New use of linc02166 for diagnosis and treatment of gastric cancer

Abstract

The present application relates to a new use of LINC02166 for the diagnosis and treatment of gastric cancer, and specifically relates to the use of LINC02166 as a diagnostic marker for gastric cancer in the preparation of a diagnostic reagent for gastric cancer and as a therapeutic target in the preparation of a therapeutic drug for gastric cancer. The present application proves that LINC02166 can be used as a diagnostic marker and a therapeutic target for gastric cancer. By detecting the expression level of LINC02166, early diagnosis of gastric cancer can be achieved. By inhibiting the expression of LINC02166, LINC02166 can be used as a drug target to assist clinical surgical treatment, improve the cure rate of patients with gastric cancer, and improve the quality of life of patients.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a new use of LINC02166 in the diagnosis and treatment of gastric cancer. 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 LINC02166 in gastric cancer tissues is significantly higher than that in adjacent normal tissues; therefore, LINC02166 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 LINC02166 in NCBI, with gene ID 105371416. The transcript sequence of LINC02166 includes the following: Genbank accession number XR_001752310.1 (798 bp in length, corresponding DNA sequence as shown in SEQ ID NO.1); Genbank accession number XR_001752312.1 (421 bp in length, corresponding DNA sequence as shown in SEQ ID NO.2); Genbank accession number XR_001752311.1 (795 bp in length, corresponding DNA sequence as shown in SEQ ID NO.3).

[0009] Furthermore, the reagents include PCR amplification primers used when detecting the expression level of LINC02166 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.4 and SEQ ID NO.5.

[0011] This invention provides a product for the diagnosis of gastric cancer, the product comprising a reagent for detecting the expression level of LINC02166.

[0012] Furthermore, the reagents include PCR amplification primers used when detecting LINC02166 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.4 and SEQ ID NO.5.

[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-LINC02166 abnormalities associated with gastric cancer through high-throughput sequencing also falls under the application of LINC02166 and is also within the scope of protection of this invention.

[0015] The kit includes reagents for detecting the expression level of LINC02166, the reagents including nucleic acids that bind to LINC02166 or its DNA sequence, the nucleic acids including PCR amplification primers used for detecting the expression level of LINC02166 using SYBR Green, TaqMan probes, molecular beacons, two-hybrid probes, or composite probes.

[0016] The chip includes reagents for detecting the expression level of LINC02166, the reagents including nucleic acids that bind to LINC02166 or its DNA sequence, and the nucleic acids including probes capable of detecting the expression level of LINC02166.

[0017] The test strip includes a reagent for detecting the expression level of LINC02166, the reagent including a nucleic acid that binds to LINC02166 or its DNA sequence, the nucleic acid including a probe capable of detecting the expression level of LINC02166.

[0018] The present invention provides a pharmaceutical composition for treating gastric cancer, the pharmaceutical composition comprising an agent that inhibits LINC02166.

[0019] Furthermore, the reagent is not limited, as long as it can inhibit the expression level of LINC02166 or inhibit the functional activity of LINC02166.

[0020] The reagents include siRNA or shRNA of LINC02166. In a specific embodiment of the present invention, the siRNA sequence of LINC02166 is shown in SEQ ID NO.8 and SEQ ID NO.9.

[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 the reagent for inhibiting the aforementioned long non-coding RNA in the preparation of a drug 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 LINC02166 in the subject samples;

[0031] (3) The measured expression level of LINC02166 was correlated with whether the subject had the disease.

[0032] (4) If the expression level of LINC02166 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 LINC02166 or inhibiting the functional activity of LINC02166.

[0034] This invention also provides a method for screening drugs for gastric cancer. The effect of the drug on improving tumor prognosis can be determined by measuring the expression level of LINC02166 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. More specifically, when the expression level of LINC02166 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 LINC02166, can be used as a novel therapeutic agent for gastric cancer. Attached Figure Description

[0044] Figure 1 This graph shows the statistical results of detecting the inhibition of LINC02166 expression using qPCR.

[0045] Specific implementation methods

[0046] 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.

[0047] Example 1: Study on Differential Expression of LncRNA

[0048] 1. Research Subjects

[0049] 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.

[0050] 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.

[0051] 2. Detection at the transcriptional level

[0052] 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) qPCR was performed on a LightCycler 480 using the reaction system and time specified in the instructions. All experimental results were repeated three times, and the results were calculated as 2 - ΔΔCT and statistically analyzed. GAPDH was used as an internal reference gene. For LINC02166, the upstream primer was 5'-TACCTTTGGAACACCCTA-3' (SEQ ID NO. 4), and the downstream primer was 5'-ACAGAGACAAGTGAATGG-3' (SEQ ID NO. 5); for GAPDH, the upstream primer was 5'-TCATGGGTGTGAACCATGAGAA-3' (SEQ ID NO. 6), and the downstream primer was 5'-GGCATGGACTGTGGTCATGAG-3' (SEQ ID NO. 7).

[0053] 3. Results

[0054] Statistical results are as follows Figure 1 The results showed that the expression level of LINC02166 in gastric cancer tissue was significantly increased (approximately 9.6-fold) compared with adjacent normal tissue, and the difference was statistically significant (P<0.05). Electronic validation using the GEO database and ROC curve analysis showed that the AUC value of LINC02166 in distinguishing between gastric cancer patients and normal individuals was 0.7814.

[0055] Example 2: Inhibition of LINC02166 expression

[0056] 1. Cell Culture and Transfection

[0057] 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.

[0058] 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.

[0059] 2. siRNA design

[0060] siRNA (small interfering RNA) design: siRNA sequences were designed from the specific sequence region of LINC02166 using BLAST search.

[0061] siRNA-LINC02166

[0062] Justice chain: 5'-AUGAUUACUGAAAUCAGUCUC-3' (SEQ ID NO.8); Antisense chain: 5'-GACUGAUUUCAGUAAUCAUCA-3' (SEQ ID NO.9).

[0063] The siRNA was synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd., which also provided a negative control siRNA (siRNA-NC).

[0064] 3. Use qPCR to detect siRNA interference.

[0065] The steps are the same as in Example 1.

[0066] 4. Results

[0067] 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 (siRNA-LncRNA, relative expression level 35.97±4.27%) was significantly lower than that in the control group (siRNA-NC, relative expression level 100%), and the difference was statistically significant (P<0.05), indicating that the siRNA-LncRNA of the present invention can significantly inhibit the expression of LINC02166 in cells.

[0068] Example 3: Determination of the effect of inhibiting LINC02166 expression on the proliferation of gastric cancer cells.

[0069] 1. Steps:

[0070] 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.

[0071] 2. Statistical Analysis

[0072] 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.

[0073] 3. Results

[0074] The results showed that 72 h after transfection, the cell growth rate of the siRNA-LncRNA group (1.119±0.032) was significantly lower than that of the siRNA-NC group (1.546±0.052) (P<0.05). These results indicate that inhibiting LINC02166 expression has an inhibitory effect on the proliferation of gastric cancer cell line BGC823.

[0075] 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> New Uses of LINC02166 in the Diagnosis and Treatment of Gastric Cancer <160> 9 <170> SIPOSequenceListing 1.0 <210> 1 <211> 798 <212> DNA <213> Human origins (Homo sapiens) <400> 1 gccttatttt agggtgaagc ccgggcagcc gacccggctg cgccgtagtg ggaggccacg 60 gcgattaccc aggtgaaggt cttccagccc agccctggaa ggtggaaaaa gaaaagttga 120 catggaatgt ttaggaagca ccattggtcc aggtccagga tcggggcagc cagcatgtcc 180 tctaatctgg cttcactgtt gaaatccagt cgagcaagca gggcccaaac cactgcgaga 240 ccagctcggt cgggcccaaa ccaccgccca ctgcgagacc agctcggtcg ggcccaaacc 300 accgcccact gcgggaccag ctcggtcggg cccaaaccac cgcccactgc gggaccagct 360 cggtcgggcc caaaccactg cccactgcgg gaccagctcg gtcgtggaga ccctaaccca 420 gcggcgctag aggaattaaa gaaacacaca cagaaatata ggtacctcgc aagggttttt 480 tgcatgtctg atacctacgg ctccaacagg acccactgac tgatgatggc tccacctgga 540 cctgccaact actcctgcag ccccacccag aagcggttca gcacacggga ggatcctgtc 600 tcacacccta cgatggcccc ccgcccccac aacccatcag cagcaagcac ccactgccaa 660 gctaccccac cccttccccc aaactacctt tggaacaccc tagtccccaa attcttgggg 720 agactgattt cagtaatcat caaattctgg tctcccattc acttgtctct gtgtgaatta 780 aaccctttct ctattgca 798 <210> 2 <211> 421 <212> DNA <213> Homo sapiens <400> 2 cttattttag ggtgaagccc gggcagccga cccggctgcg ccgtagtggg aggccacggc 60 gattacccag gtgaaggtct tccagtacct cgcaagggtt ttttgcatgt ctgataccta 120 cggctccaac aggacccact gactgatgat ggctccacct ggacctgcca actactcctg 180 cagccccacc cagaagcggt tcagcacacg ggaggatcct gtctcacacc ctacgatggc 240 cccccgcccc cacaacccat cagcagcaag cacccactgc caagctaccc caccccttcc 300 cccaaactac ctttggaaca ccctagtccc caaattcttg gggagactga tttcagtaat 360 catcaaattc tggtctccca ttcacttgtc tctgtgtgaa ttaaaccctt tctctattgc 420 a 421 <210> 3 <211> 795 <212> DNA <213> Homo sapiens <400> 3 gggcagccga cccggctgcg ccgtagtggg aggccacggc gattacccag gtgaaggtct 60 tccaggtgag acgggttccc ccagcccagc cctggaaggt ggaaaaagaa aagttgacat 120 ggaatgttta ggaagcacca ttggtccagg tccaggatcg gggcagccag catgtcctct 180 aatctggctt cactgttgaa atccagtcga gcaagcaggg cccaaaccac tgcgagacca 240 gctcggtcgg gcccaaacca ccgcccactg cgagaccagc tcggtcgggc ccaaaccacc 300 gcccactgcg ggaccagctc ggtcgggccc aaaccaccgc ccactgcggg accagctcgg 360 tcgggcccaa accactgccc actgcgggac cagctcggtc gtggagaccc taacccagcg 420 gcgctagagg aattaaagaa acacacacag aaatataggt acctcgcaag ggttttttgc 480 atgtctgata cctacggctc caacaggacc cactgactga tgatggctcc acctggacct 540 gccaactact cctgcagccc cacccagaag cggttcagca cacgggagga tcctgtctca 600 caccctacga tggccccccg cccccacaac ccatcagcag caagcaccca ctgccaagct 660 accccacccc ttcccccaaa ctacctttgg aacaccctag tccccaaatt cttggggaga 720 ctgatttcag taatcatcaa attctggtct cccattcact tgtctctgtg tgaattaaac 780 cctttctcta ttgca 795 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 tacctttgga acacccta 18 <210> 5 <211> 18 <212> DNA <213> Artificial Sequence <400> 5 acagagacaa gtgaatgg 18 <210> 6 <211> twenty two <212> DNA <213> Artificial Sequence <400> 6 tcatgggtgt gaaccatgag aa 22 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <400> 7 ggcatggact gtggtcatga g 21 <210> 8 <211> twenty one <212> RNA <213> Artificial Sequence <400> 8 augauuacug aaaucagucu c 21 <210> 9 <211> twenty one <212> RNA <213> Artificial Sequence <400> 9 gacugauuuc aguaaucauc a 21

Claims

1. The application of LINC02166 siRNA in the preparation of drugs for treating gastric cancer, wherein the sense strand of the siRNA is shown in SEQ ID NO.8 and the antisense strand of the siRNA is shown in SEQ ID NO.9.

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