Application of LncRNA LINC01748 in the diagnosis, treatment and drug sensitivity improvement of glioma

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to the application of LncRNA LINC01748 in the diagnosis, treatment and improvement of drug sensitivity of glioma. The present invention first discovered that the expression of LncRNA LINC01748 in glioma is significantly higher than that in normal brain tissue. After knocking down LncRNA LINC01748, the proliferation ability, migration ability and invasion ability of glioma cells have all been significantly reduced; at the same time, the sensitivity of glioma cells to temozolomide can be improved. The above findings can be used in the preparation of products such as the diagnosis and treatment of gliomas and the improvement of targeted drug sensitivity, and have great application prospects.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and specifically relates to the application of LncRNA LINC01748 in glioma diagnosis, treatment and improving drug sensitivity. Background Art

[0002] Glioma is the most common primary intracranial malignant tumor, accounting for more than 70% of brain tumors. Due to its invasive growth characteristics and the particularity of its anatomical location, it cannot be completely removed by surgery. Patients often show high recurrence rate, high disability rate and low cure rate, and the prognosis is poor. In recent years, although some progress has been made in the treatment of glioma in immunotherapy, gene therapy and oncolytic virus therapy, most of them are limited to clinical trials.

[0003] Temozolomide (TMZ) is the necessary first-choice chemotherapy drug for the treatment of malignant gliomas after surgical resection due to its good blood-brain barrier permeability and outstanding therapeutic effect. However, a considerable number of glioma patients still show primary or secondary resistance to temozolomide, which leads to chemotherapy failure. More importantly, more and more studies have shown that the temozolomide resistance mechanism involves DNA damage repair, abnormal cell signaling pathways, tumor microenvironment, etc., and its resistance mechanism is complex. Therefore, in-depth exploration of the molecular mechanism of temozolomide resistance in gliomas and providing a theoretical basis for individualized precision treatment of glioma patients at the molecular level have become hot spots and difficulties that need to be solved urgently.

[0004] Long non-coding RNA (lncRNA) is a type of non-coding RNA with a length greater than 200 nucleotides. More and more research results have found that the abnormal expression of various long non-coding RNAs is closely related to major human diseases. A large number of research results published recently show that lncRNA plays a pivotal regulatory role in tumor cells, and the abnormal expression of specific lncRNAs directly affects the occurrence, development, metastasis, and drug resistance of tumors. At the same time, some studies have shown that the expression of a single lncRNA can help with tumor diagnosis and prognosis. In actual clinical work, molecular markers have begun to be used in the diagnosis of some cancers.

[0005] At present, the research on the pathogenesis, diagnosis and treatment of glioma by lncRNA is still very lacking. Therefore, the research on long non-coding RNA in glioma is of great value and significance, especially the search for lncRNA related to the occurrence and development of glioma is of great significance for the diagnosis and prognosis assessment of tumors and the guidance of individualized precision treatment. Summary of the invention

[0006] The first object of the present invention is to provide an application of or its inhibitor in the preparation of a drug for treating glioma.

[0007] The sequence of lncRNA LINC01748 is recorded as follows: https: / / www.ncbi.nlm.nih.gov / nuccore / NR_14650 8.1, specifically:

[0008]

[0009]

[0010] The applicant found that knocking down / silencing LncRNA LINC01748 significantly reduced the proliferation, migration and invasion abilities of glioma cells. This application is funded by the National Natural Science Foundation of China (No. 81860664, 82060680) and the Natural Science Foundation of Jiangxi Province (NO. 20202BABL216080, 20192BAB215063).

[0011] The above findings indicate that LncRNA LINC01748 plays an important role in the pathogenesis and chemotherapy of gliomas. It can be used as a new molecular marker and drug target for the diagnosis and treatment of gliomas, thereby guiding clinicians to intervene and treat, improve the survival rate and quality of life of patients, and has great application prospects.

[0012] Among them, preferably, the inhibitor is siRNA-LINC01748; the target sequence of the siRNA-LINC01748 is GACCTTCGGAGCTGAATAA.

[0013] The second object of the present invention is to provide a use of a reagent for detecting LncRNA LINC01748 in the preparation of a glioma diagnostic reagent or a glioma diagnostic kit.

[0014] Wherein, preferably, the reagent for detecting LncRNA LINC01748 comprises a primer pair, wherein the sequence of the forward primer of the primer pair is: F: 5′-TCATCCCACATTTGGAGCAC-3′; and the sequence of the reverse primer is: R: 5′-CCCATGTAGCAGACTCCTACTCA-3′.

[0015] The third object of the present invention is to provide a glioma diagnosis and evaluation kit, which includes the above-mentioned reagent for detecting LncRNA LINC01748.

[0016] Preferably, the above kit also includes TB green, ddH 2O and cDNA template. The reaction system is 20 μL, and the concentrations of the forward primer and the reverse primer are both 10 μM. The reaction system of 20 μL is specifically: TB green 10 μL, forward primer and reverse primer 0.8 μL each, ddH 2 O 6.4 μL, cDNA template 2 μL.

[0017] The fourth object of the present invention is to provide a use of LncRNA LINC01748 or an inhibitor thereof in the preparation of a product for increasing the sensitivity of glioma cells to irinotecan. Preferably, the inhibitor is siRNA-LINC01748; the target sequence of the siRNA-LINC01748 is GACCTTCGGAGCTGAATAA. The inventors found that knocking down / silencing LncRNALINC01748 can increase the drug sensitivity of temozolomide.

[0018] The beneficial effects of the present invention are as follows: the present invention first discovered that after knocking down LncRNA LINC01748, the proliferation ability, migration ability, and invasion ability of glioma cells were significantly reduced; at the same time, the sensitivity of glioma cells to temozolomide can be improved. The above discovery can be used in the preparation of products such as diagnosis and treatment of gliomas and improving the sensitivity of targeted drugs, and has great application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown is the result graph of the expression of lncRNA LNC01748 in glioma tissue and normal brain tissue;

[0020] Figure 2 The graph shows the expression of lncRNA LINC01748 in different glioma cells HS683, U87, T98G, and U251.

[0021] Figure 3 Shown is the effect of shRNA-LINC01748 on the RNA expression of LINC01748;

[0022] Figure 4 Shown is the result of knocking down LINC01748 on the proliferation ability of glioma cells;

[0023] Figure 5 Shown is the result of interfering with LINC01748 on the proliferation ability of glioma cells;

[0024] Figure 6 Shown is the result of knocking down LINC01748 on the migration ability of glioma cells;

[0025] Figure 7Shown is the result of interfering with LINC01748 on the invasion ability of glioma U251 and T98G cells;

[0026] Figure 8 The results of interfering with LINC01748 on the drug sensitivity of temozolomide in glioma cells U251 and T98G are shown;

[0027] Fig. 9 The figure shows the effect of interfering with LINC01748 on the drug sensitivity of temozolomide in glioma cells U251 and T98G.

[0028] Fig.10 The figure shows the effect of interfering with LINC01748 on the drug sensitivity of temozolomide in glioma cells U251 and T98G. DETAILED DESCRIPTION

[0029] The following will provide a clear and complete description of the concept and technical effects of the present invention in conjunction with the embodiments and drawings, so as to fully understand the purpose, scheme and effects of the present invention.

[0030] Embodiment 1:

[0031] 1. Screening of differentially expressed lncRNAs in gliomas

[0032] 1. Analysis and screening of IncRNA in TCGA database

[0033] Methods: The applicant previously performed lncRNA sequencing on glioma and normal brain tissue (3vs3) and found that LINC0 1748 is a newly discovered lncRNA, but its role and mechanism in glioma have not yet been elucidated. To explore the expression and clinical significance of LINC01748 in glioma, the expression profile data of lncRNA in glioma and normal brain tissue (Tumor, n=689; Normal, n=1157) in the TCGA-GTEx database were analyzed by bioinformatics, and a scatter plot was drawn.

[0034] Results: Figure 1 As shown in A, the expression of LINC01748 in glioma was significantly higher than that in normal brain tissue (p<0.00 1). The median of the Normal group was 0.411 (0.214-0.66), and the median of the Tumor group was 0.566 (0.299-0.956).

[0035] 2. Clinical sample verification

[0036] Methods: RT-qPCR was used to detect the expression of LINC01748 in glioma and normal brain tissue samples (Tumor, n=68; Normal, n=20) collected in our hospital.

[0037] This embodiment discloses a LINC01748 diagnostic detection method, which collects glioma and normal brain tissue samples, and uses Q IAGEN's tissue RNA extraction kit to extract RNA samples. The specific operation is detailed in the instructions.

[0038] (1) Reverse transcription reaction:

[0039] PrimeScript RT reagent Kit with gDNA Eraser (PerfectReal Time) (Cat. No.: RR047B) from TAKARA was used for lncRNA reverse transcription. After removing genomic DNA, 1 μL of gDNA Eraser, 2 μL of 5×gDNA Eraser B, and 1 μg of Total RNA were added to the EP tube. RNase Free ddH 2 Dilution was added to make the total volume to 10 μL and placed in a PCR instrument. The reaction conditions were heating at 42°C for 2 min, 4°C, +∞.

[0040] Reverse transcription reaction: 5× Primescript Buffer 24μL, RT primer Mix 1μL, PrimescriptRT Enzyme Mix 1μL, RNase Free ddHO 2 O 5.0μL, add to the above EP tube and mix together for a total of 20μL, place in a PCR instrument, the reaction conditions are 37℃, 15min; 85℃, 5s; 4℃, +∞.

[0041] (2) Primer design

[0042] According to the sequences of LINC01748 and GAPDH genes in GCBI, RT-qPCR amplification primers were designed and synthesized by Guangzhou Ruibo Biotechnology Co., Ltd. and Shanghai Shenggong Co., Ltd. The specific primer sequences are as follows:

[0043] LINC01748 Gene:

[0044] The forward primer was: F: 5′-TCATCCCACATTTGGAGCAC-3′;

[0045] The reverse primer was: R: 5′-CCCATGTAGCAGACTCCTACTCA-3′;

[0046] GAPDH gene:

[0047] The forward primer was: F: 5′-CCCATCACCATCTTCCAGGAG-3′;

[0048] The reverse primer was: R: 5′-GTTGTCATGGATGACCTTGGC-3′;

[0049] (3) RT-qPCR amplification test

[0050] The TB green premix Ex Taq II (Cat. No.: RR820B (A×2)) kit from TAKARA was used for amplification, and the experimental operation was carried out according to the product instructions.

[0051] A 20 μL reaction system was used: 10 μL TB green, 0.8 μL each of forward and reverse primers (10 μM), ddHO 2 O 6.4μL, cDNA template 2μL.

[0052] The amplification program in the fluorescence quantitative PCR instrument is as follows: the first step is denaturation at 95℃ for 30s; the second step is PCR reaction at 95℃ for 5s, 60℃ for 30s; 40 cycles; the third step is melting curve at 95℃ for 10s, 65℃ for 5s. Three parallel tubes are set for each sample, and all amplification reactions are repeated three times to ensure the reliability of the results. -ΔCt The relative expression level was calculated.

[0053] (4) Results

[0054] The results are as follows Figure 1 As shown in B, it was found that LINC01748 was significantly expressed in glioma tissues ( Figure 1 B, Tumor, n=68; Normal, n=20, p<0.01), which is consistent with the previous bioinformatics analysis results. Therefore, it is inferred that the expression of LINC01748 in glioma is significantly higher than that in normal brain tissue.

[0055] 2. Expression of LINC01748 in glioma cell lines

[0056] 1. Cell culture

[0057] Glioma HS683, U87, T98G, and U251 cells were cultured in DMEM medium containing 10% FBS and in an atmosphere of 5% CO. 2 , cultured in a constant temperature incubator at 37°C.

[0058] 2. Transfection

[0059] The LINC01748 shRNA constructed in the present application was synthesized by Hanbio using the siRNA target sequence information of LINC01748. The siRNA target sequence information is as follows: siRNA-LINC01748, GACCTTCGGAGCTGAATAA.

[0060] Transfection: Taking a 6-well plate as an example, glioma cells U251 and T98G were plated the day before transfection and cultured with DMEM complete medium containing 10% FBS. When the cells grow to a confluency of 60%, the cells were transfected. Before transfection, remove the old cell culture medium, wash twice with PBS, and add 1.5mL complete medium to each well. Then prepare the transfection system: Add 250μL Opti-MEM to each of the three 1.5mL EP tubes, and then add 5μL HNRNPC siRNA and NC fragments respectively. The other group without interference fragments is set as the Control group, gently mix and let stand for 5min; take another 3 1.5mL EP tubes and add 250μL Opti-MEM to each, and then add 5μL lipo3000 respectively, gently mix and let stand for 5min. Finally, gently mix the two systems, let stand for 20min, and then add them to each well.

[0061] 3. RT-qPCR detection of LINC01748 expression level

[0062] (1) 48 h after cell transfection, total cellular RNA was extracted using the Trizol method;

[0063] (2) RT-qPCR detection of LINC01748 expression level, the operation steps are as follows:

[0064] The amplification was performed using TAKARA's TB green premix Ex Taq II (Cat. No.: RR820B (A×2)) kit, and the experimental operation was performed according to the product instructions.

[0065] A 20 μL reaction system was used: 10 μL TB green, 0.8 μL each of forward and reverse primers (10 μM), ddHO 2 O 6.4μL, cDNA template 2μL.

[0066] The amplification program in the fluorescence quantitative PCR instrument is as follows: the first step is denaturation at 95℃ for 30s; the second step is PCR reaction at 95℃ for 5s, 60℃ for 30s; 40 cycles; the third step is melting curve at 95℃ for 10s, 65℃ for 5s. Three parallel tubes are set for each sample, and all amplification reactions are repeated three times to ensure the reliability of the results. -ΔCt The relative expression was calculated by the method.

[0067] 4. Experimental results

[0068] Results: Figure 2 As shown in the figure, the expression of LINC01748 in high-grade glioma cells U87, T98G, and U251 was significantly higher than that in low-grade glioma cells HS683, and T98G and U251 expressed LINC01748 the highest. Therefore, these two cell lines were selected for subsequent experiments.

[0069] like Figure 3 As shown in the data, in glioma U251 and T98G cell lines, shRNA-LINC01748 could significantly downregulate the RNA expression of LINC01748 (p<0.01, p<0.05).

[0070] Silencing LINC01748 can inhibit the proliferation ability of glioma cells

[0071] 1. MTS experiment

[0072] Experimental method: Take the logarithmic growth phase glioma U251 and T98G cells cultured in DMEM, plant them in 96-well plates, 3000 cells per well, transfect NC and shRNA-LINC01748, continue to culture for 24h, 48h, 72h, 96h, and use MTS to detect cell proliferation at each time point. Take the cells at the time point, remove the original culture medium, prepare the working solution according to the ratio of MTS: DMEM = 1:9, mix well, take 100μL of the working solution and add it to each well. Place in a cell culture incubator at 37℃ for 30min, and detect the absorbance at a wavelength of 490nm with an enzyme reader.

[0073] Experimental results: Figure 4 As shown in the figure, compared with the NC group, the proliferation ability of glioma U251 and T98G cells was significantly decreased after knockdown of LINC01748 (96h, U251 p<0.001; T98G p<0.001).

[0074] 2. EDU experiment

[0075] U251 and T98G cells in logarithmic growth phase were taken and seeded in 96-well plates. After 48 hours of transfection with NC and shRNA-LINC01748, 100 μL of 50 μM EdU medium (EdU reagent A diluted at a ratio of 1000:1) was added to each well and incubated for 2 hours. Wash with PBS twice, add 1 mL of 4% paraformaldehyde to each well for 30 minutes; wash with PBS once, add 100 μL of permeabilization solution (0.5% TritonX-100 in PBS) to each well, decolorize and incubate on a shaker for 10 minutes; wash with PBS once, add 100 μL of 1×Apollo staining reaction solution to each well, incubate in a dark, room temperature, decolorize and shaker for 30 minutes; wash with PBS once, add 100 μL of 1×DAPI staining solution to each well for 30 minutes. Wash with PBS 1-3 times, and take pictures and statistics under a fluorescence microscope.

[0076] The results are as follows Figure 5 As shown in the figure, compared with the NC group, knockdown of LINC01748 can significantly inhibit the proliferation of glioma cells and reduce the proliferation ability.

[0077] Silencing LINC01748 can inhibit the migration and invasion of glioma cells

[0078] 1. Scratch test

[0079] Experimental method: Glioma U251 and T98G cells in the logarithmic growth phase cultured with DMEM were seeded in 6-well plates. When the cell confluence reached 60%, NC and shRNA-LINC01748 were transfected. When the cells grew to 90%, scratches were made. First, a straight line was drawn in the middle of the well on the back of the 6-well plate with a fine black marker, and 3 parallel straight lines were drawn perpendicular to the black line drawn before in the well with a 1mL imported Tip. The scraped free cells were gently washed twice with PBS, and the culture medium was replaced with DMEM containing 2% FBS. Three fixed positions were randomly selected in each group under an inverted microscope to record the scratch width, and photos were taken at 0 hours and 24 hours, respectively, to compare the migration speed of cells in different groups.

[0080] Experimental results: Figure 6 As shown in the figure, compared with the NC group, the migration ability of glioma U251 and T98G cells was significantly decreased after knockdown of LINC01748 (U251 p<0.001; T98G p<0.001).

[0081] 2. Transwell experiment

[0082] Experimental methods:

[0083] (1) Dilute the matrix gel and pre-cooled serum-free DMEM at a ratio of 1:8, and quickly mix them by pipetting with a pre-cooled 200 μL Tip. Evenly add 60 μL of the mixture to each traswell chamber, gently tap the four edges of the 24-well plate to evenly spread the matrix gel suspension on the bottom of the chamber, and place the 24-well plate in a constant temperature cell culture incubator;

[0084] (2) Glioma U251 and T98G cells were pre-transfected with NC and shRNA LINC01748, and digested, resuspended, and diluted with serum-free medium 24 hours later. The liquid in the chamber was discarded, and about 200 μL of glioma cell suspension containing 3000 cells of different treatment groups was added to each chamber; and 600 μL of DMEM culture medium containing 10% FBS was added to the gap between the bottom of the transwell chamber and the bottom of the 24-well plate; after culturing in a constant temperature incubator for 24 hours, the culture medium inside and outside the chamber was discarded with a Tip, and the cells in the upper chamber were gently wiped off with a cotton swab pre-moistened with PBS;

[0085] (3) The transwell chamber was gently washed twice with PBS, blown dry, and fixed with 4% paraformaldehyde for 10 minutes; the paraformaldehyde was removed, the chamber was gently washed twice with PBS, 0.1% crystal violet staining solution was added, and staining was performed for two hours, and the chamber was washed twice with PBS. Five fields of view were randomly selected under a microscope to calculate the average number of cells that passed through the basement membrane.

[0086] Experimental results: Figure 7 As shown in the figure, compared with the NC group, the invasion ability of glioma U251 and T98G cells was significantly decreased after knockdown of LINC01748 (U251 p<0.001; T98G p<0.001).

[0087] 5. Effect of silencing LINC01748 on temozolomide sensitivity in glioma

[0088] 1. MTS experiment

[0089] Experimental method: The effect of LINC01748 on the sensitivity of temozolomide in glioma cells was detected by MTS experiment (operation steps are the same as those in step 3). After 24 hours of interference with LINC01748, medium containing temozolomide (TMZ) at concentrations of 0, 100, 200, 400, and 800 μM was added, and the cells were cultured for 72 hours and the survival rate of each group was detected.

[0090] Experimental results: Figure 8 As shown in Figure 2, after interfering with LINC01748, the survival rates of glioma U251 and T98G cells were significantly decreased ( Figure 4), that is, the sensitivity of glioma cells to the first-line chemotherapy drug temozolomide is significantly increased.

[0091] 2. Flow cytometry to detect cell apoptosis

[0092] Experimental method: After glioma U251 and T98G cells were interfered with LINC01748 for 12 hours, culture medium containing temozolomide, a first-line chemotherapy drug for glioma cells, was added and cultured for 72 hours. The supernatant was collected and centrifuged, washed twice with PBS, digested with trypsin and the cells were collected and transferred to the EP tube with the collected supernatant cells, centrifuged at 3000rpm for 10 minutes, and the supernatant was discarded; after washing with PBS, 195μL of binding solution was added to each sample to resuspend the cells, and 5μL of Annexin V-FITC and 10μL of propidium iodide were added in sequence, gently mixed, incubated at room temperature in the dark for 20 minutes, and cell apoptosis was detected by flow cytometry.

[0093] Experimental results: Fig. 9 As shown in Figure 2, after interfering with LINC01748, the number of apoptotic glioma cells treated with temozolomide increased significantly ( Fig. 9 A and B). That is, the research results show that interfering with LINC01748 can significantly increase the drug sensitivity of glioma cells U251 and T98G to temozolomide.

[0094] 3. Hoechst staining to detect cell apoptosis

[0095] Experimental method: After interfering with LINC01748 for 12 hours, glioma U251 and T98G cells were added with culture medium containing temozolomide, a first-line chemotherapy drug for glioma cells, and cultured for 72 hours. The old culture medium was discarded, and an appropriate amount of 4% paraformaldehyde was added to fix the cells for 30 minutes. After discarding the paraformaldehyde, the cells were washed with PBS 2-3 times, and an appropriate amount of Hoechst 33342 fluorescent anti-quencher was added for staining for 20 minutes, and cell apoptosis was immediately observed under a fluorescence microscope.

[0096] Experimental results: Fig.10 As shown, the Hoechest staining results showed that the number of apoptotic glioma U251 and T98G cells treated with temozolomide increased significantly after interfering with LINC01748, increasing the drug sensitivity of glioma cells to temozolomide.

[0097] The results of the above MTS experiments, flow cytometry apoptosis assays, and Hoechst staining apoptosis assays all indicate that interfering with LINC01748 can increase the drug sensitivity of glioma cells to temozolomide. That is, lncRNALINC01748 plays an important role in the chemotherapy of temozolomide, a first-line chemotherapy drug for glioma.

[0098] Summary: In the early stage of this invention, high-throughput lncRNA sequencing and bioinformatics analysis of TCGA glioma-related information were used to find that lncRNA LINC01748 was significantly highly expressed in gliomas, and the results were verified by samples from our hospital's sample library. The results were consistent with the results of bioinformatics analysis, that is, the expression of LIN01748 in glioma tissue was significantly higher than that in normal brain tissue samples. And through MTS experiments, EDU experiments, scratch experiments, and Transewell experiments, it was further shown that interfering with the expression of lncRNA LINC01748 can significantly inhibit the proliferation and invasion of gliomas. In addition, the results of MTS experiments, flow cytometer cell apoptosis detection experiments, and Hoechst staining cell apoptosis detection experiments all showed that interfering with LINC01748 can increase the drug sensitivity of glioma cells to the first-line chemotherapy drug temozolomide. These results indicate that lncRNA LINC01748 plays an important role in the diagnosis, pathogenesis and chemotherapy of gliomas, and can be used as a new molecular marker and drug target for the diagnosis and treatment of gliomas, thereby guiding clinicians to intervene and treat, and improving the survival rate and quality of life of patients, which has great application prospects.

[0099] The above is only a preferred embodiment of the present invention. The present invention is not limited to the above implementation. As long as the technical effect of the present invention is achieved by the same means, it should belong to the protection scope of the present invention. Within the protection scope of the present invention, its technical scheme and / or implementation method can have various modifications and changes.

Claims

1. Application of reagents for detecting LncRNA LINC01748 in the preparation of glioma diagnostic reagents or glioma diagnostic kits, It is characterized in that The reagent for detecting LncRNA LINC01748 detects glioma by quantitatively detecting LncRNA LINC01748 in brain tissue samples; The reagent for detecting LncRNA LINC01748 includes a primer pair, wherein the sequence of the forward primer of the primer pair is: F: 5′-TCATCCCACATTTGGAGCAC-3′; the sequence of the reverse primer is: R: 5′-CCCATGTAGCAGACTCCTACTCA-3′; The sequence of the LncRNA LINC01748 is shown in SEQ ID No: 1.

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