CN110042164B

CN110042164B - Lung cancer diagnosis and treatment lncRNA marker

Info

Publication number: CN110042164B
Application number: CN201910472082.2A
Authority: CN
Inventors: 杨承刚; 高舒欣
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2020-12-25
Anticipated expiration: 2039-05-31
Also published as: CN110042164A

Abstract

The invention discloses a lncRNA marker for diagnosis and treatment, which is RP11-284F 21.10. The invention discovers that the expression of RP11-284F21.10 is remarkably up-regulated in lung adenocarcinoma for the first time, and further proves that RP11-284F21.10 can be used as a potential molecular target for clinical diagnosis of lung adenocarcinoma through a large-sample QPCR experiment. The invention also proves that RP11-284F21.10 influences the proliferation of lung adenocarcinoma cells through CCK 8.

Description

Lung cancer diagnosis and treatment lncRNA marker

Technical Field

The invention belongs to the field of biological medicines, and relates to an lncRNA marker for lung cancer diagnosis and treatment, wherein the specific marker is RP11-284F 21.10.

Background

Lung cancer is the most common cancer with incidence and mortality in the world (FERRAY J, SOERJOMATARAM I, DIKSHIT R, et al. cancer invasion and motility cancers: sources, methods and major patterns in GLOBOCAN 2012[ J ]. Int J cancer.2015,136(5): E359-386.). Lung cancer comprises four major subtypes, as well as a number of minor or very rare subtypes (BRAMBILLA E, TRAVIS W D, COLBY T V, et al, the new World Health Organization classification of lung tumors [ J ]. Eur Respir J.2001,18(6):1059-1068.), each of which possesses specific biological and clinical characteristics. They can be classified by clinical pathology into Small cell carcinoma (SCLC) and Non-Small cell carcinoma (NSCLC) (SUN S, SCHILLER J H, GAZDAR A F. Lung cancer in boiler-a differential disease [ J ] Nat Rev cancer.2007,7(10): 778-. Among them, non-small cell cancer is the most common type of lung cancer, accounting for over 80% of lung cancer. Among the non-small cell carcinomas, there are three subtypes, lung adenocarcinoma, lung squamous carcinoma and large cell carcinoma. Adenocarcinoma of the Lung and squamous carcinoma of the Lung are the most common types of non-Small Cell carcinomas (YILDIZ O, BUYUKTAS D, EKIZ E, et al. facial New Panel: An annual Presenting Feature of Small Cell Lung Cancer [ J ]. Case Reports in Oncology.2011,4(1):35-38.) which have different pathogenesis and diagnostic modalities. In contrast, lung adenocarcinoma is more likely to occur in the periphery of the lungs and is more common in people who never smoke, while squamous lung carcinoma is more likely to occur in the center of the lungs and is highly correlated with the patient's smoking history.

Lung cancer has been considered a disease that is unique to smokers. However, statistical analysis worldwide has found that 15% of lung Cancer patients in men and 53% of lung Cancer patients in women are tobacco-independent (PARKIN D M, BRAY F, FERLAY J, et al. Global Cancer statistics,2002[ J ]. CA Cancer J Clin.2005,55(2): 74-108.). It has been found that lung cancer is the seventh most common cancer in non-smoking populations, and precedes uterine, pancreatic and prostate cancer. Only less than 16% of the current lung cancer patients are diagnosed in the initial stage of lung cancer, which greatly increases the difficulty and cost of treatment. Therefore, there is a need to find biomarkers that can be identified and diagnosed at an early stage of lung cancer. This can greatly improve the efficiency of treatment and reduce the social cost of treatment.

Long non-coding RNAs (1 ncRNAs) are found with the development of cDNA chip technology, and have poor conservation and low expression level, so they are once considered as transcription noise, genome loophole transcription (LIU J.control of protein synthesis and mRNA degradation by microRNAs [ J ]. Curr Opin Cell biol.2008,20(2): 214-. With the development of sequencing technologies, they are gradually recognized and become a generation of star RNA. At present, the application research of lncRNA in early diagnosis and prognosis prediction of lung adenocarcinoma is less, and more lung cancer related 1ncRNAs are yet to be further discovered.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide a lncRNA biomarker related to the occurrence and development of lung adenocarcinoma and application thereof in diagnosis and treatment of lung adenocarcinoma.

The invention also aims to provide a method for screening a candidate drug for treating lung adenocarcinoma.

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

in a first aspect of the invention, there is provided a detection reagent which can detect the level of RP11-284F 21.10.

Further, the agent is selected from:

a probe that specifically recognizes RP11-284F 21.10; or

Primers for specifically amplifying RP11-284F 21.10.

Furthermore, the primer sequence of the specific amplification RP11-284F21.10 is shown in SEQ ID NO. 1-2.

In a second aspect the invention provides a product comprising an agent according to the first aspect of the invention.

Further, the product comprises a kit, a chip and a nucleic acid membrane strip.

In a third aspect of the invention, there is provided a composition comprising an effective amount of an inhibitor of RP11-284F 21.10. The inhibitor is selected from: an interfering molecule which uses RP11-284F21.10 or a transcript thereof as a target sequence and can inhibit the expression or gene transcription of RP11-284F21.10 gene, 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 inhibitor is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 7-12.

Preferably, the sequence of the siRNA is shown in SEQ ID NO. 7-8.

In a fourth aspect of the present invention, there is provided a method of screening for a candidate drug for treating lung adenocarcinoma, the method comprising:

treating a system expressing or containing an RP11-284F21.10 gene with a substance to be screened; and

detecting the expression of the RP11-284F21.10 gene in the system;

wherein, if the substance to be screened can reduce the level of RP11-284F21.10 gene, the substance to be screened is a candidate drug for 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 aiming at the RP11-284F21.10 gene or upstream or downstream genes thereof.

A fifth aspect of the invention provides the use of any one of:

a. use of an agent according to the first aspect of the invention in the manufacture of a means for diagnosing lung adenocarcinoma;

b. use of a product according to the second aspect of the invention in the manufacture of a means for diagnosing lung adenocarcinoma;

application of RP11-284F21.10 in constructing a calculation model for diagnosing lung adenocarcinoma;

the application of RP11-284F21.10 in preparing a medicament for treating lung adenocarcinoma;

e. use of a composition according to the third aspect of the invention in the manufacture of a medicament for the treatment of lung adenocarcinoma;

use of RP11-284F21.10 for screening a candidate drug for treating lung adenocarcinoma.

Drawings

FIG. 1 is a graph showing the detection of the expression of RP11-284F21.10 gene in lung adenocarcinoma tissue by QPCR;

FIG. 2 is a graph showing the effect of siRNA on silencing RP11-284F 21.10;

FIG. 3 is a graph showing the effect of RP11-284F21.10 on lung adenocarcinoma cell proliferation measured by the CCK8 method.

Detailed Description

The invention is widely and deeply researched, the expression of lncRNA in a lung adenocarcinoma specimen in a tumor tissue and a tissue beside the tumor is detected through a high-throughput sequencing and bioinformatics analysis method, lncRNA with obvious expression difference is found, and the relation between the lncRNA and the occurrence and development of lung adenocarcinoma is discussed, so that a better way and a better method are found for the diagnosis and the targeted therapy of the lung adenocarcinoma. Through screening, the invention discovers the significant up-regulation of RP11-284F21.10 in lung adenocarcinoma for the first time, and determines the correlation between the RP11-284F21.10 and the lung adenocarcinoma proliferation by designing siRNA aiming at RP11-284F21.10 according to the relation between the RP11-284F21.10 and the lung adenocarcinoma. Provides a new tumor marker and a new therapeutic target for early diagnosis and treatment of the lung adenocarcinoma.

The term "level of expression" or "expression level" generally refers to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information is converted into structures that are present and operational in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of a polynucleotide and/or polypeptide modification (e.g., post-translational modification of a polypeptide) should also be considered expressed, whether they are derived from transcripts generated by alternative splicing or degraded transcripts, or from post-translational processing of a polypeptide (e.g., by proteolysis). "expressed gene" includes genes that are transcribed into a polynucleotide (e.g., mRNA) and then translated into a polypeptide, as well as genes that are transcribed into RNA but not translated into a polypeptide (e.g., transport and ribosomal RNA, miRNA, IncRNA, circRNA). In a particular embodiment of the invention, an "expressed gene" refers to a gene that is transcribed into RNA but not translated into a polypeptide.

Increased expression, "increased expression level," "increased level," "elevated expression level," or "elevated level" refers to increased expression or increased level of a biomarker in an individual relative to a control, such as a median expression level of the biomarker in an individual not having a disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or a sample from one patient group/population.

"reduced expression", "reduced expression level", "reduced expression level" or "reduced level" refers to reduced expression or reduced level of a biomarker in an individual relative to a control, such as a median expression level of the biomarker in an individual or an internal control (e.g., a housekeeping biomarker) that does not have a disease or disorder (e.g., cancer), or a sample from one patient group/population. In some embodiments, the reduced expression is little or no expression.

RP11-284F21.10

The gene for transcribing RP11-284F21.10 is located on human chromosome 1, and RP11-284F21.10 in the invention includes wild type, mutant type or fragment thereof. Those skilled in the art will appreciate that in performing sequencing analysis, the original sequencing results will be aligned to the human reference genome, and therefore the RP11-284F21.10 in the screening results may contain different transcripts as long as the RP11-284F21.10 on the reference genome can be aligned. In the examples of the present invention, the nucleotide sequence of a representative transcribed RP11-284F21.10 gene is shown in ENST 00000605886.1.

The present invention may utilize any method known in the art to determine the expression level of a gene. 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.

Some methods of detection or quantification of lncRNA levels are known in the art and are all suitable for use in the methods provided herein to measure levels of biomarkers. Exemplary methods include, but are not limited to, northern blots (northern blots), ribonuclease protection assays, and PCR-based methods. When the biomarker is a lncRNA molecule, the lncRNA sequence or fragment thereof can be used to prepare a probe that is at least partially complementary. The probes can then be used to detect lncRNA sequences in the sample using any suitable assay, such as PCR-based methods, Northern blotting (Northern blotting) or dipstick assay (dipstick assay).

The assay method may vary depending on the type of lncRNA information desired. Exemplary methods include, but are not limited to, Northern blots (Northern blots) and PCR-based methods (e.g., qRT-PCR). The method of qRT-PCR and the like can also accurately quantify the amount of lncRNA in the sample.

Any suitable assay platform can be used to determine the presence of lncRNA in a sample. For example, the assay may be in the form of a dipstick (dipstick), membrane (membrane), chip (chip), disk (disk), test strip (test strip), filter (filter), microsphere (microspherole), slide (slide), multiwell plate (multiwell plate) or optical fiber (optical fiber). The assay system can have a solid support to which nucleic acids corresponding to incrnas are attached. The solid support may comprise, for example, a plastic, a silicon wafer, a metal, a resin, a glass, a membrane, a particle, a precipitate (precipitate), a gel, a polymer, a sheet (sheet), a sphere, a polysaccharide, a capillary, a film (film), a plate, or a slide. The assay components can be prepared and packaged together as a kit for detecting lncRNA.

The nucleic acids can be labeled, if desired, to make a population of labeled lncrnas. In general, the sample can be labeled using methods well known in the art. In certain embodiments, the sample is labeled with a fluorescent label. Exemplary fluorescent dyes include, but are not limited to, xanthene (xanthene) dyes, fluorescein dyes, rhodamine dyes, Fluorescein Isothiocyanate (FITC), 6-carboxyfluorescein (FAM), 6-carboxy-2 ',4',7',4, 7-Hexachlorofluorescein (HEX), 6-carboxy-4 ',5' -dichloro-2 ',7' -dimethoxyfluorescein (JOE or J), N, N, N ', N ' -tetramethyl-6-carboxyrhodamine (TAMRA or T), 6-carboxy-X-rhodamine (ROX or R), 5-carboxyrhodamine 6G (R6G5 or G5), 6-carboxyrhodamine 6G (R6G6 or G6), and rhodamine 110; cyanine dyes such as Cy3, Cy5 and Cy7 dyes; alexa dyes, such as Alexa-fluor-555; coumarin, diethylaminocoumarin, umbelliferone; a benzimine dye such as Hoechst 33258; phenanthridine dyes, such as Texas red (Texas red); ethidium dye; an acridine dye; a carbazole dye; a phenoxazine dye; a porphyrin dye; polymethine dyes, BODIPY dyes, quinoline dyes, pyrene (pyrene), fluorescein chlorotriazinyl (fluoroscein chlorotriazinyl), R110, Eosin, JOE, R6G, tetramethylrhodamine, lissamine, ROX and naphthofluorescein.

The nucleic acid may be present in a specific addressable location on the solid support; each position corresponds to at least a portion of the incrna sequence of the biomarker.

In certain embodiments, the lncRNA assay comprises the steps of: 1) obtaining surface-bound probes for one or more biomarkers; 2) hybridizing a population of lncrnas to the surface-bound probes under conditions sufficient to provide specific binding; 3) removing unbound nucleic acids from the hybridization step; and 4) detecting the hybridized lncRNA.

Hybridization can be performed under suitable hybridization conditions, the stringency of which can be varied as desired. Typical conditions are sufficient to generate a probe/target complex between complementary binding members on the solid surface, i.e., between the surface-bound probe and the complementary lncRNA in the sample.

In certain embodiments, stringent hybridization conditions are used. The choice of appropriate conditions, including temperature, salt concentration, polynucleotide concentration, hybridization time, and stringency of washing conditions, will depend on the design of the experiment, including the source of the sample, the type of capture reagent, the degree of complementarity desired, and the like, and can be determined by one of ordinary skill in the art as a routine experimentation.

After the lncRNA hybridization procedure, the surface-bound polynucleotides are washed to remove unbound nucleic acids. The washing may be carried out using any convenient washing protocol. In certain embodiments, the washing conditions are stringent. Hybridization of the target lncRNA to the probe can then be detected using standard techniques.

Kit, chip and nucleic acid membrane strip

The invention provides a kit which can be used for detecting the expression of RP11-284F 21.10.

As a preferred embodiment, the kit comprises one or more probes or primers that specifically bind to incrna of a biomarker.

As a more preferred embodiment, the kit further comprises a wash solution.

As a more preferred embodiment, the kit further comprises reagents for performing a hybridization assay, means for lncRNA isolation or purification, means for detection, and positive and negative controls.

As a further preferred embodiment, the kit further comprises instructions for using the kit. The kit may be customized for home use, clinical use, or research use.

The kit comprises a specific primer pair for amplifying RP11-284F 21.10; a standard DNA template; and (3) PCR reaction liquid.

The 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 RP11-284F 21.10.

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 nylon membrane, glass or silicon slice modified by active group (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.

The RP11-284F21.10 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 device, arranged into a predetermined sequence or array, and then fixed by standing overnight, so as to obtain the lncRNA chip of the present invention.

In the invention, the nucleic acid membrane strip comprises a substrate and an oligonucleotide probe which is fixed on the substrate and specifically recognizes RP11-284F 21.10; the substrate may be any substrate suitable for immobilizing oligonucleotide probes, such as a nylon membrane, a nitrocellulose membrane, a polypropylene membrane, a glass plate, a silica gel wafer, a micro magnetic bead, or the like.

The gene detection kit or the gene chip or the nucleic acid membrane strip can be used for detecting the expression levels of a plurality of genes (for example, a plurality of genes related to lung adenocarcinoma) including RP11-284F21.10 genes, and can simultaneously detect a plurality of markers of the lung adenocarcinoma, thereby greatly improving the accuracy of diagnosis of the lung adenocarcinoma.

In the present invention, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways, as the skilled person will know. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.

Preferably, the mathematical algorithm applied in the marker combination is a logarithmic function. Preferably, the result of applying such a mathematical algorithm or such a logarithmic function is a single value. Such values can be readily correlated with, for example, an individual's risk for lung adenocarcinoma or with other diagnostic uses of interest that are helpful in assessing lung adenocarcinoma patients, in light of the underlying diagnostic question. In a preferred manner, such a logarithmic function is obtained as follows: a) classifying individuals into groups, e.g., normal humans, individuals at risk for lung adenocarcinoma, patients with lung adenocarcinoma, etc., b) identifying markers that differ significantly between these groups by univariate analysis, c) log regression analysis to assess independent difference values of the markers that can be used to assess these different groups, and d) constructing a log function to combine the independent difference values. In this type of analysis, the markers are no longer independent, but represent a combination of markers.

The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing lung adenocarcinoma is selected from DA (i.e. linear, quadratic, regular discriminant analysis), Kernel method (i.e. SVM), non-parametric method (i.e. k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e. logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e. logarithmic regression).

Inhibitors and drugs

Based on the findings of the inventors, the present invention provides an inhibitor of RP11-284F21.10, the nature of which is not important to the present invention as long as it inhibits the functional expression of RP11-284F21.10 gene, for example, the inhibitor of the present invention may be an interfering molecule which is targeted by RP11-284F21.10 gene and is capable of inhibiting RP11-284F21.10 gene, including: 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. These inhibitors are useful as agents for down-regulating RP11-284F21.10 and are useful for treating lung adenocarcinoma.

As a preferred mode of the invention, the inhibitor of RP11-284F21.10 is a small interfering RNA molecule specific to RP11-284F 21.10. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets IncRNAs of homologous complementary sequences to degrade a particular IncRNA, a process known as RNA interference (RNA interference). 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 invention designs and synthesizes a plurality of siRNA sequences, and the siRNA sequences are respectively verified by transfecting a lung adenocarcinoma cell line with a transfection reagent, and siRNA with the best interference effect is selected. One skilled in the art will recognize that the selection of the most effective siRNA for subsequent experiments does not mean that other sirnas do not perform similarly, and that siRNA interference experiments are performed to demonstrate that the expression level of RP11-284F21.10 is indeed correlated with the proliferation and invasion and migration of lung adenocarcinoma cells, and that representative sirnas are generally selected for experiments to reduce costs.

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.

In the present invention, "drug" and "pharmaceutical composition" may be used in general. In an alternative embodiment, the pharmaceutical composition comprises an inhibitor of the RP11-284F21.10 gene and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include (but are not limited to): diluents, excipients such as lactose, sodium chloride, glucose, urea, starch, water, etc., fillers such as starch, sucrose, etc.; binders such as simple syrup, glucose solution, starch solution, cellulose derivatives, alginates, gelatin, and polyvinylpyrrolidone; humectants such as glycerol; disintegrating agents such as dry starch, sodium alginate, laminarin powder, agar powder, calcium carbonate and sodium bicarbonate; absorption accelerators quaternary ammonium compounds, sodium lauryl sulfate, and the like; surfactants such as polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, glyceryl monostearate, cetyl alcohol, etc.; humectants such as glycerin, starch, etc.; adsorption carriers such as starch, lactose, bentonite, silica gel, kaolin, and bentonite, etc.; lubricants such as talc, calcium and magnesium stearate, polyethylene glycol, boric acid powder, and the like.

In the present invention, the pharmaceutical composition may be prepared using various additives, such as buffers, stabilizers, bacteriostats, isotonizing agents, chelating agents, pH controlling agents, and surfactants.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, by lung adenocarcinoma, nasally, buccally, vaginally or via an implanted reservoir device. Oral administration or injection administration is preferred. The pharmaceutical compositions of the present invention may contain any of the usual non-toxic pharmaceutically acceptable carriers, adjuvants or vehicles. In some cases, pharmaceutically acceptable acids, bases or buffers may be used to adjust the pH of the formulation to improve the stability of the formulated compound or its dosage form in which it is administered. The term parenteral as used herein includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intracolic, intralesional, and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention may be administered to a subject by any route as long as the target tissue is reached.

The pharmaceutical compositions of the present invention may be administered orally in any oral dosage form including, but not limited to, capsules, tablets, emulsions and aqueous suspensions, dispersions and solutions. For oral tablets, carriers that are commonly used include lactose and corn starch. Lubricating agents such as magnesium stearate are also typically added. For oral administration in capsule form, suitable diluents include lactose and anhydrous corn starch. When aqueous suspensions and/or emulsions are administered orally, the active ingredient may be suspended or dissolved in the oil phase and combined with emulsifying and/or suspending agents. If desired, sweetening and/or flavouring and/or colouring agents may be added. Dosage unit formulations for oral administration may be microencapsulated, as appropriate. The formulations may also be prepared to provide extended or sustained release, for example, by coating or embedding the particulate material in a polymer, wax, or the like. The pharmaceutical composition can be used for reducing the endogenous RP11-284F21.10 overexpression and reducing the expression of RP11-284F21.10, thereby treating lung adenocarcinoma caused by the up-regulation of the expression of RP11-284F 21.10.

In the present invention, a compound that inhibits expression of RP11-284F21.10 can be administered to a subject as naked RNA along with a delivery agent as a nucleic acid (e.g., a recombinant plasmid or viral vector) comprising a sequence that inhibits expression of RP11-284F 21.10. The delivery agent may be a lipophilic agent, a polycation, a liposome, or the like.

In the present invention, the term "effective amount" means an amount sufficient to treat the disease at a reasonable benefit/risk ratio applicable to any medical treatment. The effective dosage level of the composition may be determined according to the type of the subject, the severity of the disease, the age and sex of the subject, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration, the excretion rate, the treatment time, the drug to be used in combination with the composition, and other known factors in the medical field. The pharmaceutical compositions of the present invention may be used alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. The compositions may be administered in one or more dosage forms. In view of all the above factors, it is important to administer the composition at the minimum amount capable of exhibiting the maximum effect without causing side effects, which can be readily determined by one skilled in the art.

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.

Statistical analysis

In the specific embodiment of the present invention, the experiments were performed by repeating at least 3 times, the data of the results are expressed as mean ± standard deviation, and the statistical analysis is performed by using SPSS18.0 statistical software, and the difference between the two is considered to have statistical significance by using t test when P is less than 0.05.

The present invention is further illustrated below with reference to specific examples, which are provided only for the purpose of illustration and are not meant to limit the scope of the present invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 screening of Gene markers associated with Lung adenocarcinoma

1. Sample collection

35 lung adenocarcinoma tissues and corresponding paraneoplastic tissues were collected, and all patients received no other treatment prior to surgery, from which 4 lung adenocarcinoma tissues and corresponding paraneoplastic tissues were selected for high throughput sequencing.

2. Preparation and quantitative analysis of RNA samples

Tissue RNA extraction was performed using a tissue RNA extraction kit from QIAGEN, and the specific procedures were performed according to the instructions.

The RNA extracted above was subjected to agarose gel electrophoresis, the concentration and purity of the extracted RNA were determined using Nanodrop2000, RNA integrity was determined by agarose gel electrophoresis, and RIN value was determined by Agilent 2100. The total amount of RNA required for single library construction is 5 mug, the concentration is more than or equal to 200 ng/mug, and the OD260/280 is between 1.8 and 2.2.

3. Construction of cDNA library and sequencing

The construction and sequencing of the cDNA library are completed by the Huada gene, and the steps are as follows:

1) removal of rRNA

Removing ribosomal RNA from the total RNA using a Ribo-Zero kit;

2) fragmented RNA

For the complete RNA sequence, metal ions are utilized to randomly break the RNA into small fragments of about 200 bp.

3) Reverse Synthesis of cDNA

Constructing cDNA library by using Truseq RNA sample Prep Kit of Illumina, reversely synthesizing single-strand cDNA by using lncRNA as a template and random primer under the action of reverse transcriptase, and replacing dTTP with dUTP in dNTPs reagent when performing double-strand synthesis to make the base in the second strand of cDNA contain A/U/C/G.

4) Connection adapter

The sticky End of the double stranded cDNA is made blunt by adding End Repair Mix, followed by an A base at the 3' End for ligation to the Y-shaped adaptor.

5) UNG enzyme digestion of cDNA double strand

The second strand of the cDNA was digested with UNG enzyme, so that only the first strand of the cDNA was contained in the library.

6) 2X 150bp sequencing was performed using the Illumina X-Ten sequencing platform.

4. High throughput transcriptome sequencing data analysis

After deleting the non-detectable lncRNA (i.e. the number of samples with the read count value of 0 in case is more than 20% of the total case sample amount or the number of samples with the read count value of 0 in normal is more than 20% of the total normal sample amount), performing differential expression analysis by using DESeq2 of R-3.3.3 tool, differential expression lncRNA screening standard: FDR<0.05，abs(log₂FC)>2。

5. Results

The results show that the expression level of RP11-284F21.10 is significantly up-regulated in lung adenocarcinoma tissue compared to paracarcinoma tissue.

Example 2 QPCR sequencing verification of differential expression of the RP11-284F21.10 Gene

1. The 35 samples collected were verified for differential expression of the RP11-284F21.10 gene.

2. RNA extraction

RNA samples were extracted using QIAGEN's tissue RNA extraction kit, and the specific procedures are described in the specification.

3、QPCR

1) Reverse transcription reaction

Using FastQ μ ant cDNA first strand synthesis kit (cat # KR106) from Tiangen corporation to perform lncRNA reverse transcription, genomic DNA reaction was removed first, 5 XgDNA B μ ffer 2.0 μ l, total RNA1 μ g, RNase Free ddH were added to a test tube₂O to make the total volume to 10 μ l, heating in water bath at 42 deg.C for 3min.

10 XFast RT B. mu.ffer 2.0. mu.l, RT Enzyme Mix 1.0. mu.l, FQ-RT Primer Mix 2.0. mu.l, RNase Free ddH₂O5.0 μ l, mixing, adding into the above test tube, mixing to give 20 μ l, heating in water bath at 42 deg.C for 15min, and heating at 95 deg.C for 3min.

2) Primer design

QPCR amplification primers were designed based on the coding sequences of RP11-284F21.10 gene and GAPDH gene from Genebank and were synthesized by Bomeide Bio Inc. The specific primer sequences are as follows:

RP11-284F21.10 gene:

the forward primer is 5'-TTAGCCATAGCAACATAG-3' (SEQ ID NO. 1);

the reverse primer was 5'-CAGATGATGACAGTAAGAT-3' (SEQ ID NO. 2).

GAPDH gene:

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

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

3) QPCR amplification assay

Amplification was carried out using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions.

A20. mu.l reaction was used: 2 XSuperReal PreMix Plus 10. mu.l, forward and reverse primers (10. mu.M) 0.6. mu.l each, 5 XROX Reference Dye^△2. mu.l, DNA template 2. mu.l, sterilized distilled water 4.8. mu.l. Each sample was provided with 3 parallel channels and all amplification reactions were repeated three more times to ensure the reliability of the results.

The amplification procedure was: 95 ℃ 15min, (95 ℃ 10s, 55 ℃ 30s, 72 ℃ 32s) x 40 cycles, 95 ℃ 15s, 60 ℃ 60s, 95 ℃ 15 s).

4) Screening for cDNA template concentration

Mixing cDNA of each sample, diluting the cDNA by 10 times gradient (10 times, 100 times, 1000 times, 10000 times and 100000 times) by taking the cDNA as a template, taking 2 mu l of each diluted sample as the template, respectively amplifying by using a target gene primer and an internal reference gene primer, simultaneously carrying out melting curve analysis at 60-95 ℃, and screening the concentration of the template according to the principle of high amplification efficiency and single peak of the melting curve.

From the dissolution curve, it can be seen that when 10-fold dilution of cDNA was performed, the amplification efficiency of PCR was high and the single peak of the dissolution curve was good.

5) Sample RealTime PCR detection

After 10-fold dilution of each sample cDNA, 2. mu.l of each sample cDNA was used as a template, and the target gene primer and the reference gene primer were used for amplification. Simultaneously performing dissolution curve analysis at 60-95 deg.C, and determining target band by dissolution curve analysis and electrophoresis, 2^-ΔΔCTThe method is used for relative quantification.

4. Results

The QPCR result is shown in figure 1, compared with the tissue beside the cancer, the RP11-284F21.10 is up-regulated in the lung adenocarcinoma tissue, the difference has statistical significance (P <0.05), and the RP11-284F21.10 is suggested to have higher application value in the diagnosis of the lung adenocarcinoma.

Example 3 silencing of the RP11-284F21.10 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%. Changing the solution for 1 time in 2-3 days, and using 0.25% EPassage by trypsin digestion of DTA.

2. Design of siRNA

siRNA is designed aiming at the sequence of RP11-284F21.10 gene, and the designed siRNA sequence is shown as follows:

sequence of negative control siRNA-NC:

sense strand: 5'-UUCUCCGAACGUGUCACGU-3' (SEQ ID NO.5),

antisense strand: 5'-ACGUGACACGUUCGGAGAA-3' (SEQ ID NO. 6);

siRNA1：

sense strand: 5'-AUUGUGUAGGCUCAUGUUGCU-3' (SEQ ID NO.7),

antisense strand: 5'-CAACAUGAGCCUACACAAUAU-3' (SEQ ID NO. 8);

siRNA2：

sense strand: 5'-AUUGCAAUUACUUCUACAGGC-3' (SEQ ID NO.9),

antisense strand: 5'-CUGUAGAAGUAAUUGCAAUCA-3' (SEQ ID NO. 10);

siRNA3：

the sense strand is 5'-AUUUCAGAUUCUAUACGAGAG-3' (SEQ ID NO.11),

the antisense strand is 5'-CUCGUAUAGAAUCUGAAAUGU-3' (SEQ ID NO.12)

3. Transfection

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%.

The experiment was divided into three groups: a control group (A549), a negative control group (siRNA-NC) and an experimental group (siRNA1-3), wherein the negative control group siRNA-NC has no homology with the sequence of the RP11-284F21.10 gene and has the concentration of 20 nM/hole, and is transfected respectively. Transfection was performed using the Lipofectamine3000 kit from Invitrogen, and the specific transfection method was performed according to the instructions.

4. QPCR detection of transcript level of RP11-284F21.10 gene

1) Extraction of Total RNA from cells

Total RNA in cells was extracted using QIAGEN cell RNA extraction kit, the detailed steps are described in the specification.

2) The reverse transcription procedure was as in example 2.

3) The QPCR amplification procedure was as in example 2.

5. Results

The results are shown in fig. 2, compared with the control group A549 and the siRNA-NC group, the experimental group (siRNA1-3) can reduce the level of RP11-284F21.10, wherein the effect of siRNA1 is most significant, so that siRNA1 is selected for subsequent experiments.

Example 4 Effect of RP11-284F21.10 on Lung adenocarcinoma cell proliferation

1. Lung adenocarcinoma cells A549 were inoculated in 6-well plates and cultured, and when the cell density reached 85% -90%, siRNA1 was transfected by using liposome 3000. Replacing the new culture medium after the culture in the serum-free culture medium for 4-6 h.

2. After siRNA1 transfection, the cells of the interference group and the cells of the control group are digested for 24h, and the transfected A549 cell suspension and 100 μ l (1X 10) of each control group are inoculated in a 96-well plate⁴One/well), detection was performed 12h, 24h, 48h, 72h after transfection.

3. To each well was added 10. mu.l of CCK8 solution.

4. The culture plate is placed in an incubator to be cultured for 1-4 h.

5. The absorbance at 450nm was measured using a microplate reader, and a cell growth activity curve was plotted with the absorbance value as the vertical axis and time as the horizontal axis.

6. Results

The results are shown in fig. 3, compared with the control group, the cell activity of the siRNA1 transfected group is obviously reduced along with the increase of the growth time, the difference has significant statistical significance (P <0.05), and the RP11-284F21.10 is related to the proliferation of lung adenocarcinoma cells, and can be used as a potential target for being applied to the treatment of 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

<110> Beijing, the deep biometric information technology GmbH

<120> lncRNA marker for diagnosis and treatment of lung cancer

<160> 12

<170> SIPOSequenceListing 1.0

<210> 1

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttagccatag caacatag 18

<210> 2

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

cagatgatga cagtaagat 19

<210> 3

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aatcccatca ccatcttcca g 21

<210> 4

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gagccccagc cttctccat 19

<210> 5

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

uucuccgaac gugucacgu 19

<210> 6

<211> 19

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

acgugacacg uucggagaa 19

<210> 7

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

auuguguagg cucauguugc u 21

<210> 8

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

caacaugagc cuacacaaua u 21

<210> 9

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

auugcaauua cuucuacagg c 21

<210> 10

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cuguagaagu aauugcaauc a 21

<210> 11

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

auuucagauu cuauacgaga g 21

<210> 12

<211> 21

<212> RNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cucguauaga aucugaaaug u 21

Claims

1. A method of screening for a drug candidate that inhibits lung adenocarcinoma cell proliferation, said method comprising:

detecting the expression level of the RP11-284F21.10 gene in the system;

wherein, if the substance to be screened can reduce the expression level of the RP11-284F21.10 gene, the substance to be screened is a candidate drug for inhibiting the proliferation of lung adenocarcinoma cells.

2. Any one of the following applications:

a. the application of the reagent for detecting the expression level of RP11-284F21.10 in preparing a tool for diagnosing lung adenocarcinoma;

b. use of a product comprising an agent for detecting the expression level of RP11-284F21.10 in the manufacture of a means for diagnosing lung adenocarcinoma;

c. application of RP11-284F21.10 in constructing a calculation model for diagnosing lung adenocarcinoma;

d. use of an inhibitor of RP11-284F21.10 in the manufacture of a medicament for inhibiting lung adenocarcinoma cell proliferation, wherein the inhibitor inhibits the expression level of RP11-284F 21.10;

e. use of a composition comprising an inhibitor of RP11-284F21.10 in the manufacture of a medicament for inhibiting lung adenocarcinoma cell proliferation, wherein the inhibitor inhibits the expression level of RP11-284F 21.10;

f. application of RP11-284F21.10 in screening candidate drugs for inhibiting lung adenocarcinoma cell proliferation.

3. The use according to claim 2, wherein the agent of a or b is selected from the group consisting of:

a probe that specifically recognizes RP11-284F 21.10; or

Primers for specifically amplifying RP11-284F 21.10.

4. The use according to claim 3, wherein the primer sequence for specific amplification of RP11-284F21.10 is shown in SEQ ID No. 1-2.

5. The use of claim 2, wherein the product of b comprises a kit, a chip, a nucleic acid membrane strip.

6. The use of claim 2, wherein the inhibitor of d or e is an siRNA.

7. The use of claim 6, wherein the siRNA has the sequence shown in SEQ ID No. 7-12.