CN107058499B

CN107058499B - Molecular marker for diagnosis and treatment of lung adenocarcinoma

Info

Publication number: CN107058499B
Application number: CN201710055722.0A
Authority: CN
Inventors: 田子强; 李振华; 温士旺
Original assignee: Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
Current assignee: Fourth Hospital of Hebei Medical University Hebei Cancer Hospital
Priority date: 2017-01-25
Filing date: 2017-01-25
Publication date: 2020-03-13
Anticipated expiration: 2037-01-25
Also published as: CN107058499A

Abstract

The invention discloses a molecular marker for diagnosis and treatment of lung adenocarcinoma, which has specificity and sensitivity for diagnosis and treatment of lung adenocarcinoma. The invention discovers that the molecular marker LOC101926969 is highly expressed in lung adenocarcinoma patients, and siRNA is used for interfering the expression of the gene, so that the invention can inhibit the proliferation of cells and reduce the invasion number and migration number of cancer cells.

Description

Molecular marker for diagnosis and treatment of lung adenocarcinoma

Technical Field

The invention belongs to the field of biological medicines, and relates to a molecular marker for diagnosis and treatment of lung adenocarcinoma, wherein a specific target is LOC 101926965.

Background

Lung cancer is one of the most common malignancies in humans and is also the leading cause of cancer-related death. Despite the increasing updating of lung cancer diagnosis and treatment methods and techniques, the overall prognosis is still not ideal, the mortality rate is still high at the first of the cancers, and the 5-year survival rate is less than 15%. While non-small cell lung cancer accounts for over 85% of all lung cancer patients, and lung adenocarcinoma is the most prominent part of the non-small cell lung cancer patients, the most effective treatment method is total pneumonectomy plus a proper adjuvant radiotherapy and chemotherapy strategy, but even in stage I patients, after the tumor is completely removed, about more than 25% of the patients relapse in a short time, and the serious influence is caused to the health and the life of the human beings.

In recent years, with the intensive research on the occurrence and development mechanism of lung cancer, some therapeutic targets are found, and the attempt of a new targeted therapeutic method brings a new breakthrough for the diagnosis and treatment of lung cancer, but the current research finds that the number of drug-resistant targets is small and the abnormal distribution population range is narrow, so that the corresponding therapeutic method has the problems of narrow applicable population range, treatment resistance and the like, and the therapeutic effect faces the challenge. Therefore, screening new drug-like targets related to non-small cell lung cancer and developing new treatment strategies from molecular level become continuous hot spots in the field of lung cancer research.

With the advent of genomic microarrays and whole genome and transcriptome sequencing technologies, the human transcriptome was found to have a large number of antisense genes, overlapping genes, and non-coding RNA expressions (non-coding RNAs, ncRNAs). IncRNAs are a class of noncoding RNAs with a length of more than 200bp nucleotides, sometimes up to 100 kb. lncRNA is widely expressed in humans and plays an important role in multiple physiological processes of global regulation of genes. IncRNA has been shown to be closely associated with a variety of human diseases, particularly neoplastic diseases. Some lncRNA presents obvious positive correlation with the occurrence of diseases, namely the corresponding lncRNA presents an over-expression state in the occurrence and development process of the diseases; some lncRNA presents a certain negative correlation with the occurrence of diseases, namely the corresponding lncRNA expression level is obviously reduced in the process of the occurrence and the development of the diseases compared with normal tissues or normal people. However, since lncRNA does not itself encode a protein, it has been studied very rarely for its specific role in biological processes. The method finds the lncRNA related to the lung adenocarcinoma, discusses the mechanism of the lncRNA in the lung adenocarcinoma pathogenesis process, and has great significance for early diagnosis and targeted treatment of diseases and scientific research of lncRNA genes.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide a molecular marker for diagnosis and treatment of lung adenocarcinoma.

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

the invention provides an application of a reagent for detecting LOC101926969 gene expression in preparing a product for diagnosing early lung adenocarcinoma.

Further, the agent is selected from:

a probe specifically recognizing LOC 101926969; or

A primer for specifically amplifying LOC 101926969.

Preferably, the primer sequence of the specific amplification LOC101926969 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 LOC 101926965 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 an application of LOC101926969 gene in preparing a pharmaceutical composition for preventing or treating lung adenocarcinoma.

Further, the pharmaceutical composition comprises a down-regulator of LOC 101926969. The down-regulating agent is selected from: an interfering molecule targeting LOC101926969 or its transcript and capable of inhibiting LOC101926969 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 an application of LOC101926969 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 a system expressing or containing the LOC101926969 gene with a candidate substance; and

detecting the expression of LOC101926969 gene in the system;

wherein, if the candidate substance can reduce the expression or activity of LOC 101926965 gene, (preferably significantly reduced, such as more than 20% lower, preferably more than 50% lower, more preferably more than 80% lower), it indicates 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.

The candidate substances include (but are not limited to): interfering molecules, nucleic acid inhibitors, small molecule compounds and the like designed against the LOC101926969 gene or its upstream or downstream genes.

The invention has the advantages and beneficial effects that:

the invention discovers that the differential expression of LOC101926969 is related to the occurrence and development of lung adenocarcinoma for the first time, and suggests that the gene can be used as an index for diagnosing the lung adenocarcinoma.

Experiments prove that the siRNA silences the expression of the LOC101926969 gene, can inhibit the proliferation of lung adenocarcinoma cells, reduce the migration and invasion rate of the lung adenocarcinoma cells, and prompt that the LOC101926969 gene can be used as a drug target for treating lung adenocarcinoma and metastasis thereof.

Drawings

FIG. 1 is a graph showing the detection of the expression of LOC 101926965 gene in lung adenocarcinoma tissue by QPCR;

FIG. 2 is a graph showing the detection of transfection of LOC 101926965 in lung adenocarcinoma cells using QPCR;

FIG. 3 is a graph showing the effect of LOC101926969 gene on lung adenocarcinoma cell proliferation measured by the CCK-8 method;

FIG. 4 is a graph showing the effect of LOC 101926965 gene on apoptosis of lung adenocarcinoma cells by flow cytometry;

FIG. 5 is a graph of the effect of LOC 101926965 on migration and invasion of lung adenocarcinoma cells using a Transwell chamber; wherein Panel A is a graph of the effect of LOC 101926965 on migration of lung adenocarcinoma cells; panel B is a graph of the effect of LOC101926969 on lung adenocarcinoma cell invasion.

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 that LOC 101926965 is remarkably upregulated in lung adenocarcinoma for the first time. Experiments prove that siRNA interference silences LOC101926969, can effectively inhibit proliferation and invasion of lung adenocarcinoma cells, and provides a new way for accurate 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. In some embodiments, it has a cDNA sequence at least 85% identical or similar to the listed sequences, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical or similar to the listed sequences.

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.

LOC101926969 gene

LOC101926969 is located in the short arm 1 region of human chromosome 2, and the nucleotide sequence of a representative human LOC101926969 gene is shown in SEQ ID NO. 1. LOC 101926965 in the present invention includes wild type, mutant type or a fragment 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.

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 known as PCR, uses multiple cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of the target nucleic acid sequence, transcription-mediated amplification of TMA (autocatalytically synthesizing multiple copies of the target nucleic acid sequence under substantially constant conditions of temperature, ionic strength, and pH, where 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 β 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 for detecting the expression level of LOC 101926965 gene in the detection, and the products comprise (but are not limited to) preparations, chips or kits. 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 by LOC 101926969.

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.

The invention provides a kit which can be used for detecting the expression of LOC 101926965. 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 down-regulator LOC 101926965 in preparing a pharmaceutical composition for inhibiting lung adenocarcinoma. As used herein, the down-regulator of LOC 101926965 includes, but is not limited to, inhibitors, antagonists, blockers, nucleic acid inhibitors, and the like.

The LOC101926969 down-regulator refers to any substance which can down-regulate the expression of the LOC101926969 gene or inhibit the transcription of the LOC101926969 gene, and the substance can be used for preventing or treating the lung adenocarcinoma as a substance which is useful for down-regulating the LOC 101926969.

As a preferred mode of the invention, the down-regulator of LOC101926969 is a LOC101926969 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, a process known as RNA interference (RNAInterferce). 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 LOC101926969 gene in cells and the proliferation of 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 containing an effective amount of the LOC 101926965 down-regulator and a pharmaceutically acceptable carrier. The composition can be used for inhibiting lung adenocarcinoma. Any of the foregoing down-regulators of LOC 101926967 may 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, the down-regulator of LOC 101926965 can be administered directly to a subject by a method such as injection; alternatively, an expression unit (such as an expression vector or virus, etc., or siRNA or shRNA) carrying the down regulator of LOC101926969 can be delivered to a target site in a certain way and allowed to express the active LOC101926969 down regulator, depending on the type of the down regulator, which is well known to 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 that inhibits the expression of the LOC 101926965 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 tested is added into a cell culture system, and the expression level of LOC 101926965 is measured; in a control group, no test compound is added to the same culture system, and the expression level of LOC 101926965 is determined; wherein, if the expression level of LOC101926969 in the experimental group is greater than that in the control group, the candidate compound is a down-regulator of LOC 101926969.

In the present invention, the method further comprises: the candidate compound obtained in the above step is further tested for its effect of inhibiting liver cancer, 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 tissues adjacent to and adjacent to lung adenocarcinoma were collected for 8 cases each. 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 e.z.n.a.

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 Technologies2100Bioanalyzer 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 are analyzed by using Agilent GeneSpring software, and lncRNA with significant difference (standard is that the difference of the expression quantity of lncRNA in cancer and nearby cancer is more than 2 times, and p is less than 0.05) in expression quantity is screened.

6. Results

The results show that the expression level of LOC101926969 in lung adenocarcinoma tissues is significantly higher than that in paracarcinoma tissues.

Example 2QPCR sequencing verification of differential expression of the LOC 101926965 Gene

1. Large sample QPCR validation was performed on LOC101926969 gene differential expression. 50 cases of the 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 the LOC101926969 gene and GAPDH gene from Genebank and were synthesized by Bomader Biotech. The specific primer sequences are as follows:

LOC101926969 gene:

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

the reverse primer was 5'-GCAAGTTCTGATGTAAGTG-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, compared with the tissues beside the lung adenocarcinoma, LOC 101926965 is up-regulated in the lung adenocarcinoma tissues, and the difference has statistical significance (P <0.05), which is consistent with the chip detection results.

Example 3 silencing of the LOC101926969 Gene

1. Cell culture

Human lung adenocarcinoma cell line A549 prepared by culturing RPMI1640 medium containing 10% fetal calf serum and 1% P/S at 37 deg.C and 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. Overnight, the next day the cell density was observedTransfection can be carried out at a cell density of 70% or more.

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'-UACAGAAAAUAAGUCAUAGAA-3' (SEQ ID NO.8)

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

siRNA-2：

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

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

3. Transfection

The experiment was divided into three groups: a control group (A549), a negative control group (siRNA-NC) and an experimental group (siRNA1, siRNA2), wherein the siRNA of the negative control group has no homology with the sequence of the LOC101926969 gene and has the concentration of 20 nM/hole, and the transfection is carried out respectively.

4. QPCR detection of transcription level of LOC101926969 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 lml 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) 25 μ 1 RNase-free DEPC water was added and stored at-70 ℃.

4.2 reverse transcription procedure as in example 2.

4.3 QPCR amplification step as in example 2.

5. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± standard deviation, statistically analyzed using SPSS18.0 statistical software, and the difference between the experimental group and the control group of the LOC 101926965 gene was determined to be statistically significant when P <0.05 using the t test.

6. Results

The results are shown in fig. 2, where the expression level of LOC 101926965 was significantly reduced in the experimental group compared to the non-transfected group and the transfected siRNA-NC group, and the difference was statistically significant (P < 0.05).

Example 4 Effect of LOC101926969 Gene on Lung adenocarcinoma cell proliferation

CCK-8 experiment is adopted to detect the influence of LOC 101926965 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 concentration was diluted to 15000 cells/ml, and then the cells were seeded in a 96-well plate, 200. mu.1 of the cell suspension was added to each well, and the number of cells was controlled to about 3000, and 8 replicate 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 1 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 has obviously inhibited cell proliferation after siRNA-1 transfection, and the difference has statistical significance (P <0.05), which indicates that LOC101926969 has the function of promoting cell proliferation.

Example 5 Effect of LOC101926969 Gene on apoptosis of Lung adenocarcinoma cells

The influence of the LOC101926969 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 100. mu.1 and added to the 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.1 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, and the apoptosis rate of the experimental group is significantly changed compared with the control group (P <0.05), which indicates that LOC 101926965 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

Cell suspension 200. mu.1 (migration assay 100. mu.1, invasion assay 200. mu.1) was added to the Transwell chamber. 500 μ 1 of FBS-containing 1640 medium 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 LOC101926969 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

1. Application of a reagent for detecting LOC101926969 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 specifically recognizing LOC 101926969; or

A primer for specifically amplifying LOC 101926969.

3. The use according to claim 1, wherein the product comprises a reagent for detecting the expression level of the LOC 101926965 gene in the 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 and nucleic acid sequence based amplification.

The application of a down regulator of LOC101926969 gene in preparing a pharmaceutical composition for preventing or treating lung adenocarcinoma is characterized in that the down regulator is siRNA, and the sequences of the siRNA are SEQ ID NO.8 and SEQ ID NO. 9.

6. 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 LOC101926969 gene in screening potential substances for preventing or treating lung adenocarcinoma.

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

detecting the expression of LOC101926969 gene in the system;

wherein, if the candidate substance can reduce the expression or activity of the LOC 101926965 gene, the candidate substance is a potential substance for preventing or treating lung adenocarcinoma.