Application of XRCC5 gene inhibitor in preparing medicine for treating T-ALL

Abstract

The present invention discloses the application of XRCC5 gene inhibitors in the preparation of drugs for treating T-ALL, belonging to the field of biomedicine. The present invention discovers for the first time that down-regulating the expression of XRCC5 can significantly inhibit the proliferation of T-ALL tumor cells and effectively promote the apoptosis of T-ALL tumor cells. Specifically, based on the RNA interference technology, the siRNA targeting the above gene is artificially synthesized to achieve the reduction of the expression of XRCC5. The present invention has important significance for developing new gene drugs for treating T-ALL and improving the treatment effect of T-ALL, and has great application prospects and economic value.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of an XRCC5 gene inhibitor in the preparation of a drug for treating T-ALL. Background Art

[0002] Acute T-lymphoblastic leukemia (T-ALL) is a malignant and invasive hematological tumor, in which the malignant transformation of developing T-cell precursors is caused by the abnormal accumulation of oncogenes. Although current combination chemotherapy has significantly improved the cure rate of T-ALL, approximately 20% of children and 50% of adult patients still die from drug resistance or recurrence. In addition, compared with B-lymphocyte tumors, T-ALL has high heterogeneity and lacks targeted and immunotherapeutic means. Therefore, there is an urgent need to find new treatment strategies effectively targeting T-ALL.

[0003] In recent years, using RNA interference technology to transduce chemically synthesized small interfering RNA (siRNA) into specific cells to silence the expression of related genes is one of the most effective means for studying gene function and also one of the most attractive methods for targeted gene therapy. Selecting specific target genes to synthesize effective siRNAs and choosing appropriate transfection methods to make them act efficiently in host cells are the key points for implementing this targeted treatment measure. The rapid development of high-throughput sequencing technology has provided more and more accurate predictions for the diagnosis and prognosis of T-ALL patients, thereby enabling more effective precision treatment for patients. Summary of the Invention

[0004] The primary object of the present invention is to provide the application of an XRCC5 gene inhibitor in the preparation of a drug for treating T-ALL.

[0005] Another object of the present invention is to provide an siRNA capable of inhibiting the expression of the XRCC5 gene.

[0006] The objects of the present invention are achieved by the following technical solutions:

[0007] The application of an XRCC5 gene inhibitor in the preparation of a drug for treating T-ALL.

[0008] The inhibitor is a molecule or preparation prepared or screened using XRCC5 as a target and having an inhibitory effect thereon, and can be at least one of siRNA, shRNA, dsRNA, miRNA, cDNA, antisense RNA / DNA, low molecular weight compounds, peptides, or antibodies.

[0009] Preferably, the inhibitor is siRNA, and among them, the sense strand sequences of the siRNAs inhibiting XRCC5 expression are as follows:

[0010] si-XRCC5-1, 5'-CGUGGGCUUUACCAUGAGUAATT-3';

[0011] si-XRCC5-2, 5'-CCUCAUAUCAAGCAUAACUAUTT-3'.

[0012] An siRNA capable of inhibiting XRCC5 expression, and the sense strand sequence is one of the following:

[0013] si-XRCC5-1, 5'-CGUGGGCUUUACCAUGAGUAATT-3';

[0014] si-XRCC5-2, 5'-CCUCAUAUCAAGCAUAACUAUTT-3'.

[0015] A drug for treating T-ALL, comprising the above siRNA capable of inhibiting XRCC5 expression.

[0016] The present invention has the following advantages and effects compared with the prior art:

[0017] 1. The present invention first proposes the application of XRCC5 as a target in the preparation of drugs for treating T-ALL, providing a new and effective treatment strategy for T-ALL.

[0018] 2. The siRNA of the present invention capable of inhibiting XRCC5 expression can efficiently inhibit the expression of the XRCC5 gene. The down-regulation degree of the mRNA of the siRNA capable of efficiently inhibiting XRCC5 reaches 50% - 75%, and significantly inhibits the protein level of XRCC5.

[0019] 3. The siRNA of the present invention capable of inhibiting XRCC5 expression can significantly promote the apoptosis of T-ALL tumor cells.

[0020] 4. The siRNA sequence of the present invention is of great significance for the development of new gene drugs for treating T-ALL and improving the treatment effect of T-ALL, can significantly reduce the drug resistance problem of traditional treatment drugs, and has great application prospects and economic value. Brief Description of the Drawings

[0021] Figure 1It is a partial experimental result diagram of Example 1; among them, A&B are the gene expression conditions of XRCC5 in T-ALL specimens and healthy control specimens (A, the specimen library of Jinan University; B, the GEO database); C&D are the XRCC5 protein expressions of the T-ALL cell lines CCRF and JURKAT cells treated with the chemotherapeutic drugs doxorubicin (DOX) and vincristine (VCR) at different time points; E&F are the expression level results of XRCC5 in JURKAT cells; G is the statistical result of the apoptosis rate analyzed in three repeated experiments after siRNA transfection of CCRF and JURKAT cells. Detailed implementation mode

[0022] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation modes of the present invention are not limited thereto.

[0023] Example 1

[0024] (1) Add 1 ml of TRIzol reagent to 5 - 10×10 6 personal T-ALL leukemia cells (cell lines CCRF-CEM and JURKAT).

[0025] (2) Total RNA extraction

[0026] 2.1 Place the treated sample on ice. After reacting for 5 minutes, add 0.2 ml of chloroform, mix well (for 15 seconds), then place on ice for 2 - 3 minutes, and centrifuge at low temperature and high speed (2 - 8°C, 12000g) for 15 - 30 minutes;

[0027] 2.2 Carefully aspirate the upper layer liquid (about 50% of the total volume) and transfer it to a new 1.5 ml tube. Add 0.5 ml of isopropanol and mix well. After placing on ice for 10 minutes, centrifuge at low temperature and high speed for 10 - 20 minutes;

[0028] 2.3 Discard the supernatant and wash twice with 1 ml of 75% ethanol (-20°C) (2 - 8°C, 10000g). Each time, mix well for 30 seconds and then centrifuge for 5 - 10 minutes;

[0029] 2.4 Discard the supernatant and centrifuge once more to remove the remaining ethanol;

[0030] 2.5 Dry the washed sample in a low-temperature vacuum centrifuge for 5 - 10 minutes;

[0031] 2.6 Add 50 μl of ultrapure water (Biotecx BL-5700) (can be appropriately increased or decreased according to the situation) and mix well.

[0032] 2.7 Dilute 2 μL of the RNA solution to 400 μL, and detect the optical density of the sample at a wavelength of 260 nm in an ultraviolet spectrophotometer to estimate the purity and content of the sample (1 OD A260nm = 40 μg / mL), total RNA amount = OD number × 400 μg;

[0033] 2.8 After adding 0.1 volume (5 μL) of 3M NaAC (sodium acetate) and 2 volumes of ethanol (100 μL) to the RNA sample, store it at -70 °C for later use.

[0034] (3) RT-PCR

[0035] 3.1 Mix 500 ng of RNA, 0.5 μL of oligo(dT) (0.5 μg / reaction), 0.5 μL of random primers (0.5 μg / reaction) and RNase-free double-distilled water (ddH2O) (up to 5 μL) in proportion, incubate at 70 °C for 5 minutes, and then quickly cool on ice for 5 minutes;

[0036] 3.2 Add 4.0 μL of GoScriptTM 5× reaction buffer, 1.7 μL of MgCl2 (final concentration 2.0 mM), 1.0 μL of 0.5 mM dNTP, 0.3 μL of ribonuclease inhibitor (20 U), 1.0 μL of reverse transcriptase and ddH2O, with a total volume of 15 μL;

[0037] 3.3 After mixing, incubate 20 μL of the sample at 42 °C for 60 minutes, and then inactivate at 70 °C for 15 minutes;

[0038] 3.4 Finally, add 80 μL of ddH2O for dilution and store at -20 °C.

[0039] (4) qRT-PCR

[0040] 4.1 Prepare a 20 μL system for qRT-PCR as follows: 10 μL of 2× Mix, 0.5 μL of forward primer (10 μM), 0.5 μL of reverse primer (10 μM), 1 μL of cDNA and 8 μL of RNase-free ddH2O;

[0041] The sequences of the primers used are as follows:

[0042] XRCC5-F: 5'-GTTCTAAAGGTCTTTGCAGCAAGA-3'

[0043] XRCC5-R: 5'-AAAAGCCACGCCGACTTGAGGA-3'

[0044] ACTB-F: 5'-TTGTTACAGGAAGTCCCTTGCC-3'

[0045] ACTB-R: 5'-ATGCTATCACCTCCCCTGTGTG-3'.

[0046] 4. The program settings for qRT-PCR are as follows: After treatment at 95°C for 15 minutes, 45 cycles are carried out with 10 s at 95°C to 20 s at 60°C.

[0047] (5) siRNA transfection

[0048] 5.1 Design and synthesis of siRNA

[0049] Search for the XRCC5 gene sequence (ACCESSION NM_021141.4) in GenBank (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and then design siRNA targeting XRCC5 online according to the StealthTM RNAi design rules provided by Invitrogen (www.invitrogen.com). The specific sense strand sequences of the siRNA involved in the present invention are respectively:

[0050] si-XRCC5-1, 5'-CGUGGGCUUUACCAUGAGUAATT-3';

[0051] si-XRCC5-2, 5'-CCUCAUAUCAAGCAUAACUAUTT-3'.

[0052] The above siRNA and siNC (siN0000001, sequence: UUC UCC GAA CGU GUC ACG UTT) are chemically synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0053] 5.2 Prepare the T-ALL leukemia cell line CCRF or JURKAT in the logarithmic growth phase

[0054] Inoculate CCRF-CEM or JURKAT cells in RPMI1640 medium containing 10% (v / v) newborn bovine serum, 100 U / mL penicillin and 100 U / mL streptomycin, and continuously culture at 37°C in an incubator containing 5% (v / v) CO2. Passage once every 2 - 3 days to obtain CCRF-CEM or JURKAT cells in the logarithmic growth phase.

[0055] 5.3 Neon electroporation of siRNA

[0056] A. Collect 2×10 5The CCRF-CEM or JURKAT cells in the logarithmic growth phase prepared in step (2) were centrifuged at 200 g for 10 min to completely remove the supernatant;

[0057] B. Use 10 μL of Buffer R and 1 μL of 100 μM CCRF-CEM or JURKAT cells;

[0058] C. Start the JURKAT electroporation program (1450 V, 10 ms, 3 pulses);

[0059] D. After the program is completed, inoculate the transfected cell solution into a 6-well plate with a final volume of 3 mL;

[0060] E. Incubate in an incubator for 48 hours.

[0061] (6) Detect the expression level of XRCC5 in the transfected cells

[0062] Refer to step (1).

[0063] (7) Detect cell apoptosis

[0064] 7.1 Centrifuge and wash with pre-cooled PBS to collect the electroporated cells.

[0065] 7.2 Dilute 5×Binding Buffer with double-distilled water to 1× working solution, and resuspend the cells with 500 μL of 1× Binding Buffer.

[0066] 7.3 Add 5 μL of Annexin V-APC and 10 μL of PI to each tube.

[0067] 7.4 Gently vortex and mix well, then incubate at room temperature in the dark for 5 minutes.

[0068] 7.5 Analyze on the machine.

[0069] 7.6 Export the flow cytometry results through Flowjo software.

[0070] (8) Detect protein level expression

[0071] 8.1 Gently wash once with sterile PBS, centrifuge at 250 g for 5 min to collect the cells, add lysis buffer (containing protease inhibitor) at 1×10 7 cells / mL RIPA, place on ice for sufficient lysis, centrifuge at high speed at 4 °C for 10 min, and collect the supernatant to obtain the total protein;

[0072] 8.2 Prepare a 10% SDS-PAGE gel (Table 1)

[0073] Table 1. 10% SDS-PAGE gel formula

[0074]

[0075] 8.3 SDS-PAGE Electrophoresis: The sample loading volume per well is 10 μg. Keep the voltage constant at 80 V for 20 min; then change to 110 V for 70 min.

[0076] 8.4 Semi-dry Transfer Blotting: Cut a 6 cm * 8 cm PVDF membrane in advance and activate it with methanol. Place it in the order of sponge paper, PVDF membrane, separating gel, and sponge paper to avoid generating air bubbles; the transfer blotting conditions are: keep the voltage constant at 20 V for 30 min.

[0077] 8.5 Blocking: After the transfer blotting is completed, take out the membrane and place it in the blocking solution of 5% skim milk powder, shake it on a shaker at room temperature for 1 h;

[0078] 8.6 Primary Antibody Incubation: Cut the PVDF membrane according to the position indicated by the protein Maker, and then put it into the corresponding diluted primary antibody, incubate it on a shaker at 4 °C overnight;

[0079] 8.7 Secondary Antibody Incubation: Recover the primary antibody on the second day, wash the membrane 4 times with 1×TBST, 8 min each time; incubate with the corresponding secondary antibody (diluted 1:10000), incubate it on a shaker at room temperature for 1 h;

[0080] 8.8 ECL Color Development: Recover the secondary antibody, wash the membrane 3 times with 1×TBST, 8 min each time; Prepare the developing solution according to A solution: B solution: ddH2O = 1:1:2, evenly drip the developing solution on the membrane and put it into the UVI Alliance.

[0081] 8.9 Develop and take pictures in the gel imager.

[0082] The results are as Figure 1As shown. Among them, A&B show the gene expression of XRCC5 in T-ALL specimens and healthy control specimens. It can be seen that XRCC5 is significantly highly expressed in T-ALL (A, Specimen Bank of Jinan University; B, GEO database); C&D show the XRCC5 protein expression in T-ALL cell lines CCRF and JURKAT cells treated with chemotherapy drugs doxorubicin (DOX) and vincristine (VCR) at different time points. It can be seen that the expression level of XRCC5 decreases with the prolongation of the treatment time of T-ALL cell lines induced by chemotherapy drugs, and the down-regulation degrees reach 100% and 90% respectively; E&F are the result graphs of the expression level of XRCC5 in JURKAT cells. Compared with the si-NC group, the mRNA level and protein level of the si-XRCC5-1 and si-XRCC5-2 groups are significantly down-regulated, and the down-regulation degrees reach 48±3% and 70±6% respectively; G is the statistical result graph of the apoptosis rate analyzed in three repeated experiments after siRNA transfection of CCRF-CEM and JURKAT cells. It can be seen that inhibiting the gene expression of XRCC5, the inhibition rate of CCRF-CEM can reach 30.6±10.6% and 45.7±15.1%, and the inhibition rate of JURKAT can reach 71.7±4.3% and 72.5±5.5%, which can significantly promote the apoptosis of T-ALL cells.

[0083] The above experimental results illustrate that the XRCC5-specific siRNA provided by the present invention can efficiently inhibit the expression of the XRCC5 gene, and inhibiting the expression of the XRCC5 gene has an obvious function of inhibiting the proliferation of T-ALL cells and inducing cell apoptosis.

[0084] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of an XRCC5 gene inhibitor in the preparation of a drug for treating T-ALL, characterized in that: The T-ALL refers to acute T-lymphoblastic leukemia; The inhibitor is siRNA. Among them, the sense strand sequence of the siRNA that inhibits XRCC5 expression is one of the following: si-XRCC5-1, 5'- CGUGGGCUUUACCAUGAGUAATT -3'; si-XRCC5-2, 5'- CCUCAUAUCAAGCAUAACUAUTT -3'.

2. An siRNA capable of inhibiting XRCC5 expression, characterized in that: The sense strand sequence is one of the following: si-XRCC5-1, 5'- CGUGGGCUUUACCAUGAGUAATT -3'; si-XRCC5-2, 5'- CCUCAUAUCAAGCAUAACUAUTT -3'.

3. A drug for treating T-ALL, characterized in that: It contains the siRNA that can inhibit XRCC5 expression described in claim 2.

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