Disclosure of Invention
The invention aims to provide a long-chain non-coding RNA marker for diagnosing and treating gastric cancer. Experiments prove that the expression level of LINCR-0001 in gastric cancer tissues is obviously higher than that in para-cancer tissues, so that the LINCR-0001 can be used as a molecular marker for diagnosing and treating gastric cancer.
In order to test the purpose, the invention adopts the following technical scheme:
the invention provides an application of a reagent for detecting long-chain non-coding RNA expression in preparation of gastric cancer diagnosis products.
The long-chain non-coding RNA of the invention is named LINCR-0001 in NCBI, and the gene ID: 101929191, the transcript sequence for LINCR-0001 is Genbank accession No. NR _120604.1 (having a length of 545bp, corresponding to the DNA sequence shown in SEQ ID NO. 1).
Further, the reagent comprises a PCR amplification primer used for detecting the expression level of LINCR-0001 by using SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a composite probe.
In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.2 and SEQ ID NO. 3.
The invention provides a product for gastric cancer diagnosis, which comprises an agent for detecting the expression level of LINCR-0001.
Further, the reagent comprises SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a PCR amplification primer used for detecting the expression level of LINCR-0001 by a composite probe.
In a specific embodiment of the invention, the primer sequences are shown as SEQ ID NO.2 and SEQ ID NO. 3.
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 gastric 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 knowledge that the abnormality of non-LINCR-0001 is associated with gastric cancer in high-throughput sequencing is also included in the use of LINCR-0001 and is also within the scope of the present invention.
The kit comprises a reagent for detecting the expression level of LINCR-0001, wherein the reagent comprises a nucleic acid combined with the LINCR-0001 or a DNA sequence thereof, and the nucleic acid comprises SYBR Green, a TaqMan probe, a molecular beacon, a double-hybrid probe or a PCR amplification primer used when a composite probe is used for detecting the expression level of LINCR-0001.
The chip comprises a reagent for detecting the expression level of LINCR-0001, wherein the reagent comprises a nucleic acid combined with LINCR-0001 or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of LINCR-0001.
The test strip comprises a reagent for detecting the expression level of LINCR-0001, wherein the reagent comprises a nucleic acid combined with LINCR-0001 or a DNA sequence thereof, and the nucleic acid comprises a probe capable of detecting the expression level of LINCR-0001.
The present invention provides a pharmaceutical composition for treating gastric cancer, comprising an agent that inhibits LINCR-0001.
Further, the agent is not limited as long as it can inhibit the expression level of LINCR-0001 or inhibit the functional activity of LINCR-0001.
The reagent comprises siRNA or shRNA of LINCR-0001. In a specific embodiment of the invention, the siRNA sequence of LINCR-0001 is shown as SEQ ID NO.6 and SEQ ID NO. 7.
The pharmaceutical composition of the present invention may be administered alone or together with other drugs as a medicine. The other drug that can be administered together with the pharmaceutical composition of the present invention is not limited as long as it does not impair the effect of the therapeutic or prophylactic pharmaceutical composition of the present invention.
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. 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 the application of the long-chain non-coding RNA in preparing a medicament for treating gastric cancer.
The invention also provides the application of the reagent for inhibiting the long-chain non-coding RNA in preparing the medicine for treating gastric cancer.
Further, the reagent is as defined above.
The present invention also provides a method for diagnosing gastric cancer, comprising the steps of:
(1) obtaining a sample from a subject;
(2) detecting the expression level of LINCR-0001 in the subject sample;
(3) correlating the measured expression level of LINCR-0001 with the presence or absence of disease in the subject.
(4) If the expression level of LINCR-0001 is increased compared to the control, the subject is judged to have gastric cancer, or the subject is at high risk of having gastric cancer, or the gastric cancer patient is judged to have poor prognosis.
The invention also provides a method for treating gastric cancer, which comprises reducing the expression level of LINCR-0001 or inhibiting the functional activity of LINCR-0001.
The present invention also provides a method for screening a gastric cancer drug, which can determine the effect of a tumor drug on improving tumor prognosis by measuring the expression level of LINCR-0001 after adding a test drug to gastric cancer cells or at a certain period after administering the test drug to a gastric cancer model animal. More specifically, when the expression level of LINCR-0001 is decreased or normal level is restored after addition or administration of the test drug, the drug can be selected as a therapeutic drug for improving the prognosis of the tumor.
In the context of the present invention, "diagnosing gastric cancer" includes determining whether a subject has gastric cancer, determining whether a subject is at risk of having gastric cancer, determining responsiveness of a gastric cancer patient to drug treatment, or determining a prognosis for a gastric 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 discloses a molecular marker for diagnosing gastric cancer, which can be used for judging the early stage of gastric cancer occurrence and provides the survival rate of patients.
The therapeutic agent comprising an agent inhibiting LINCR-0001 of the present invention can be used as a novel therapeutic agent for gastric cancer.
Example 3 measurement of ability to inhibit LINCR-0001 expression to proliferate gastric cancer cells
1. The method comprises the following steps:
BGC-823 cells in logarithmic growth phase are taken and inoculated on a 96-well culture plate, after the cells are cultured for 12 hours, siRNA-LncRNA and siRNA-NC are transfected respectively, and after 4-6 hours, DMEM containing 10% FBS is replaced to continue culturing for 72 hours; and adding 10 mu LCCK8 to each well of each plate, incubating for 2h in an incubator, and detecting the absorbance value with the wavelength of 450nm by using a microplate reader. Each group of cells was set with 5 replicate wells, and the experiment was repeated 3 times to plot cell growth curves.
2. Statistical analysis
The experimental data are expressed as mean-squared-off standard deviations (MS-SD) using SPSS15.0 statistical software, and subjected to one-way analysis of variance (ANOVA) or t-test. P <0.05 is statistically significant for the differences.
3. Results
The results showed that 72h after transfection, the cell growth rate of siRNA-LncRNA group (1.022. + -. 0.047) was significantly decreased compared to siRNA-NC (1.546. + -. 0.052) (P < 0.05). The result shows that the inhibition of the expression of LINCR-0001 has an inhibition effect on the proliferation capacity of a gastric cancer cell strain BGC 823.
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> Beijing, the deep biometric information technology GmbH
Application of LINCR-0001 in preparation of gastric cancer diagnosis product and treatment drug
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