CN111850130B - Application of long-chain non-coding RNA in lung cancer diagnosis and treatment - Google Patents
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Abstract
The invention provides application of long-chain non-coding RNA in lung cancer diagnosis and treatment, wherein the long-chain non-coding RNA is RP11-720L 2.4. The research of the invention shows that the expressions of RP11-720L2.4 in lung cancer tissues and tissues beside the lung cancer tissues are obviously different, and the change of RP11-720L2.4 can regulate the proliferation of lung cancer cells, so that RP11-720L2.4 can be used as a lung cancer diagnosis and treatment marker for clinical application.
Description
Technical Field
The invention belongs to the field of biomedicine, and relates to application of long-chain non-coding RNA in lung cancer diagnosis and treatment.
Background
According to the data published in 2018 of the global Cancer statistics report (SIEGEL R L, ILLER K D, JEMAL A. Cancer statistics, 2018[ J ]. CA: a Cancer journal for clinicians, 2018, 68(1):7-30), new lung Cancer cases rank second in tumors and death cases rank first. Currently, lung cancer still lacks effective and accurate early-screening biomarkers and preventive targets, has a low early diagnosis rate and a high late-stage recurrence and metastasis rate, and lacks sensitive and stable clinical drugs, whereby the overall survival rate of patients is low (CHEN W J, GAN T Q, QIN H, et al. Imperial of downward diagnosis and pro-active pathway signaling of microRNA-375in long squamous cell cancer [ J ]. Pathology, research and practice, 2017, 213(4): 364-72).
The majority of lung cancers are non-small cell lung cancers (NSCLC), which comprise two major histological subtypes: squamous cell carcinoma of the Lung (LUSC) and adenocarcinoma of the Lung (LUAD) (TORRE L A, SIEGEL R L, JEMAL A. Lung Cancer Statistics [ J ]. Advances in experimental media and biology, 2016, 893: 1-19).
Tumor development is a multifactorial, long-term, and multistage process, and like other tumor characteristics, the development of squamous cell lung carcinoma is regulated by multifactorial factors, such as apoptosis-related gene expression regulation, posttranslational protein modification, molecular interaction, signal pathway and feedback, DNA damage repair and damaged cell clearance, oncogene activation and oncogene suppressor silencing. In recent years, the role of epigenetic molecular regulation in the development of squamous cell lung carcinoma has been more and more emphasized, and epigenetics is the study of gene expression changes based on non-gene sequence changes. Researches find that in the occurrence and development process of the squamous cell lung carcinoma, epigenetic key gene/protein changes exist, so that related pathways are influenced, and finally, different degrees of influences are generated on the proliferation, apoptosis and cycle of cells. The research in the field of epigenetic regulation includes DNA methylation, micrornas (mirnas), long non-coding RNAs (incrnas), circular RNAs (circrnas), and the like.
Based on the knowledge, the research progress and early-stage research foundation of the latest lung cancer markers at home and abroad are integrated, and lncRNA is used as an entry point to explore lncRNA related to lung cancer diagnosis and treatment and explore the possible role of lncRNA in lung cancer pathogenesis.
Disclosure of Invention
The invention provides application of a reagent for detecting the expression of RP11-720L2.4 in preparing a product for diagnosing lung cancer.
The RP11-720L2.4 sequence can be queried at the Ensembl website.
Further, the reagent comprises PCR amplification primers used for detecting the expression quantity of RP11-720L2.4 by using SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes or composite probes.
In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.
The invention provides a product for lung cancer diagnosis, which comprises a reagent for detecting the expression level of RP11-720L 2.4.
Further, the reagent comprises a PCR amplification primer used for detecting the expression quantity of RP11-720L2.4 by SYBR Green, a TaqMan probe, a molecular beacon, a double-hybridization probe or a composite probe.
In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.1 and SEQ ID NO. 2.
Further, the aforementioned products include, but are not limited to, chips, kits, test strips, or high throughput sequencing platforms; the high-throughput sequencing platform is a special tool for diagnosing lung cancer, and with the development of a high-throughput sequencing technology, the construction of an RNA expression profile of a person becomes very convenient work. By comparing the RNA expression profiles of patients with disease and normal populations, it is easy to identify which RNA abnormalities are associated with disease. Therefore, the fact that the expression abnormality of RP11-720L2.4 is related to lung cancer in high-throughput sequencing also belongs to the application of RP11-720L2.4 and is also within the protection scope of the invention.
The kit comprises a reagent for detecting the expression quantity of RP11-720L2.4, wherein the reagent comprises nucleic acid combined with RP11-720L2.4 or a DNA sequence thereof, and the nucleic acid comprises a PCR amplification primer used when SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a composite probe is used for detecting the expression quantity of RP11-720L 2.4.
The chip comprises a reagent for detecting the expression level of RP11-720L2.4, wherein the reagent comprises nucleic acid combined with RP11-720L2.4 or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of RP11-720L 2.4.
The test paper comprises a reagent for detecting the expression level of RP11-720L2.4, the reagent comprises nucleic acid combined with RP11-720L2.4 or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of RP11-720L 2.4.
The invention provides a pharmaceutical composition for treating lung cancer, which comprises an agent promoting the expression of RP11-720L 2.4.
Further, the agent is not limited as long as it can promote the expression level of RP11-720L 2.4.
Still further, the agent comprises an RP11-720L2.4 overexpression vector.
The pharmaceutical composition of the present invention may be administered alone or together with other drugs as a medicine. Other drugs that can be administered with the pharmaceutical composition of the present invention are not limited as long as it does not impair the effect of the therapeutic or prophylactic pharmaceutical composition of the present invention, and preferably, drugs for treating or preventing tumors may include, for example, alkylating agents such as ifosfamide, cyclophosphamide, dacarbazine, temozolomide, nimustine, busulfan, procarbazine, melphalan, and ramustine; antimetabolites such as enocitabine, capecitabine, carmofur, cladribine, gemcitabine, cytarabine octadecylphosphate (cytarabine ocfosfate), tegafur-uracil, tegafur-gimeracil oteracil potassium, doxifluridine, hydroxyurea, fluorouracil, fludarabine, pemetrexed, pentostatin, mercaptopurine, and methotrexate; plant alkaloids such as irinotecan, etoposide, sobuzolff, docetaxel, nogitecan, paclitaxel, vinorelbine, vindesine, and vinblastine; anticancer antibiotics such as actinomycin D, aclarubicin, amrubicin, idarubicin, epirubicin, netostatin stimalamer, daunorubicin, doxorubicin, pirarubicin, bleomycin, pellomycin, mitomycin C, and mitoxantrone; platinum-based drugs such as oxaliplatin, carboplatin, cisplatin, and nedaplatin; hormonal agents such as anastrozole, exemestane, estramustine, ethinylestradiol, chlormadinone, goserelin, tamoxifen, dexamethasone, toremifene, bicalutamide, flutamide, prednisolone, fosfestrol, mitotane, methyltestosterone, medroxyprogesterone, meindroxane, leuprorelin, and letrozole; biological response modifiers such as interferon alpha, interferon beta, interferon gamma, interleukin, ubenimex, dry BCG, and lentinan; and molecularly targeted drugs such as imatinib (imatinib), gefitinib (gefitinib), gemumab, ozomicin, tamibarotene, trastuzumab, tretinoin, bortezomib (bortezomib), and rituximab, and the like.
The pharmaceutical composition of the invention can be prepared into various dosage forms according to requirements. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.
The route of administration of the pharmaceutical composition of the present invention is not limited as long as it can exert the desired therapeutic or prophylactic effect, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.
The dosage of the pharmaceutical composition of the present invention is not limited as long as the desired therapeutic effect or prophylactic effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like. The dose of the therapeutic or prophylactic pharmaceutical composition of the present invention can be determined using, for example, the therapeutic effect or prophylactic effect on a disease as an index.
The invention also provides application of RP11-720L2.4 in constructing a calculation model for lung cancer diagnosis.
As the skilled person will be aware, the step of associating a marker level with a certain likelihood or risk may be carried out and carried out in different ways. Preferably, the measured concentrations of the marker and one or more other markers are mathematically combined and the combined value is correlated to the underlying diagnostic problem. The determination of marker values may be combined by any suitable prior art mathematical method.
Preferably, the mathematical algorithm applied in the marker combination is a logarithmic function. Preferably, the result of applying such a mathematical algorithm or such a logarithmic function is a single value. Such values can be readily correlated, based on underlying diagnostic questions, with, for example, an individual's risk for lung cancer or with other intentional diagnostic uses that help in assessing patients with early stage lung cancer. In a preferred manner, such a logarithmic function is obtained as follows: a) classifying individuals into groups, e.g., normal, at risk for lung cancer, patients with lung cancer, etc., b) identifying markers that differ significantly between these groups by univariate analysis, c) logistic regression analysis to assess independent difference values of the markers that can be used to assess these different groups, and d) constructing a logistic function to combine the independent difference values. In this type of analysis, the markers are no longer independent, but represent a combination of markers.
The logarithmic function used to correlate marker combinations with disease preferably employs algorithms developed and obtained by applying statistical methods. For example, suitable statistical methods are Discriminant Analysis (DA) (i.e., linear, quadratic, regular DA), Kernel methods (i.e., SVM), nonparametric methods (i.e., k-nearest neighbor classifiers), PLS (partial least squares), tree-based methods (i.e., logistic regression, CART, random forest methods, boosting/bagging methods), generalized linear models (i.e., logistic regression), principal component-based methods (i.e., SIMCA), generalized additive models, fuzzy logic-based methods, neural network-and genetic algorithm-based methods. The skilled person will not have problems in selecting a suitable statistical method to evaluate the marker combinations of the invention and thereby obtain a suitable mathematical algorithm. In one embodiment, the statistical method used to obtain the mathematical algorithm used in assessing lung cancer is selected from DA (i.e., linear, quadratic, regular discriminant analysis), Kernel method (i.e., SVM), non-parametric method (i.e., k-nearest neighbor classifier), PLS (partial least squares), tree-based method (i.e., logistic regression, CART, random forest method, boosting method), or generalized linear model (i.e., logarithmic regression).
The invention also provides application of RP11-720L2.4 in preparing a medicament for treating lung cancer.
Further, the medicament is defined as the pharmaceutical composition as described above.
The invention also provides application of RP11-720L2.4 in screening candidate drugs for treating lung cancer.
Further, the screening method is as follows:
1) treating a system expressing or containing RP11-720L2.4 with a test substance;
2) detecting the expression of RP11-720L2.4 in the system;
3) selecting a test agent that increases the expression level of RP11-720L2.4 as compared to the expression level detected in the absence of the test agent, i.e., the test agent promotes the expression level of RP11-720L2.4, indicates that the test agent is a candidate for treating lung cancer.
Further, the system is selected from: a cell system, a subcellular system, a solution system, a tissue system, an organ system, or an animal system.
The present invention also provides a method for diagnosing lung cancer, comprising the steps of:
(1) obtaining a sample from a subject;
(2) detecting the expression level of RP11-720L2.4 in the sample of the subject;
(3) the measured expression level of RP11-720L2.4 was correlated with the presence or absence of disease in the subject.
(4) A significant reduction in the expression level of RP11-720L2.4 as compared to a normal control, indicates that the subject is judged to have, or is judged to be at high risk for having, lung cancer.
The invention also provides a method for treating lung cancer, which comprises the step of promoting the expression level of RP11-720L 2.4.
In the context of the present invention, "diagnosing lung cancer" includes determining whether a subject has had lung cancer, determining whether a subject is at risk for having lung cancer, determining whether a lung cancer patient has relapsed and metastasized, determining responsiveness of a lung cancer patient to drug treatment, or determining a prognosis for a lung cancer patient.
As used herein, "treatment" encompasses treatment-related diseases or disease states in a mammal, such as a human, having the associated disease or disorder, and includes:
(1) preventing the occurrence of a disease or condition in a mammal, particularly when the mammal is susceptible to said disease condition but has not been diagnosed as having such a disease condition;
(2) inhibiting a disease or disease state, i.e., preventing its occurrence; or
(3) Alleviating the disease or condition, i.e., causing regression of the disease or condition.
The term "treatment" generally refers to the treatment of a human or animal (e.g., as applied by a veterinarian) wherein some desired therapeutic effect is achieved, e.g., inhibiting the progression of a condition (including slowing the progression, stopping the progression), ameliorating the condition, and curing the condition. Treatment as a prophylactic measure (e.g., prophylaxis) is also included. The use of a patient who has not yet developed a condition but who is at risk of developing the condition is also encompassed by the term "treatment".
The invention has the advantages and beneficial effects that:
the invention discovers that the differential expression of RP11-720L2.4 is related to the occurrence and the development of lung cancer for the first time, and whether a subject suffers from early lung cancer can be judged by detecting the expression level of RP11-720L 2.4.
The invention discloses a method for screening a candidate drug for treating lung cancer, which judges whether a test substance is the candidate drug for treating lung cancer by detecting whether the test substance can up-regulate the expression level of RP11-720L 2.4.
Drawings
FIG. 1 shows a ROC plot of RP11-720L2.4 for distinguishing lung adenocarcinoma from normal;
FIG. 2 shows a ROC plot of RP11-720L2.4 distinguishing squamous cell lung carcinoma from normal;
FIG. 3 shows a statistical chart of the detection of RP11-720L2.4 overexpression using QPCR;
FIG. 4 is a graph showing the results of using CCK8 to examine the effect of RP11-720L2.4 expression on lung cancer cell proliferation.
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.
Example 1 lncRNA screening for association with Lung cancer
1. Screening using TCGA database
TCGA squamous cell carcinoma LUSC data tumor tissue 220 cases, and paracarcinoma tissue 17 cases;
TCGA lung adenocarcinoma LUAD data 488 tumor tissues and 58 paracancerous tissues.
And obtaining the differentially expressed lncRNA through bioinformatics analysis.
2. Results
666 IncRNAs (P <0.05, ROC >0.8) which are abnormally expressed in lung squamous carcinoma and lung adenocarcinoma are found together, wherein 224 IncRNAs are abnormally expressed in tumor tissues and 442 IncRNAs are abnormally expressed in the tumor tissues.
Wherein, RP11-720L2.4 is expressed and down-regulated in lung squamous carcinoma and lung adenocarcinoma tissues, and the expression level is shown in Table 1.
TABLE 1 RP11-720L2.4 expression
| Average expression level in cancer tissue | Mean expression level in para-carcinoma tissue | P | |
| Adenocarcinoma of lung | 1.081486 | 2.437983 | P<0.05 |
| Squamous cell carcinoma of lung | 0.622646 | 2.470347 | P<0.05 |
The results of ROC curve analysis of the diagnostic efficacy of RP11-720L2.4 against lung adenocarcinoma are shown in FIG. 1, with an AUC value of 0.935, sensitivity at the optimal cut-off of 0.828, and specificity of 0.948.
The results of ROC curve analysis of the diagnostic efficacy of RP11-720L2.4 against squamous cell lung carcinoma are shown in FIG. 2, with an AUC value of 0.994, a sensitivity of 0.941 at the optimal cut-off point and a specificity of 1.
Example 2 RP11-720L2.4 overexpression Lung cancer cell line construction
1. Construction of RP11-720L2.4 overexpression cell line
The long non-coding RNA RP11-720L2.4 sequence was found in public databases and submitted to pcDNA3.1 by Shanghai Jima to construct LINC00324 overexpression plasmids. Coli DH5 α competent cells were used for transformation and plasmid extraction.
2. Cell culture and plasmid transfection
A549 cells were inoculated in whole medium (89% RPMI1640 medium, 10% fetal bovine serum and 1% double antibody), and cultured in incubator (incubator conditions of 37 deg.C, 5% CO)2And saturated humidity). The cell line in logarithmic growth phase was digested with Trypsin and inoculated in 6-well plate at appropriate density. Using LipofectamineTM2000 the empty plasmid pcDNA3.1 (control group) and the overexpression plasmid pcDNA3.1-RP11-720L2.4 (experimental group) were transfected.
3. Extraction of Total RNA from cells
After transfection for 48 hours, total RNA of cells was extracted by TRIzol, cells were digested with Trypsin and centrifuged to remove the supernatant, and PBS was added to resuspend the cells and centrifuged again to remove the supernatant. Adding 750 mu L of TRIzol lysis cells into the precipitate to extract total RNA, quantifying the obtained total RNA by using a spectrophotometer, detecting the purity and checking whether organic solvent pollution or degradation exists in the total RNA.
4. Detection of RP11-720L2.4 gene expression level
The reverse transcription was performed using a reverse transcription kit of TaKaRa to obtain cDNA. Then, qRT-PCR is carried out by taking the cDNA as a template to detect the gene expression quantity. The upstream primer sequence of the RP11-720L2.4 gene is 5'-AAACAAACTTTCCAGGTA-3' (SEQ ID NO.1), and the downstream primer sequence is 5'-TACAGAGCCAACATTAAC-3' (SEQ ID NO. 2). The sequence of an upstream primer of the reference gene beta-actin is as follows: 5'-TGACGTGGACATCCGCAAAG-3' (SEQ ID NO. 3); the sequence of the downstream primer is as follows: 5'-CTGGAAGGTGGACAGCGAGG-3' (SEQ ID NO. 4). The reaction system adopts a 25 mu L system, and the reaction conditions are as follows: 30s at 95 ℃; 95 ℃ 5s, 60 ℃ 30s, 40 cycles). Relative expression of lncRNA RP11-720L2.4 was analyzed by 2- Δ Δ Ct method, and Δ Ct of the experimental group and the control group was calculated and substituted to calculate 2- Δ Δ Ct (Δ Ct ═ Ct target gene — Ct reference gene, Δ Δ Ct ═ Δ Ct experimental group — Δ Ct control group).
5. Statistical analysis
Data analysis was performed using SPSS 22.0 statistical software, with the data measured in x + -s, and independent sample t-test was used for comparison between the two groups.
6. Results
The qRT-PCR technology is used for detecting the expression level of RP11-720L2.4 in the transfected cell line, which shows that the expression level of RP11-720L2.4 in the A549 cell transfected with pcDNA3.1-RP11-720L2.4 (experimental group) is obviously higher than that of the pcDNA3.1 empty plasmid control group, and the average is 79 times (P <0.05), thereby proving that the construction of the RP11-720L2.4 overexpression cell line is successful (figure 3).
Example 3 Effect of RP11-720L2.4 overexpression on proliferation of non-Small cell Lung cancer cells
1. Method of producing a composite material
Cells were plated at appropriate densities in 6-well plates using Lipofectamine, operating according to CCK-8 instructionsTM2000 transfecting pcDNA3.1-LINC00324 over-expression plasmid and pcDNA3.1 empty plasmid into A549 cells respectively, and incubating and culturing 4Cells were collected after 8h and plated at appropriate density into 96-well plates (3 replicates per group). Adding CCK-8 reagent at 1, 2, 3, 4, and 5d, respectively, dyeing and incubating for 2h, and measuring optical density value [ OD (450) at 450nm]. The experiment was repeated 3 times.
2. Statistical analysis
Data analysis was performed using SPSS 22.0 statistical software, with the data measured in x + -s, and independent sample t-test was used for comparison between the two groups.
3. Results
The results are shown in fig. 4, and after RP11-720L2.4 gene was overexpressed (experimental group), the proliferation potency of non-small cell lung cancer cell a549 was reduced compared to the empty vector control group (control group), and the difference was statistically significant (P < 0.05).
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> Tianjin City first central hospital
Application of <120> long-chain non-coding RNA in lung cancer diagnosis and treatment
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaacaaactt tccaggta 18
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
tacagagcca acattaac 18
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Claims (3)
- Application of RP11-720L2.4 in preparing a medicament for treating non-small cell lung cancer.
- 2. The use of claim 1, wherein the medicament comprises an agent that promotes the expression of RP11-720L 2.4.
- 3. The use of claim 2, wherein said agent comprises an RP11-720L2.4 overexpression vector.
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| CN110205385A (en) * | 2019-05-31 | 2019-09-06 | 北京泱深生物信息技术有限公司 | Molecular marker and its application for adenocarcinoma of lung diagnosis |
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