CN110951888A - Reagent for detecting and targeting AL845472.2 and application of reagent in lung cancer diagnosis and treatment - Google Patents

Abstract

The invention relates to a reagent for detecting and targeting AL845472.2 and application thereof in lung cancer diagnosis and treatment, in particular to a reagent for detecting AL845472.2 by using a QPCR method. The invention also relates to the use of a double-stranded molecule targeting AL845472.2 to transfect squamous lung cancer cells to reduce the proliferative activity and migratory capacity of the cancer cells.

Description

Reagent for detecting and targeting AL845472.2 and application of reagent in lung cancer diagnosis and treatment

Technical Field

The invention belongs to the field of biological medicines, and relates to a reagent for detecting and targeting AL845472.2 and application thereof in lung cancer diagnosis and treatment.

Background

In the global scope, the incidence and the mortality of lung cancer are the first rank among various malignant tumors, and the incidence and the mortality of lung cancer in China also show the trend of increasing year by year. Data of Chinese tumor registration center in 2017 show that lung cancer is the first of malignant tumors in incidence and mortality (Wangwetong 2016. J. clinical medicine literature journal of Chinese malignant tumor incidence and mortality analysis, 2017,4(19): 3604.). Although the country has increased the investment in lung Cancer prevention and treatment, the majority of medical workers have made great efforts, but most of the diagnosed lung Cancer patients are in a relatively advanced stage (Fordepm, ethiger ds. targeted therapy for non-transformed cell lung Cancer: past, present and future [ J ]. Expert Rev Anticancer Ther,2013,13:745758.), which is one of the main reasons for the low long-term survival rate of the patients in China, and the five-year survival rate of the lung Cancer patients is only 18% (Siegel RL, Miller KD, Jemal A. Cancer statistics,2017[ J. CA Cancer J CIin,2017,67(1): 7-30.). Therefore, it is necessary to further enhance the popularization and education of lung cancer prevention measures, and research personnel and clinicians in medical field are required to develop new lung cancer screening and diagnosis methods to improve the lung cancer, especially the diagnosis efficiency of early lung cancer, and thus improve the therapeutic effect of lung cancer.

Generally, when lung cancer is clinically classified, it is classified into non-small cell lung cancer (NSCLC) and Small Cell Lung Cancer (SCLC). The former accounts for about 80-85% of the total number of lung cancer patients, and the latter accounts for about 15-20%. The traditional methods for treating lung cancer include surgery, radiotherapy, chemotherapy, immunotherapy and traditional Chinese medicine. In recent years, a significant advance in the treatment of NSCLC has been the advent of molecularly targeted drugs, which significantly extend the survival of patients with advanced lung cancer, achieving a favorable therapeutic effect (Antonia S, Goldberg SB, Balmonukan A, et al.safety and anti activity of a reduced cancer tremelimumab in non-small cell lung cancer: a multiple, phase 1b study [ J ]. Lancet Onco1,2016,17(3): 299-308), although the overall survival rate for patients with lung cancer remains low for a long period of time. Clinical evidence shows that the 5-year survival rate of the patients with stage I lung cancer can reach 60-70 percent after operation, which is obviously higher than that of the patients with stage II or III lung cancer. Therefore, the early discovery of lung cancer and the search of accurate and reliable biological tumor markers become important tasks in clinical diagnosis and treatment. The molecular biological mechanism of the lung cancer in the occurrence and development process is thoroughly and clearly elucidated, and the method is of great importance for early screening, diagnosis and treatment, prognosis judgment, new medicine research and development, long-term survival rate improvement, life quality improvement and the like of the lung cancer.

Many findings suggest that lncRNA plays an important role in the development and progression of malignant tumors. Although conventional methods widely used clinically at present have certain sensitivity and accuracy for detecting early malignant tumors, lncRNA expression level detection can complement these conventional methods and has shown potential to be a novel high-sensitivity biological oncology marker.

Disclosure of Invention

In order to overcome the defects of the prior art, the invention aims to provide a molecular marker for diagnosing and treating lung cancer.

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

the invention provides application of a reagent for detecting AL845472.2 in preparation of a product for diagnosing lung cancer.

Further, the expression level of AL845472.2 was significantly up-regulated in cancer samples.

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 AL 845472.2.

Further, the reagent comprises a probe or primer directed to the transcript of the AL845472.2 gene,

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

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

The invention provides application of AL845472.2 in preparing a pharmaceutical composition for treating lung cancer and/or lung cancer metastasis.

Further, the pharmaceutical composition includes an inhibitor of AL 845472.2.

Further, the inhibitor is a double-stranded molecule.

Further, the double-stranded molecule is siRNA.

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

The present invention provides a pharmaceutical composition for treating lung cancer and/or lung cancer metastasis, comprising an inhibitor of AL 845472.2. The inhibitor down-regulates the expression level of AL 845472.2.

Wherein the inhibitor is selected from: an interfering molecule targeting AL845472.2 or its transcript and capable of inhibiting AL845472.2 gene expression or gene transcription, comprising: shRNA (small hairpin RNA), small interfering RNA (sirna), dsRNA, microrna, antisense nucleic acid, or a construct capable of expressing or forming said shRNA, small interfering RNA, dsRNA, microrna, antisense nucleic acid.

Further, the inhibitor is a double-stranded molecule.

Further, the double-stranded molecule is siRNA.

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

The present invention provides a pharmaceutical composition for treating lung cancer and/or lung cancer metastasis, comprising an inhibitor of AL 845472.2.

Further, the inhibitor is a double-stranded molecule.

Further, the double-stranded molecule is siRNA.

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

The present invention provides a method for screening a candidate substance for treating or preventing lung cancer or inhibiting the growth of cancer cells, the method comprising the steps of:

1) contacting a test agent with a cell expressing an AL845472.2 gene; and are

2) Selecting a test agent that reduces the expression level of the AL845472.2 gene as compared to the expression level detected in the absence of the test agent.

Drawings

FIG. 1 is a graph showing the detection of the expression of AL845472.2 gene in lung cancer tissues by QPCR;

FIG. 2 is a graph showing the effect of siRNA on AL845472.2 expression;

FIG. 3 is a graph of the effect of scratch test assay AL845472.2 on lung cancer cell migration.

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 "biomarker" refers to an indicator molecule that can be detected in a sample and includes, for example, AL845472.2 or a collection thereof with other molecules (e.g., predictive, diagnostic, and/or prognostic indicators). A biomarker may be a predictive biomarker and serve as an indicator for a patient with a particular disease or disorder, such as a proliferative cell disorder (e.g., cancer). Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA (e.g., mRNA)), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides, polypeptide and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers. In some embodiments, the biomarker is a gene.

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.

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

By "differential expression increase" or "upregulation" is meant that gene expression (as measured by RNA expression or protein expression) exhibits an increase of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more or 1.1-fold, 1.2-fold, 1.4-fold, 1.6-fold, 1.8-fold or more, of the gene relative to a control.

By "differential expression reduction" or "down-regulation" is meant a gene whose expression (as measured by RNA expression or protein expression) exhibits a reduction in gene expression relative to a control of at least 10% or more, e.g., 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or less than 1.0-fold, 0.8-fold, 0.6-fold, 0.4-fold, 0.2-fold, 0.1-fold or less. For example, an up-regulated gene includes a gene that has an increased level of expression of RNA or protein in a sample isolated from an individual characterized as having lung cancer, as compared to the expression of RNA or protein isolated from a normal individual. For example, a down-regulated gene includes a gene that has a reduced level of RNA or protein expression in a sample isolated from an individual characterized as having lung cancer, as compared to a sample isolated from a normal individual.

AL845472.2

The gene for transcription of AL845472.2 is located on human chromosome 9, and AL845472.2 in the present invention includes wild type, mutant type or fragment thereof. One skilled in the art will appreciate that in performing sequencing analysis, the original sequencing results will be aligned to the human reference genome, and therefore AL845472.2 in the screening results may contain different transcripts as long as it can be aligned to AL845472.2 on the reference genome. In the examples of the present invention, the nucleotide sequence of a representative transcribed AL845472.2 gene is shown in ENST 00000633881.1.

According to the present invention, the expression level of AL845472.2 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 probe can be used to quantitate RNA of AL845472.2 by hybridization methods (e.g., Northern hybridization). The detection may be performed on a chip or array. For detecting the expression level of AL845472.2, the use of an array is preferred. The skilled artisan can use the sequence information of AL845472.2 to prepare such probes. For example, cDNA of AL845472.2 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 AL845472.2 RNA 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.

In the present invention, the term "double-stranded molecule" refers to a nucleic acid molecule that inhibits expression of a target gene, and includes, for example, short interfering RNAs (siRNAs; e.g., double-stranded ribonucleic acids (dsRNA) or small hairpin RNAs (shRNAs)) and short interfering DNAs/RNAs (siD/R-NA; e.g., a double-stranded chimera of DNA and RNA (dsD/R-NA) or a small hairpin chimera of DNA and RNA (shD/R-NA)). Herein, "double-stranded molecule" is also referred to as "double-stranded nucleic acid", "double-stranded nucleic acid molecule", "double-stranded polynucleotide molecule", "double-stranded oligonucleotide", and "double-stranded oligonucleotide molecule".

The term "target sequence" refers to a stretch of nucleotide sequence within the RNA or cDNA sequence of a target gene that, if a double-stranded molecule targeted to that sequence is introduced into a cell expressing the target gene, results in repression of transcription of the entire gene of the target gene. A certain nucleotide sequence of a gene can be identified as a target sequence if a double-stranded molecule comprising a sequence corresponding to the target sequence inhibits expression of the gene in a cell expressing the gene. Double-stranded polynucleotides that repress gene expression may consist of a target sequence and a 3' overhang (e.g., uu) of 2 to 5 nucleotides in length.

When the target sequence is revealed by the cDNA sequence, the sense strand sequence of the double-stranded cDNA (i.e., the sequence from which the RNA sequence is converted to the DNA sequence) is used to define the target sequence. A double-stranded molecule comprises a sense strand (which has a sequence corresponding to a target sequence) and an antisense strand (which has a sequence complementary to the target sequence), and the antisense strand hybridizes to the sense strand at the complementary sequence to form the double-stranded molecule.

The term "siRNA" as used herein refers to the prevention of overexpression of a target gene. Standard techniques for introducing siRNA into cells are used, including those in which RNA is transcribed using DNA as a template. The siRNA includes a sense nucleic acid sequence (also referred to as "sense strand"), an antisense nucleic acid sequence (also referred to as "antisense strand"), or both. The siRNA can be constructed such that a single transcript has an antisense nucleic acid sequence to which the sense nucleic acid sequence of the target gene is complementary, e.g., a hairpin structure. The siRNA may be dsRNA or shRNA.

The term "dsRNA" as used herein refers to a construct comprising two RNA molecules of mutually complementary sequences that anneal through the complementary sequences to form a double stranded RNA molecule.

In the present invention, the term "shRNA" means: an siRNA having a stem-loop structure comprising a first region and a second region (i.e., a sense strand and an antisense strand) that are complementary to each other. The degree and orientation of complementarity of the two regions is sufficient to allow base pairing to occur between the two regions, the first and second regions being joined by a loop region formed by the lack of base pairing between nucleotides (or nucleotide analogs) within the loop region. The loop region of the shRNA is a single-stranded region between the sense strand and the antisense strand, and may also be referred to as an "intervening single-strand".

Can be prepared by contacting the cell with a double-stranded molecule directed against the AL845472.2 gene, a vector expressing the molecule or a vector comprising the molecule

The composition of the same molecule is contacted to inhibit the growth of a cell expressing the AL845472.2 gene. The cells may be further contacted with a transfection agent. Suitable transfection agents are known in the art. The phrase "inhibiting cell growth" means that the cell proliferates at a lower rate or has reduced viability compared to a cell not exposed to the molecule. Cell growth can be determined by techniques known in the art, for example using CCK-8, MTT cell proliferation assays.

Any kind of cell can be repressed according to the present method, so long as the cell expresses or overexpresses the target gene of the double-stranded molecule of the present invention. Exemplary cells include lung cancer cells, such as NSCLC and SCLC.

According to the methods of the invention, to inhibit cell growth and thereby treat cancer, when a plurality of such double-stranded molecules (or vectors expressing the same or compositions containing the same) are administered, each such molecule may have a different structure, but act on RNA that matches the same target sequence. Alternatively, multiple double stranded molecules can act on RNAs that match different target sequences of the same gene, or on RNAs that match different target sequences of different genes. For example, the methods may use double stranded molecules directed against different target sequences of the AL845472.2 gene. Alternatively, for example, the method may utilize double stranded molecules directed against one, two or more target sequences of the AL845472.2 gene and other genes.

To inhibit cell growth, the double-stranded molecules of the invention can be introduced directly into the cell in a form that permits binding of the molecule to the corresponding RNA transcript. Alternatively, as described above, DNA encoding the double-stranded molecule can be introduced into cells as a vector. To introduce the double-stranded molecule and the vector into the cell, a transfection-enhancing agent may be used.

A treatment is considered "effective" when it results in a clinical benefit, such as a decrease in AL845472.2 gene expression, a decrease in the size, prevalence (prevalence), or metastatic potential of the cancer in the subject. When applied prophylactically to a treatment, "effective" means that it delays or prevents the formation of cancer, or prevents or alleviates the clinical symptoms of cancer. Effectiveness is determined in conjunction with any known diagnostic or therapeutic method for a particular tumor type.

Treating and/or preventing cancer and/or preventing postoperative recurrence thereof includes any of the steps such as surgically removing cancer cells, inhibiting cancerous cell growth, tumor regression or regression, inducing cancer regression and suppressing carcinogenesis, tumor regression, and reducing or inhibiting metastasis. Effective treatment and/or prevention of cancer can reduce mortality and improve prognosis in individuals with cancer, reduce the levels of tumor markers in their blood, and reduce detectable symptoms associated with cancer. For example, a reduction or amelioration of symptoms constitutes an effective treatment and/or prevention, including a 10%, 20%, 30% or more reduction, or a stable condition.

For the treatment of cancer, the double-stranded molecules of the invention may also be administered to a subject in combination with an agent different from the double-stranded molecule. Alternatively, the double-stranded molecules of the invention may also be administered to a subject in combination with other therapeutic methods intended for the treatment of cancer. For example, the double-stranded molecules of the invention can be administered in combination with current therapeutic methods for treating cancer or preventing cancer metastasis (e.g., radiation therapy, surgery, and treatment with chemotherapeutic agents such as cisplatin, carboplatin, cyclophosphamide, 5-fluorouracil, doxorubicin, daunorubicin, or tamoxifen, etc.).

In the methods of the invention, the double-stranded molecule may be administered to the subject as a naked double-stranded molecule, in combination with a delivery substance, or as a recombinant plasmid or viral vector expressing the double-stranded molecule.

Suitable delivery materials for administration in combination with a double stranded molecule of the invention include a Mirus Transit TKO lipophilic material, Lipofectin, Lipofectamine, Cellffectin, or a polycation (e.g.polylysine), or a liposome. One preferred delivery material is a liposome.

Liposomes can help deliver the duplex to a specific tissue, such as lung tumor tissue, and can also increase the blood half-life of the duplex. Liposomes suitable for use in the present invention are formed from conventional vesicle-forming lipids (vesicular-forming lipids), which typically include neutral or negatively charged phospholipids, as well as sterols, such as cholesterol. Consideration of several factors may generally provide guidance for the selection of lipids, such as the desired liposome size and half-life of the liposomes in the bloodstream.

The double-stranded molecules of the invention can be administered to a subject by any means suitable for delivering the double-stranded molecule to a cancer site. For example, the double-stranded molecule can be administered by gene gun, electroporation, or other suitable parenteral or enteral routes of administration.

Suitable enteral routes of administration include oral, rectal, or intranasal delivery.

Suitable routes of parenteral administration include intravascular administration (e.g., intravenous bolus, intravenous infusion, intra-arterial bolus, intra-arterial infusion, and catheter instillation to the vascular network), peri-and intra-tissue injection (e.g., peri-and intra-tumoral injection), subcutaneous injection or deposition, including subcutaneous infusion (e.g., using an osmotic pressure pump), direct application to the site of cancer or an area near it, e.g., via a catheter or other placement device (e.g., a suppository or implant comprising a porous, non-porous, or gelatinous material), and inhalation. Preferably, the double-stranded molecule or vector is administered to or near the cancer site by injection or infusion.

The double-stranded molecules of the invention may be administered in a single dose or in divided doses. When the administration of the double-stranded molecule of the invention is by infusion, the infusion may be a single continuous dose, or may be administered by multiple infusions. It is preferred to inject the agent directly into the tissue at or near the site of cancer. It is particularly preferred to inject the agent multiple times into the tissue at or near the site of the cancer.

In the present invention, "drug" and "pharmaceutical composition" may be used in general. The pharmaceutical compositions of the present invention are characterized as being at least sterile and pyrogen free.

As a preferred embodiment, the pharmaceutical composition of the invention comprises at least one double-stranded molecule of the invention or a carrier encoding them (for example, from 0.1% to 90% by weight), or a physiologically acceptable salt of said molecule, in admixture with a physiologically acceptable carrier medium. The physiologically acceptable carrier medium is preferably water, buffered water, physiological saline, 0.4% saline, 0.3% glycine, hyaluronic acid and the like.

According to the invention, the composition may comprise a plurality of double-stranded molecules, each of which may be directed against a different target sequence of the same gene or a different target sequence of a different gene. For example, the composition may comprise a double-stranded molecule directed against an AL845472.2 gene target sequence. Alternatively, for example, the composition may comprise a double-stranded molecule directed against one, two or more target sequences of the AL845472.2 gene and other genes.

The composition of the invention may be a pharmaceutical composition. The pharmaceutical compositions of the present invention may also contain conventional pharmaceutical excipients and/or additives. Suitable pharmaceutical excipients include stabilizers, antioxidants, osmotic pressure regulators, buffers, and pH regulators. Suitable additives include: a physiologically biocompatible buffer (e.g., trometamol hydrochloride), supplemental chelator (e.g., DTPA or DTPA-bisamide, etc.), or a calcium chelator complex (e.g., calcium DTPA, CaNaDTPA-bisamide), or, optionally, supplemental calcium or sodium salt (e.g., calcium chloride, calcium ascorbate, calcium gluconate, or calcium lactate). The pharmaceutical compositions of the present invention may be packaged for use as a liquid or may be lyophilized.

For solid compositions, conventional non-toxic solid carriers may be used; for example, pharmaceutical grades of mannitol, lactic acid, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.

For example, any of the carriers and excipients listed above, as well as 10-95%, preferably 25-75% of one or more double stranded molecules of the invention may be included in a solid pharmaceutical composition for oral administration. Pharmaceutical compositions for aerosol (inhalation) administration may comprise 0.01-20 wt%, preferably 1-10 wt%, of one or more of the double-stranded molecules of the invention coated in liposomes as described above, and a propellant. Carriers such as lecithin for intranasal delivery and the like may also be included as desired.

In addition to the above, other pharmaceutically active ingredients may be included in the present compositions, as long as they do not inhibit the in vivo function of the present double-stranded molecule. For example, the above composition may contain a chemotherapeutic agent conventionally used for cancer treatment.

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 AL845472.2 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

A QPCR amplification primer is designed according to coding sequences of AL845472.2 genes and GAPDH genes in Genebank, wherein a primer sequence for amplifying AL845472.2 genes is shown as SEQ ID NO. 1-2 (F: 5'-CACCTCCTTAGCAACATT-3'; R: 5'-CTCCGAAGAAGATACACAA-3'), and a primer pair sequence for amplifying reference genes 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 32s) multiplied by 45 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 AL845472.2 to determine the diagnostic potency, sensitivity and specificity of the gene.

5. Results

The QPCR results are shown in fig. 1, and compared with the paracarcinoma tissues, AL845472.2 was up-regulated in lung cancer tissues by about 9.3-fold, and the difference was statistically significant (P <0.05), suggesting that AL845472.2 has a higher application value in the diagnosis of lung cancer.

ROC curve analysis shows that AL845472.2 can be used as a biomarker for diagnosing squamous cell lung carcinoma. The area under the curve is 0.917, and patients with squamous cell lung carcinoma can be effectively distinguished.

Example 2 silencing of AL845472.2 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. Design of siRNA

Synthesis of siRNA directed against AL845472.2 gene, siRNA-AL845472.2 and general negative control siRNA-NC were purchased from Shanghai Jima Genenco chemical technology, Inc. The sequence of the siRNA against AL845472.2 is F: 5'-AAAAUGCAAGACAAAACCCAA-3' (SEQ ID NO. 5); r: 5'-GGGUUUUGUCUUGCAUUUUAU-3' (SEQ ID NO.6) (siRNA 1); sense strand: 5'-AUACACAAAUGUGUAAGACGG-3' (SEQ ID NO.7), antisense strand: 5'-GUCUUACACAUUUGUGUAUCU-3' (SEQ ID NO.8) (siRNA2)

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 siRNA-NC), and an experimental group (siRNA1, siRNA2), and after labeling groups, the cells were placed at 37 ℃ in 5% CO₂The cells are cultured in the incubator overnight, the growth state of the cells is observed the next day, and when the fusion degree of the cells reaches 70-80%, the transfection is carried out. The medium in the 24-well plate was removed and washed 2 times with 1 × PBS. Then 0.5ml serum-free is addedDMEM complete medium, cells were starved for 3 h. One of the tubes was filled with Opti-MEM Medium 50. mu.l + RNAIMAMAX 3. mu.l, and the other tube was filled with Opti-MEM Medium 50. mu.l + siRNA 1. mu.l, carefully mixed well, and left to stand for 5 min. The solutions in the two EP tubes were then mixed well and incubated for 5min at room temperature. Then the mixture was uniformly poured into a 24-well plate and left at 37 ℃ with 5% CO₂Culturing for 4-6 h in a cell culture box, and continuously culturing by replacing with a conventional culture medium containing serum and antibiotics.

4. QPCR detection of transcription level of AL845472.2 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, compared with the blank control group and the negative control group, the experimental group (siRNA 1-2) can significantly reduce the level of AL845472.2 (P <0.05), and the blank control group and the negative control group have no significant difference; among them, siRNA2 was the most effective, and therefore siRNA2 was selected for subsequent experiments.

Example 3 Effect of AL845472.2 on Lung cancer cells

1) MTT assay to examine the effect of AL845472.2 on proliferation of lung squamous carcinoma cells:

washing transfected cell strain with PBS solution, removing dead cell, digesting with pancreatin to prepare single cell suspension,the cell suspension was diluted to a density of 5X 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.

MTT results show that the cell growth and proliferation rate (0.413 +/-0.026) of the transfected siRNA2 group is remarkably lower than that of a blank control group (0.598 +/-0.022) and a transfected siRNA-NC (0.586 +/-0.017) group (P <0.05), and no remarkable difference exists between the blank control group and the transfected siRNA-NC group, which indicates that the proliferation of the lung squamous carcinoma cells can be remarkably inhibited by down-regulating the expression level of AL 845472.2.

2) Scratch test the effect of AL845472.2 on the migratory capacity of squamous cell lung 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 × 10⁵And each cell/ml is obtained by drawing a transverse line on the back of each culture dish by using a marking pen as a mark, adding 1ml of cell suspension into each culture dish, shaking uniformly, putting the culture dish into an incubator for culture, performing the next operation when the cell coverage area is 95%, marking a linear scratch in the middle of a monolayer by using a 100 mu l flash pipette tip, washing the cells for 3 times by using PBS (phosphate buffer solution), adding a DMEM (dimethyl ether medium) culture medium, and putting the cells into the incubator for culture. 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.

As shown in fig. 3, compared with the blank control group and the siRNA-NC group, the cell migration ability of squamous cell lung carcinoma was significantly decreased after the group transfected with siRNA2 down-regulated the expression level of AL845472.2 in squamous cell lung carcinoma cells (P <0.05), suggesting that interfering with the expression of AL845472.2 could significantly inhibit the migration ability of squamous cell lung 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

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Claims (10)

1. Use of a reagent for detecting AL845472.2 in the manufacture of a product for diagnosing lung cancer.

2. The use according to claim 1, wherein the expression level of AL845472.2 is significantly upregulated in cancer samples.

3. Use according to claim 2, wherein the sample comprises tissue, sputum, blood, pleural effusion or urine, preferably the sample is tissue.

4. A product for diagnosing lung cancer, comprising reagents for detecting AL 845472.2.

5. The product according to claim 4, wherein the reagent comprises a probe or primer directed against the transcript of the AL845472.2 gene, preferably wherein the sequence of the primer is as shown in SEQ ID No. 1-2.

6. The product of claim 4 or 5, wherein the product comprises a chip, a kit or a nucleic acid membrane strip.

Use of AL845472.2 in the preparation of a pharmaceutical composition for the treatment of lung cancer and/or lung cancer metastases.

8. The use according to claim 7, wherein the pharmaceutical composition comprises an inhibitor of AL845472.2, preferably wherein the inhibitor is a double-stranded molecule; preferably, the double-stranded molecule is siRNA; preferably, the sequence of the siRNA is shown in SEQ ID NO. 5-6.

9. A pharmaceutical composition for treating lung cancer and/or lung cancer metastasis, comprising an inhibitor of AL845472.2, preferably wherein the inhibitor is a double-stranded molecule; preferably, the double-stranded molecule is siRNA; preferably, the sequence of the siRNA is shown in SEQ ID NO. 5-6.

10. A method of screening a candidate substance for treating or preventing lung cancer or inhibiting the growth of cancer cells, comprising the steps of:

1) contacting a test agent with a cell expressing an AL845472.2 gene; and are

2) Selecting a test agent that reduces the expression level of the AL845472.2 gene as compared to the expression level detected in the absence of the test agent.

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