CN106729756B

CN106729756B - Application of biomarker as target in diagnosis and treatment of lung adenocarcinoma

Info

Publication number: CN106729756B
Application number: CN201710112636.9A
Authority: CN
Inventors: 边洋; 李曙光
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2017-02-28
Filing date: 2017-02-28
Publication date: 2021-05-25
Anticipated expiration: 2037-02-28
Also published as: CN106729756A

Abstract

The invention discloses application of a biomarker as a target in diagnosis and treatment of lung adenocarcinoma, and particularly relates to the biomarker CLINT 1. The incidence of lung adenocarcinoma continues to increase worldwide, and there is a need for effective biomarkers to diagnose and/or treat lung adenocarcinoma. Experiments prove that the expression of the CLINT1 is up-regulated in lung adenocarcinoma patients, and the reduction of the expression of the CLINT1 gene can inhibit the proliferation and invasion of lung adenocarcinoma cells. The CLINT1 disclosed by the invention can be used as a marker for early diagnosis of lung adenocarcinoma; meanwhile, the gene can also be used as a potential drug target for treating the lung adenocarcinoma, and a foundation is provided for the precise medical treatment of the lung adenocarcinoma.

Description

Application of biomarker as target in diagnosis and treatment of lung adenocarcinoma

Technical Field

The invention belongs to the field of biomedicine, and relates to application of a biomarker as a target in diagnosis and treatment of lung adenocarcinoma, wherein the biomarker is CLINT 1.

Background

Lung cancer is one of the most common tumors with morbidity and mortality worldwide, and seriously harms human health. Lung cancer can be classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC). Among them, NSCLC accounts for about 80% -85% of the total lung cancer, and has rapid disease development, rapid metastasis rate and high recurrence rate. Most patients have been found to have progressed to the middle and late stages with poor prognosis and a 5-year survival rate of less than 15%. Early treatment of NSCLC patients has a recurrence rate of 40%, and more than 70% of patients are found to be locally invasive or distant metastases and inoperable.

At present, surgery, chemotherapy and radiotherapy are the main treatment modes of lung cancer. In recent years, with the development of multidisciplinary combination therapy (MDT), the treatment of lung cancer has been advanced greatly. However, 5-year survival rate of lung cancer is still around 15%. The precise medical model is the trend and direction of tumor treatment today, and it is critical to utilize effective biomarkers for specific individualized treatment. Therefore, it is very important to find new effective molecular markers and potential therapeutic targets for lung cancer.

With the completion of human genome project, the new understanding of lung cancer at gene level is provided. Squamous lung carcinoma and adenocarcinoma of the lung at the molecular gene level are considered "two diseases". The existing data also suggest that biomarkers may be more readily found in lung adenocarcinoma and may benefit from it.

The advocation of an accurate medical model opens a new way for the diagnosis and treatment of lung cancer. The precise medicine is a medical mode based on molecular bioinformatics, and the key point of the precise medicine is to utilize effective biomarkers to distinguish target groups so as to carry out specific individual treatment. Therefore, finding new effective molecular biomarkers and signal paths of lung cancer, further clarifying the occurrence and development mechanism of lung cancer, and providing new theoretical basis for individual precise treatment of lung cancer becomes a current research hotspot and direction.

Disclosure of Invention

In order to remedy the deficiencies of the prior art, it is an object of the present invention to provide a biomarker associated with the development of lung adenocarcinoma.

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

the invention provides an application of a CLINT1 gene in preparing a pharmaceutical composition for preventing or treating lung adenocarcinoma.

Further, the pharmaceutical composition comprises a down-regulator of CLINT 1. The down-regulating agent is selected from: an interfering molecule that targets CLINT1 or its transcript and is capable of inhibiting CLINT1 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; or a binding molecule that specifically binds to a protein encoded by CLINT1 (e.g., an antibody or ligand capable of inhibiting the activity of CLINT1 protein).

Further, the down regulator is selected from the group consisting of siRNA of the following sequences: SEQ ID NO.8, SEQ ID NO. 9.

The present invention provides a down-regulator of CLINT1 for use in the prevention or treatment of lung adenocarcinoma, said down-regulator being selected from the group consisting of:

nucleic acid inhibitors, protein inhibitors, proteolytic enzymes, protein binding molecules capable of down-regulating the expression or activity of CLINT1 gene or its encoded protein at the protein or gene level.

In the present invention, said down-regulator of CLINT1 is also useful for inhibiting the invasion and proliferation of lung adenocarcinoma cells.

The present invention provides a pharmaceutical composition for preventing or treating lung adenocarcinoma, comprising:

the downregulation of CLINT1 described above; and

a pharmaceutically acceptable carrier.

The invention provides an application of a CLINT1 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 comprising the CLINT1 gene or a protein encoded thereby with a candidate substance; and

detecting expression or activity of CLINT1 gene or its encoded protein in the system;

wherein, if the candidate substance can reduce the expression or activity of the CLINT1 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.

Such candidate substances include, but are not limited to: interfering molecules, nucleic acid inhibitors, binding molecules (such as antibodies or ligands), small molecule compounds and the like designed aiming at the CLINT1 gene or protein encoded by the CLINT1 gene or upstream or downstream genes or proteins of the CLINT1 gene.

In the present invention, the method further comprises: further cell experiments and/or animal experiments are performed on the obtained potential substances to further select and identify substances useful for preventing, alleviating or treating lung adenocarcinoma from the candidate substances.

The invention provides an application of a CLINT1 gene in preparation of a product for diagnosing lung adenocarcinoma. Wherein the product includes, but is not limited to, a chip, a formulation or a kit.

Further, the product comprises a reagent for detecting the expression of the CLINT1 gene.

Further, the agent is selected from:

a probe that specifically recognizes CLINT 1; or

Primers for specific amplification of CLINT 1; or

An antibody or ligand that specifically binds to a protein encoded by CLINT 1.

Preferably, the primer for specifically amplifying the CLINT1 gene is a primer pair, and the sequence is shown as SEQ ID NO.3 and SEQ ID NO. 4.

Drawings

FIG. 1 is a graph showing the detection of the expression of CLINT1 gene in lung adenocarcinoma tissue by QPCR;

FIG. 2 is a graph of CLINT1 transfection in lung adenocarcinoma cells using QPCR;

FIG. 3 is a graph showing the effect of CLINT1 gene on the proliferation of lung adenocarcinoma cells measured by the CCK-8 method;

FIG. 4 is a graph showing the effect of the CLINT1 gene on apoptosis of lung adenocarcinoma cells measured by flow cytometry;

FIG. 5 is a graph of the effect of CLINT1 on migration and invasion of lung adenocarcinoma cells using a Transwell chamber; wherein panel a is a graph of the effect of CLINT1 on migration of lung adenocarcinoma cells; panel B is a graph of the effect of CLINT1 on lung adenocarcinoma cell invasion.

Detailed Description

The invention is widely and deeply researched, the expression of genes in a lung adenocarcinoma specimen in tumor tissues and tissues beside the lung adenocarcinoma is detected by adopting a gene chip which covers the most wide database at present through a high-throughput method, gene segments with obvious expression difference are found, and the relationship between the gene segments and the occurrence of the lung adenocarcinoma is discussed, so that a better way and a better method are found for the early detection and the targeted therapy of the lung adenocarcinoma. Through screening, the invention discovers that CLINT1 is remarkably upregulated in lung adenocarcinoma for the first time. Experiments prove that the CLINT1 can be effectively inhibited from growing and invading lung adenocarcinoma cells by reducing the expression level of the CLINT1, the detection of the expression level of the CLINT1 gene can be one of auxiliary diagnostic indexes for early diagnosis of lung adenocarcinoma, and the interference of the expression of the CLINT1 gene can be a new way for preventing or treating lung adenocarcinoma or lung adenocarcinoma metastasis.

CLINT1 gene

CLINT1 is located on 3 band of long arm 3 of human chromosome 5, and the nucleotide sequence and amino acid sequence of a representative human CLINT1 gene are shown as SEQ ID NO.1 and SEQ ID NO. 2. CLINT1 in the present invention includes wild type, mutant or fragments thereof.

The full-length nucleotide sequence or the fragment of the human CLINT1 can be obtained by a PCR amplification method, a recombinant method or an artificial synthesis method. For the PCR amplification method, the sequence can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template based on the known nucleotide sequence. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

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.

Down-regulating agent and pharmaceutical composition

Based on the discovery of the inventor, the invention provides the application of a down-regulating agent of CLINT1 in preparing a pharmaceutical composition for inhibiting lung adenocarcinoma. As used herein, said down-regulator of CLINT1 includes, but is not limited to, inhibitors, antagonists, blockers, nucleic acid inhibitors, and the like.

The CLINT1 gene or protein down-regulator refers to any substance which can reduce the activity of the CLINT1 protein, reduce the stability of the CLINT1 gene or protein, down-regulate the expression of the CLINT1 protein, reduce the effective action time of the CLINT1 protein, or inhibit the transcription and translation of the CLINT1 gene, and the substances can be used for the invention, can be used as substances which are useful for down-regulating the CLINT1, and can be used for preventing or treating lung adenocarcinoma. For example, the inhibitor is: nucleic acid inhibitors, protein inhibitors, antibodies, ligands, proteolytic enzymes, protein binding molecules, as long as they are capable of down-regulating the expression of CLINT1 protein or its encoding gene at the protein or gene level.

As an alternative of the invention, said down-regulator of CLINT1 is an antibody that specifically binds CLINT 1. The antibody may be a monoclonal antibody or a polyclonal antibody. Animals, such as rabbits, mice, rats, etc., can be immunized with CLINT1 protein to produce polyclonal antibodies; various adjuvants may be used to enhance the immune response, including but not limited to Freund's adjuvant and the like. Similarly, cells expressing CLINT1 or an antigenic fragment thereof can be used to immunize animals to produce antibodies. The antibody may also be a monoclonal antibody, and such monoclonal antibodies may be prepared using hybridoma technology. By "specific" of an antibody is meant that the antibody is capable of binding to the CLINT1 gene product or fragment. Preferably, those antibodies that bind to the CLINT1 gene product or fragment, but do not recognize and bind to other unrelated antigenic molecules. The antibodies of the invention can be prepared by a variety of techniques known to those skilled in the art. The invention encompasses not only intact monoclonal or polyclonal antibodies, but also immunologically active antibody fragments, such as Fab' or (Fab)²A fragment; an antibody heavy chain; an antibody light chain; a genetically engineered single chain Fv molecule; or a chimeric antibody. The antibody against the CLINT1 protein can be used in immunohistochemical technology to detect the CLINT1 protein content in biopsy specimens, and can also be used as a specific therapeutic agent for preventing liver cancer metastasis and invasion. The direct measurement of CLINT1 protein in blood sample or urine can be used as the auxiliary diagnosis of tumor and the observation index after healing, and can also be used as the basis for early diagnosis of tumor. Antibodies can be blotted by ELISA, Western BlotAnalysis, or coupling with a detection group, detection by chemiluminescence, isotopic tracing, and the like.

As a preferred mode of the invention, the down-regulator of CLINT1 is a small interfering RNA molecule specific for CLINT 1. 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 the siRNA with the best interference effect, has the sequences shown in SEQ ID NO.8 and SEQ ID NO.9 respectively, further performs experiments at a cell level, and proves that the inhibition efficiency is very high for cell experiments.

As an alternative of the present invention, the CLINT1 down-regulator may be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, and the Small hairpin RNA can inhibit gene expression via RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

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 pharmaceutical composition, which contains an effective amount of the CLINT1 down-regulator and a pharmaceutically acceptable carrier. The composition can be used for inhibiting lung adenocarcinoma. Any of the foregoing sint 1 down-regulators may be used in the preparation of the compositions.

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 effective amount of the down-regulator may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the down-regulator of CLINT1 gene such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like.

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 down-regulator of CLINT1 can be administered directly to a subject by a method such as injection; alternatively, expression units (such as expression vectors or viruses, etc., or siRNA or shRNA) carrying a down-regulator of CLINT1 can be delivered to a target in a manner that allows expression of the active CLINT1 down-regulator, depending on the type of down-regulator, as is well known to those of skill in the art.

The term "host cell" may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: coli, bacterial cells of the genus streptomyces; fungal cells such as yeast; a plant cell; insect cells of Drosophila S2 or Sf 9; CHO, COS, or 293 cell.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂. If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The pharmaceutical composition comprises a down-regulator of CLINT1, and/or other medicines compatible with the down-regulator, and a pharmaceutically acceptable carrier and/or auxiliary materials.

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 CLINT1 is measured; in a control group, no test compound is added into the same culture system, and the expression level of CLINT1 is measured; wherein, if the expression level of CLINT1 in the experimental group is greater than that in the control group, the candidate compound is a down-regulator of CLINT 1.

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.

Chip and kit

The gene chip of the invention comprises: 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 the CLINT 1.

Specifically, suitable probes can be designed according to the genes of the present invention, and immobilized on a solid support to form an "oligonucleotide array". By "oligonucleotide array" is meant an array having addressable locations (i.e., locations characterized by distinct, accessible addresses), each addressable location containing a characteristic oligonucleotide attached thereto. The oligonucleotide array may be divided into a plurality of subarrays as desired.

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 oligonucleotide probe of the invention directed against the CLINT1 gene may be DNA, RNA, DNA-RNA chimeras, PNA or other derivatives. The length of the probe is not limited, and any length may be used as long as specific hybridization and specific binding to the target nucleotide sequence are achieved. The length of the probe may be as short as 25, 20, 15, 13 or 10 bases in length. Also, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or more, even for the entire gene. Since different probe lengths have different effects on hybridization efficiency and signal specificity, the length of the probe is usually at least 14 base pairs, and at most, usually not more than 30 base pairs, and the length complementary to the nucleotide sequence of interest is optimally 15 to 25 base pairs. The probe self-complementary sequence is preferably less than 4 base pairs so as not to affect hybridization efficiency.

The solid phase carrier of the present invention can be made of various materials commonly used in the field of gene chip, such as but not limited to plastic products, microparticles, membrane carriers, etc. The plastic products can be combined with antibodies or protein antigens through a non-covalent or physical adsorption mechanism, and the most common plastic products are small test tubes, small beads and micro reaction plates made of polystyrene; the micro-particles are microspheres or particles polymerized by high molecular monomers, the diameter of the micro-particles is more than micron, and the micro-particles are easy to form chemical coupling with antibodies (antigens) due to the functional groups capable of being combined with proteins, and the combination capacity is large; the membrane carrier comprises microporous filter membranes such as a nitrocellulose membrane, a glass cellulose membrane, a nylon membrane and the like.

The CLINT1 chip can be prepared by conventional methods for manufacturing biochips known in the art. For example, if a modified glass slide or silicon wafer is used as the solid support, and the 5' end of the probe contains a poly-dT string modified with an amino group, the oligonucleotide probe can be prepared into a solution, and then spotted on the modified glass slide or silicon wafer using a spotting apparatus, arranged into a predetermined sequence or array, and then fixed by standing overnight, thereby obtaining the gene chip of the present invention.

The invention provides a kit which can be used for detecting the expression of CLINT1 gene or protein. 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.

The components of the kit may be packaged in aqueous medium or in lyophilized form. Suitable containers in the kit generally include at least one vial, test tube, flask, pet bottle, syringe, or other container in which a component may be placed and, preferably, suitably aliquoted. Where more than one component is present in the kit, the kit will also typically comprise a second, third or other additional container in which the additional components are separately disposed. However, different combinations of components may be contained in one vial. The kit of the invention will also typically include a container for holding the reactants, sealed for commercial sale. Such containers may include injection molded or blow molded plastic containers in which the desired vials may be retained.

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 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 high concentration, and can be used for gene expression profiles and screening experiments of chips.

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 a human whole genome expression profile chip of the agent company. According to the steps of the chip use instruction.

5. Data analysis

Chip results were analyzed by using Agilent GeneSpring software, and genes with significant differences in expression levels (standard is that the difference between the expression levels of the gene at the cancer site and the expression level at the cancer site is more than 2 times, and p is less than 0.05) were selected.

6. Results

The results show that the expression level of CLINT1 in lung adenocarcinoma tissues is significantly higher than that in paracarcinoma tissues.

Example 2 verification of differential expression of CLINT1 Gene by QPCR sequencing

1. Large sample QPCR validation was performed on differential expression of the CLINT1 gene. 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 CLINT1 gene and GAPDH gene in Genebank and synthesized by Bomeide Bio Inc. The specific primer sequences are as follows:

CLINT1 gene:

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

the reverse primer was 5'-GGACCATATCTGTATTCT-3' (SEQ ID NO. 4).

GAPDH gene:

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

the reverse primer was 5'-GAGCCCCAGCCTTCTCCAT-3' (SEQ ID NO. 6).

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

Results as shown in fig. 1, compared with the tissues beside the lung adenocarcinoma, CLINT1 was up-regulated in the lung adenocarcinoma tissues, and the difference was statistically significant (P <0.05), which is consistent with the results of the chip detection.

Example 3 silencing of CLINT1 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. 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.7)

The antisense strand is 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO.8)

siRNA-1：

The sense strand is 5'-UUAUCACUGAAUGGAAAAGCA-3' (SEQ ID NO.9)

The antisense strand is 5'-CUUUUCCAUUCAGUGAUAAAU-3' (SEQ ID NO.10)

siRNA-2：

The sense strand is 5'-UCCAAUUCUUUUUGUUGUCUU-3' (SEQ ID NO.11)

The antisense strand is 5'-GACAACAAAAAGAAUUGGAGA-3' (SEQ ID NO.12)

siRNA-3：

The sense strand is 5'-AUACUUGUCUUUGUUCUUCUU-3' (SEQ ID NO.13)

The antisense strand is 5'-GAAGAACAAAGACAAGUAUGU-3' (SEQ ID NO.14)

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 and siRNA-3), wherein the siRNA of the negative control group has no homology with the sequence of the CLINT1 gene and the concentration is 20 nM/hole, and the transfection is carried out respectively.

4. QPCR detection of transcript level of CLINT1 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, statistically analyzed using SPSS18.0 statistical software, and the differences between the CLINT1 gene experimental group and the control group were determined by t-test to be statistically significant when P < 0.05.

6. Results

Results as shown in figure 2, expression level of CLINT1 was significantly reduced in the experimental group compared to the non-transfected group and the transfected siRNA-NC group, with the difference being statistically significant (P < 0.05).

Example 4 Effect of the CLINT1 Gene on Lung adenocarcinoma cell proliferation

CCK-8 experiment is adopted to detect the effect of CLINT1 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

The results are shown in figure 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 CLINT1 has the effect of promoting cell proliferation.

Example 5 Effect of the CLINT1 Gene on apoptosis of Lung adenocarcinoma cells

The effect of CLINT1 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 represented as mean ± sd, and the statistical analysis was performed using SPSS18.0 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 remarkably increased compared with that of the control group (P <0.05), and the results show that the over-expression of CLINT1 inhibits the apoptosis of lung adenocarcinoma 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 CLINT1 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

Use of a down-regulator of CLINT1 gene, which is an siRNA, for the preparation of a pharmaceutical composition for the prevention or treatment of lung adenocarcinoma.
2. The use of claim 1, wherein the down-regulating agent is an siRNA selected from the group consisting of: SEQ ID NO.9, SEQ ID NO. 10.
3. Application of a reagent for specifically detecting the expression level of the CLINT1 gene in preparing a product for diagnosing lung adenocarcinoma.
4. Use according to claim 3, wherein said agent is selected from:

a probe that specifically recognizes CLINT 1; or

Primers for specific amplification of CLINT 1; or

An antibody or ligand that specifically binds to a protein encoded by CLINT 1.