Application of linc01909 in preparation of medicine for treating cancer and diagnostic kit

Abstract

The application discloses application of LINC01909 in preparation of a cancer treatment drug and a diagnostic kit, and belongs to the technical field of biological medicines. The application has a promoting effect on clinical diagnosis of gastric cancer and preparation of a treatment drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to the application of LINC01909 in the preparation of cancer treatment drugs and diagnostic reagent kits. Background Technology

[0002] Gastric cancer (GC) is the second leading cause of cancer death worldwide. Its rapid progression and metastasis result in a poor prognosis. To improve early diagnosis and targeted therapy for GC, it is urgent to understand its underlying molecular mechanisms and identify new, effective biomarkers and therapeutic targets.

[0003] Long noncoding RNAs (lncRNAs) are a class of RNA molecules with transcript lengths exceeding 200 nt. Unlike other small noncoding RNAs, they can regulate RNA metabolism, protein function and activity, histone modification, and chromatin remodeling through cis (in cis) or trans (in trans) actions, and are widely involved in cell biological processes at the epigenetic, transcriptional, and post-transcriptional levels. Noncoding RNAs (ncRNAs) are RNAs that cannot be translated into proteins. They are divided into two main categories: small RNAs (<200 nt) and lncRNAs. Small ncRNAs, including small RNA molecules, siRNAs, and DNA, have been shown to play an important role in the development of gastric cancer. In recent years, many studies have shown that lncRNAs are involved in the development of various diseases and have multiple functions in a wide range of biological processes, particularly in relation to the pathogenesis of human cancer. Research shows that, in addition to these widely studied coding genes, aberrant expression of long noncoding RNAs (lncRNAs) also participates in the malignant progression of tumors. A growing body of research confirms that lncRNAs not only play a crucial regulatory role in maintaining normal physiological functions and development, but their abnormal expression is also closely linked to disease development, particularly the malignant progression of tumors. One well-known oncogenic lncRNA involved in tumor pathogenesis is HOTAIR (HOX-transcribed antisense RNA), which has been consistently upregulated and identified as a strong prognostic marker for patients, influencing the survival rate of various human cancers.

[0004] Recent studies have shown that lncRNAs are highly expressed in gastric cancer tissues. Therefore, there is an urgent need to find new lncRNAs and explore their biological effects on gastric cancer in order to identify potential therapeutic targets for diagnosis and gene therapy of gastric cancer. Summary of the Invention

[0005] The purpose of this invention is to provide a long non-coding RNA biomarker for the diagnosis and treatment of gastric cancer. This invention experimentally demonstrates that the expression level of LINC01909 in gastric cancer tissues is significantly higher than that in adjacent normal tissues; therefore, LINC01909 can be used as a molecular biomarker for the diagnosis and treatment of gastric cancer.

[0006] To achieve the above objectives, the present invention employs the following technical solution:

[0007] This invention provides the application of reagents for detecting the expression of long non-coding RNAs in the preparation of gastric cancer diagnostic products.

[0008] The long non-coding RNA of this invention is named LINC01909 in NCBI, with gene ID: 101927481. The transcript sequence of LINC01909 is Genbank accession number NR_126336.1 (with a length of 815 bp, and the corresponding DNA sequence is shown in SEQ ID NO.1).

[0009] Furthermore, the reagents include PCR amplification primers used when detecting the expression level of LINC01909 using SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or composite probes.

[0010] In a specific embodiment of the present invention, the primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0011] This invention provides a product for the diagnosis of gastric cancer, the product comprising a reagent for detecting the expression level of LINC01909.

[0012] Furthermore, the reagents include PCR amplification primers used when detecting LINC01909 expression levels using SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or composite probes.

[0013] In a specific embodiment of the present invention, the primer sequences are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0014] Furthermore, the aforementioned products include, but are not limited to, chips, reagent kits, test strips, or high-throughput sequencing platforms. High-throughput sequencing platforms are specialized tools for diagnosing gastric cancer, and with the development of high-throughput sequencing technology, constructing an individual's RNA expression profile will become very convenient. By comparing the RNA expression profiles of disease patients and healthy individuals, it is easy to analyze which RNA abnormalities are associated with the disease. Therefore, identifying non-LINC01909 abnormalities associated with gastric cancer through high-throughput sequencing also falls under the application of LINC01909 and is also within the scope of protection of this invention.

[0015] The kit includes reagents for detecting the expression level of LINC01909, the reagents including nucleic acids that bind to LINC01909 or its DNA sequence, the nucleic acids including PCR amplification primers used for detecting the expression level of LINC01909 using SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or composite probes.

[0016] The chip includes a reagent for detecting the expression level of LINC01909, the reagent including a nucleic acid that binds to LINC01909 or its DNA sequence, the nucleic acid including a probe capable of detecting the expression level of LINC01909.

[0017] The test strip includes a reagent for detecting the expression level of LINC01909, the reagent including a nucleic acid that binds to LINC01909 or its DNA sequence, the nucleic acid including a probe capable of detecting the expression level of LINC01909.

[0018] The present invention provides a pharmaceutical composition for treating gastric cancer, the pharmaceutical composition comprising an agent that inhibits LINC01909.

[0019] Furthermore, the reagent is not limited, as long as it can inhibit the expression level of LINC01909 or inhibit the functional activity of LINC01909.

[0020] The reagents include siRNA or shRNA of LINC01909. In a specific embodiment of the present invention, the siRNA sequence of LINC01909 is shown in SEQ ID NO.6 and SEQ ID NO.7.

[0021] The pharmaceutical compositions of the present invention can be administered alone or in combination with other drugs. Other drugs that can be administered with the pharmaceutical compositions of the present invention are not limited, as long as they do not impair the efficacy of the therapeutic or preventative pharmaceutical compositions of the present invention.

[0022] The pharmaceutical compositions of the present invention can be prepared into various dosage forms as needed. These include, but are not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols, or suppositories for use via the skin, mucous membranes, nose, cheeks, sublingually, or orally.

[0023] The routes of administration of the pharmaceutical compositions of the present invention are not limited, as long as they achieve the desired therapeutic or preventative effect, including but not limited to intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesical, intramuscular, intratracheal, subcutaneous, through the skin, through the pleura, locally, inhaled, through mucous membranes, skin, gastrointestinal tract, intra-articular, intraventricular, rectal, vaginal, intraskull, intraurethral, ​​and intrahepatic administration. In some cases, systemic administration may be used. In some cases, local administration may be used.

[0024] The dosage of the pharmaceutical composition of the present invention is not limited, as long as the desired therapeutic or preventive effect is achieved, and can be appropriately determined based on symptoms, gender, age, etc. The dosage of the therapeutic or preventive pharmaceutical composition of the present invention can be determined using, for example, the therapeutic or preventive effect on a disease as an indicator.

[0025] The present invention also provides the application of the aforementioned long non-coding RNA in the preparation of drugs for treating gastric cancer.

[0026] The present invention also provides the use of reagents for inhibiting the aforementioned long non-coding RNAs in the preparation of medicaments for treating gastric cancer.

[0027] Furthermore, the limitations of the reagents are the same as before.

[0028] The present invention also provides a method for diagnosing gastric cancer, the method comprising the following steps:

[0029] (1) Obtain samples from the subjects;

[0030] (2) Detect the expression level of LINC01909 in the subject samples;

[0031] (3) The measured expression level of LINC01909 was correlated with whether the subject had the disease.

[0032] (4) If the expression level of LINC01909 is increased compared with 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 a poor prognosis.

[0033] The present invention also provides a treatment method for gastric cancer, the method comprising reducing the expression level of LINC01909 or inhibiting the functional activity of LINC01909.

[0034] This invention also provides a method for screening drugs for gastric cancer. The method involves measuring the expression level of LINC01909 at a certain time after adding the test drug to gastric cancer cells or after administering the test drug to a gastric cancer model animal to determine the effect of the drug on improving tumor prognosis. More specifically, when the expression level of LINC01909 decreases or returns to normal after adding or administering the test drug, the drug can be selected as a therapeutic agent to improve tumor prognosis.

[0035] In the context of this invention, "diagnosing gastric cancer" includes determining whether a subject already has gastric cancer, determining whether a subject is at risk of developing gastric cancer, determining the responsiveness of a gastric cancer patient to drug treatment, or determining the prognosis of a gastric cancer patient.

[0036] The term "treatment" as used in this article encompasses the treatment of related diseases or disease states in mammals, such as humans, suffering from related diseases or conditions, and includes:

[0037] (1) To prevent the occurrence of disease or disease state in mammals, especially when the mammal is susceptible to the disease state but has not yet been diagnosed with the disease state;

[0038] (2) To suppress the disease or disease state, that is, to prevent its occurrence; or

[0039] (3) Relieve the disease or disease state, even if the disease or disease state subsides.

[0040] The term "treatment" generally refers to the treatment of humans or animals (e.g., by veterinarians) in which certain desired therapeutic effects are achieved, such as inhibiting the development of a disease (including slowing its progression, stopping its development), improving the condition, and curing the disease. It also includes treatment as a preventative measure (e.g., prevention). Use in patients who have not yet developed the disease but are at risk of developing it is also included in the term "treatment."

[0041] Advantages and beneficial effects of the present invention:

[0042] The present invention has discovered a molecular marker for diagnosing gastric cancer. Using this molecular marker can help identify gastric cancer at an early stage, thus improving patient survival rates.

[0043] The therapeutic agent of the present invention, which includes a reagent that inhibits LINC01909, can be used as a novel therapeutic agent for gastric cancer. Attached Figure Description

[0044] Figure 1 A statistical graph showing the inhibition of LINC01909 expression detected by qPCR;

[0045] Figure 2This is a statistical graph showing the effect of LINC01909 expression on cancer cell proliferation detected using CCK8.

[0046] Specific implementation methods

[0047] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the embodiments are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer.

[0048] Example 1: Study on Differential Expression of LncRNA

[0049] 1. Research Subjects

[0050] Surgical specimens (gastric cancer tissue and corresponding adjacent normal tissue) were collected from 50 patients with primary gastric cancer who underwent radical gastrectomy at the hospital's oncology surgery department. Of the 50 patients, 30 were male and 20 were female; their ages ranged from 38 to 79 years, with a mean age of 61 years. None of the patients had received adjuvant therapy prior to surgery. RNA was extracted from the tissue specimens and immediately stored at -80°C.

[0051] Inclusion criteria: a. Preoperative diagnosis of primary gastric cancer, and no prior tumor treatment; b. No coexisting malignant tumors; c. No other complications, such as gastric perforation, gastrointestinal bleeding, or gastrointestinal obstruction, and the patient's general condition was good preoperatively; d. No other chronic diseases, such as hypertension or diabetes.

[0052] 2. Detection at the transcriptional level

[0053] Total RNA was extracted from the experimental group (gastric cancer tissue) and the control group (adjacent normal gastric tissue) using RNAisoPlus (TaKaRa, Shiga, Japan). The concentration and purity of the RNA were detected using a spectrophotometer. PrimeScript reverse transcriptase (TaKaRa, Shiga, Japan) was used to reverse transcribe the RNA into complementary DNA (cDNA). Using cDNA as a template, SYBRGREEN EX Taq was used... TM (TaKaRa, Shiga, Japan) The reaction system and reaction time specified in the manufacturer's instructions were followed for qPCR on a LightCycler 480. All experimental results were repeated three times, and the results were presented as follows: －△△CT was calculated and statistically analyzed. GAPDH was used as an internal reference gene in the experiment. For LINC01909, the upstream primer was 5'-CTTGCTTACAATGAGGTTAT-3' (SEQ ID NO.2), and the downstream primer was 5'-TGTATTCAGTCATCAATCAC-3' (SEQ ID NO.3); for GAPDH, the upstream primer was 5'-TCATGGGTGTGAACCATGAGAA-3' (SEQ ID NO.4), and the downstream primer was 5'-GGCATGGACTGTGGTCATGAG-3' (SEQ ID NO.5).

[0054] 3. Results

[0055] Statistical results are as follows Figure 1 The results showed that the expression level of LINC01909 was significantly increased in gastric cancer tissue compared with adjacent normal tissue, approximately 12.2 times higher, with a statistically significant difference (P<0.05). Electronic validation using the GEO database and ROC curve analysis showed that the AUC value of LINC01909 in distinguishing between gastric cancer patients and normal individuals was 0.8156.

[0056] Example 2: Inhibition of LINC01909 expression

[0057] 1. Cell Culture and Transfection

[0058] Cell culture: The gastric cancer cell line BGC-823 was cultured in DMEM containing 10% FBS in a 5% CO2, saturated humidity, 37°C CO2 incubator. The culture medium was changed every two days, and cells were digested with 0.25% trypsin during cell passage.

[0059] siRNA transfection: The day before transfection, digest and seed cells into culture dishes or plates. The seeding density should be sufficient to achieve 30-50% confluency for transfection the following day. Strictly follow the Lipofectamine protocol during siRNA transfection. TM Follow the instructions in the 2000 manual. After 4-6 hours, replace the medium with fresh culture medium containing 10% FBS and continue culturing for another 48 hours.

[0060] 2. siRNA design

[0061] siRNA (small interfering RNA) design: siRNA sequences were designed from the specific sequence region of LINC01909 using BLAST search.

[0062] siRNA-LINC01909

[0063] Chain of Justice: 5'-AACUUUGUCACUGUUUCUGAA-3' (SEQ ID NO.6);

[0064] Antisense chain: 5'-CAGAAACAGUGACAAAGUUCA-3' (SEQ ID NO.7).

[0065] The siRNA was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., which also provided a negative control siRNA (siRNA-NC).

[0066] 3. Use qPCR to detect siRNA interference.

[0067] The steps are the same as in Example 1.

[0068] 4. Results

[0069] After transfecting the gastric cancer cell line BGC823 with siRNA, the changes in LncRNA transcription levels were detected by qPCR. The results showed that the expression level of LncRNA in the experimental group cells (siRNA-LncRNA, relative expression level 31.99±2.32%) was significantly lower than that in the control group cells (siRNA-NC, relative expression level 100%), with an inhibition rate of approximately 68%, and the difference was statistically significant (P<0.05), indicating that the siRNA-LncRNA of the present invention can significantly inhibit the expression of LINC01909 in cells.

[0070] Example 3: Determination of the effect of inhibiting LINC01909 expression on the proliferation of gastric cancer cells.

[0071] 1. Steps:

[0072] Logarithmic growth phase BGC-823 cells were seeded into 96-well plates. After 12 hours of culture, the cells were transfected with siRNA-lncRNA and siRNA-NC, respectively. After 4-6 hours, the plates were replaced with DMEM containing 10% FBS and cultured for another 72 hours. 10 μL CCK8 was added to each well of each plate, and the plates were incubated for 2 hours. The absorbance was then measured at 450 nm using a microplate reader. The experiment was repeated three times, and cell growth curves were plotted.

[0073] 2. Statistical Analysis

[0074] All experimental data are expressed as mean ± standard deviation. SPSS 15.0 statistical software was used for one-way ANOVA or t-tests. P < 0.05 was considered statistically significant.

[0075] 3. Results

[0076] The results are as follows Figure 2The results showed that 72 h after transfection, the cell growth rate of the siRNA-LncRNA group (0.944±0.042) was significantly lower than that of the siRNA-NC group (1.546±0.052) (P<0.05). These results indicate that inhibiting LINC01909 expression has an inhibitory effect on the proliferation of gastric cancer cell line BGC823.

[0077] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention. sequence list <110> Beijing Yangshen Biotechnology Co., Ltd. <120> Application of LINC01909 in the preparation of therapeutic cancer drugs and diagnostic reagent kits <160> 7 <170> SIPOSequenceListing 1.0 <210> 1 <211> 815 <212> DNA <213> Human origins (Homo sapiens) <400> 1 ttgggtcctg tcttggtgat aaagataaaa ctaaaggaca atcaaatctc atcaaacttc 60 tctctgagga gctgatgacg tttcagaaac agtgacaaag ttcagaatcc cattcttcca 120 tcctagatgg gaaacatcaa ctttcctgat gatgctgaaa aagcaagata ctcatttttt 180 ccagccaccc ttgcagagcc agaaagccca tgagattcaa tagatgtaag actgagag 240 attttcattc ctggatgaaa ggagacaggc ctgggagaag agttttctct tgccatcttt 300 gcagtctctc cactcctgac gtgtacaggg tcaggaaagg ccatgtgaaa acatggcgag 360 aagacagtca tctgcaaccc aaggagagag acttcaccag acactgaccc tgctggcacc 420 ttgatcttgg acttccagtg ttcagaacta taagaaataa attcctgtta tgtaaggtgc 480 ccagtccatg gtattttgtt gtaacagcct gagctgttga agatggattt tcctttcaag 540 atcagatctt gggcctcaga gtcagcctca ctatagaaag caagggctag tctcagcttc 600 ccacagaaga cttggtttta ttggaggcaa gttttgtctt gcttacaatg aggttataca 660 aagcataaca aaattcacat tttgttaagt gactaccata tgatcactct gaattttatt 720 ttacctgtca tttctcggta tcaataggtt ttacttcccc ttgttctgga tttcagtgat 780 tgatgactga atacaataaa tatttattga gcgcc 815 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cttgcttaca atgaggttat 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <400> 3 tgtattcagt catcaatcac 20 <210> 4 <211> twenty two <212> DNA <213> Artificial Sequence <400> 4 tcatgggtgt gaaccatgag aa 22 <210> 5 <211> twenty one <212> DNA <213> Artificial Sequence <400> 5 ggcatggact gtggtcatga g 21 <210> 6 <211> twenty one <212> RNA <213> Artificial Sequence <400> 6 aacuuuguca cuguuucuga a 21 <210> 7 <211> 20 <212> RNA <213> Artificial Sequence <400> 7 cagaaacagu gacaaaguuc 20

Claims

1. Application of LINC01909 siRNA in the preparation of drugs for treating gastric cancer; the sequence of the sense strand of the siRNA is shown in SEQ IN NO.6, and the sequence of the antisense strand of the siRNA is shown in SEQ ID NO.7.

Images