MiRNA molecular sponge and application thereof in preparation of drugs for treatment or adjuvant treatment of brain glioma

Abstract

The invention discloses a miRNA molecular sponge and application thereof in preparation of drugs for treatment or adjuvant treatment of brain glioma, PLK1 gene is taken as a treatment target gene, mir-1301-3p is taken as a target spot, the miRNA molecular sponge absorbs mir-1301-3p to block interaction between the mir-1301-3p and mRNA of the PLK1 gene so as to down-regulate PLK1 protein expression, and the miRNA molecular sponge can be used for treating brain glioma. Therefore, the effect of treating or adjunctively treating brain glioma is achieved. Bioinformatics analysis and experimental verification show that hsa-mir-1301-3p can be specifically combined with a 3UTR (Untranslated Region) region of a PLK1 gene, and is different from the down-regulation effect of conventional microRNA (Ribonucleic Acid) on the expression of a target gene protein, and the has-mir-1301-3p has a promotion effect on the expression of the PLK1 gene; according to the present invention, the combination of has-mir-1301-3p and the 3UTR region of the mRNA of the PLK1 gene is blocked by using the microRNA sponge technology, such that the expression of the PLK1 protein can be down-regulated, and the growth amplification of the brain glioma cells, the formation of the adherent form and the drug resistance are inhibited so as to achieve the treatment effect. The invention provides a new thought and method for the treatment of brain glioma.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a miRNA molecular sponge and application thereof in preparing a drug for treating or assisting in treating brain glioma. Background Art

[0002] Gliomas are caused by the canceration of glial cells in the brain or spinal cord. They are the most common malignant tumors of the central nervous system in clinical practice. They are characterized by high incidence, high recurrence rate, easy metastasis, high mortality rate and low cure rate. According to the WHO classification, gliomas are divided into types I-IV according to the increasing degree of tumor malignancy. Among them, WHO type IV is the most malignant and is also called glioblastoma multiform (GBM). Although some progress has been made in the treatment of gliomas in recent years, such as surgical resection, radiotherapy and chemotherapy, there is still a lack of sufficient treatment methods overall. Surgical resection is still the most effective treatment for gliomas, but it is difficult to completely remove gliomas due to the characteristics of easy spread of gliomas and unclear boundaries between tumors and normal tissues, which leads to a high recurrence rate. With the progress of tumor molecular biology research, molecular targeted therapy targeting genes has gradually become a method with great potential for the clinical treatment of malignant tumors. Unlike traditional chemotherapy, molecular targeted therapy prevents the growth of cancer cells by interfering with specific target molecules in the occurrence and development of malignant tumors. It has the advantages of strong specificity, obvious efficacy and few adverse reactions. The Polo-like kinase family (PLKs) is a highly conserved serine / threonine protein kinase, including PLK1, PLK2, PLK3 and PLK4, which play a vital regulatory role in each stage of the cell cycle. Among them, PLK1 is closely related to the mitotic process of the cell cycle, can regulate the separation of chromosomes during cell division, the conversion of the cell cycle G2 / M, and can positively regulate the migration and drug resistance of cells. It is an important target for cancer treatment, so it is very meaningful to develop more drugs with it as a target for cancer treatment. miRNA (microRNA, also known as microRNA) is a class of endogenous small single-stranded hairpin RNA with a length of about 18-25 nucleotides (nt) (Eacker et al., 2009). MiRNA plays a role by complementary binding to the mRNA transcript of protein-coding genes. More and more studies have shown that microRNA plays an important role in the occurrence and development of diseases. Among them, miRNA "sponges", also known as miRNA sponges, are one of the most effective tools for studying miRNA functions. It can be used to continuously inhibit the function of miRNA in cell lines or animals. miRNA sponges contain complementary binding sites to the target miRNA. On the one hand, they are designed for single or multiple miRNAs, and can inhibit the expression of single or multiple miRNAs; on the other hand, they are designed for miRNAs with the same seed region, and can effectively inhibit the function of the miRNA family with the same seed region. Therefore, its development as an inhibitor for targeted genes is very promising. However, there are few studies on the treatment of brain gliomas using miRNA sponges. Summary of the invention

[0003] Therefore, based on the above background, the present invention provides a miRNA molecular sponge and its use in the preparation of a drug for treating or assisting the treatment of brain glioma, which targets mir-1301-3p and utilizes the miRNA molecular sponge to adsorb mir-1301-3p, blocking its interaction with the mRNA of the PLK1 gene, thereby downregulating the expression of the PLK1 protein, to achieve the effect of treating or assisting the treatment of brain glioma.

[0004] The technical solution provided by the present invention is:

[0005] A miRNA molecular sponge, wherein the nucleotide sequence of the encoding DNA of the miRNA molecular sponge is shown as SEQ ID NO: 2.

[0006] Further,

[0007] It can adsorb microRNA hsa-mir-1301-3p, block the binding of microRNA hsa-mir-1301-3p to the target gene, and thus downregulate the protein level expression;

[0008] The nucleotide sequence of the microRNA hsa-mir-1301-3p is shown in SEQ ID NO:1.

[0009] Furthermore, the target gene is the PLK1 gene, and the protein is the PLK1 protein.

[0010] Based on the same inventive concept, the present invention applies the above-mentioned miRNA molecular sponge to the preparation of drugs for treating or assisting in the treatment of brain glioma.

[0011] Based on the same inventive concept, the present invention also provides a recombinant lentiviral vector, which contains the nucleotide sequence of the DNA encoding the miRNA molecule sponge.

[0012] Based on the same inventive concept, the present invention applies the above-mentioned recombinant lentiviral vector to the preparation of drugs for treating or assisting in the treatment of brain glioma.

[0013] Based on the same inventive concept, the present invention also provides a recombinant lentivirus, which comprises the nucleotide sequence of the DNA encoding the miRNA molecule sponge, or the recombinant lentiviral vector.

[0014] Furthermore, the above-mentioned recombinant lentiviral vector is co-transfected with the lentiviral packaging plasmids psPAX2 and pMD2G into HEK293 cell line, and then packaged and concentrated to obtain the result.

[0015] Based on the same inventive concept, the present invention also applies the above-mentioned recombinant lentivirus to the preparation of drugs for treating or assisting in the treatment of brain glioma.

[0016] Based on the same inventive concept, the present invention also provides a drug for treating or assisting in treating brain glioma, which comprises an effective amount of a recombinant lentivirus and a pharmaceutically acceptable excipient.

[0017] The above technical solution has the following beneficial effects:

[0018] The present invention uses PLK1 gene as a therapeutic target gene and mir-1301-3p as a target, and utilizes miRNA molecular sponge to adsorb mir-1301-3p, thereby blocking its interaction with the mRNA of the PLK1 gene and downregulating the expression of PLK1 protein, thereby achieving the effect of treating or assisting in the treatment of brain glioma.

[0019] The present invention has found through bioinformatics analysis and experimental verification that hsa-mir-1301-3p can specifically bind to the 3UTR region of the PLK1 gene, and unlike the conventional microRNA that downregulates the expression of the target gene protein, has-mir-1301-3p has a promoting effect on the expression of the PLK1 gene; the present invention blocks the binding of has-mir-1301-3p to the 3UTR region of the PLK1 gene mRNA through the microRNA sponge technology, thereby downregulating the expression of the PLK1 protein, and inhibiting the growth and proliferation of brain glioma cells, the formation of the wall morphology and the drug resistance, thereby achieving a therapeutic effect.

[0020] The present invention can provide new ideas and methods for the treatment of brain glioma. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Attached Figure 1 This is the hsa-mir-1301-3p inhibition lentiviral vector plasmid map and the control lentiviral map of the embodiment of the present invention.

[0022] Attached Figure 2 The cell viability and immunofluorescence staining results of cells treated with the PLK1 specific inhibitor GSK461364A according to Example 2 of the present invention.

[0023] Attached Figure 3 This is the bioinformatics analysis result of Example 3 of the present invention:

[0024] in

[0025] A: The sequence and secondary structure of mir-1301-3p including the precursor;

[0026] B: High-throughput sequencing quantitative results of PLK1 mRNA and microRNA mir-1301-3p in five cell samples;

[0027] C: Prediction results of the binding target of mir-1301-3p and the 3UTR region of PLK1.

[0028] Attached Figure 4 The sponge sequence design result of Example 4 of the present invention is:

[0029] in

[0030] Figure A: Design principle of sponge sequence and schematic diagram of qPCR detection principle;

[0031] Figure B: Relative quantitative results of sponge sequences detected by qPCR;

[0032] Figure C: Melting curve of the detection primers for qPCR;

[0033] Figure D: Cell viability test results of five cell lines infected with sponge sequence inhibition lentivirus

[0034] Attached Figure 5 These are the results of immunofluorescence staining of PLK1 after the five cell lines of Example 4 of the present invention were infected with mir-1301-3p inhibition lentivirus. DETAILED DESCRIPTION

[0035] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0036] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0037] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0038] The present invention provides a miRNA molecular sponge and its application in the preparation of a drug for treating or assisting in the treatment of brain glioma. In the present invention, the microRNA hsa-mir-1301-3p is a new target for drugs for treating brain glioma. The microRNA sponge adsorbs microRNA, blocks its interaction with the PLK1 gene, downregulates the protein level expression of PLK1, reduces the growth rate of cancer cells, the ability to form an adherent state, and drug resistance, thereby inhibiting cancer cells. In the present invention, the nucleotide sequence of the hsa-mir-1301-3p is shown in SEQ ID NO: 1, specifically:

[0039] 5-UUGCAGCCUGCCUGGGAGUGACUUC-3.

[0040] In the present invention, the nucleotide sequence of the DNA encoding the microRNA sponge is shown in SEQ ID NO: 2. Specifically, the positive strand of the DNA encoding the microRNA sponge is:

[0041] 5-GAAGTCACTCCCGCCAGCTGCAAtatacGAAGTCACTCCCGCCAGCTGCAAacatcGAAGTCA CTCCCGCCAGCTGCAAtcttcaGAAGTCACTCCCGCCAGCTGCAA-3;

[0042] The capital bases are the reverse complementary sequence of hsa-mir-1301-3p with partial mismatch, which plays a role in directly adsorbing microRNA hsa-mir-1301-3p and blocking its binding to the mRNA of the PLK1 gene in the cell;

[0043] The purpose of introducing mismatches is to avoid forming a completely complementary double-stranded RNA structure, thereby avoiding triggering the classic RNA interference pathway in the cell and ensuring the stability of the sponge sequence; the lowercase base part is the connecting sequence; the four mismatched reverse complementary sequences form a tandem structure, and finally the coding DNA sequence of the microRNA sponge shown in SEQ ID NO:2.

[0044] In the present invention, SEQ ID NO: 2 is transcribed to produce mRNA, which adsorbs mir-1301-3p in brain glioma cells, blocks its binding to 3UTR of PLK1 mRNA, downregulates the expression of PLK1 at the protein level, and inhibits the proliferation, adhesion, drug resistance and other characteristics of cancer cells, thereby playing a role in treating cancer.

[0045] The present invention also provides a recombinant lentiviral vector containing the nucleotide sequence of SEQ ID NO:2.

[0046] It can be made by using pHBLV-CMV-MCS-mCherry-T2A-PURO or pHBLV-CMV-MCS-T2A-PURO as the skeleton and inserting the coding DNA sequence of the microRNA sponge.

[0047] In the present invention, the microRNA sponge is expressed by a CMV promoter.

[0048] The pHBLV-CMV-MCS-mCherry-T2A-PURO or pHBLV-CMV-MCS-T2A-PURO is from conventional commercial sources. In the present invention, the insertion sites of the shRNA are preferably XhoI and MluI. The present invention has no particular restrictions on the construction method of the recombinant lentiviral vector, and the conventional recombinant lentiviral vector construction method in the art can be used.

[0049] In actual application, the backbone of the recombinant lentiviral vector is not limited to the above

[0050] pHBLV-CMV-MCS-mCherry-T2A-PURO or pHBLV-CMV-MCS-T2A-PURO is the backbone, and any commonly used plasmid backbone on the market can be used.

[0051] The present invention also provides a recombinant lentivirus, which is obtained by transfecting a cell line with the recombinant lentivirus vector described in the above scheme and packaging it.

[0052] In the present invention, the method for constructing the recombinant lentivirus preferably comprises the following steps: co-transfecting the recombinant lentiviral vector, the lentiviral envelope vector and the lentiviral packaging vector into the HEK293 cell line, and collecting the cell supernatant. In the present invention, the lentiviral envelope vector is preferably pMD2.G; the lentiviral packaging vector is preferably psPAX2; and the cell line is preferably 293T cells. The present invention does not specifically limit the transfection method, which is a conventional technique in the art.

[0053] The recombinant lentivirus of the present invention inhibits the proliferation, adhesion, metastasis, drug resistance and other abilities of cancer cells by downregulating the expression of PLK1 protein level, thereby playing a role in treating cancer.

[0054] The present invention also provides a drug for treating brain glioma, wherein the drug comprises the recombinant lentivirus and a pharmaceutically acceptable excipient. The present invention has no special requirements on the dosage form of the drug, and a conventional dosage form in the art can be used.

[0055] In the present invention, the brain glioma cell lines are preferably SW1088, CCFSTTG1, A172, SF188 and U87.

[0056] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0057] Example 1: Construction of microRNA sponge lentivirus

[0058] 1. Construction of lentiviral vector plasmid.

[0059] The nucleotide sequence of the coding DNA of the miRNA sponge that can adsorb microRNA hsa-mir-1301-3p (hereinafter referred to as the sponge sequence fragment) is shown in SEQ ID NO: 2; the vector plasmid pHBLV-CMV-MCS--mCherry-T2A-PURO was double-digested with XhoI and MluI of Takara Company, and the required reagents are shown in Table 1. After reacting in a 37°C water bath for 3 hours, 10 μL 6×Loading Buffer was added to each centrifuge tube to terminate the enzyme digestion. The vector DNA was purified using QIAquick GelExtraction Kit of QIAGEN Company.

[0060] Table 1 Vector restriction enzyme digestion reaction system

[0061]

[0062] 2. Acquisition of target fragments of sponge sequences

[0063] This example uses fusion PCR to obtain sponge sequences.

[0064] LV-hsa-miR-1301-3p sponge-1F:

[0065] gcctgaatctaggtcgacaatcacgcgtGAAGTCACTCCCGCCAGCTGCAAtatacG

[0066] LV-hsa-miR-1301-3p sponge-1R:

[0067] GTGACTTCgatgtTTGCAGCTGGCGGGAGTGACTTCgtataTTGCAGCTGGCGGG

[0068] LV-hsa-miR-1301-3p sponge-2F:

[0069] GCTGCAAacatcGAAGTCACTCCCGCCAGCTGCAAtcttcaGAAGTCACTC

[0070] LV-hsa-miR-1301-3p sponge-2R:

[0071] ccagaggttgattgttccagctcgagTTGCAGCTGGCGGGAGTGACTTCtgaagaTTGC

[0072] Use PCR method to amplify the target fragment, prepare the following system, mix gently, and place in PCR instrument for reaction:

[0073] Table 2: PCR reaction system

[0074]

[0075] The PCR program is as follows:

[0076] Table 3: PCR condition settings

[0077]

[0078] The enzyme connection system of the sponge sequence fragment and the vector is as follows:

[0079] Table 4: Connection conditions

[0080]

[0081] Ligate overnight at 16°C. Figure 1 shown.

[0082] Transform the ligated plasmid into E. coli DH5α as follows:

[0083] 1) After taking out the DH5α competent cells from the -80℃ refrigerator, put them on ice to thaw immediately;

[0084] 2) After the competent cells melt, add 5 μL of the ligation product to 50 μL of the competent cells and place on ice for 30 minutes;

[0085] 3) Heat shock at 42℃ for 90s, and immediately incubate on ice for 2-3min;

[0086] 4) In a clean bench, add 500 μL of SOC medium and gently invert 3-5 times;

[0087] 5) Incubate at 37°C, 230 rpm with shaking for 45-60 min;

[0088] 6) Apply the bacterial solution to the solid plate with ampicillin resistance, spread it evenly, then invert the plate and culture it in a 37℃ incubator for 16h. After a single colony grows, inoculate it into 5ml LB medium containing ampicillin, and culture it at 37℃, 220rpm shaking for 18h. A small amount of bacteria is mixed with glycerol and stored at -80℃ for later use. Take 3ml of bacterial solution to extract the plasmid. After DNA sequencing confirms that the inserted sequence is correct, the glycerol strain is inoculated into 10mL LB medium containing ampicillin resistance, and culture it at 37℃, 220rpm shaking for 18h. Take 3mL of overnight bacteria and add them to 200mL LB medium containing ampicillin resistance, and culture it at 37℃, 220rpm shaking for 16h. After harvesting the bacterial solution, use the QIAGENQIAfilter large-scale plasmid extraction kit to extract the plasmid, and the final harvested plasmid concentration is not less than 1μg / μl.

[0089] 2. Packaging of lentivirus.

[0090] 1) Cell pre-culture. Subculture 293T cells in advance to a 10 cm diameter round culture dish and place in a 37°C, 5% CO2 incubator overnight; observe the cell density the next day, and transfection can be performed when the confluence rate reaches 70% to 80%; before starting transfection, wash 293T cells twice with preheated DMEM and place in a 37°C incubator for later use.

[0091] 2) Transfection. This example uses liposome transfection. The ingredients are as follows:

[0092] Table 5: Liposome transfection system (10 cm diameter dish)

[0093]

[0094] 3) Virus collection. Collect the virus supernatant 48 hours after transfection. Add fresh complete culture medium containing 2% fetal bovine serum (FBS) 48 hours after virus collection, place in a constant temperature incubator at 37°C and 5% CO2 for further culture, and collect the virus again 72 hours after transfection.

[0095] 4) Ultracentrifugation. Place the virus supernatant collected in step (3) in a 50 mL centrifuge tube, centrifuge at 4°C, 2000 × g for 10 min to remove cell debris; then collect the virus stock solution supernatant in an ultracentrifuge tube, centrifuge at 4°C, 82700 × g for 120 min, remove the supernatant, resuspend the lentivirus ultracentrifugation precipitate in PBS, dispense into sterilized virus tubes to obtain virus stock solution, and store in a -80°C refrigerator.

[0096] 5) Titer detection. Lentivirus titer detection uses the dilution counting method. Titer unit: TU / mL, refers to the number of biologically active virus particles contained in each milliliter. "TU" is the abbreviation of "transducing units", which means "transduction unit" in Chinese, indicating the number of viral genomes that can infect and enter the target cells. IU / mL refers to the number of integration-active virus particles contained in each milliliter. "IU" is the abbreviation of integration units, which means integration unit in Chinese. The titer detection steps of fluorescent lentivirus are as follows:

[0097] I. Cell Preparation

[0098] Digest and count the 293T cells that are growing well and dilute them to 1x10 5 / mL, add to 96-well plate, 100 μL / well, prepare 6 wells for each virus, and culture in a 37°C, 5% CO2 incubator.

[0099] II. Adding virus

[0100] On the second day, prepare 6 1.5mL EP tubes, add 10μL of virus solution to the first EP tube, and then make 3-fold gradient dilutions in complete medium containing 10% FBS, for a total of 6 dilutions. Add 100μL of medium containing different dilutions to 293T cells.

[0101] III. Adding culture medium

[0102] On the third day, remove 100 μL of virus-containing medium and add 100 μL of fresh 10% FBS complete medium.

[0103] IV. Observe the results and calculate the titer

[0104] On the fifth day, the results were observed under a fluorescence microscope. Fresh 10% FBS complete medium was replaced 6 hours before the results were observed. 80 μL of medium was aspirated from the wells, and then 80 μL of fresh 10% FBS complete medium was added. The wells were cultured in a 37°C, 5% CO2 incubator. The results were observed under a fluorescence microscope after 6 hours, and the virus titer was calculated for the wells with a fluorescence percentage of 10-50%.

[0105] Titer (TU / mL) = number of cells × percentage of positive clones × MOI (1) × virus dilution times × 10 3 TU / mL

[0106] Titer detection steps for non-fluorescent lentivirus:

[0107] I. Cell Preparation

[0108] Digest and count the 293T cells that are growing well and dilute them to 1x10 5 / mL, add to 96-well plate, 100 μL / well, prepare 12 wells for each virus, 6 of which are puromycin screening wells and 6 are control wells. Culture in a 37°C, 5% CO2 incubator.

[0109] II. Adding virus

[0110] On the second day, prepare 6 1.5mL EP tubes, add 10μL of virus solution to the first EP tube, and then make 3-fold gradient dilutions in complete medium containing 10% FBS, for a total of 6 dilutions. Add 100μL of medium containing different dilutions to all 293T cell wells.

[0111] III. Adding culture medium

[0112] On the third day, first remove 100 μL of virus-containing culture medium, add 100 μL of 10% FBS complete culture medium containing 1.5 μg / ml puromycin to the 6 puro selection wells, and add 100 μL of normal 10% FBS complete culture medium to the 6 control groups.

[0113] IV. Observe the results and calculate the titer

[0114] On the fifth day, the results were observed under a fluorescence microscope. Fresh 10% FBS complete medium was replaced 6 hours before the results were observed. 80 μL of medium was aspirated from the wells, and then 80 μL of fresh 10% FBS complete medium was added and placed in a 37°C, 5% CO2 incubator for culture. The results were observed under a fluorescence microscope for 6 hours, and the cell count was used to calculate the ratio of live cells in the purine screening group to live cells in the control group. The titer was calculated as follows:

[0115] Titer (TU / mL) = number of cells × percentage of positive clones × MOI (1) × virus dilution times × 10 3 TU / mL

[0116] Example 2: Functional verification of PLK1 protein levels in brain glioma cells

[0117] PLK1 is a therapeutic target gene for a variety of cancers, including glioma. For the purpose of rigor and setting up a control group, this example repeatedly verifies the inhibitory effect of downregulating PLK1 expression on cancer cells from the protein level in CCFSTTG1 and SW1088 cell lines. All cell lines used have been identified by STR to confirm that the strains are correct. The reagents and methods used are conventional techniques in the field, so except for key information such as basic principles, reagent item numbers, concentrations, experimental conditions, etc., the detailed experimental steps will not be repeated.

[0118] Culture medium components: SW1088 is DMEM culture medium + 10% FBS cell culture grade fetal bovine serum + 1% penicillin / streptomycin double antibody; CCFSTTG1 is RPMI1640 culture medium + 10% FBS cell culture grade fetal bovine serum + 1% penicillin / streptomycin double antibody.

[0119] CCFSTTG1 and SW1088 cells were revived; after growing to 90% confluence, they were trypsinized and transferred to 24-well plates, and the number of cells was counted using a hemocytometer to ensure that the number of cells inoculated between different treatment groups of the same cell line was the same; after 24 hours, the culture medium was replaced, and the PLK1-specific inhibitor GSK461364A (MCE Catalog No.: HY-50877) was added to a final concentration of 30 nM / ml.

[0120] Among them, GSK461364A is a PLK1-specific ATP-competitive inhibitor, which directly inhibits the phosphorylation level of PLK1, and then promotes the degradation of dephosphorylated PLK1, resulting in downregulation of protein levels.

[0121] After 48 hours of PLK1 inhibitor treatment, the culture was terminated, and the cell viability was detected by measuring OD450 with CCK8 reagent, and P-PLK1 and PLK1 were detected by immunofluorescence staining. The primary antibody number was MCE HY-P80474 (working concentration 1: 50) and abcam ab189139 (working concentration 1: 200), and the secondary antibody number was abcam ab150077 (working concentration 1: 500). 40 mg / ml BSA + 10% blocking goat serum (Solebo SL038) was used for blocking and antibody dilution. 10% formaldehyde PBS solution was used for cell fixation, and 0.2% tritonX-100 PBS solution was used for cell permeabilization. A fluorescent inverted microscope was used for photography. The experimental group and the control group of the same cell line and the same batch had the same fluorescence photography conditions, that is, the same sampling mode of the photo, the same black balance background subtraction area, and the same exposure time of the green fluorescence field. The cell immunofluorescence experimental conditions involved in the subsequent Example 4 are consistent with this section.

[0122] The results are as follows Figure 2 As shown. It can be seen that the growth of cells treated with GSK461364A was inhibited and the cell viability was significantly reduced; the phosphorylation level was inhibited, resulting in a significant downregulation of P-PLK, and the total amount of PLK1 protein was also significantly downregulated. In particular, fragmented nuclei can be observed in the SW1088 inhibitor treatment group. These results all prove the important role of PLK1 in the growth of cancer cells, and downregulating its expression can be used as a treatment method for brain glioma cancer cells.

[0123] Example 3: Next-generation sequencing results of brain glioma cell samples and prediction of microRNA targets

[0124] The cell samples were sequenced by next-generation sequencing to detect the overall expression of mRNA and microRNA hsa-mir-1301-3p. The experiment was outsourced to a sequencing company, and the steps used, including total RNA extraction, mRNA or microRNA purification, library construction, quality inspection, and sequencing, were all commercial routine methods and will not be repeated here. Both sequencing methods were performed using the NovaSeq 6000 platform (Illumina).

[0125] Data analysis software workflow

[0126] The installation process and operating instructions of the required software, as well as the specific Linux commands or R language programming codes for implementing the analysis process, are all available on the Internet.

[0127] mRNA data analysis:

[0128] mRNA quantitative analysis and PLK1 expression display

[0129] The original data from the sequencing platform [fastq format file] was removed and data quality checked by fastp software to obtain CleanData [fastq format file]; then the hisat2 software was used to map the measured sequence with the human reference genome index (the human reference genome index was directly downloaded from the hisat2 official website, [ht2 format file]); the samtools software was used to convert the file format, reorder and merge genes to obtain [bam format file]; the stringtie software was used to combine the human genome reference gene annotation file (the human genome reference gene annotation was downloaded from ensemble, [gtf format file]) to obtain the gene expression quantitative results with reference sequences [gtf format file], which included three quantitative methods: readcount, FPKM and TPM; the R language "rtracklayer" package was used to read the quantitative result gtf format file, and the expression information of PLK1 was selected for drawing using TPM quantification.

[0130] MicroRNA data analysis workflow:

[0131] Preparation:

[0132] First, download the human genome reference sequence GRCH38 [fasta format file] from the ensemble website, enter the bowtie2 software to establish the human reference genome index; download the human reference genome annotation file [gtf format file] from the ensemble; download the sequence information of mature and precursor forms of all known human microRNAs from the mirBase website [two fasta format files].

[0133] Quantitative analysis of microRNA and display of expression, structure and sequence

[0134] The original data from the sequencing platform [fastq format file] was quality checked, removed from the linker and converted to [fasta format file] by fastp software; this file, together with the human reference genome index [ht2 format file], human reference genome annotation [gtf format file], microRNA precursor and mature body sequence files [both fasta format files] prepared above, were input into miRDeep2 software for microRNA quantitative analysis to obtain microRNA readcount and CPM quantitative results [excel format file], and the sequence / secondary structure diagram of all detected microRNAs [html web link]; the CPM quantitative results and secondary structure diagram results of hsa-mir-1301-3p were selected for plotting. The results are as follows Figure 2 As shown in A and B

[0135] MicroRNA target prediction and result display

[0136] Use the R language "rtracklayer" package to read the human reference genome annotation [gtf format file] downloaded above, select the region with the annotation category of "3UTR" and output it as 3UTR coordinates [bed format file] for backup; use the bedtools software, take the human genome reference sequence GRCH38 [fasta format file] and 3UTR coordinates [bed format file] as input information, and obtain the 3UTR sequence information of all human mRNA [fasta format file] for backup; use the R language programming method to extract the sequence information fasta format file of hsa-mir-1301-3p from the microRNA mature sequence file, and input this file together with the aforementioned "whole mRNA 3UTR sequence information" file into the miRanda software to obtain the microRNA-mRNA 3UTR interaction results [txt format file], which can be opened with professional text editing tools such as ultraedit, and independent drawings can be made according to the displayed information, such as Figure 2 As shown in C.

[0137] Note: In theory, the microRNA interacts with the 3UTR of mRNA. In the drawings of the specification of this embodiment, "T" is used instead of "U" in the mRNA, which is the fixed format of the prediction results of the miRanda software. It is specially noted here and will not be repeated in the following figures.

[0138] Figure 3A shows the RNA sequence of mir-1301-3p and the secondary structure formed by its synthetic precursor; Figure 3 B shows the expression levels of PLK1 mRNA and mir-1301-3p microRNA in five glioma cell lines; Figure 3 C used software calculations to prove that mir-1301-3p and PLK1 have base complementary binding containing mismatches. The binding free energy of this process is -25.87 kcal / mol, which is a spontaneous process, indicating that PLK1 is a target of mir-1301-3p.

[0139] Example 4: Verification of the effect of mir-1301-3p inhibition lentivirus infection on cell growth rate and target protein PLK1 expression in brain glioma cells

[0140] The cell lines used are five brain glioma cell lines, U87, A172, CCFSTTG1, SF188 and SW1088, all of which have been confirmed by STR identification. Except for the CCFSTTG1 culture medium group, which was replaced with RPMI1640 culture medium in equal amounts, the culture medium of the other four cell lines was DMEM + 10% FBS fetal bovine serum + 1% penicillin / streptomycin double antibody. 0.25% trypsin-EDTA was used for digestion and passage. During the experiment, the cells were inoculated into a 24-well plate to ensure that the number of cells of the same type inoculated in each well was the same; after 24 hours, the microRNA inhibitory lentivirus prepared in Example 1 was added, and the MOI of the lentivirus was controlled at 10 to 50, which varied according to the cell line; the control group was added with an equal amount of control lentivirus packaged with an empty vector plasmid without a microRNA inhibitory sponge sequence connected.

[0141] The culture medium was changed every 2-3 days. CCK8 cell viability test was performed on day 5 or day 6. The results were as follows: Figure 4 As shown in C; the expression of the predicted target gene PLK1 protein level was detected by cell immunofluorescence staining experiment. The results are as follows Figure 5 As shown, the primary antibody for PLK1 staining is abcam ab189139 product, with a dilution ratio of 1:200, the secondary antibody is abcam ab150077 product, with a dilution ratio of 1:500, and other experimental parameters are the same as described in Example 2.

[0142] The design principle and detection principle of microRNA inhibitory sponge sequence are shown in the following figure. Figure 4As shown in A, the specific adsorption of mir-1301-3p is achieved while avoiding the RNAi interference pathway by mismatching to ensure the stability of the sponge sequence. The presence of the sponge sequence is detected by qPCR. Here, the sponge sequence and the sequences at both ends in the lentiviral vector plasmid are first detected by the first-generation sequencing method, as shown below, where the black bold base part is the positive strand of the sponge sequence:

[0143] Then, primers were designed at both ends of the sponge sequence for qPCR detection. The detection primers spanned the connection point between the sponge sequence and the vector plasmid backbone to ensure the specificity of the primers for the sponge sequence template detection. The F primer sequence is as follows:

[0144] The primer sequence of 5-CGACAATCACGCGTGAAGTCA-3R is as follows:

[0145] 5-TCCAGCTCGAGTTGCAGCTG-3 uses human β-actin as the quantitative internal reference gene, and the primers used are commercially available conventional primers, which will not be described here.

[0146] The results of QPCR are as follows Figure 4 B, and the corresponding qPCR detection melting curve is as follows Figure 4 C. According to the melting curve of 4C, the melting curves of the miRNA sponge samples have a single main peak in a consistent position, indicating that the qPCR amplified fragment is unique and specific; the qPCR detection of the control group has a large number of miscellaneous peaks, indicating that there is actually more than one non-specific amplification, and the peak shape is significantly different between different cell lines, indicating that the five cell lines have differences in RNA expression background. Figure 4 From the quantitative results of qPCR in B, it can be seen that the expression level of the single amplified miRNA sponge experimental group is much higher than that of the control group with multiple non-specific amplifications. Therefore, it can be seen that a large number of sponge sequences were successfully expressed in the experimental group.

[0147] Comprehensive consideration Figure 4 and Figure 5The results showed that in the five glioma cell lines, the cell viability decreased significantly after expressing the sponge sequence. At the same time, the PLK1 protein level, which is a key therapeutic target and a predicted target of hsa-mir-1301-3p, decreased significantly, indicating that expressing the sponge sequence and inhibiting mir-1301-3p can indeed inhibit the growth of glioma. This inhibitory effect is effective in five different cell lines, indicating that it has a certain universality. At the same time, it is very novel that the classical theory holds that in animals, including cancer cells, the interaction of microRNA with the 3UTR of mRNA inhibits protein translation and silences gene expression, so inhibiting the binding of microRNA to mRNA should promote the protein expression of the target gene; however, according to the results in this embodiment, the use of sponge sequences inhibited the interaction of mir-1301-3p with the 3UTR of PLK1, which in turn significantly reduced the protein expression of PLK1 in five brain glioma cell lines, indicating that the interaction of mir-1301-3p with PLK1 mRNA promotes, or at least maintains, the expression of PLK1, which is an innovative point of the present invention.

[0148] also, Figure 4 The schematic diagram of the sponge sequence design of A shows that the combination of the sponge sequence and microRNA must have a certain mismatch to resist the degradation pathway of RNA interference. This means that mir-1301-3p is not the only microRNA that will be adsorbed and inhibited by the sponge sequence. There must be other microRNAs whose sequences are similar to the sponge sequence of SEQ ID NO: 2 of the present invention, or mir-1301-3p, and whose functions are also inhibited; in addition, microRNA regulation itself allows mismatched binding with the target gene mRNA so that a microRNA can interact and regulate the protein expression of more than one gene. Although the present invention predicts and verifies that PLK1 is a protein regulated by mir-1301 and is also a key target for cancer treatment through bioinformatics methods, based on the above two reasons, there must be more than one key target protein for cancer treatment that will be affected by the sponge sequence shown in SEQ ID NO: 2 of the present invention.

[0149] Based on the principle that microRNA binds to target gene mRNA by base mismatch, and the feature that the sponge sequence and microRNA also have base mismatch to resist RNA interference, the microRNA sponge with nucleotide sequence as shown in SEQ ID NO:2 in the present invention can interact and regulate the coding gene of protein level expression is not unique; it can adsorb and inhibit the function of microRNA, which also has diversity, and then leads to a richer diversity of affected protein expression. In addition to the verified PLK1 mentioned in the present invention, all changes in the protein level of coding genes that can be triggered by it that are beneficial to inhibiting cancer cells are within the scope of the claims of this patent.

[0150] The present invention and its implementation methods are described above, and such description is not restrictive, and the actual methods are not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design implementation methods and embodiments similar to the technical solution without creativity, which should all fall within the protection scope of the present invention.

Claims

1. A miRNA molecular sponge, characterized in that: The nucleotide sequence of the DNA encoding the miRNA molecule sponge is shown in SEQ ID NO:

2.

2. A miRNA molecular sponge according to claim 1, characterized in that: It can adsorb microRNA hsa-mir-1301-3p, block the binding of microRNA hsa-mir-1301-3p to the target gene, and thus downregulate the protein level expression; The nucleotide sequence of the microRNA hsa-mir-1301-3p is shown in SEQ ID NO:

1.

3. A miRNA molecular sponge according to claim 2, characterized in that, The target gene is the PLK1 gene, and the protein is the PLK1 protein.

4. Use of a miRNA molecular sponge according to any one of claims 1 to 3 in the preparation of a drug for treating or assisting in the treatment of brain glioma.

5. A recombinant lentiviral vector, characterized in that: It contains the nucleotide sequence of the DNA encoding the miRNA molecular sponge according to claim 1.

6. Use of a recombinant lentiviral vector according to claim 5 in the preparation of a drug for treating or assisting in the treatment of brain glioma.

7. A recombinant lentivirus, characterized in that: It comprises the nucleotide sequence of the DNA encoding the miRNA molecule sponge described in any one of claims 1 to 3, or the recombinant lentiviral vector described in claim 5.

8. A recombinant lentivirus according to claim 7, characterized in that: The recombinant lentiviral vector according to claim 5 is co-transfected with lentiviral packaging plasmids psPAX2 and pMD2G into HEK293 cell line, and then packaged and concentrated to obtain the vector.

9. Use of a recombinant lentivirus according to claim 7 or 8 in the preparation of a drug for treating or assisting in the treatment of brain glioma.

10. A drug for treating or assisting in treating brain glioma, characterized in that: It comprises an effective amount of the recombinant lentivirus according to claim 7 or 8 and a pharmaceutically acceptable excipient.

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