CN110923327A

CN110923327A - Application of differentially expressed lncRNA in diagnosis and treatment of lung cancer

Info

Publication number: CN110923327A
Application number: CN201911422742.2A
Authority: CN
Inventors: 杨承刚; 高舒欣; 魏琳
Original assignee: Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-03-27

Abstract

The invention discloses application of lncRNA (long non-coding ribonucleic acid) with differential expression in diagnosis and treatment of lung cancer. The invention discloses application of a reagent for detecting AC105046.1 in preparing a product for diagnosing lung cancer and a corresponding product for diagnosing lung cancer based on the expression down-regulation of AC105046.1 in lung cancer. The invention discloses application of an AC105046.1 promoter in preparing a pharmaceutical composition for treating lung cancer and a corresponding pharmaceutical composition based on that the proliferation and migration of cells can be influenced by regulating the expression level of AC 105046.1.

Description

Application of differentially expressed lncRNA in diagnosis and treatment of lung cancer

Technical Field

The invention belongs to the field of biomedicine, and relates to application of lncRNA (long non-coding ribonucleic acid) with differential expression in diagnosis and treatment of lung cancer.

Background

According to the data published in 2018 of the global cancer statistics report (SIEGEL R L, MILLER K D, JEMALA. cancer statistics, 2018[ J ]. CA: a cancer juoural for clinicians, 2018, 68(1):7-30.), new lung cancer cases rank second in tumors and death cases rank first. Currently, lung cancer still lacks effective and accurate early-screening biomarkers and preventive targets, has a low early diagnosis rate and a high late-stage recurrence and metastasis rate, and lacks sensitive and stable clinical drugs, whereby the overall survival rate of patients is low (CHEN W J, GAN T Q, QIN H, ethyl. immunity and reactive pathway signaling of microRNA-375in long squamous cell cancer [ J ]. Pathology, research and diagnosis, 2017, 213(4): 364-72.). The majority of lung cancers are non-small cell lung cancers (NSCLC), which comprise two major histological subtypes, squamous cell lung carcinoma (LUSC) and adenocarcinoma of the Lung (LUAD) (TORRE L A, SIEGEL R L, JEMALA. Lung Cancer Statistics [ J ]. Advances in experimental medicine and biology, 2016, 893: 1-19.). Squamous cell carcinoma of the lung, also known as squamous cell carcinoma of the lung, occurs well in men and is closely associated with smoking, accounting for about 30% of non-small cell lung cancers (BECK J T, ISMAIL A, TOLOMEO C. targeting the refractory resin 3-kinase (PI 3K)/AKT/refractory target of rapamycin (mTOR) pathway: an empirical treatment for squaring cell lung Cancer [ J ]. Cancer treatment reviews 2014, 40(8): 980-9.). Many studies have now found novel biomarkers for lung adenocarcinoma to improve the prognosis, diagnosis and prognosis (PU W, GENG X, CHEN S, et al. Abserrant methylation of CDH13 can a diagnostic biomarker for lung adenocarcinoma [ J ]. Journal of Cancer 2016, 7(15):2280-9.), such as the presence of Epidermal Growth Factor Receptors (EGFRs), Anaplastic Lymphoma Kinase (ALK) and Vascular Endothelial Growth Factor (VEGF) inhibitors (PIPERDI B, MERLA A, PEREZ-SOLER. Targeting angiogenesis in squamous non-spherical cell Cancer [ J ]. Drugs, 2014, 74(4):403-13 ], increased survival rate in patients with lung adenocarcinoma, but relatively lacking the lung adenocarcinoma biomarker (YAG J, nutritional biomarkers J.: said anode J.: Cancer J.), 2017, 5: e 4101.). Therefore, it is important to find a lung squamous carcinoma biomarker which can be used for early diagnosis and prognosis improvement.

Tumor development is a multifactorial, long-term, and multistage process, and like other tumor characteristics, the development of squamous cell lung carcinoma is regulated by multifactorial factors, such as apoptosis-related gene expression regulation, posttranslational protein modification, molecular interaction, signal pathway and feedback, DNA damage repair and damaged cell clearance, oncogene activation and oncogene suppressor silencing. In recent years, the role of epigenetic molecular regulation in the development of squamous cell lung carcinoma has been more and more emphasized, and epigenetics is the study of gene expression changes based on non-gene sequence changes. It was found that there are alterations in epigenetic key genes/proteins during development of squamous cell lung carcinoma, which in turn affect the relevant pathways and ultimately affect the proliferation, apoptosis and cycle of cells to varying degrees (WANG Y, ZHOU J, XU Y J, et al Long non-coding RNA LINC00968acts as an oncogene in NSCLC by activating the Wnt signaling pathway [ J ]. journal of cellular physiology, 2018, 233(4): 3397-. The research in the field of epigenetic regulation includes DNA methylation, micrornas (mirnas), long non-coding RNAs (incrnas), circular RNAs (circrnas), and the like.

Based on the knowledge, the research progress and early stage research foundation of the latest lung squamous cell carcinoma marker at home and abroad are integrated, the lncRNA is used as an entry point to explore lncRNA related to diagnosis and treatment of lung squamous cell carcinoma, and the possible effect of lncRNA in pathogenesis of lung squamous cell carcinoma is explored.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide application of the lncRNA with differential expression in lung cancer diagnosis and treatment.

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

the invention provides application of a reagent for detecting AC105046.1 in preparation of a product for diagnosing lung cancer.

Further, determining expression levels of AC105046.1 in a sample obtained from the patient, wherein decreased expression levels of AC105046.1 in the sample as compared to a reference expression level identifies the patient as a patient for lung cancer.

Further, the expression level of AC105046.1 was determined by RNA sequencing, PCR, qPCR, reverse transcription PCR, gene hybridization, gene expression profiling, gene approximation to series analysis, or microarray analysis.

Further, the sample comprises tissue, sputum, blood, pleural effusion or urine,

further, the sample is a tissue.

The invention provides a product for diagnosing lung cancer, which comprises a reagent for detecting AC 105046.1.

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

Further, the reagent comprises a probe or primer for the transcript of the AC105046.1 gene,

furthermore, the sequence of the primer is shown as SEQ ID NO. 1-2.

The invention provides application of AC105046.1 in constructing a calculation model for predicting lung cancer.

The invention provides application of AC105046.1 in preparing a pharmaceutical composition for treating lung cancer and/or lung cancer metastasis.

Further, the pharmaceutical composition comprises an accelerant of AC105046.1,

further, the promoter is an agent that promotes the expression level of AC 105046.1.

Further, the promoter is a vector overexpressing AC 105046.1.

The invention provides a pharmaceutical composition for treating lung cancer, which comprises an agent for promoting the expression of AC 105046.1.

Further, the agent is a vector overexpressing AC 105046.1.

The invention has the advantages and beneficial effects that:

the invention discovers that the expression level of AC105046.1 in a lung cancer patient is obviously reduced, the expression level of AC105046.1 can be detected to judge whether a subject suffers from lung cancer, and ROC curve analysis shows that the AUC value is 0.956, so that the kit has high specificity and sensitivity and high diagnosis efficiency.

Given the significant down-regulation of AC105046.1 expression levels in lung cancer patients, it was found that the proliferative activity as well as the migratory capacity of cancer cells can be altered by over-expressing AC105046.1 into the cells, thereby providing a new target for the treatment of lung cancer.

Drawings

FIG. 1 is a graph showing the detection of the expression of AC105046.1 gene in lung cancer tissues by QPCR;

FIG. 2 is a graph showing the detection of AC105046.1 expression after transfection.

Detailed Description

Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention, the preferred methods, devices, and materials are now described. However, before the present materials and methods are described, it is to be understood that this invention is not limited to the particular sizes, shapes, dimensions, materials, methods, protocols, etc. described herein as these may vary according to routine experimentation and optimization. It is also to be understood that the terminology used in the description is for the purpose of describing the particular versions or embodiments only, and is not intended to limit the scope of the present invention.

The term "sample" refers to a subset of an intact organism or a tissue, cell, or component thereof (e.g., a bodily fluid, including but not limited to blood, mucus, lymph, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, umbilical cord blood, urine, vaginal fluid, and semen). "sample" further refers to a homogenate, lysate, extract, cell culture or tissue culture, or portion thereof prepared from the whole organism or a subset of its cells, tissues or components. Finally, "sample" refers to a medium containing cellular components (such as proteins or polynucleotides), such as a nutrient broth or gel in which an organism has been propagated.

By "tissue sample" or "cell sample" is meant a collection of similar cells obtained from a tissue of a subject or individual. The source of the tissue or cell sample may be a solid tissue, such as from a fresh, frozen and/or preserved organ, a tissue sample, a biopsy, and/or an aspirate; blood or any blood component, such as plasma; body fluids such as cerebrospinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from a subject at any time during pregnancy or development. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a diseased tissue/organ. For example, a "tumor sample" is a tissue sample obtained from a tumor or other cancerous tissue. The tissue sample may contain a mixed population of multiple cell types (e.g., tumor cells and non-tumor cells, cancerous cells and non-cancerous cells). Tissue samples may contain compounds that are not naturally intermixed with the tissue in nature, such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, and the like.

As used herein, the term "AC 105046.1" refers to any native AC105046.1 from any vertebrate source, including mammals, such as primates (e.g., humans) and rodents (e.g., mice and rats), unless otherwise indicated. The term encompasses "full length," unprocessed AC105046.1 as well as any form of AC105046.1 that results from processing in the cell. The term also encompasses naturally occurring variants of AC105046.1, such as splice variants or allelic variants. An exemplary nucleic acid sequence of human AC105046.1 is shown in ENST 00000566457.1.

As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length and includes DNA and RNA. The nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or their analogs, or any substrate that can be incorporated into the polymer by DNA or RNA polymerase, or by synthetic reaction. Thus, for example, polynucleotides as defined herein include, but are not limited to, single and double stranded DNA, DNA comprising single and double stranded regions, single and double stranded RNA, and RNA comprising single and double stranded regions, hybrid molecules comprising DNA and RNA, which may be single stranded, or more typically double stranded, or comprise single and double stranded regions. In addition, the term "polynucleotide" as used herein refers to a triple-stranded region comprising RNA or DNA or both RNA and DNA. The chains in such regions may be from the same molecule or from different molecules. The region may comprise the entire molecule or molecules, but more typically is a region involving only some molecules. One of the molecules of the triple-helical region is often an oligonucleotide. The term "polynucleotide" specifically includes cDNA.

As used herein, "oligonucleotide" generally refers to a short, single-stranded polynucleotide that is less than about 250 nucleotides in length, although this is not required. The oligonucleotide may be synthetic. The terms "oligonucleotide" and "polynucleotide" are not mutually exclusive. The above description for polynucleotides is equally and fully applicable to oligonucleotides.

The term "primer" refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing polymerization of the complementary nucleic acid, typically by providing a free 3' -OH group.

As used herein, a biomarker or "amount" or "level" of a marker is a detectable level in a biological sample. These can be measured by methods known to those skilled in the art and also disclosed herein.

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 (e.g., gene-encoded and/or epigenetic information) is converted into structures present and operating 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 polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they are derived from transcripts generated or degraded by alternative splicing, or from post-translational processing of polypeptides, e.g., by proteolysis. "expressed genes" include those that are transcribed into a polynucleotide that is an mRNA and then translated into a polypeptide, as well as those that are transcribed into RNA that is not translated into a polypeptide (e.g., transport and ribosomal RNA, lncRNA).

"elevated expression," "elevated expression level," "elevated expression level," "elevated level," or "up-regulation" refers to elevated expression or elevated level of a biomarker in an individual relative to a control, such as the median expression level of the biomarker in one or more individuals without a disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or a sample from a group/population of patients.

"reduced expression", "reduced expression level", "reduced expression level", "reduced level" or "down-regulation" refers to reduced expression or reduced level of a biomarker in an individual relative to a control, such as the median expression level of the biomarker in one or more individuals without a disease or disorder (e.g., cancer), an internal control (e.g., a housekeeping biomarker), or a sample from a group/population of patients. In some embodiments, the reduced expression is little or no expression.

The term "housekeeping gene" refers herein to a gene or set of genes that encode a protein whose activity is essential for the maintenance of cellular function and which typically occurs similarly in all cell types.

According to the present invention, the expression level of AC105046.1 in cancer cells or tissues obtained from a subject is determined and the expression level can be determined at the level of the transcription (nucleic acid) product using methods known in the art. For example, the RNA of AC105046.1 can be quantified using probes by hybridization methods (e.g., Northern hybridization). The detection may be performed on a chip or array. For detecting the expression level of AC105046.1, the use of an array is preferred. The sequence information of AC105046.1 can be used by those skilled in the art to prepare the above probes. For example, cDNA of AC105046.1 can be used as a probe. If desired, the probe may be labeled with a suitable label such as a dye, a fluorescent substance and an isotope, and the expression level of the gene may be detected as the intensity of the label to which hybridization has occurred.

The probes or primers used in the method hybridize to RNA of AC105046.1 under stringent, medium or low stringency conditions. As used herein, the phrase "stringent (hybridization) conditions" refers to conditions under which a probe or primer will hybridize to its target sequence, but not to other sequences. Stringent conditions are sequence dependent and will be different in different circumstances. Specific hybridization of longer sequences is observed at higher temperatures than shorter sequences. Generally, the temperature of stringent conditions is selected to be about 5 ℃ lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of probes complementary to their target sequence hybridize to the target sequence at equilibrium. Since the target sequence is generally present in excess, at Tm, 50% of the probes are occupied at equilibrium. Typically, stringent conditions will be such that: wherein the salt concentration is less than about 1.0M sodium ion, typically about 0.01-1.0M sodium ion (or other salt), pH7.0-8.3, and the temperature is at least about 30 ℃ for shorter probes or primers (e.g., 10-50 nucleotides) and at least about 60 ℃ for longer probes or primers. Stringent conditions may also be achieved with the addition of destabilizing agents, such as formamide.

A pharmaceutical composition as used herein, comprising an effective amount of said promoter of AC105046.1, and/or a pharmaceutically acceptable carrier, said medicament being useful for the treatment of lung cancer.

As a preferred mode of the invention, the promoter of AC105046.1 is an expression vector of AC 105046.1. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

The medicament of the present invention may be in a form suitable for administration by injection, in a form suitable for oral ingestion (e.g., capsules, tablets, caplets, elixirs), in the form of an ointment, cream or lotion suitable for topical administration, in a delivery form suitable for use as eye drops, in an aerosol form suitable for administration by inhalation (e.g., by intranasal or oral inhalation), in a form suitable for parenteral administration, i.e., subcutaneous, intramuscular or intravenous injection.

The medicament of the invention can also be used in combination with other medicaments for treating lung cancer, and other therapeutic medicaments can be simultaneously administered with the main active ingredients, even in the same composition. Other therapeutic compounds may also be administered alone in a composition or dosage form different from the main active ingredient.

Preferably, it can be carried out by means of gene therapy. For example, the AC105046.1 promoter may be administered directly to the subject by a method such as injection; alternatively, the AC 105046.1-carrying regulatory-promoting expression unit (e.g., expression vector or virus) may be delivered to the target site by any route, depending on the type of promoter, as is well known to those skilled in the art.

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 QPCR detection of AC105046.1 expression in Lung cancer

1. Collecting a sample

Samples of squamous cell lung carcinoma tissue and its corresponding paraneoplastic tissue were collected 46.

Inclusion criteria were:

1) pathologically diagnosed as squamous cell lung carcinoma, 2) the RNA expression amount and clinical pathological information of the sample are complete.

Exclusion criteria:

1) the patients with other malignant tumors except for lung squamous carcinoma, 2) the cases are treated by chemoradiotherapy and targeted drugs before specimen collection.

2. Preparation and quantitative analysis of RNA samples

Extracting RNA in sample tissues by using Trizol reagent, and the steps are as follows:

adding 1mL of Trizol into a glass homogenizing bottle in a super clean bench, weighing 50-100mg of tissues into the glass homogenizing bottle, adjusting the rotation speed to about 1500 turns, starting homogenizing in an ice-water bath, stopping 30s every 30s of grinding, repeating for 3-4 times until the sample volume does not exceed 10% of the Trizol volume, placing the sample added with Trizol at room temperature for 10min to completely separate nucleic acid-protein complexes, adding chloroform (Trizol: 5:1), shaking vigorously for 2min, shaking for two times every 1 min, standing for 5-6 times, standing for 7min, 4 ℃, 12000rpm, centrifuging for 15min, transferring the upper aqueous phase into a new EP tube (about 400 μ L, and not sucking the middle layer as much as possible to avoid pollution), adding 500 μ L of isopropanol, placing at room temperature for 10min, 4 ℃, 12000rpm, centrifuging for 15min, centrifuging to generate white precipitate at the bottom of the tube after centrifugation, the supernatant was carefully removed by pipette, 1mL of 75% precooled ethanol was added, and the pellet was washed with shaking. Centrifuging at 7500rpm for 5min at 4 deg.C, and carefully discarding the supernatant; turning over the EP tube on a filter paper to absorb excessive water, carefully sucking the liquid in the tube by using a 10-microliter gun head (the gun head does not contact RNA), and placing the EP tube at room temperature for 5 min; adding 50 μ L RNase-free water (DEPC water), detecting OD value and concentration with naodrop, and marking on the tube; storing in a refrigerator at-80 deg.C.

3、QPCR

1) Reverse transcription reaction

The reverse transcription of lncRNA was carried out using the FastQ μ ant cDNA first strand synthesis kit (cat # KR106) from Tiangen as follows:

first, remove the genomic DNA reaction, add 5 XgDNA B. mu.ffer 2.0. mu.l, total RNA 1. mu.g, RNase Free ddH to the tube₂O to make the total volume 10. mu.l, heating in a water bath at 42 ℃ for 3min, and adding 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 3 min.

2) Primer design

QPCR amplification primers are designed according to coding sequences of an AC105046.1 gene and a GAPDH gene in Genebank, wherein the sequence of a primer for amplifying the AC105046.1 gene is shown as SEQ ID NO. 1-2 (F: 5'-CCTCCTACATCTGAATCT-3'; R: 5'-AGCAATGATAAGACAAGAG-3'), and the sequence of a primer pair for amplifying an internal reference gene is shown as SEQ ID NO. 3-4 (F: 5'-AATCCCATCACCATCTTCCAG-3', R: 5'-GAGCCCCAGCCTTCTCCAT-3').

3) QPCR amplification assay

Using ABI 7300 type fluorescent quantitative PCR instrument, adopting 2^-△△CTThe method performs a relatively quantitative analysis of the data.

Amplification was performed using SuperReal PreMix Plus (SYBR Green) (cat # FP205) and the experimental procedures were performed according to the product instructions. The Real time reaction system is as follows: 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 ℃ for 15min, (95 ℃ for 10s, 55 ℃ for 30s, 72 ℃ for 35s) x 40 cycles, and a melting point curve is drawn at 60-95 ℃.

4. Statistical method

The experiment was repeated 3 times, the data were expressed as mean ± sd, and the statistical analysis was performed using SPSS18.0 statistical software, and the difference between the two was considered statistically significant when P <0.05 using the t-test. ROC curve analysis was performed on variable AC105046.1 to determine the diagnostic potency, sensitivity and specificity of the gene.

5. Results

The QPCR results are shown in fig. 1, compared to the para-cancer tissues, AC105046.1 was down-regulated in lung cancer tissues by about 44.3-fold, the differences were statistically significant (P <0.05), suggesting that AC105046.1 has a higher application value in the diagnosis of lung cancer.

ROC curve analysis indicates that AC105046.1 can be used as a biomarker for diagnosis of squamous cell lung carcinoma. The area under the curve is 0.956, and patients with lung squamous carcinoma can be effectively distinguished.

Example 2 overexpression of the AC105046.1 Gene

1. Cell culture

The lung squamous carcinoma cell line H2170 cell was cultured in DMEM medium supplemented with 10% fetal bovine serum and P/S. Cells were grown adherent to the wall and placed under conditions of 5% CO₂And culturing in a constant-temperature incubator with the humidity of 37 ℃.

2. Construction of Gene overexpression vectors

Specific PCR amplification primers were synthesized based on the sequence of AC 105046.1. The cDNA sequence is double digested with restriction endonucleases KpnI and XhoI and inserted into eukaryotic cell expression vector pcDNA3.1(+) double digested with KpnI and XhoI, and the obtained recombinant vector pcDNA3.1(+) -1 is connected for subsequent experiment.

3. Transfection

The day before transfection, cells in logarithmic growth phase were diluted at a density of 2X 10⁵Each/ml, the diluted cells were inoculated in a 24-well plate at 0.5ml per well, and the cells were divided into a blank control group, a negative control group (transfection-empty group), and an experimental group (transfection-pcDNA3.1 (+) -1), and after labeling groups, the cells were left at 37 ℃ with 5% CO₂The cells were cultured overnight in the incubator of (1), the growth state of the cells was observed the next day, and when the degree of fusion of the cells reached 70% to 80%, transfection of the vector was carried out using liposome 2000. Diluting pcDNA (3.1+) -1 and Lipo2000 with opti-MEM, mixing, blowing diluted pcDNA (3.1+) -1 and Lipo2000, mixing, incubating at room temperature for 20min to form complex, adding the complex dropwise into 24-well plate with fresh culture medium, mixing at 37 deg.C with 5% CO₂After culturing for 6h in the culture phase, the culture is continued by replacing the culture phase with a conventional medium containing serum and antibiotics.

4. QPCR detection of transcript level of AC105046.1 Gene

Extraction of total RNA of cells:

removing the culture medium in a 24-well plate, adding PBS to wash for 3 times, removing the PBS, adding 1mL Trizol reagent, mixing uniformly, repeatedly blowing and beating the vortex cells by using a pipette gun, transferring the obtained cell suspension into a 1.5mL centrifuge tube, standing for 5min at room temperature, adding chloroform, shaking up to milk white by intense oscillation, then standing for 5min at room temperature, centrifuging for 15min at 12000g at 4 ℃, taking the supernatant into a new 1.5mL centrifuge tube, adding 0.5-1.0mL of isopropanol precooled in advance into the centrifuge tube, slowly shaking up, and standing for 10min at room temperature. 12000g, centrifuging at 4 ℃ for 10min, discarding the supernatant, adding precooled 75% ethanol 1 mL/tube into the centrifuge tube, 7500g, centrifuging at 4 ℃ for 5min, discarding the supernatant, drying, and adding 20 mu L of enzyme-free water to the precipitate.

The reverse transcription and QPCR detection steps were the same as in example 2.

5. Results

The results are shown in fig. 2, where the experimental group significantly promoted the level of AC105046.1 (P <0.05) compared to the blank control group and the negative control group, while there was no significant difference between the blank control group and the transfection-unloaded group.

Example 3 Effect of AC105046.1 on Lung cancer cells

1) The MTT method detects the influence of AC105046.1 on the proliferation of squamous cell carcinoma of lung:

washing transfected cell strain with PBS, removing dead cell, digesting with pancreatin to prepare single cell suspension, diluting the cell suspension, and adjusting density to 5 × 10⁴One cell per ml, inoculating into 96-well culture plate, adding 100 μ l cell suspension per well, placing 96-well culture plate at 37 deg.C and 5% CO₂The cell culture box is continuously cultured for 48 hours, 1 XMTT is added into each hole, the culture is continuously carried out for 4 hours, supernatant is sucked and removed, DMSO is added into each inner hole, the shaking is carried out for 10min, and the OD value of each hole is measured by a microplate reader when the wavelength is 450 nm.

The MTT results show that the cell growth and proliferation rate (0.376 +/-0.043) of the experimental group over-expressing AC105046.1 is remarkably lower than that of the blank control group (0.598 +/-0.022) and the negative control group (0.576 +/-0.032) with no transfection load (P <0.05), and the difference between the blank control group and the negative control group with no transfection load is not remarkable, which indicates that the expression level of the over-expressing AC105046.1 can remarkably inhibit the proliferation of the lung squamous carcinoma cells.

2) Scratch test the effect of AC105046.1 on the migratory capacity of lung squamous carcinoma cells:

washing transfected cells with good logarithmic growth state with 1 × PBS for 3 times, removing dead cells, digesting with pancreatin, preparing single cell suspension, diluting the cell suspension, and adjusting density to 5 × 10⁵Marking a transverse line on the back of each culture dish by using a marking pen, adding 1ml of cell suspension into each culture dish, shaking uniformly, and placing into an incubatorCulturing, when the cell coverage area is 95%, carrying out the next operation, scratching a line-shaped scratch in the middle of a monolayer of cells by using a 100-microliter flash pipette tip, washing the cells for 3 times by using PBS (phosphate buffer solution), adding a DMEM (dimethyl ether methane) culture medium, and culturing in an incubator. The culture dish was taken out for 24 hours, the medium was removed, the culture dish was washed 3 times with PBS solution, the condition of the cells was observed, and the distance of the scar was measured and recorded.

Compared with a blank control group (percentage of healing: 71.00 +/-7.55) and a transfection-unloaded negative control group (percentage of healing: 66.67 +/-2.08), the experimental group (percentage of healing: 48.67 +/-3.05) which over-expresses AC105046.1 has a certain reduction of the cell migration capacity of the lung squamous carcinoma after over-expressing the expression level of AC105046.1 in the lung squamous carcinoma cells, and the suggestion that the expression of AC105046.1 has a certain influence on the migration capacity of the lung squamous carcinoma cells.

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> application of lncRNA differentially expressed in diagnosis and treatment of lung cancer

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>18

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>1

cctcctacat ctgaatct 18

<210>2

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

agcaatgata agacaagag 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

Claims

1. Application of a reagent for detecting AC105046.1 in preparing a product for diagnosing lung cancer.

2. The use of claim 1, wherein the expression level of AC105046.1 is determined in a sample obtained from the patient, wherein a decreased expression level of AC105046.1 in the sample as compared to a reference expression level identifies the patient as a patient for lung cancer.

3. The use of claim 2, wherein the expression level of AC105046.1 is determined by RNA sequencing, PCR, qPCR, reverse transcription PCR, gene hybridization, gene expression profiling, gene approximation to series analysis, or microarray analysis.

4. The use according to any one of claims 1 to 3, wherein the sample comprises tissue, sputum, blood, pleural effusion or urine, preferably the sample is tissue.

5. A product for diagnosing lung cancer, comprising a reagent for detecting AC105046.1, preferably, the product comprises a chip, a kit or a nucleic acid membrane strip.

6. The product of claim 5, wherein the reagent comprises a probe or primer for the transcript of the AC105046.1 gene, preferably the primer has the sequence shown in SEQ ID No. 1-2.

Use of AC105046.1 in the construction of a computational model for predicting lung cancer.

Use of AC105046.1 in the preparation of a pharmaceutical composition for the treatment of lung cancer and/or lung cancer metastases.

9. The use according to claim 7, wherein the pharmaceutical composition comprises an enhancer of AC105046.1, preferably the enhancer is an agent that enhances the expression level of AC105046.1, preferably the enhancer is a vector that overexpresses AC 105046.1.

10. A pharmaceutical composition for treating lung cancer, comprising an agent that promotes the expression of AC105046.1, preferably a vector that overexpresses AC 105046.1.