Long-chain non-coding RNA Pnky and application thereof

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to a long-chain non-coding RNAPnky and application thereof. According to the application, long-chain non-coding RNAPnky is used as a molecular intervention target, migration and invasion of glioma cells can be remarkably inhibited by down-regulating or inhibiting expression of Pnky, migration and invasion of the glioma cells can be promoted by up-regulating expression of Pnky, and meanwhile, expression of migration and invasion related factors such as MMP9 and MMP2 is influenced by differential expression of Pnky. It is shown that the long-chain non-coding RNAPnky can regulate migration and invasion of human glioma cells, and a new target spot is provided for targeted treatment of glioma. The invention also discloses two reagents for detecting cancer cells, which are used for detecting the expression level of the long-chain non-coding RNAPnky through fluorescent quantitative PCR (Polymerase Chain Reaction) and fluorescence in situ hybridization (FISH) so as to identify whether the tissue (or cell) is a glioma tissue (or cell) or not and evaluate the malignant degree of the tissue (or cell). The invention provides a new viewpoint for diagnosis and treatment of glioma, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a long non-coding RNA Pnky and its application. Background Art

[0002] As one of the most common malignant primary tumors in the human central nervous system, gliomas seriously threaten the survival and health of patients with their high heterogeneity, invasiveness and lethality. Among various glioma types, glioblastoma (GBM) is the most lethal one, accounting for 70% of all diffuse glioma cases. Unfortunately, the median overall survival of glioblastoma patients is very short, only 15 months. Although some positive progress has been made in the field of glioma treatment in recent years, the recurrence rate and mortality of gliomas remain high. Therefore, finding new treatment targets and developing more effective detection methods are of crucial significance for improving the treatment effect of gliomas and the prognosis of patients.

[0003] Long non-coding RNAs (lncRNAs) have become the focus of cancer gene research. In-depth exploration of lncRNAs has revealed that they play key roles in the formation and progression of various tumors, involving aspects such as the growth, movement, invasion of tumor cells, and epithelial-mesenchymal transition. In various cancer types, such as lung cancer, gastric cancer, liver cancer and colorectal cancer, differential expressions of multiple lncRNAs have been identified. The expression levels of some lncRNAs are closely related to the malignancy, spread range and lymph node metastasis of cancers. In addition, some lncRNAs are also considered potential biomarkers for cancer treatment and prognosis assessment. Summary of the Invention

[0004] In view of this, the present invention provides a non-coding RNA Pnky, and the nucleotide sequence of the non-coding RNA Pnky is shown as SEQ ID NO.1.

[0005] The long non-coding RNA Pnky can regulate the migration and invasion processes of human glioma cells. Specifically, knockdown of Pnky can significantly inhibit the migration and invasion of glioma cells, overexpression of Pnky promotes their migration and invasion, and at the same time, the differential expression of Pnky affects the expression of migration- and invasion-related factors such as MMP9 and MMP2. Therefore, its high expression can be used as a treatment target for human gliomas.

[0006] Furthermore, the present application provides the application of the above long non-coding RNA Pnky as a treatment target for human gliomas in the preparation of glioma-targeted drugs and a preparation method of a glioma-targeted drug, including using fluorescence quantitative qPCR to amplify primers for the long non-coding RNA Pnky and primers for the internal reference β-actin;

[0007] Among them, the upstream primer for qPCR amplification of RNA Pnky is: 5’-AGGTCCGGAGGAACCTCTAC-3’;

[0008] The downstream primer for qPCR amplification of RNA Pnky is: 5’-TGTGTCAAGGTACTGGGTGC-3’;

[0009] The downstream primer for qPCR amplification of Pnky is: 5’-TGTGTCAAGGTACTGGGTGC-3’;

[0010] The upstream primer for qPCR amplification of Pnky is 5’-GCCTGGATAGCAACGTACAT-3’.

[0011] Furthermore, the preparation method of the drug further includes amplifying an interfering sequence targeting long non-coding RNA Pnky. The interfering sequence Pnky siRNA-Sense is: 5’-GGGAAACAUCCACGAAUAATT-3’; the interfering sequence PnkysiRNA-Antisense is 5’-UUAUUCGAGGAUGUUUCCCTT-3’.

[0012] The interfering sequence in this technical solution is a sequence that inhibits the expression level of long non-coding RNA Pnky.

[0013] The drug obtained through the above preparation method can inhibit the migration and invasion of human glioma tissues or cells.

[0014] Another technical solution of this application provides a reagent for detecting human glioma tissues / cells, including non-coding RNA Pnky primers and β-actin internal reference primers for fluorescence quantitative qPCR detection.

[0015] Among them, the upstream primer for qPCR amplification of RNA Pnky is: 5’-AGGTCCGGAGGAACCTCTAC-3’;

[0016] The downstream primer for qPCR amplification of RNA Pnky is: 5’-TGTGTCAAGGTACTGGGTGC-3’;

[0017] The downstream primer for qPCR amplification of Pnky is: 5’-TGTGTCAAGGTACTGGGTGC-3’;

[0018] The upstream primer for qPCR amplification of Pnky is 5’-GCCTGGATAGCAACGTACAT-3’.

[0019] The technical solution of the present application also provides another reagent for detecting human glioma tissues / cells, including a non-coding RNA Pnky fluorescent probe mixture for fluorescence in situ hybridization detection, wherein the mixture includes the following sequences:

[0020] 5’-AGAAAAGAAGUUCCACUCAGAGGUU-3’;

[0021] 5’-CGUUCAAACAUGAUCCUCUAAGGUG-3’;

[0022] 5’-UGAAAGUGUGAGAGAUAACAUGGGU-3’;

[0023] 5’-GUUCCUUAUAAAUGCAUCCCCCAAG-3’.

[0024] Furthermore, the above-mentioned reagent detects the expression level of long non-coding RNA Pnky in human glioma tissues or cells by fluorescence quantitative PCR detection and fluorescence in situ hybridization detection methods respectively.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses long non-coding RNA Pnky as a molecular intervention target. Down-regulating or inhibiting the expression of Pnky can significantly inhibit the migration and invasion of glioma cells, while up-regulating the expression of Pnky promotes their migration and invasion. At the same time, the differential expression of Pnky affects the expression of migration and invasion-related factors such as MMP9 and MMP2. It shows that the long non-coding RNA Pnky of the present invention can regulate the migration and invasion of human glioma cells, providing a new target for glioma targeted therapy.

[0026] The present invention also discloses two reagents for detecting cancer cells. By fluorescence quantitative PCR and fluorescence in situ hybridization (FISH), the expression level of long non-coding RNA Pnky is detected to identify whether the tissue (or cell) is a glioma tissue (or cell) and to evaluate the malignancy degree of the tissue (or cell). The present invention provides a new perspective for the diagnosis and treatment of glioma and has good clinical application prospects. Description of the Drawings

[0027] Figure 1 It is for detecting the expression level of long non-coding RNA Pnky in human neurons, microglia (HMC3), astrocytes (SVG P12) and glioma cells (LN229 and U87) by fluorescence quantitative PCR.

[0028] Figure 2To detect the interference effect of siRNA on long non-coding RNA Pnky by fluorescence quantitative PCR. Among them, (A) shows the interference effect of siRNA on Pnky in human glioma cell line LN229; (B) shows the interference effect of siRNA on Pnky in human glioma cell line U87.

[0029] Figure 3 To knockdown long non-coding RNA Pnky and inhibit the migration of glioma cell lines LN229 and U87. Among them, (A) shows the effect of siRNA interfering with Pnky expression on the migration of LN229 cells detected by scratch assay; (B) shows the effect of siRNA interfering with Pnky expression on the migration of U87 cells detected by scratch assay.

[0030] Figure 4 To knockdown long non-coding RNA Pnky and inhibit the invasion of glioma cell lines LN229 and U87. Among them, (A) shows the effect of siRNA interfering with Pnky expression on the invasion of LN229 cells detected by transwell assay; (B) shows the relative invasion rate of LN229 cells in each group of Figure A analyzed by Image J software; (C) shows the effect of siRNA interfering with Pnky expression on the invasion of U87 cells detected by transwell assay; (D) shows the relative invasion rate of U87 cells in each group of Figure C analyzed by Image J software.

[0031] Figure 5 To overexpress long non-coding RNA Pnky and promote the migration of glioma cell lines LN229 and U87. Among them, (A) shows the effect of Pnky overexpression on the migration of LN229 cells detected by scratch assay; (B) shows the effect of Pnky overexpression on the migration of U87 cells detected by scratch assay.

[0032] Figure 6 To overexpress long non-coding RNA Pnky and promote the invasion of glioma cell lines LN229 and U87. Among them, (A) shows the effect of Pnky overexpression on the invasion of LN229 cells detected by transwell assay; (B) shows the relative invasion rate of LN229 cells in each group of Figure A analyzed by Image J software; (C) shows the effect of Pnky overexpression on the invasion of U87 cells detected by transwell assay; (D) shows the relative invasion rate of U87 cells in each group of Figure C analyzed by Image J software.

[0033] Figure 7Effect of differential expression of long non-coding RNA Pnky on the expression of glioma cell migration / invasion-related factors. Among them, (A) shows the effect of interfering with the expression of Pnky on the expression of migration / invasion-related factors detected by Western Blot in LN229 cells; (B) shows the effect of interfering with the expression of Pnky on the expression of migration / invasion-related factors detected by Western Blot in U87 cells; (C) shows the effect of overexpressing Pnky on the expression of migration / invasion-related factors detected by Western Blot in LN229 cells; (D) shows the effect of overexpressing Pnky on the expression of migration / invasion-related factors detected by Western Blot in U87 cells.

[0034] Figure 8 To detect the expression levels of long non-coding RNA Pnky in astrocytes (SVG P12) and glioma cells (LN229) by fluorescence in situ hybridization (FISH). Among them, (A) shows the detection of the expression level of Pnky in SVG P12 cells by fluorescence in situ hybridization (FISH); (B) shows the detection of the expression level of Pnky in LN229 cells by fluorescence in situ hybridization (FISH). Detailed implementation mode

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present invention.

[0036] The experimental methods in the following embodiments are all conventional methods unless otherwise specified. The test materials used in the following embodiments are all obtained from commercial channels unless otherwise specified.

[0037] Example 1: Detection of the expression levels of long non-coding RNA Pnky in human neurons, microglia (HMC3), astrocytes (SVG P12) and glioma cells (LN229 and U87) by fluorescence quantitative PCR.

[0038] A: Collection of cells and extraction of Total RNA

[0039] (1) Take human neurons, HMC3, SVG P12, LN229 and U87 cells in the logarithmic growth phase to make single-cell suspensions, add 1 ml of Trizon and pipette and mix well to lyse the cells, and let stand at room temperature for 5 min for lysis.

[0040] (2) Then add 200 μl of chloroform, invert and mix well, and let stand at room temperature for 5 min for reaction.

[0041] (3) Centrifuge at 4°C at 12,000 rpm for 15 min. After centrifugation, the solution will be layered.

[0042] (4) Transfer the supernatant to a new EP tube, add 500 μl of isopropanol, invert the tube up and down to mix well, and let it stand at room temperature for 10 min.

[0043] (5) Centrifuge at 4°C at 12,000 rpm for 10 min, discard the supernatant, and wash the precipitate with ethanol.

[0044] (6) Centrifuge at 4°C at 7,500 rpm for 5 min, discard the supernatant and retain the precipitate, and air-dry the precipitate.

[0045] (7) Add 20 - 100 μl of RNase-free ddH 2 O to dissolve the precipitate, and proceed to the next experiment or store it at -80°C for later use.

[0046] B: Reverse transcription of RNA into cDNA

[0047] Use the HiscriptⅡQ RT SuperMix for qPCR (+gDNA wiper) reverse transcription kit to perform the reverse transcription experiment. The procedure is as follows:

[0048] (1) Measure the concentration of the extracted RNA and make good marks. Prepare the following mixture in a RNase-free centrifuge tube.

[0049] Table 1 Removal of genomic DNA

[0050]

[0051] Gently pipette to mix well and incubate at 42°C for 2 min.

[0052] (1) Directly add 4 μl of 5×HiScriptⅡqRT SuperMixⅡ to the mixture obtained in the first step, and pipette to mix well. At this time, the total volume of the mixture is 20 μl.

[0053] (2) Incubate in a water bath at 50°C for 15 min and at 85°C for 5 sec. The obtained product can be immediately used for qPCR reaction or stored at -20°C for later use.

[0054] C: RT-qPCR reaction

[0055] (1) Prepare the reaction system according to the experimental groups as shown in the following table; set three replicates for each group of experiments, and use β-actin as the internal reference gene.

[0056] Table 2 RT-qPCR reaction system

[0057]

[0058] (2) Perform RT-qPCR reaction according to the reaction program in the following table

[0059] Table 3 RT-qPCR reaction program

[0060]

[0061] Analyze the results using Bio-Rad CFX Manager software.

[0062] The experimental results are as Figure 1 , long non-coding RNA Pnky is significantly highly expressed in human glioma cells LN229 and U87, while its expression level is extremely low in human neurons, microglia (HMC3), and astrocytes (SVG P12), suggesting that long non-coding RNA Pnky can be used as a potential diagnostic marker for human glioma.

[0063] Example 2: Detection of the interference effect of siRNA on long non-coding RNA Pnky by fluorescence quantitative PCR.

[0064] Among them, the interference sequence Pnky siRNA-Sense is: 5'-GGGAAACAUCCACGAAUAATT-3'; the interference sequence Pnky siRNA-Antisense is 5'-UUAUUCGAGGAUGUUUCCCTT-3'.

[0065] A: siRNA interference experiment

[0066] (1) Seed the cells of each group into six-well plates and wait for 24 hours until the cells grow stably and adhere to the bottom of the plate. The confluence rate of the cells should be maintained between 30% and 50% during the transfection operation.

[0067] (2) Dilute 5 μl of siRNA (20 μM / L stock solution) with 100 μL of DMEM medium without antibiotics and serum. After pipetting and mixing evenly, add 5.0 μL of Lipofectamine RNAi MAX transfection reagent, pipette and mix again, and incubate at room temperature for 20 min.

[0068] (3) Add 50 μL of the transfection reagent-siRNA mixture to each well of the six-well cell plate evenly, and gently shake the cell plate back and forth to mix evenly.

[0069] (4) Place the cell plate in an incubator at 37 °C and 5% CO 2 for 48 h.

[0070] (5) Effect detection: Cells in each group after transfection were collected, and the fluorescence quantitative PCR detection method as in Example 1 was used to evaluate the silencing effect of long non-coding RNA Pnky.

[0071] As Figure 2 The experimental results shown indicate that compared with the control group, after transfection with Pnky siRNA, the expression levels of Pnky in glioma cells LN229 and U87 were significantly reduced. Specifically, the interference efficiency of Pnky siRNA in LN229 cells reached 81%; while in U87 cells, the knockdown efficiency was 78%. It shows that transfection with Pnky siRNA in glioma cells LN229 and U87 can indeed significantly interfere with the expression of Pnky.

[0072] Example 3: Knockdown of long non-coding RNA Pnky inhibits the migration of glioma cells LN229 and U87.

[0073] Glioma cells LN229 and U87 in the logarithmic growth phase were seeded in 6-well plates (1.5×10 5 cells / well). After the cells adhered for 24 h, the siRNA interference experiment was carried out according to the method described in Example 2. After 48 h, the culture medium was discarded, and a sterile 200 μL pipette tip was used to scratch vertically at the bottom of the plate. PBS was added to wash the floating cells, repeated 2 - 3 times, and after discarding PBS, serum-free medium was replaced. Inverted microscopy was used to take pictures at different time points (0 h, 24 h, 48 h) to record the wound healing of cells in different groups.

[0074] The experimental results are as Figure 3 shown. Compared with the control group, after knockdown of long non-coding RNA Pnky, the wound healing ability of human glioma cells LN229 and U87 was significantly reduced, indicating that silencing of Pnky can significantly inhibit the migration of glioma cells LN229 and U87.

[0075] Example 4: Knockdown of long non-coding RNA Pnky inhibits the invasion of glioma cells LN229 and U87.

[0076] Glioma cells LN229 and U87 in the logarithmic growth phase were seeded in 6-well plates (1.5×10 5(cells / well). After the cells adhered to the wall for 24 h, the siRNA interference experiment was carried out according to the method described in Example 2. After 48 h, the Transwell technique was used to evaluate the differences in cell invasion among groups. The specific method was as follows: a) Slowly spread 50 μL of the diluted Matrigel along the wall of the upper chamber and place it in a 37 °C cell culture incubator until the gel completely solidified. b) Add 500 μL of complete medium containing 20% FBS to a 24-well plate and place the upper chamber into the 24-well plate. Collect cells from different treatment groups and make a single-cell suspension with serum-free medium (density: 2.5×10 5 cells / mL). Take 200 μL of the cell suspension and inoculate it into the upper chamber, and culture it in a 37 °C, 5% CO 2 incubator for 24 h. c) Take out the upper chamber, wash the upper chamber 2-3 times with PBS, add 500 μL of pre-cooled 4% paraformaldehyde to the lower chamber, and drop one drop onto the upper chamber to fix for 20 min. d) Discard the fixing solution, wash the upper chamber 2-3 times with PBS, gently wipe the cells on the inoculation side of the upper chamber with a cotton swab, dry the upper chamber, and add 1× crystal violet staining solution to stain for 30 min. e) Discard the staining solution, wash the upper chamber 2-3 times with PBS, and dry the upper chamber. Take pictures with an inverted microscope, and use ImageJ to count the number of cells that entered the lower chamber and calculate the relative cell invasion rate.

[0077] The experimental results were as Figure 4 shown: Compared with the control group, after knocking down the long non-coding RNA Pnky, the number of human glioma cells LN229 and U87 that penetrated through the Matrigel and reached the lower chamber was significantly reduced; among them, the invasion rate of the Pnky siRNA treatment group of LN229 cells was about (25.57±2.43)% of the control group; the invasion rate of the Pnky siRNA treatment group of U87 cells was about (27.89±2.34)% of the control group. It was shown that knocking down the long non-coding RNA Pnky could significantly inhibit the invasion of glioma cells LN229 and U87.

[0078] Example 5: Overexpression of long non-coding RNA Pnky promotes the migration of glioma cells LN229 and U87.

[0079] A. Lentivirus packaging

[0080] (1) Culture HEK293T in a 6-cm dish with DMEM complete medium until the confluence reaches 70-80%. Transfect the plv-CMV-Pnky-eGFP, PH1, PH2 plasmids containing the target gene and the control vector using Lipo3000.

[0081] (2) After 24 h, replace the medium with fresh DMEM complete medium and continue to culture it in a cell culture incubator for 48-72 h.

[0082] (3) After 48 - 72 h, collect the supernatant culture medium, filter it through a 0.45 μm filter membrane, and use ELISA to measure the virus titer of the obtained lentiviral vector.

[0083] B: Lentiviral transfection

[0084] (1) Take glioma cells LN229 and U87 in the logarithmic growth phase, inoculate them into a 6 - well plate (1.5×10 5 cells / well), and after the cells adhere for 24 h, change the medium and add 1 mL of complete DMEM medium.

[0085] (2) After the virus is thawed on ice, add the corresponding volume of virus solution and polybrene, mix well, and then place it in an incubator at 37 °C for further culture.

[0086] (3) After 4 h, supplement 1 mL of medium, and change the medium after 14 h.

[0087] (4) After 72 h of virus infection, observe the fluorescence under an inverted microscope to monitor the infection efficiency. When there is more fluorescence, add 5 μg / ml Puromycin to the transfected cells for screening.

[0088] (5) Select monoclonal cell lines for further culture to obtain a satisfactory cell line stably expressing Pnky.

[0089] (6) Use qRT - PCR to detect whether the expression level of the target gene is significantly increased.

[0090] C: Scratch assay

[0091] Take the Pnky - stably expressing and control LN229 (and U87) cell lines in the logarithmic growth phase, inoculate them into a 6 - well plate (3×10 5 cells / well), and after the cells adhere for 24 h, scratch vertically at the bottom of the plate with a sterile 200 μL pipette tip. Add PBS to wash the floating cells, repeat 2 - 3 times, discard the PBS, and then change to serum - free medium. Take pictures at different time points (0 h, 24 h, 48 h) using an inverted microscope to record the wound healing of cells in different groups.

[0092] The experimental results are as Figure 5 shown. Compared with the control group, after overexpressing the long non - coding RNA Pnky, the wound - healing ability of human glioma cells LN229 and U87 is significantly improved, indicating that overexpression of Pnky can significantly promote the migration of glioma cells LN229 and U87.

[0093] Example 6: Overexpression of long non - coding RNA Pnky promotes the invasion of glioma cells LN229 and U87.

[0094] Lentivirus packaging and lentivirus transfection were carried out according to the method described in Example 5 to obtain glioma cells LN229 and U87 with stable Pnky expression and control cell lines. The Transwell technique was used to evaluate the differences in the invasion ability of cells in each group. The specific method is as follows: a) Slowly spread 50 μL of diluted Matrigel along the wall of the upper chamber and place it in a 37 °C cell culture incubator until the gel completely solidifies. b) Add 500 μL of complete medium containing 20% FBS to a 24-well plate and place the upper chamber into the 24-well plate. Collect cells from different treatment groups and make a single-cell suspension with serum-free medium (density: 2.5×10 5 cells / mL). Take 200 μL of the cell suspension and inoculate it into the upper chamber, and culture it in a 37 °C, 5% CO 2 incubator for 24 h. c) Take out the upper chamber, wash the upper chamber 2 - 3 times with PBS, add 500 μL of pre-cooled 4% paraformaldehyde to the lower chamber, and drop one drop onto the upper chamber to fix for 20 min. d) Discard the fixing solution, wash the upper chamber 2 - 3 times with PBS, gently wipe the cells on the inoculation side of the upper chamber with a cotton swab, dry the upper chamber, and add 1× crystal violet staining solution to stain for 30 min. e) Discard the staining solution, wash the upper chamber 2 - 3 times with PBS, and dry the upper chamber. Take a photo with an inverted microscope, and use Image J to count the number of cells that enter the lower chamber and calculate the relative invasion rate of the cells.

[0095] The experimental results are as Figure 6 shown: In LN229 cells, the number of cells in the Pnky overexpression group that passed through the Matrigel and reached the lower layer was approximately (198.21 ± 12.76)% of that in the control group; while in U87 cells, the number of cells in the overexpression group that passed through the Matrigel and reached the lower layer was approximately (187.89 ± 7.16)% of that in the control group. This indicates that overexpression of the long non-coding RNA Pnky significantly enhances the invasion ability of glioma cells LN229 and U87.

[0096] Example 7: Effect of differential expression of long non-coding RNA Pnky on the expression of glioma cell migration / invasion-related factors.

[0097] (1) Protein extraction: Collect glioma cells LN229 and U87 with Pnky knockdown, stable expression and control cells, wash them once with 1× PBS, centrifuge and discard the supernatant, add an appropriate amount of 1× protein lysate, shake and mix well, lyse at room temperature for 10 min, continue to boil in a 100 °C metal bath for 10 min, and store at -20 °C for later use.

[0098] (2) Preparation of SDS polyacrylamide gel: Wash and dry the gel-making rack, sample comb, long and short glass plates and assemble them. According to the size of the target protein, prepare a 10% separating gel. The components of the separating gel are as follows in the table:

[0099] Table 4 Composition table of 10% separating gel

[0100]

[0101] Inject the prepared separating gel into the middle of the long and short glass plates, about to the 2 / 3 position, and then inject 1 ml of isopropanol to remove air bubbles and prevent the separating gel from drying out. Wait until the separating gel is completely solidified, discard the isopropanol, wash with pure water, and prepare 3 ml of stacking gel according to the following table formula.

[0102] Table 5 Composition Table of 5% Stacking Gel

[0103]

[0104] Slowly inject the prepared stacking gel into the middle of the glass plates, avoiding the generation of air bubbles, gently insert the sample comb, and wait until the stacking gel is completely solidified before performing gel electrophoresis.

[0105] (3) Electrophoresis: Remove the prepared glass plates and gel from the gel rack and install them in the electrophoresis tank, clamp them, pour 1×SDS running buffer into the liquid surface of the electrophoresis tank until the outer liquid surface reaches the 1 / 2 position, gently pull out the sample comb with both hands in parallel, and then load the Marker and protein samples. Perform electrophoresis at 80 V for the stacking gel and 120 V for the separating gel.

[0106] (4) Transfer: When the bromophenol blue reaches the bottom of the glass plate, electrophoresis can be ended. Take out the gel from the glass plate, cut a NC membrane of the same size as the gel, immerse it in 1× protein transfer solution containing 20% for activation and standby, prepare two sponges and eight filter papers for standby, and assemble the transfer cassette in the order of the black rubber surface of the transfer cassette, sponge, four layers of filter paper, gel, NC membrane, four layers of filter paper, sponge, and white rubber surface of the transfer cassette. Pay attention to pressing during the assembly process to avoid air bubbles in the transfer cassette. Then, put the assembled transfer cassette and ice box into the transfer box and perform transfer at 300 mA for 90 min (the transfer time can be adjusted according to the size of the target band).

[0107] (5) Blocking: After the transfer is completed, wash the NC membrane with 1×PBS to wash away the gel attached to the membrane, and add an appropriate amount of skim milk to block it on a shaker at room temperature for 30 min.

[0108] (6) Incubate with antibodies: Place the blocked NC membrane in a self-sealing bag, add an appropriate amount of skim milk and the appropriate antibodies (the antibody dilution ratios are as follows: anti-paxillin 1:1000, anti-integrin 1:1000, anti-MMP2 1:500, anti-MMP9 1:1000, GAPDH 1:2000), incubate overnight on a shaker at 4°C. After the incubation with the primary antibody, wash three times with 1×PBS for 10 minutes each time. Place it in an incubation box, add an appropriate amount of skim milk and the corresponding secondary antibody, and incubate on a shaker at room temperature for 2 hours. After the incubation with the secondary antibody, wash three times with 1×PBS on the shaker for 10 minutes each time.

[0109] (7) Chemiluminescence detection: Drop 200 μl of the pre-prepared ECL luminescent solution (solution A and solution B at a ratio of 1:1) onto the NC membrane, and use the ChemiDoc XRS+ imaging system of the Bio-RAD chemiluminescence imager to collect signals and perform chemiluminescence detection.

[0110] The experimental results are as Figure 7 shown: In glioma cell lines LN229 and U87 cells, compared with the control cells, interfering with the expression of Pnky can significantly inhibit the expression levels of migration / invasion-related factors such as MMP2, MMP9, Paxillin, and Intergrin. Overexpression of Pnky, on the other hand, promotes the expression of these proteins, indicating that the differential expression of long non-coding RNA Pnky affects the expression of glioma cell migration / invasion-related factors.

[0111] Example 8: Fluorescence in situ hybridization (FISH) was used to detect the expression levels of long non-coding RNA Pnky in astrocytes (SVG P12) and glioma cells (LN229).

[0112] The fluorescence in situ hybridization (FISH) experiment was carried out according to the instructions of the RiboTM Fluorescent In Situ Hybridization Kit of Guangzhou Ribobio Co., Ltd. The specific steps are as follows: a) Cell culture: Place the cell slides at the bottom of a 24-well plate, inoculate an appropriate amount of SVG P12 and LN229 cells, and make the cell density reach about 70% before the experiment. b) Cell fixation and permeabilization: Wash SVG P12 and LN229 cells with 1×PBS for 5 min, fix with 4% paraformaldehyde for 20 min. After fixation, wash with PBS for 5 min, add 1 ml of pre-cooled permeabilization solution, and let it stand at 4°C for 10 min. Discard the permeabilization solution, and wash the cells with PBS 3 times, 5 min each time. c) Probe detection: Add 200 μl of pre-hybridization solution to each well, block at 37°C for 30 min, add 2.5 μl of 20 μM Probe Mix to 100 μl of hybridization solution, and hybridize overnight at 37°C in the dark. After hybridization, wash the cells with hybridization solution 1 3 times, 5 min each time, wash the cells with hybridization solution 2 once, wash the cells with hybridization solution 3 once, and wash the cells with 1×PBS once. d) Nucleus staining: Stain with 1×DAPI for 10 min, and wash the cells with 1×PBS three times, 5 min each time. e) Mounting: Carefully take out the cell slides, fix them on the glass slides with mounting medium, and perform fluorescence detection.

[0113] The experimental results are as Figure 8 shown: In human glioma cell line LN229, long non-coding RNA Pnky showed a significantly high expression level and was mainly localized in the cytoplasm. In contrast, in normal astrocyte SVG P12, this RNA was hardly detectable. This indicates that the expression level of long non-coding RNA Pnky can be used as an indicator for diagnosing human glioma.

[0114] As mentioned above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A long non-coding RNA Pnky, characterized in that The nucleotide sequence of the non-coding RNA Pnky is shown in SEQ ID NO.

1.

2. Use of the long non-coding RNA Pnky as claimed in claim 1 as a therapeutic target for human glioma in the preparation of glioma targeted drugs.

3. A method for preparing a glioma targeting drug, characterized in that: It includes using fluorescent quantitative qPCR to amplify long non-coding RNA Pnky primers and β-actin internal reference primers.

4. The preparation method according to claim 3, characterized in that: It also includes amplifying an interfering sequence targeting the long noncoding RNA Pnky.

5. A glioma targeting drug obtained according to the preparation method of claim 3 or 4.

6. A reagent for detecting human glioma tissue / cells, characterized in that: Includes non-coding RNA Pnky primers and β-actin internal reference primers for fluorescent quantitative qPCR detection.

7. A reagent for detecting human glioma tissue / cells, characterized in that: Includes a cocktail of noncoding RNA Pnky fluorescent probes for fluorescence in situ hybridization detection.

8. The reagent according to claim 6 or 7, characterized in that The detection sample of the reagent is located in human glioma tissue or cells.

Images