanol. Centrifuge at 7500g for 5min at 4 ℃.

5) And pouring out the cleaned 75% ethanol, and airing at room temperature for 5-10 min.

6) Add 25. mu.l RNase-free DEPC water and store at-70 ℃.

4.2 reverse transcription procedure as in example 2.

4.3QPCR amplification step as in example 2.

5. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± sd, and statistically analyzed using SPSS18.0 statistical software, and the differences between the LINC02014 gene experimental group and the control group were considered statistically significant when P <0.05 using the t-test.

6. Results

The results are shown in FIG. 2, where the expression level of LINC02014 is significantly reduced in the experimental group compared to the non-transfected group and the transfected siRNA-NC group, whereas the silencing effect of transfected siRNA-1 is the best and the difference is statistically significant (P < 0.05).

Example 4 Effect of LINC02014 Gene on Lung adenocarcinoma cell proliferation

CCK-8 experiment is adopted to detect the influence of LINC02014 gene on the proliferation capacity of lung adenocarcinoma cells.

1. Cell culture and transfection procedures were as in example 3, and the medium was changed 6h after transfection and placed in a cell incubator overnight.

2. Taking out the cells the next day, observing the growth condition of the cells under a microscope, adding pancreatin containing EDTA into 1 ml/hole, digesting the cells, removing the pancreatin after digestion is finished, adding a cell culture medium, uniformly mixing to suspend the cells, and then counting the cells.

3. The cell suspension was diluted to a concentration of 15000 cells/ml, and then seeded in a 96-well plate, 200. mu.l of the cell suspension was added to each well, and the number of cells was controlled to about 3000, and 8 wells were seeded. The siRNA-1 experimental group and the siRNA-NC control group were set. A total of 4 96 well plates were plated for 4 detection time points of 24h, 48h, 72h, and 96h, respectively.

4. And after 24h, taking out the first 96-well plate, adding 10 mu l of CCK-8 detection solution into each well, continuously putting the 96-well plate into a cell culture box, incubating for about 4h, detecting the absorbance value of each well at the wavelength of 450nm by using an enzyme-labeling instrument, and recording data.

5. And (5) repeating the operation in the step (4) after 48h, 72h and 96h respectively, and finally counting the absorbance values of all time points to make a growth curve graph.

6. Statistical analysis

The experiments were performed in 3 replicates using SPSS18.0 statistical software for statistical analysis, and the differences between the two were considered statistically significant when P <0.05 using the t-test.

7. Results

As shown in FIG. 3, compared with the control, the experimental group showed that the cell proliferation was significantly inhibited after siRNA-1 transfection, and the statistical significance of the difference (P <0.05) indicates that LINC02014 has the effect of promoting cell proliferation.

Example 5 Effect of LINC02014 Gene on apoptosis of Lung adenocarcinoma cells

The effect of LINC02014 gene on apoptosis was examined using flow cytometry.

1. The cell culture procedure was as in example 3.

2. The cell transfection procedure was as in example 3.

3. Step (ii) of

1) 3m 110 Xloading buffer was diluted with 27m1 distilled water.

2) Cell samples were collected and washed with pre-cooled PBS.

3) Cells were added to lml 1 Xloading buffer, centrifuged at 300g for 10min and buffer aspirated.

4) The lml 1 Xloading buffer was added again to adjust the cell concentration in the cell suspension to 1X 10⁶One per ml.

5) The cell suspension was removed in 100. mu.l and added to an EP tube.

6) Add 5. mu.l Annexin V FITC to the EP tube, mix the liquid in the EP tube, incubate for 10min at room temperature in the dark.

7) Add 5. mu.l PI stain to the EP tube and protect from light for 5min at room temperature.

8) Add 500. mu.l PBS solution to EP tube, mix gently, and detect by up-flow cytometry within 1 h.

3. Statistical method

The experiments were performed in 3 replicates, the results were expressed as mean ± sd, and the statistical analysis was performed using SPSS13.0 statistical software, and the differences between the two were statistically significant using the t-test, which was considered to be when P < 0.05.

4. As a result:

the results are shown in fig. 4, where there was a significant change in the apoptosis rate (P <0.05) in the experimental group compared to the control group, indicating that LINC02014 inhibits apoptosis of the cells.

Example 6 cell migration and invasion assay

1. Transwell cell preparation

The Matrigel was thawed in an ice bath under sterile conditions, diluted 20-fold with PBS and applied to a polycarbonate membrane in a Transwell chamber at a volume of 50. mu.l/well. Standing at 37 deg.C for 4 hr, taking out after Matrigel gel polymerizes into gel, and sucking out supernatant liquid gently. 50 μ l of serum-free BSA-containing culture medium was added to each well to hydrate the basement membrane, and the membrane was left at 37 ℃ for 30 min.

2. Preparing a cell suspension

Starving the cells for 12-24h, digesting the cells, centrifuging after digestion is stopped, and removing the upper culture solution. The pelleted cells were washed with PBS and resuspended by adding serum-free medium containing BSA. Adjusting the cell density to 5 xl 0⁵One per ml.

3. Cell seeding

200. mu.l of cell suspension (100. mu.l for migration experiments and 200. mu.l for invasion experiments) was taken and added to the Transwell chamber. 500. mu.l of 1640 medium containing FBS was added to the lower chamber of the 24-well plate. The cells were placed in a cell incubator for 24 h.

4. Dyeing process

Cells were stained with DAPI after the end of the culture. The cell of the chamber is rinsed 2 times with PBS and then placed in DAPI working solution for staining for 5-20min at room temperature. Rinsed 2 times with PBS, placed under a fluorescent microscope for observation and counted.

5. Results

The results are shown in fig. 5, after the lung adenocarcinoma cells are transfected with the interfering RNA, the migration and invasion capacities of the experimental group are obviously reduced compared with those of the control group, and the results show that LINC02014 can promote the migration and invasion of the lung adenocarcinoma.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Sequence listing

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Claims (9)

1. Application of a reagent for detecting LINC02014 gene expression in preparation of products for diagnosing early lung adenocarcinoma.

2. The use according to claim 1, wherein the agent is selected from the group consisting of:

a probe that specifically recognizes LINC 02014; or

And (3) a primer for specifically amplifying LINC 02014.

3. The use of claim 1, wherein said product comprises detection of the expression level of LINC02014 gene in a sample by sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology.

4. Use according to claim 3, wherein the nucleic acid amplification technique is selected from the group consisting of polymerase chain reaction, reverse transcription polymerase chain reaction, transcription mediated amplification, ligase chain reaction, strand displacement amplification.

5. Use of a down-regulator of the LINC02014 gene for the preparation of a pharmaceutical composition for the prevention or treatment of lung adenocarcinoma.

6. The use of claim 5, wherein said down-regulating agent is an siRNA.

7. The use of claim 5, wherein the pharmaceutical composition further comprises other drugs compatible with the down-regulator and a pharmaceutically acceptable carrier and/or adjuvant.

Application of LINC02014 gene in screening potential substances for preventing or treating lung adenocarcinoma.

9. A method of screening for potential agents for preventing or treating lung adenocarcinoma, the method comprising:

treating the system expressing or containing the LINC02014 gene with a candidate substance; and

detecting the expression of the LINC02014 gene in the system;

wherein, if the candidate substance can reduce the expression or activity of the LINC02014 gene, the candidate substance is a potential substance for preventing or treating lung adenocarcinoma.

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