CN106929577B

CN106929577B - lncRNA biomarker related to lung adenocarcinoma

Info

Publication number: CN106929577B
Application number: CN201710128322.8A
Authority: CN
Inventors: 陈钢; 杨承刚
Original assignee: Shandong Provincial Hospital; Beijing Medintell Bioinformatic Technology Co Ltd
Current assignee: Shandong Provincial Hospital; Beijing Medintell Bioinformatic Technology Co Ltd
Priority date: 2017-03-06
Filing date: 2017-03-06
Publication date: 2020-05-26
Anticipated expiration: 2037-03-06
Also published as: CN106929577A

Abstract

The invention discloses an lncRNA biomarker related to lung adenocarcinoma, wherein the marker is LOC105371720, and the expression of LOC105371720 is verified to be down-regulated in lung adenocarcinoma tissues through QPC detection. Furthermore, biological experiments prove that promoting the expression of the LOC105371720 gene can promote the apoptosis of lung adenocarcinoma cells and inhibit the migration and invasion of the lung adenocarcinoma cells. Based on this, LOC105371720 as a potential molecule can be applied to diagnosis and targeted therapy of lung adenocarcinoma.

Description

lncRNA biomarker related to lung adenocarcinoma

Technical Field

The invention belongs to the field of biomedicine, and relates to an lncRNA biomarker related to lung adenocarcinoma, in particular to LOC105371720 serving as the lncRNA biomarker.

Background

Lung cancer is the leading cause of tumor-related death. Lung cancer can be divided into two major classes, non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC), with NSCLC accounting for 80-85% of the total lung cancer cases. Most patients with non-small cell lung cancer have advanced treatment, the operation is difficult to be radically cured, and the disease is easy to recur after the operation. Radiotherapy and chemotherapy are often used as postoperative auxiliary means, and cannot kill tumor cells specifically, and meanwhile, great toxic and side effects are generated. The research on new ways for treating non-small cell lung cancer, especially the elucidation of the mechanism of occurrence, development and evolution of non-small cell lung cancer from the gene level, and the search for effective gene therapy targets are the hot spots in recent years.

The research on the potential molecular biological mechanism and the treatment of the non-small cell lung cancer are greatly improved, but the overall survival time of the non-small cell lung cancer is limited. Due to the lack of sensitive diagnostic tools, many patients are already late in the diagnosis and therefore miss the best treatment opportunities. Many early stage non-small cell lung cancer patients may also develop tumor metastasis after surgery. The molecular targeted therapy aiming at the lung adenocarcinoma patient can obviously improve the survival rate of the non-small cell lung cancer patient; however, there are still a large number of patients who are not effective for treatment. Thus, patients with a survival time of more than 5 years do not exceed 20%. The molecular mechanism of non-small cell lung cancer is not fully understood, and therefore, it is important to further study the development of non-small cell lung cancer in order to make early diagnosis and early treatment possible and obtain better prognosis.

Previous molecular mechanisms of tumorigenesis have focused on protein-encoding genes. With the development of high throughput transcriptome sequencing technology, researchers have found that over 90% of transcriptomes in the human genome are non-coding RNAs-micrornas (mirnas) among which have been thought to be involved in a number of biological processes, including malignant behavior of lung cancer. With the demonstration of lung cancer-associated mirnas, attention has been focused on other non-coding RNAs, i.e., long non-coding RNAs (lncRNAs). Long non-coding RNAs are over 200 nucleotide units in length and have no or only limited protein coding capacity. The long non-coding RNA has the functions of regulating chromatin and regulating genes according to different positions in cells. Up to now more than 10000 long non-coding RNAs have been found, however, less than 1% are annotated. Although the potential functions of long non-coding RNAs remain mysterious, several studies have found that they can regulate various cellular processes, such as proliferation, cell growth and apoptosis. The abnormal expression of long-chain non-coding RNA is closely related to the occurrence of tumors, and more evidences indicate that lncRNAs participate in the mechanism of the occurrence and development of non-small cell lung cancer. The study of the function of lncRNAs opens up a new approach for the biological study of non-small cell lung cancer and offers new possibilities for the development of new more effective therapies.

Disclosure of Invention

In order to make up the defects of the prior art, the invention aims to provide the lncRNA biomarker for diagnosis and treatment of the lung adenocarcinoma, and the lncRNA biomarker provided by the invention has higher sensitivity and specificity for the human lung adenocarcinoma and can be used as a novel biomarker for detection and treatment of the lung adenocarcinoma.

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

the invention provides an application of LOC105371720 in preparation of a product for diagnosing lung adenocarcinoma.

Further, the sequence of LOC105371720 is shown in SEQ ID NO. 1.

Further, the above-described product can diagnose whether or not a patient has lung adenocarcinoma by detecting the expression level of the LOC105371720 gene in a sample, and when the expression of LOC105371720 gene in the sample is down-regulated, the patient has lung adenocarcinoma or is at risk of having lung adenocarcinoma.

Further, the product for diagnosing lung adenocarcinoma comprises a chip, a preparation or a kit.

Wherein, the chip includes: a solid support; and oligonucleotide probes immobilized on the solid phase carrier in an ordered manner, wherein the oligonucleotide probes specifically correspond to a part or all of the sequence shown by LOC 105371720; the kit comprises a chip or a primer specific to LOC 105371720.

The invention provides a chip, a preparation or a kit, which contains the LOC 105371720.

Further, the chip can be used to detect the expression levels of a plurality of genes including the LOC105371720 gene (e.g., a plurality of genes associated with lung adenocarcinoma); the kit can be used to detect the expression levels of a plurality of genes including the LOC105371720 gene (e.g., a plurality of genes associated with lung adenocarcinoma).

The invention provides the application of the chip, the preparation or the kit in preparing products for diagnosing lung adenocarcinoma.

The invention provides an application of LOC105371720 in screening of human lung adenocarcinoma diagnosis and treatment medicines.

The invention provides an application of LOC105371720 in preparation of a pharmaceutical composition for preventing or treating lung adenocarcinoma.

Further, the pharmaceutical composition comprises an accelerator of the LOC105371720 gene and/or an expression product thereof.

The present invention provides a pharmaceutical composition for preventing or treating lung adenocarcinoma, comprising a therapeutically effective amount of the above-described enhancer.

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

The medicament of the invention can also be used in combination with other medicaments for treating lung adenocarcinoma, and other therapeutic compounds can be administered simultaneously with the main active ingredient, 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. 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.

The invention has the advantages and beneficial effects that:

the invention discovers that the differential expression of LOC105371720 is related to the occurrence and the development of lung adenocarcinoma for the first time, and whether the subject suffers from the lung adenocarcinoma or is at risk of suffering from the lung adenocarcinoma can be judged by detecting the expression of LOC105371720 in a sample of the subject, so that a clinician is guided to provide a prevention scheme or a treatment scheme for the subject.

lncRNA LOC105371720 as a potential molecular target provides new possibilities for clinical diagnosis and treatment of lung adenocarcinoma.

The discovery of the invention provides a theoretical basis for the mechanism research of the lung adenocarcinoma and provides a new way for the personalized treatment of the lung adenocarcinoma patients.

Drawings

FIG. 1 is a graph showing the detection of the expression of LOC105371720 gene in lung adenocarcinoma tissue by QPCR;

FIG. 2 is a graph showing the detection of transfection of LOC105371720 in lung adenocarcinoma cells using QPCR;

FIG. 3 is a graph showing the effect of LOC105371720 gene on lung adenocarcinoma cell proliferation measured by the CCK-8 method;

FIG. 4 is a graph showing the effect of LOC105371720 gene on apoptosis of lung adenocarcinoma cells detected by flow cytometry;

FIG. 5 is a graph showing the effect of LOC105371720 on migration of lung adenocarcinoma cells;

FIG. 6 is a graph showing the effect of LOC105371720 on lung adenocarcinoma cell invasion.

Detailed Description

The invention is widely and deeply researched, and the LOC105371720 in the lung adenocarcinoma is found to be specifically and lowly expressed for the first time through a large amount of screening. Experiments prove that the growth and invasion of lung adenocarcinoma cells can be effectively inhibited by specifically improving the activity of the expression level or the expression product of LOC105371720, so that the effect of inhibiting lung adenocarcinoma is achieved

Biomarkers

The term "biomarker" is any gene whose expression level in a tissue or cell is altered compared to the expression level of a normal or healthy cell or tissue.

One skilled in the art will recognize that the utility of the present invention is not limited to quantifying gene expression of any particular variant of the marker genes of the present invention. As a non-limiting example, the marker gene may have the nucleotide sequence specified in SEQ ID NO. 1. In some embodiments, it has a cDNA sequence at least 85% identical or similar to the listed sequences, such as at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical or similar to the listed sequences.

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

In some embodiments, the expression level of the biomarker is detected at the transcriptional level. Various methods for specific DNA and RNA measurements using nucleic acid hybridization techniques are known to those skilled in the art. Some methods involve electrophoretic separation (e.g., Southern blots for detecting DNA and Northern blots for detecting RNA), but measurements of DNA and RNA can also be made without electrophoretic separation (e.g., by dot blotting). Southern blots of genomic DNA (e.g., from humans) can be used to screen for Restriction Fragment Length Polymorphisms (RFLPs) to detect the presence of a genetic disorder affecting a polypeptide of the invention. All forms of RNA can be detected.

Chip and method for manufacturing the same

The lncRNA chip 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 LOC 105371720.

Specifically, suitable probes can be designed according to the lncRNA of the present invention, and are 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.

In the present invention, the solid phase carrier includes plastic products, microparticles, membrane carriers, and the like. 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 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 term "complementary" or "complementarity" is used to refer to polynucleotides (i.e., sequences of nucleotides) related by the base-pairing rules. For example, the sequence "5 '-A-G-T-3'" is complementary to the sequence "3 '-T-C-A-5'". Complementarity may be "partial," in which only some of the nucleic acids' bases are matched according to the base pairing rules. Alternatively, "complete" or "total" complementarity may also exist between nucleic acids. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between nucleic acid strands. This is particularly important in amplification reactions and detection methods that rely on binding between nucleic acids.

The term "stringency" is used to refer to the conditions under which nucleic acid hybridization is performed: temperature, ionic strength, and the presence of other compounds such as organic solvents. Under "low stringency conditions," a nucleic acid sequence of interest will hybridize to its exact complement, sequences with single base mismatches, closely related sequences (e.g., sequences having 90% or greater homology), and sequences with only partial homology (e.g., sequences having 50-90% homology). Under "medium stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement, sequences with single base mismatches, and closely related sequences (e.g., 90% or greater homology). Under "high stringency conditions," a nucleic acid sequence of interest will hybridize only to its exact complement and (depending on conditions such as temperature) sequences with a single base mismatch. In other words, under high stringency conditions, the temperature can be raised to exclude hybridization to sequences with single base mismatches.

The oligonucleotide probe of the present invention against the LOC105371720 gene may be DNA, RNA, DNA-RNA chimera, 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 nylon membrane, glass or silicon slice modified by active group (such as aldehyde group, amino group, etc.), unmodified glass slice, plastic slice, etc.

The LOC105371720 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.

Reagent kit

The present invention provides a kit useful for detecting the expression of LOC 105371720.

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.

Drug screening

The invention provides an application of LOC105371720 in screening of human lung adenocarcinoma diagnosis and treatment medicines. Namely: treating a system expressing LOC105371720 with a candidate substance; and detecting the expression or activity of LOC105371720 in said system; if the candidate substance can promote the expression or activity of LOC105371720, the candidate substance is a potential substance for inhibiting lung adenocarcinoma. The system for expression of LOC105371720 may be, for example, a cell (or cell culture) system, and the cell may be a cell endogenously expressing LOC 105371720; or may be a cell recombinantly expressing LOC 105371720. The system for expressing LOC105371720 can also be a subcellular system, a solution system, a tissue system, an organ system or an animal system (such as an animal model, preferably a non-human mammal animal model, such as a mouse, a rabbit, a sheep, a monkey, etc.), and the like.

Accelerator and pharmaceutical composition of LOC105371720

Based on the findings of the present invention, the present invention provides a pharmaceutical composition comprising an enhancer of LOC 105371720.

The promoter of LOC105371720 refers to any substance that can improve the stability of the LOC105371720 gene or expression product, up-regulate the expression of LOC105371720, increase the effective action time of lncRNA LOC105371720, or promote the transcription of the LOC105371720 gene, and these substances can be used in the present invention, and can be used as a substance useful for up-regulating the expression of the LOC105371720 gene, and thus can be used for preventing or treating lung adenocarcinoma.

In a preferred embodiment of the present invention, the promoter of LOC105371720 is an expression vector containing LOC 105371720. The expression vector usually further contains a promoter, an origin of replication, and/or a marker gene.

Methods well known to those skilled in the art can be used to construct the expression vectors required by the present invention. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The expression vector preferably comprises one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells, such as kanamycin, gentamicin, hygromycin, ampicillin resistance.

In the present invention, there are various vectors known in the art, such as commercially available vectors, including plasmids, cosmids, phages, viruses, and the like. The expression vector can be introduced into the host cell by a known method such as electroporation, calcium phosphate method, liposome method, DEAE dextran method, microinjection, viral infection, lipofection, or binding to a cell membrane-permeable peptide.

The term "host cell" includes prokaryotic and eukaryotic cells. Examples of commonly used prokaryotic host cells include E.coli, Bacillus subtilis, and the like. Commonly used eukaryotic host cells include yeast cells, insect cells, and mammalian cells. Preferably, the host cell is a eukaryotic cell, such as a CHO cell, a COS cell, or the like.

Pharmaceutical composition

The pharmaceutical composition comprises the accelerant of LOC105371720, and/or other medicines compatible with the accelerant, and a pharmaceutically acceptable carrier and/or auxiliary materials.

The pharmaceutically acceptable carrier may be one or more, and includes, but is not limited to, diluents such as lactose, sodium chloride, glucose, urea, starch, water, and the like; binders such as starch, pregelatinized starch, dextrin, maltodextrin, sucrose, acacia, gelatin, methyl cellulose, carboxymethyl cellulose, ethyl cellulose, polyvinyl alcohol, polyethylene glycol, polyvinyl pyrrolidone, alginic acid and alginates, xanthan gum, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, 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 zinc stearate, glyceryl monostearate, polyethylene glycol, talc, calcium stearate and magnesium stearate, polyethylene glycol, boric acid powder, hydrogenated vegetable oil, sodium stearyl fumarate, polyoxyethylene monostearate, monolaurocyanate, sodium lauryl sulfate, magnesium lauryl sulfate, etc.; fillers such as mannitol (granular or powder), xylitol, sorbitol, maltose, erythrose, microcrystalline cellulose, polymeric sugar, coupling sugar, glucose, lactose, sucrose, dextrin, starch, sodium alginate, laminarin powder, agar powder, calcium carbonate, sodium bicarbonate, etc.; disintegrating agent such as crosslinked vinylpyrrolidone, sodium carboxymethyl starch, low-substituted hydroxypropyl methyl, crosslinked sodium carboxymethyl cellulose, soybean polysaccharide, etc.

The pharmaceutical composition of the present invention may further comprise additives such as stabilizers, bactericides, buffers, isotonizing agents, chelating agents, pH control agents, and surfactants.

The term "effective amount" refers to an amount that produces a function or activity in and is acceptable to humans and/or animals. "pharmaceutically acceptable carrier" refers to a carrier for administration of a 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 transfection reagent.

In the present invention, the promoter or its transcription gene, or its pharmaceutical composition can be administered to mammals by various methods well known in the art. Including but not limited to: subcutaneous injection, intramuscular injection, transdermal administration, topical administration, implantation, sustained release administration, and the like; preferably, the mode of administration is parenteral.

Preferably, it can be carried out by means of gene therapy. For example, the promoter of LOC105371720 can be directly administered to a subject by a method such as injection; alternatively, the expression unit (e.g. expression vector or virus) carrying LOC105371720 to facilitate dispensing 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.

The effective amount of the accelerator of LOC105371720 described in the present invention 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: the pharmacokinetic parameters of the promoter of LOC105371720 such as bioavailability, metabolism, half-life and the like; 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. For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

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

miRNA kit for operation)

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

1) and (3) RNA isolation:

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

2) RNA precipitation

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

3) RNA elution

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

4) RNA resolubilization

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

5) Mass analysis of RNA samples

And (3) detection by a spectrophotometer:

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

And (3) agarose gel electrophoresis detection:

the extracted RNA is subjected to agarose gel electrophoresis, Agilent Technologies2100Bioanalyzer detects the quality of an RNA sample, and the RNA sample is observed to have obvious main bands of 28S rRNA and 18S rRNA, no degradation, qualified RNA integrity index and concentration meeting the requirements, so that the RNA sample can be used for lncRNA expression profile and screening experiment of a chip.

3. Reverse transcription and labelling

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

4. Hybridization of

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

5. Data analysis

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

6. Results

The results show that the expression level of LOC105371720 in lung adenocarcinoma tissue is significantly lower than that in para-carcinoma tissue.

Example 2 QPCR sequencing verification of differential expression of the LOC105371720 Gene

1. Large sample QPCR validation was performed on LOC105371720 gene differential expression. 50 cases of the lung adenocarcinoma paracancerous tissue and lung adenocarcinoma tissue were selected according to the sample collection method in example 1.

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

3. Reverse transcription

1) Reaction system:

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

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

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

2) conditions for reverse transcription

25℃5min，42℃60min，70℃5min。

3) Polymerase chain reaction

Designing a primer:

QPCR amplification primers were designed based on the sequences of LOC105371720 gene and GAPDH gene in Genebank and synthesized by Bomader Biotech. The specific primer sequences are as follows:

LOC105371720 gene:

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

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

GAPDH gene:

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

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

Preparing a PCR reaction system:

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

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

5. Statistical method

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

6. Results

As shown in fig. 1, LOC105371720 has a statistically significant difference (P <0.05) in the expression of lung adenocarcinoma tissue, which is lower than that of the tissue adjacent to lung adenocarcinoma, and is consistent with the results of the chip assay.

Example 3 LOC105371720 overexpression

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. Construction of Gene overexpression vectors

Specific PCR amplification primers were synthesized based on the cDNA sequence of LOC105371720 (XR-947271.2.1, shown in SEQ ID NO. 1), and HindIII and XhoI restriction sites were added to the 5 'and 3' primers, respectively. cDNA obtained by blood extraction and reverse transcription of a patient with lung adenocarcinoma is used as an amplification template, the cDNA sequence is inserted into a eukaryotic cell expression vector pcDNA3.1 which is subjected to double enzyme digestion by restriction enzymes HindIII and XhoI, and the obtained recombinant vector pcDNA3.1-1 is connected for subsequent experiments.

3. Transfection

The lung adenocarcinoma cells were divided into 3 groups, which were respectively a control group (A549), a blank control group (transfected pcDNA3.1-NC), and an experimental group (transfected pcDNA3.1-1). Transfection of the vector was performed using liposome 2000, and the specific transfection method was performed as indicated in the specification. The transfection concentrations of the pcDNA3.1 empty vector and pcDNA3.1-1 were 0.5. mu.g/ml.

4. QPCR detection of transcription level of LOC105371720 Gene

4.1 extraction of Total RNA from cells

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

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

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

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

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

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

4.2 reverse transcription procedure as in example 2.

4.3QPCR amplification step as in example 2.

5. Statistical method

The experiments were performed in 3 replicates, the data were expressed as mean ± sd, and statistically analyzed using SPSS18.0 statistical software, and the difference between the LOC105371720 gene overexpression group and the control group was determined to be statistically significant when P <0.05 using t-test.

6. Results

The results are shown in fig. 2, where LOC105371720 was overexpressed in the transfected LOC105371720 group compared to the non-transfected group and the transfected empty protein granule group, the difference was statistically significant (P < 0.05).

Example 4 Effect of LOC105371720 Gene on Lung adenocarcinoma cell proliferation

CCK-8 experiment is adopted to detect the influence of LOC105371720 gene on the proliferation capacity of lung adenocarcinoma cells.

1. The cell culture and transfection procedures were the same as in example 3.

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 pcDNA3.1-1 experimental group and the pcDNA3.1-NC control group were set. A total of 4 96 well plates were plated for 24h, 48h, 72h, 96h4 test time points, respectively.

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

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

6. Statistical analysis

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

7. Results

As shown in FIG. 3, compared with the control, the experimental group was significantly inhibited in cell proliferation after transfection of pcDNA3.1-1, and the statistical significance of the difference (P <0.05) indicates that LOC105371720 has the effect of inhibiting cell proliferation.

Example 5 Effect of LOC105371720 Gene on apoptosis of Lung adenocarcinoma cells

The influence of the LOC105371720 gene on apoptosis was examined using flow cytometry.

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

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

3. Step (ii) of

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

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

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

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

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

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

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

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

3. Statistical method

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

4. As a result:

the results are shown in fig. 4, and the apoptosis rate of the experimental group has a significant change (P <0.05) compared with the control group, and the results show that LOC105371720 has a significant effect on 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

As shown in fig. 5 and fig. 6, after the lung adenocarcinoma cells were transfected with the plasmids, the migration and invasion abilities of the experimental group were significantly reduced compared to the control group, and the results indicate that LOC105371720 can inhibit the migration and invasion 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> Shandong provincial Hospital Beijing @ is a deep bioinformatics technology GmbH

<120> lncRNA biomarker related to lung adenocarcinoma

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<170>PatentIn version 3.5

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Claims

Application of LOC105371720 in preparation of products for diagnosing lung adenocarcinoma.
2. The use according to claim 1, wherein the sequence of LOC105371720 is as set forth in SEQ ID No. 1.
3. Use according to claim 1 or 2, wherein the product comprises a formulation.
4. The use of claim 3, wherein the formulation comprises a chip or kit.
5. Application of a preparation containing a reagent for detecting the expression level of LOC105371720 in preparation of a product for diagnosing lung adenocarcinoma.
6. The use of claim 5, wherein the formulation comprises a chip or kit.
7. The use of LOC105371720 of claim 1 for screening human lung adenocarcinoma diagnostic and treatment drugs for non-disease treatment purposes.
Use of LOC105371720 for the preparation of a pharmaceutical composition for the prevention or treatment of lung adenocarcinoma.
9. The use according to claim 8, wherein the pharmaceutical composition comprises an enhancer of the LOC105371720 gene and/or expression products thereof.
10. A pharmaceutical composition for preventing or treating lung adenocarcinoma, comprising a therapeutically effective amount of the enhancer of claim 9, which is a vector overexpressing LOC 105371720.
11. The pharmaceutical composition of claim 10, further comprising other drugs compatible with the enhancer and a pharmaceutically acceptable carrier and/or adjuvant.