CN111518905A

CN111518905A - Application of lncRNA in diagnosis and treatment of lung adenocarcinoma

Info

Publication number: CN111518905A
Application number: CN202010387807.0A
Authority: CN
Inventors: 徐红玉; 杜泽东; 廖洪飞; 刘贤国; 蒙荣钦; 贺亮; 李光华; 张光宇
Original assignee: 363 Hospital
Current assignee: 363 Hospital
Priority date: 2020-05-09
Filing date: 2020-05-09
Publication date: 2020-08-11
Anticipated expiration: 2040-05-09
Also published as: CN111518905B

Abstract

The invention discloses application of lncRNA in diagnosis and treatment of lung adenocarcinoma, wherein the lncRNA is AC018890.6, AC018890.6 is highly expressed in lung adenocarcinoma tissues, and the change of the expression level of AC018890.6 can influence the proliferation, migration and invasion of lung adenocarcinoma cells, which suggests that whether a subject has lung adenocarcinoma or is at risk of having lung adenocarcinoma can be judged by detecting the expression level of AC018890.6 in a subject, and the targeted AC018890.6 can treat the lung adenocarcinoma.

Description

Application of lncRNA in diagnosis and treatment of lung adenocarcinoma

Technical Field

The invention belongs to the field of biomedicine, and relates to application of lncRNA in diagnosis and treatment of lung adenocarcinoma.

Background

Globally, lung cancer is the first malignancy with morbidity and mortality, among which non-small cell lung cancer (NSCLC) accounts for 80% and its 5-year survival rate is less than 15%. The main reason for the high mortality of NSCLC is that patients have no obvious clinical symptoms in early stage, and are diagnosed at a later stage of cancer, and tumor cells have invaded and metastasized. The current standard therapies for treating NSCLC comprise surgical resection, radiation therapy, a platinum-based combined chemotherapy scheme, targeted therapy and the like, but the therapies can hardly achieve the aim of curing NSCLC, so that the NSCLC patients have poor treatment effect and poor prognosis. Therefore, for the treatment of lung cancer, the most important thing is to find effective markers for early diagnosis and treatment, and provide theoretical basis for resisting invasion and metastasis of cancer cells. At present, the diagnosis of lung cancer is mainly based on histopathology, an effective diagnostic molecular marker is urgently needed clinically, and in order to improve the survival rate of NSCLC patients, the pathogenesis of the NSCLC patients needs to be deeply researched, so that the processes of malignant proliferation, invasion and metastasis and the like of tumors are inhibited as early as possible. LncRNA (long non-codingRNA), a genetic molecule involved in cancer biology, has emerged as a new approach in recent scientific research.

Long non-coding RNAs (lncrnas), which are RNAs with a length greater than 200 nucleotides but do not code for proteins, have been considered as "dark substances" for gene transcription, but lncrnas have been found to have important biological functions, such as regulation of cell proliferation, cell cycle, cell differentiation, apoptosis, etc. In addition, aberrant expression of lncRNA is strongly associated with human disease, especially in tumors. The lncRNA is found to be abnormally expressed in a plurality of tumors such as lung cancer, liver cancer, breast cancer, gastric cancer and the like, and the lncRNA mainly participates in the processes of tumor occurrence, growth, invasion, metastasis, relapse, drug resistance and the like, and the lncRNA is suggested to possibly play the role of oncogene or cancer suppressor gene in the process of tumor occurrence and development. The lncRNA can be reasonably predicted to be a new marker for diagnosing, judging prognosis and treating the tumor, and can be a new target point for tumor gene therapy. Currently, lncRNA has become a new focus and frontier for tumor research following mirnas.

Disclosure of Invention

In order to make up for the defects of the prior art, the invention aims to provide lncRNA related to the occurrence and development of lung adenocarcinoma, and the early diagnosis and the accurate treatment of the lung adenocarcinoma are realized by using the lncRNA.

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

the invention provides an application of an AC018890.6 gene in preparing a product for diagnosing lung adenocarcinoma.

Further, the product comprises: products for diagnosing lung adenocarcinoma by RT-PCR, real-time quantitative PCR, in-situ hybridization or gene chip.

Further, the product for diagnosing lung adenocarcinoma by RT-PCR at least comprises a pair of primers for specifically amplifying the AC018890.6 gene; the product for diagnosing the lung adenocarcinoma in real time quantitatively comprises at least one pair of primers for specifically amplifying the AC018890.6 gene; the product for diagnosing lung adenocarcinoma by in situ hybridization comprises a probe hybridized with a nucleic acid sequence of AC018890.6 gene; the product for diagnosing lung adenocarcinoma by using the gene chip comprises a probe hybridized with a nucleic acid sequence of the AC018890.6 gene.

The invention provides a product for diagnosing lung adenocarcinoma, which comprises a reagent for detecting the expression level of AC018890.6 gene.

Further, the reagents include probes or primers specific for AC 018890.6.

Further, the primer sequence is shown in SEQ ID NO. 1-2.

Further, the product comprises a chip or a kit.

Further, the chip can be used to detect the expression levels of a plurality of genes including the AC018890.6 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 AC018890.6 gene (e.g., a plurality of genes associated with lung adenocarcinoma).

The invention provides application of AC018890.6 in screening of a medicament for treating lung adenocarcinoma.

Further, the step of screening for a drug for treating lung adenocarcinoma comprises:

treating a system expressing or containing AC018890.6 with a substance to be screened; and detecting expression of AC018890.6 in the system; wherein, if the substance to be screened can reduce the expression level of AC018890.6, the substance to be screened is a candidate drug for treating lung adenocarcinoma.

The invention provides a method for screening a medicament for treating lung adenocarcinoma, which comprises the following steps:

The invention provides an application of an AC018890.6 gene in preparing a pharmaceutical composition for treating lung adenocarcinoma.

Further, the pharmaceutical composition comprises an inhibitor of AC 018890.6.

Further, the inhibitor reduces the expression level of AC018890.6, including but not limited to nucleic acid molecules, carbohydrates, small molecule compounds, or interfering lentiviruses.

Further, the inhibitor is a nucleic acid molecule.

Further, the nucleic acid molecule is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 5-6.

Furthermore, the pharmaceutical composition also comprises other medicines compatible with the inhibitor and a pharmaceutically acceptable carrier and/or auxiliary material.

The invention provides a pharmaceutical composition for treating lung adenocarcinoma, which comprises an inhibitor of AC 018890.6.

Further, the inhibitor is a nucleic acid molecule. The nucleic acid molecule is selected from: antisense oligonucleotides, double-stranded RNA, small interfering RNA or short hairpin RNA.

Further, the nucleic acid molecule is siRNA.

Further, the sequence of the siRNA is shown in SEQ ID NO. 5-6.

The invention has the advantages and beneficial effects that:

the lncRNA marker for diagnosing the human lung adenocarcinoma, provided by the invention, can realize molecular diagnosis of the lung adenocarcinoma.

The human lung adenocarcinoma lncRNA marker provided by the invention can be used as a potential drug target and applied to the targeted therapy of lung adenocarcinoma in clinic.

Drawings

FIG. 1 is a graph showing the detection of the expression of AC018890.6 gene in lung adenocarcinoma tissue by QPCR.

Detailed Description

According to the invention, through extensive and intensive research, the expression of lncRNA in a lung adenocarcinoma sample in a tumor tissue and a tissue beside the tumor is detected, lncRNA fragments with obvious differential expression are found, and the relationship between the lncRNA fragments and the occurrence of lung adenocarcinoma is discussed, so that a better way and a better method are found for early detection and targeted treatment of lung adenocarcinoma. Through research, the invention discovers that AC018890.6 is remarkably upregulated in lung adenocarcinoma for the first time.

The AC018890.6 in the present invention includes wild type, mutant type or fragment thereof, as long as it can be aligned to the known gene AC018890.6 when performing sequence alignment. There are two transcripts of AC018890.6 that have been published so far, and the sequences are shown in ENST00000442996.1 and ENST00000412835.1, respectively. In a specific embodiment of the invention, the sequence of the AC018890.6 is shown in ENST 00000442996.1.

The full-length AC018890.6 nucleotide sequence or its fragment of the present invention can be obtained by PCR amplification, recombination, or artificial synthesis. For the PCR amplification method, primers can be designed based on the disclosed nucleotide sequences, particularly open reading frame sequences, and the sequences can be amplified using a commercially available cDNA library or a cDNA library prepared by a conventional method known to those skilled in the art as a template. When the sequence is long, two or more PCR amplifications are often required, and then the amplified fragments are spliced together in the correct order.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. This is usually done by cloning it into a vector, transferring it into a cell, and isolating the relevant sequence from the propagated host cell by conventional methods. In addition, the sequence can be synthesized by artificial synthesis, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

A method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention. The primers used for PCR can be appropriately selected based on the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method. The amplified DNA/RNA fragments can be isolated and purified by conventional methods, such as by gel electrophoresis.

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, so long as quantitation of AC018890.6 can be achieved.

Chip and kit

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 the AC 018890.6. "Probe" refers to a molecule that binds to a particular sequence or subsequence or other portion of another molecule. "probes" are intended to include nucleic acid oligomers or aptamers that specifically hybridize to a target sequence in a nucleic acid or its complement under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection can be direct (i.e., generated by probes that directly hybridize to the target or amplified sequence) or indirect (i.e., generated by probes that hybridize to an intermediate molecular structure linking the probes and the target or amplified sequence). The "target" of a probe generally refers to a sequence of an amplified nucleic acid sequence that specifically hybridizes to at least a portion of the probe sequence through standard hydrogen bonding or "base pairing". Sequences that are "sufficiently complementary" allow for stable hybridization of the probe sequence to the target sequence even if the two sequences are not fully complementary. The probe may be labeled or unlabeled. Probes may be produced by molecular cloning of a particular DNA sequence, or may be synthesized. One skilled in the art to which the invention pertains can readily determine the variety of primers and probes that can be designed and used in the context of the present invention.

"hybridization" or "nucleic acid hybridization" or "hybridization" generally refers to the hybridization of two single-stranded nucleic acid molecules having complementary base sequences that, under the appropriate conditions, will form a thermodynamically stable double-stranded structure. The term "hybridization" as used herein may refer to hybridization under stringent or non-stringent conditions. The setting of the conditions is within the skill of the person skilled in the art and can be determined according to the experimental protocols described in the art. The term "hybridizing sequence" preferably refers to a sequence showing a sequence identity of at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, particularly preferably at least 80%, more particularly preferably at least 90%, more particularly preferably at least 95%, and most preferably at least 97% identity. In the case of hybridization to nitrocellulose filters (or other such supports such as nylon), such as the well-known Southern blotting procedure, nitrocellulose filters can be incubated with labeled probes in overnight solutions containing high salt (6 XSSC or 5 XSSPE), 5 XDenhardt's solution, 0.5% SDS and 100. mu.g/ml denatured carrier DNA (e.g., salmon sperm DNA) at temperatures representing the conditions of desired stringency (high stringency 60-65 ℃, medium stringency 50-60 ℃, low stringency 40-45 ℃). Non-specifically bound probes can be detected by binding in 0.2 XSSC/0.1% SDS at a temperature selected according to the desired stringency: the filter was eluted from the wash several times at room temperature (low stringency), 42 ℃ (medium stringency) or 65 ℃ (high stringency). The salt and SDS concentrations of the wash solution may also be adjusted to suit the desired stringency. The temperature and salt concentration selected are based on the melting temperature (Tm) of the DNA hybrid. Of course, RNA-DNA hybrids can also be formed and detected. In such cases, the conditions for hybridization and washing may be varied by those skilled in the art according to well-known methods. Preferably stringent conditions are used. Other protocols utilizing different annealing and washing solutions or commercially available hybridization kits (e.g., ExpressHybTM from BD Biosciences Clonetech) may also be used, as is well known in the art. It is well known that the length of the probe and the composition of the nucleic acid to be determined determine other parameters of the hybridization conditions. It is noted that variations of the above conditions can be achieved by the addition and/or substitution of alternative blocking reagents for suppressing background in hybridization experiments. Common blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA and commercially available proprietary formulations. Due to compatibility issues, the addition of specific blocking reagents may require modification of the hybridization conditions described above. Hybrid nucleic acid molecules also include fragments of the above molecules. In addition, nucleic acid molecules that hybridize to any of the above-described nucleic acid molecules also include complementary fragments, derivatives, and allelic variants of these molecules. In addition, a hybridization complex refers to a complex between two nucleic acid sequences that relies on the formation of hydrogen bonds between complementary G and C bases and between complementary A and T bases; these hydrogen bonds may be further stabilized by base stacking interactions. Two complementary nucleic acid sequences form hydrogen bonds in an antiparallel configuration. Hybridization complexes can be formed in solution (e.g., Cot or Rot assays), or between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., a membrane, filter, chip, pin, or slide that has, for example, immobilized cells).

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

The invention provides a kit which can be used for detecting the expression of AC 018890.6. Preferably, 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.

The kit of the invention can be also attached with an instruction manual of the kit, wherein the instruction manual describes how to adopt the kit for detection, how to judge the tumor development by using the detection result and how to select a treatment scheme.

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

Inhibitors and pharmaceutical compositions

Based on the findings of the present invention, the present invention provides a pharmaceutical composition comprising an inhibitor of AC 018890.6. The nature of the inhibitor is not critical to the present invention as long as it inhibits the level of AC018890.6 gene, and these inhibitors are useful as agents for down-regulating AC018890.6, and can be used for the prevention or treatment of lung adenocarcinoma.

As an alternative of the present invention, the inhibitor of AC018890.6 may be a "Small hairpin RNA (shRNA)" which is a non-coding Small RNA molecule capable of forming a hairpin structure, and the Small hairpin RNA can inhibit gene expression via an RNA interference pathway. As described above, shRNA can be expressed from a double-stranded DNA template. The double-stranded DNA template is inserted into a vector, such as a plasmid or viral vector, and then expressed in vitro or in vivo by ligation to a promoter. The shRNA can be cut into small interfering RNA molecules under the action of DICER enzyme in eukaryotic cells, so that the shRNA enters an RNAi pathway. "shRNA expression vector" refers to some plasmids which are conventionally used for constructing shRNA structure in the field, usually, a "spacer sequence" and multiple cloning sites or alternative sequences which are positioned at two sides of the "spacer sequence" are present on the plasmids, so that people can insert DNA sequences corresponding to shRNA (or analogues) into the multiple cloning sites or replace the alternative sequences on the multiple cloning sites in a forward and reverse mode, and RNA after the transcription of the DNA sequences can form shRNA (short Hairpin) structure. The "shRNA expression vector" is completely available by the commercial purchase of, for example, some viral vectors.

As a preferred mode of the invention, the inhibitor of AC018890.6 is a small interfering RNA molecule specific for AC 018890.6. As used herein, the term "small interfering RNA" refers to a short segment of double-stranded RNA molecule that targets mRNA of homologous complementary sequence to degrade a specific mRNA, a process known as RNA interference (RNAInterferce). Small interfering RNA can be prepared as a double-stranded nucleic acid form, which contains a sense and an antisense strand, the two strands only in hybridization conditions to form double-stranded. A double-stranded RNA complex can be prepared from the sense and antisense strands separated from each other. Thus, for example, complementary sense and antisense strands are chemically synthesized, which can then be hybridized by annealing to produce a synthetic double-stranded RNA complex.

In the present invention, 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.

The pharmaceutical compositions of the present invention may be administered orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir. 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 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 QPCR detection of AC018890.6 Gene expression

1. 33 lung adenocarcinoma tissues and matched paracarcinoma tissues were collected separately, and all patients received no chemotherapy, radiotherapy, targeted therapy, tumor immunotherapy and other treatments before surgery, and had no other neoplastic diseases, autoimmune diseases and severe chronic diseases.

2. RNA extraction

Cutting tissue with scissors, adding 1ml Trizol, and shaking on oscillator for 1 min; standing at room temperature for 10min to completely decompose nucleoprotein; then adding 200 mul of chloroform (chloroform), covering a pipe cover tightly, violently shaking for 15s, standing for 10min at normal temperature, centrifuging for 15min at the temperature of 4 ℃ and the rpm of 11000; transferring the water sample layer into a new centrifuge tube, and adding 500 mul of isopropanol; after being reversed and mixed evenly, the mixture is stood for 10min at normal temperature, and then centrifuged for 15min at 11000rpm after being 4 ℃; carefully sucking away the liquid with a gun, leaving the precipitate at the bottom of the tube, adding 1ml of 75% ethanol, shaking on an oscillator for 5s, washing the precipitate once, and centrifuging at 4 ℃ and 8000rpm for 5 min; then carefully removing the supernatant, drying the precipitate for 10min, and adding a proper amount of water to dissolve the precipitate for 10 min.

3、QPCR

1) Reverse transcription reaction

Using the FastQ μ ant cDNA first strand synthesis kit (cat # KR106), reverse transcription of lncRNA was performed, and the reaction system and reaction conditions were as shown in Table 1.

TABLE 1 reverse transcription reaction System and reaction conditions

2) Primer design

QPCR amplification primers were designed based on the coding sequences of the AC018890.6 gene and GAPDH gene in Genebank, and were synthesized by Bomeide Bio Inc., and the specific primer sequences are shown in Table 2.

TABLE 2 primer sequences

3) QPCR amplification assay

Amplification was performed using SuperReal PreMix Plus (SYBR Green) (cat # FP205), the experimental procedures were performed according to the product instructions, and the reagents and reaction systems are shown in Table 3.

TABLE 3 QPCR amplification reaction System and reaction conditions

4) Sample RealTime PCR detection

And amplifying each sample by using a target gene primer and an internal reference gene primer respectively. 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

QPCR results as shown in figure 1, AC018890.6 was up-regulated in lung adenocarcinoma tissue compared to paracarcinoma tissue, with the difference being statistically significant (P < 0.05); AC018890.6 was up-regulated in 31 samples, 26 of which were up-regulated in lung adenocarcinoma tissue and 5 of which were up-regulated in paracarcinoma tissue. Expression up-regulation was considered as positive (+), no significant change or down-regulation was considered as negative (-), and specific statistics are shown in table 4.

TABLE 4 expression of genes in samples

Example 3 functional verification of AC018890.6

1. Cell culture

Human lung cancer cell strain A549 prepared by culturing 10% fetal calf serum and 1% P/S in DMEM medium at 37 deg.C and 5% CO₂Culturing in a constant temperature incubator.

2. Transfection

2.1 design of siRNA

Interfering siRNA against AC018890.6, AC018890.6, was designed and synthesized by Shanghai Ji code pharmaceutical technology, Inc., and the sequence is shown in Table 5, and the control was general siRNA-NC.

TABLE 5 siRNA sequences

2.2 transfection

According to the steps of the specification operation of the invitrogen Lipo2000 transfection reagent, cells are evenly paved in a 6-well plate after being digested conventionally, after being attached to the wall on the next day, the cells are starved for 12 hours by changing to a culture medium without serum, the Lipo2000 and siRNA are respectively incubated for about 5min by using a serum-free culture medium according to the amount of each hole on the next day, then the Lipo2000 and the siRNA are gently mixed and evenly stirred for 20min, then the mixed solution is evenly added into the 6-well plate and evenly shaken, after being transfected for 4 hours, a culture medium containing 10% fetal bovine serum is changed, and the cells are continuously cultured. The experiment was divided into three groups: control group (A549), negative control group (siRNA-NC) and experimental group (siRNA-AC018890.6)

3. QPCR detection

3.1 extraction of cellular RNA procedure as in example 1

3.2 fluorescent quantitative PCR detection procedure same as example 1

4. Cell proliferation Activity assay (CCK-8)

And taking lung adenocarcinoma cells of a control group and an experimental group with good growth states, digesting and centrifuging, adding a DMEM (DMEM) culture medium, blowing and beating to form single cell suspension, and counting. Each well of a 96-well plate is inoculated with 1000 cells, and each group of cells is provided with 5 multiple wells. After 72h, CCK-8 detection was carried out. Before the assay, the old medium in the wells was discarded, and 90. mu.L of medium and 10. mu.L of CCK8 were added to each well, protected from light. 37 ℃ and 5% CO₂Was incubated for 2 h. The microplate reader was set to a single wavelength of 450nm, absorbance values were determined, and then statistical analysis was performed.

5. Transwell chamber for detecting cell movement ability

5.1 Transwell cell migration assay

Counting the number of cells, adjusting the cell density to 5 × l0⁵Adding 200 mu L of cell suspension into the upper chamber, adding 500 mu L of culture medium containing 20% fetal calf serum into the lower chamber, continuously culturing for 48h, taking out the small chamber, wiping off cells which do not pass through the membrane on the inner side of the small chamber, fixing with methanol for 30min, dip-dyeing with crystal violet staining solution for at least 30min, washing with tap water, and naturally air-drying at room temperature. The number of cells that crossed the membrane was observed and counted for each group under the microscope.

5.2 Transwell cell invasion assay

The method and procedure were identical to the Transwell cell migration experiment except that the upper chamber was first lined with matrigel on the side of the polycarbonate membrane chamber. The glue spreading process is carried out on ice, all gun heads used need to be precooled in advance, and the Matrigel glue is dissolved in ice at 4 ℃ overnight in advance.

6. Statistical analysis

The experimental results were statistically analyzed using graphpad, and P <0.05 was considered statistically significant using bilateral tests.

7. Results

The expression level of AC018890.6 in the blank control group was set as 1, and the transfection results showed that the siRNA of the experimental group had better silencing effect on AC018890.6 as shown in table 6.

TABLE 6 relative expression of AC018890.6

Note: p value is siRNA-NC/siRNA-AC018890.6vs blank control group

The CCK-8 detection results are shown in Table 7, the OD value of the experimental group (siRNA-AC018890.6) is obviously reduced compared with that of the negative control group, and AC018890.6 plays an important role in the proliferation of lung adenocarcinoma cells.

TABLE 7 OD values

The results of Transwell detection are shown in table 8, after lung adenocarcinoma cells are transfected with siRNA-AC018890.6, the migration and invasion capacity of the cells is significantly reduced, which indicates that the expression of AC018890.6 affects the migration and invasion of lung adenocarcinoma cells, and suggests that AC018890.6 can be applied to the treatment of lung adenocarcinoma metastasis and infiltration.

TABLE 8 cell migration and invasiveness assays

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> three hospitals

Application of <120> lncRNA in diagnosis and treatment of lung adenocarcinoma

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<170>SIPOSequenceListing 1.0

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<213> Artificial Sequence (Artificial Sequence)

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<210>2

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<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>2

tctctgcctt agttcttc 18

<210>3

<211>21

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

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aatcccatca ccatcttcca g 21

<210>4

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<213> Artificial Sequence (Artificial Sequence)

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<210>5

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<213> Artificial Sequence (Artificial Sequence)

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<213> Artificial Sequence (Artificial Sequence)

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Claims

1. Use of the AC018890.6 gene in the preparation of a product for diagnosing lung adenocarcinoma.

2. Use according to claim 1, characterized in that the product comprises: products for diagnosing lung adenocarcinoma by RT-PCR, real-time quantitative PCR, in-situ hybridization or gene chip.

3. The use according to claim 2, wherein the product for diagnosing lung adenocarcinoma by RT-PCR comprises at least one pair of primers for specifically amplifying AC018890.6 gene; the product for real-time quantitative diagnosis of lung adenocarcinoma comprises at least one pair of primers for specifically amplifying AC018890.6 gene; products for diagnosing lung adenocarcinoma by in situ hybridization include a probe that hybridizes to a nucleic acid sequence of the AC018890.6 gene; the product for diagnosing lung adenocarcinoma by using the gene chip comprises a probe hybridized with a nucleic acid sequence of the AC018890.6 gene.

4. A product for diagnosing lung adenocarcinoma, comprising a reagent for detecting the expression level of AC018890.6 gene.

5. The product according to claim 4, wherein the reagent comprises a probe or primer specific to AC018890.6, preferably the primer sequence is shown in SEQ ID No. 1-2.

6. The product of claim 5, wherein the product comprises a chip or a kit.

7. Use of the AC018890.6 gene in screening a candidate drug for treating lung adenocarcinoma.

8. Use of the AC018890.6 gene for preparing a pharmaceutical composition for preventing or treating lung adenocarcinoma.

9. The use according to claim 8, wherein the pharmaceutical composition comprises an inhibitor of AC018890.6, preferably wherein the inhibitor reduces the expression level of AC 018890.6; preferably, the inhibitor is a nucleic acid molecule; preferably, the nucleic acid molecule is siRNA; preferably, the sequence of the siRNA is shown in SEQ ID NO. 5-6.

10. A pharmaceutical composition for treating lung adenocarcinoma, comprising the inhibitor of claim 9.