阅读说明：本技术 环状rna作为诊治肺癌的分子靶标 (Circular RNA as molecular target for diagnosis and treatment of lung cancer ) 是由 李磊 李晓平 李明江 张亮 廉洁 杨波 杨盼 张卫东 于 2019-12-18 设计创作，主要内容包括：本发明公开了环状RNA作为诊治肺癌的分子靶标。本发明实验证明与对照相比,hsa_circ_0089974在肺癌组织中的表达上调,故其可作为诊断肺癌的标志物。本发明利用体外细胞实验证明干扰hsa_circ_0089974可抑制癌细胞增殖,故可根据环状RNA的性质开发治疗肺癌的药物。(The invention discloses a circular RNA as a molecular target for diagnosing and treating lung cancer. The experiment of the invention proves that hsa _ circ _0089974 is up-regulated in lung cancer tissue compared with the control, so that hsa _ circ _0089974 can be used as a marker for diagnosing lung cancer. In vitro cell experiments prove that the interference hsa _ circ _0089974 can inhibit the proliferation of cancer cells, so that the medicine for treating lung cancer can be developed according to the property of circular RNA.)

Use of hsa _ circ _0089974 in the preparation of a lung cancer diagnostic product.

2. The use of claim 1, wherein hsa _ circ _0089974 is upregulated in lung cancer patients.

3. The use of claim 1 or 2, wherein the diagnostic product comprises a diagnostic reagent of hsa _ circ _ 0089974.

4. The use of claim 3 wherein the diagnostic reagents comprise reagents for detecting the expression level of hsa _ circ _0089974 by real-time fluorescent quantitative PCR.

5. A kit for detecting lung cancer, comprising primers that specifically amplify hsa _ circ _ 0089974.

6. The kit of claim 5, wherein the primers comprise an upstream primer shown as SEQ ID No.1 and a downstream primer shown as SEQ ID No. 2.

7. The kit of claim 5 or 6, wherein the kit further comprises PCR buffer, Taq enzyme, and dNTPs.

8. A pharmaceutical composition for the treatment of lung cancer, comprising an agent that inhibits hsa _ circ _ 0089974.

9. The pharmaceutical composition of claim 8, wherein the agent comprises interfering RNA.

Use of hsa _ circ _0089974 in the manufacture of a medicament for the treatment of lung cancer.

Technical Field

The invention relates to the field of biomedicine, in particular to a circular RNA serving as a molecular target for diagnosing and treating lung cancer.

Background

Circular RNA (circRNA) is a recently discovered special class of non-coding RNA, which is abundant in the eukaryotic cell cytoplasm and is produced from pre-mRNA by variable splicing.

Unlike traditional linear RNA, circRNA is a closed loop structure without a5 'terminal cap and a 3' terminal poly (a) tail, and most of circRNA consists of exon sequences and is conserved in different species. The research on circRNA has been receiving attention from more and more researchers.

With the research on circRNA, the relevance of circRNA to diseases is receiving more and more attention from researchers. The tumor is an abnormal lesion formed by clonal abnormal hyperplasia caused by that a certain cell of a local tissue loses normal regulation and control on the growth of the local tissue on the gene level under the action of carcinogenic factors of an organism. At present, tumors are one of the most important global fatalities of diseases, so the related research on the pathogenesis and treatment of the tumors is still one of the major medical topics.

In 2013, researchers of Hansen et al have proved that the circRNAs as miRNA sponges can be combined with miRNAs and inhibit the functions of the miRNAs, and further research of Ghosal et al finds that the interaction between the circRNAs and the miRNAs can influence the biological behaviors of a large number of downstream mRNAs, and related diseases can be more than 90, wherein the proportion of tumor related diseases is very high. For example, CDR1as can regulate and control the functions of miR-7 to affect the occurrence and development of tumors of important tumor-related proteins downstream of miR-7, including Epidermal Growth Factor Receptor (EGFR), Insulin receptor substrate-1 (Insulin receptor substrate 1, IRS-1), Insulin receptor substrate-2 (Insulin receptor substrate2, IRS-2), Raf1 protein kinase, P21 protein Activated kinase 1(P21-Activated kinase-1, Pak1), Activated CDC42 kinase 1(Activated CDC42 kinase 1, Ack1), proteasome activator PA28 gamma, transcription factor Yin-Yang1(YY1), Insulin-like growth factor 1receptor (Insulin-Activated factor 1receptor, IGF1R), phosphatidylkinase-3 subunit (rapamycin 3kinase, mammalian delta kinase 3) and mammalian delta kinase target (mammalian kinase 3, delta kinase 3kinase, delta kinase target of mammalian kinase, delta kinase 3, delta kinase 3, delta kinase (mammalian 3) and delta kinase target, mTOR). In addition, researchers such as Zheng Q and the like also find that the expression abundance of circHIPK3 in tumor tissues such as breast cancer, colon cancer, liver cancer, stomach cancer, kidney cancer and the like is obviously higher than that of normal tissues, the circHIPK3 can be combined with miR-124 so as to regulate and control the proliferation of tumors, and the proliferation of tumor cells can be obviously inhibited by knocking down the circRNA.

Based on the important role of circRNA tumor, the application aims to research the circRNA related to lung cancer so as to search the circRNA which can be used as a lung cancer diagnosis and treatment marker and provide a theoretical basis for clinical diagnosis of lung cancer and treatment of lung cancer.

Disclosure of Invention

One of the purposes of the invention is to provide an application of hsa _ circ _0089974 in preparing lung cancer diagnosis products.

The invention also aims to provide a kit for diagnosing lung cancer.

The invention also aims to provide a medicament for treating lung cancer.

The fourth objective of the present invention is to provide a method for diagnosing lung cancer.

In order to achieve the above object, the present invention firstly provides a method for detecting the use of hsa _ circ _0089974 in the preparation of a lung cancer diagnosis product.

Preferably, hsa _ circ _0089974 is up-regulated in lung cancer patients.

Further, the diagnostic product comprises a diagnostic reagent of hsa _ circ _ 0089974.

Still further, the diagnostic reagent includes a reagent for detecting the expression level of hsa _ circ _0089974 by a real-time fluorescent quantitative PCR method.

Preferably, the real-time fluorescent quantitative PCR method includes a SYBRGreen method and a TaqMan probe method.

The invention also provides a kit for detecting lung cancer, which comprises a primer for specifically amplifying hsa _ circ _ 0089974.

Preferably, the primers comprise an upstream primer shown as SEQ ID NO.1 and a downstream primer shown as SEQ ID NO. 2.

Preferably, the kit further comprises PCR buffer, Taq enzyme, and dNTPs.

Preferably, the kit further comprises a negative control and a positive control.

The invention also provides a pharmaceutical composition for treating lung cancer. The pharmaceutical composition comprises an agent that inhibits the expression of hsa _ circ _ 0089974.

Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier, such carriers including (but not limited to): diluents, buffers, suspensions, emulsions, granules, encapsulating agents, excipients, fillers, adhesives, sprays, transdermal absorbents, wetting agents, disintegrants, absorption enhancers, surfactants, colorants, flavors, or adsorptive carriers.

The pharmaceutical composition of the present invention can be prepared into various dosage forms as required, including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories.

The route of administration of the pharmaceutical composition of the present invention is not limited as long as it can exert a desired therapeutic or prophylactic effect, and includes, but is not limited to, oral, intravenous, intramuscular, subcutaneous, sublingual, rectal infusion, nasal spray, oral spray, transdermal topical or systemic administration to the skin.

The pharmaceutical composition can also be used together with other drugs for treating lung cancer, and the combination of a plurality of drugs can greatly improve the success rate of treatment.

Preferably, the agent that inhibits expression of hsa _ circ _0089974 comprises an interfering RNA against hsa _ circ _0089974, including but not limited to siRNA, shRNA.

The agent for inhibiting expression of hsa _ circ _0089974 can be used for inhibiting expression of endogenous hsa _ circ _0089974, and treating lung cancer caused by overexpression of endogenous products by reducing expression of endogenous products.

The present invention provides a method of diagnosing lung cancer, the method comprising detecting the level of hsa _ circ _0089974 expression in a sample from a subject.

The invention also provides a method of treating lung cancer, the method comprising inhibiting hsa _ circ _0089974 expression.

The invention also provides application of hsa _ circ _0089974 in preparation of a medicament for treating lung cancer.

Definition of

The term "detection" is used herein in the broadest sense and includes both qualitative and quantitative measurements of a target molecule. Detection includes merely identifying the presence of the target molecule in the sample and determining whether the target molecule is present at a detectable level in the sample.

The term "biomarker" as used herein refers to an indicator that can be detected in a sample, e.g., predictive, diagnostic, and/or prognostic. Biomarkers can serve as indicators of particular subtypes of disease or disorder (e.g., cancer) characterized by certain molecular, pathological, histological, and/or clinical features. In some embodiments, the biomarker is a gene. Biomarkers include, but are not limited to, polynucleotides (e.g., DNA and/or RNA), polynucleotide copy number alterations (e.g., DNA copy number), polypeptides and polynucleotide modifications (e.g., post-translational modifications), carbohydrates, and/or glycolipid-based molecular markers.

The term "expression level" refers to the amount of a biomarker in a biological sample. "expression" generally refers to the process by which information (e.g., gene-encoded and/or epigenetic) is converted into structures present and operating in a cell. Thus, as used herein, "expression" may refer to transcription into a polynucleotide, translation into a polypeptide, or even polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of a polypeptide). Transcribed polynucleotides, translated polypeptides, or fragments of polynucleotide and/or polypeptide modifications (e.g., post-translational modifications of polypeptides) should also be considered expressed, whether they are derived from transcripts generated or degraded by alternative splicing, or from post-translational processing of polypeptides, e.g., by proteolysis.

"amplification" as used herein generally refers to the process of generating multiple copies of a desired sequence. "multiple copies" means at least two copies. "copy" does not necessarily imply complete sequence complementarity or identity to the template sequence. For example, the copies may include nucleotide analogs such as deoxyinosine, intentional sequence alterations (such as sequence alterations introduced via primers comprising sequences that are hybridizable but not complementary to the template), and/or sequence errors that occur during amplification.

As used interchangeably herein, "polynucleotide" or "nucleic acid" refers to a polymer of nucleotides of any length, and includes DNA and RNA, the nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and/or analogs thereof, or any substrate that can be incorporated into the polymer by DNA or RNA polymerases or by synthetic reactionsPolynucleotides may also contain analog forms of the ribose or deoxyribose commonly known in the art, including, for example, 2 '-O-methyl-, 2' -O-allyl-, 2 '-fluoro-or 2' -azido-ribose, carbocyclic sugar analogs, α -anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, glycopyranose, glycofuranose, sedoheptulose, acyclic analogs, and alkali-free nucleoside analogs such as methylribonucleosides ₂("amide ester"), P (O) R, P (O) OR', CO OR CH ₂(formacetal)) wherein each R or R' is independently H or a substituted or unsubstituted hydrocarbyl group (1-20C), optionally containing an ether (-O-) linkage, aryl, alkenyl, cycloalkyl, cycloalkenyl or aryl hydrocarbyl (araldyl). Not all linkages in a polynucleotide need be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

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

The term "diagnosis" is used herein to refer to the identification or classification of a molecular or pathological state, disease or condition.

As used herein, the term "sample" refers to a composition obtained or derived from a subject and/or individual of interest that contains cells and/or other molecular entities to be characterized and/or identified, e.g., based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase "disease sample" or variants thereof refers to any sample obtained from a subject of interest that is expected or known to contain the cellular and/or molecular entities to be characterized. Samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, serum, plasma, vitreous humor, lymph, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, and tissue culture fluids, tissue extracts such as homogenized tissue, cell extracts, and combinations thereof.

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

As used herein, "treatment" refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and may be performed during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviating symptoms, attenuating any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the rate of disease progression, ameliorating or palliating the disease state, and remission or improving prognosis.

Drawings

FIG. 1 shows the detection of hsa _ circ _0089974 expression in lung cancer patients and normal persons using QPCR;

FIG. 2 shows a statistical graph of interference efficiency for siRNA detection using QPCR;

FIG. 3 is a graph showing the effect of MTT method on lung cancer cell proliferation of interfering with hsa _ circ _0089974 expression.

Detailed Description

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.