CN115192601B - Application of PPFIA1 gene in preparation of drug-resistant CML drug for treating independent BCR-ABL1 - Google Patents
Application of PPFIA1 gene in preparation of drug-resistant CML drug for treating independent BCR-ABL1 Download PDFInfo
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
- A61P35/02—Antineoplastic agents specific for leukemia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
- C12Y301/03048—Protein-tyrosine-phosphatase (3.1.3.48)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
- C12N2310/141—MicroRNAs, miRNAs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an application of a PPFIA1 gene in preparing a medicine for treating drug-resistant chronic granulocytic leukemia independent of BCR-ABL1, wherein the drug-resistant chronic granulocytic leukemia independent of BCR-ABL1 refers to an imatinib resistant cell strain without any mutation: T315I, E255K, E255V, G250E, Q252H, Y253H. In the invention, the weight of the B-NDG mice after PPFIA1-siRNA treatment is reduced slowly, the proliferation of K562-IMR/luciferas cells in vivo is inhibited, and the PPFIA1-siRNA can overcome CML drug resistance in vivo and in vitro.
Description
Technical Field
The invention belongs to the field of biological medicines, and particularly relates to application of a PPFIA1 gene in preparation of a medicine for treating drug-resistant chronic granulocytic leukemia (CML) independent of BCR-ABL 1.
Background
Chronic myeloid leukemia is a hematological malignancy, and BCR-ABL1 fusion protein is the central role in the pathogenesis of chronic myeloid leukemia, leading to clonal expansion of hematopoietic cells.
In the treatment of Chronic Myelogenous Leukemia (CML), patients are prone to resistance to imatinib, which presents challenges for clinical treatment.
Depending on whether ABL1 is mutated or not, resistance in CML patients can be divided into two cases, BCR-ABL resistance-dependent and BCR-ABL resistance-independent. The reasons for BCR-ABL dependent drug resistance may include: (1) ABL1 in BCR-ABL1 is mutated, most commonly T315I; (2) BCR-ABL1 overexpression.
After the second and third generation Tyrosine Kinase Inhibitors (TKI) drugs are marketed, this BCR-ABL1 dependent drug resistant CML clinical treatment has achieved good results. However, in the drug-resistant CML treatment independent of BCR-ABL1, clinical treatment is not an effective unified treatment regimen due to the complex and diverse causes of the disease, and thus the clinical challenges of such drug-resistant symptoms are even greater.
Disclosure of Invention
The invention aims to provide an application of a PPFIA1 gene in preparing a medicine for treating drug-resistant chronic granulocytic leukemia independent of BCR-ABL 1.
The aim of the invention is achieved by the following technical scheme:
application of PPFIA1 (PTPRF Interacting Protein Alpha 1, receptor type protein tyrosine phosphatase f polypeptide alpha 1) gene in preparing medicines for treating chronic granulocytic leukemia independent of BCR-ABL1 drug resistance;
application of an agent for inhibiting PPFIA1 gene expression in preparing a medicine for treating drug-resistant chronic granulocytic leukemia independent of BCR-ABL 1;
the drug-resistant chronic granulocytic leukemia independent of BCR-ABL1 refers to an imatinib-resistant cell strain without any mutation: T315I, E255K, E255V, G250E, Q252H, Y253H;
the imatinib-resistant cell strain refers to K562-IMR and KCL22-IMR;
the agent for inhibiting the expression of the PPFIA1 gene comprises siRNA (PPFIA 1-siRNA) targeting the PPFIA1 gene or an expression vector containing the siRNA;
the sequence of the PPFIA1-siRNA is as follows:
sense:5’-CCACAAAGCUCUGGAUGAAdTdT-3’
antisense:5’-UUCAUCCAGAGCUUUGUGGdTdT-3’；
the medicine also contains other active ingredients and auxiliary materials (carriers);
the auxiliary materials (carriers) are preferably sustained release agents, excipients, fillers, adhesives, wetting agents, disintegrating agents, absorption promoters, adsorption carriers, surfactants or lubricants and the like;
the dosage forms of the medicine are aerosol, tablet, capsule, dripping pill, powder, solution, suspension, emulsion, granule, lipid agent, transdermal agent, buccal agent, suppository or freeze-dried powder injection, etc.
The transfected PPFIA1-siRNA can inhibit the proliferation of chronic granulocytic leukemia drug-resistant cells, can form soft agar clone, and can inhibit CML drug resistance in vitro and in vivo in experiments such as B-NDG mouse model of transplanted human K562-IMR/luciferase. PPFIA1 is probably the role and molecular basis in the progression of IM resistance in CML, and IM resistance in IM-resistant CML cells can be inhibited by targeting PPFIA1.
Leukemia stem cells are thought to be one of the major causes of the emergence of imatinib resistance and the recurrence of chronic granulocytic leukemia. CML resistant cells are treated by PPFIA1-siRNA, and the proportion of c-kit positive cells is reduced. Overexpression of PPFIA1 typically activates the KIT pathway, causing malignant proliferation of cancer cells. CD34 is a common marker for leukemia stem cells. CML resistant cells were treated with PPFIA1-siRNA and the proportion of CD34 positive cells was reduced.
Compared with the prior art, the invention has the following advantages and effects:
in the invention, the weight of the B-NDG mice after PPFIA1-siRNA treatment is reduced slowly, the proliferation of K562-IMR/luciferas cells in vivo is inhibited, and the PPFIA1-siRNA can overcome CML drug resistance in vivo and in vitro.
Drawings
FIG. 1 is a technical route for SILAC quantitative proteomics experiments and gene chip experiments.
FIG. 2 is a combination of gene expression profiling chip, SILAC quantitative proteomics and mirFOCUS bioinformatics prediction for identifying miR-181a candidate target genes.
FIG. 3 is a graph of luciferase reporter assay for targeted binding of miR-181a and PPFIA 1' UTR.
FIG. 4 is the expression level of PPFIA1 at the mRNA and protein levels in K562-IMR and KCL22-IMR cells after transfection of miR-181a and PPFIA 1-siRNA.
FIG. 5 shows the expression level of PPFIA1 mRNA in the RIP experiment.
FIG. 6 is a signal pathway associated with flag-PPFIA1 IP products integrated by KEGG database.
FIG. 7 is a graph showing the detection of drug resistance in CML resistant and susceptible strains.
FIG. 8 is SNP sequencing results of K562-IMR and KCL22-IMR; wherein A: K562-IMR; b: KCL22-IMR.
FIG. 9 is a graph showing the inhibition of K562-IMR and KCL22-IMR cells by PPFIA1-siRNA detected by CCK-8 method; wherein A: K562-IMR; b: KCL22-IMR.
FIG. 10 is a graph showing the effect of PPFIA1-siRNA on human CML bone marrow specimen cells at 72h as measured by CCK-8.
FIG. 11 is the number of CML drug resistant cell clones after PPFIA1-siRNA treatment; wherein A: cloning and forming; b: cloning of K562-IMR forms statistics; c: cloning of KCL22-IMR resulted in statistics.
FIG. 12 is a stable expression of luciferase by K562-IMR/luciferas cells wherein A: correspondence between luminous intensity and cell number; b: imaging brightness and cell number correspondence.
FIG. 13 is a model of a human K562-IMR/luciferas cell transplantation B-DNG mouse for constructing independent drug resistance CML mice; wherein A: mice appear as the backs of bows; b: weight change in B-NDG mice; c: fluorescence intensity display of B-NDG mice; d: comparison of the survival time of B-NDG mice.
FIG. 14 shows that PPFIA1-siRNA reduced the phosphorylation level of KIT as detected by phosphorylated protein chip analysis.
FIG. 15 is the ratio of c-kit+ cells in CML sensitive and drug resistant strain cells.
FIG. 16 is a graph showing the change in c-kit+ cell fraction after PPFIA1-siRNA treatment.
FIG. 17 shows the change in the expression level of c-kit protein after PPFIA1-siRNA treatment.
FIG. 18 is the ratio of CD34+ cells in CML sensitive and drug resistant strain cells.
FIG. 19 is a plot of CD34+ cell duty cycle change following PPFIA1-siRNA treatment.
FIG. 20 is a graph showing the change in proportion of early apoptotic cells after PPFIA1-siRNA treatment; wherein A: K562-IMR; b: KCL22-IMR.
*P<0.05，**P<0.01，***P<0.001，mean±SD。
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but embodiments of the present invention are not limited thereto.
Example 1
Multiunit identification of miR-181a targets
1.1 Gene expression profiling chip, proteomics and bioinformatics predictions
The sequence of hsa-miR-181a mic is 5'-aacauucaacgcugucggugagu-3';
the PPFIA1-siRNA sequence is as follows:
sense:5’-CCACAAAGCUCUGGAUGAAdTdT-3’
antisense:5’-UUCAUCCAGAGCUUUGUGGdTdT-3’；
the above sequences were all synthesized by Guangzhou Ruibo.
In order to predict the target gene of miR-181a, the accuracy of target gene prediction can be greatly improved by increasing proteomic detection on the basis of combining a gene expression profile chip and bioinformatics prediction.
The technical route of gene chip and SILAC quantitative proteomics is shown in FIG. 1. The experiments used Agilent Human 1A whole genome oligonucleotide chips, classified according to hybridization signal intensity, screened differentially expressed genes, which were likely target genes for miR-181A (fig. 2). miRFocus software (http:// mirsurface. Org), incorporate commonly used miRNA target gene prediction software and allow for integrated analysis of the signal pathway of the target gene.
1.2 miR-181a targeted inhibition of luciferase reporting containing PPFIA1-3' UTR fragment
RNA duplex (NC) of random sequence was designed and synthesized by guangzhou sharp:
sense:5’-uucuccgaacgugucacgutt-3’
antisense:5’-acgugacacguucggagaatt-3’
AMO-miR-181a：5′-actcaccgacagcgttgaatgtt-3′
the dual luciferase reporter plasmid contained two putative miR-181a recognition sequences, respectively from the 3' UTR and mutated versions of PPFIA1 mRNA (psi-CHECK-PPFIA 1-3' UTR and psi-CHECK-PPFIA1-mut-3' UTR, respectively). With these plasmids, a double luciferase reporter assay was performed. Transfection with the miR-181a mimic significantly reduced the luciferase activity of the psi-CHECK-PPFIA1-3'UTR, but not the psi-CHECK-PPFIA1-mut-3' UTR (FIG. 3). miR-181a and PPFIA 1' UTR have targeting binding effect, and PPFIA1 is one of target genes of miR-181 a.
1.3 detection of the expression level of PPFIA1 at mRNA level and protein level
K562-IMR and KCL22-IMR (CML imatinib resistant strain) were constructed by the present laboratory; after the human chronic granulocytic leukemia Imatinib resistant cell strain is constructed by the method of increasing the concentration of Imatinib, the laboratory maintains drug resistance in Imatinib with the final concentration of 1 mu M and stores the cell strain in liquid nitrogen in the laboratory.
In the same number (5X 10) 3 cell/well), total RNA and total protein of each group were extracted after transfection of miR-181a and PPFIA1-siRNA (at a concentration of 100 nM) in K562-IMR and KCL22-IMR cells, respectively. The expression level of PPFIA1 was detected at the mRNA level and the protein level, respectively.
Wherein BK is a blank control group, NC is a negative control (transfection NC);
the results showed that the expression level of PPFIA1 was decreased in both miR-181a and PPFIA1-siRNA groups compared with NC groups (FIG. 4).
1.4 AGO2 immunoprecipitation enrichment method for verifying miR-181a targeted PPFIA1
In the process of miRNA interaction with target genes, argonaute2 (Ago 2) is an important component of RISC, promoting binding of miRNA to target sites on mRNA, and then cleaving miRNA-mRNA duplex with its endonuclease activity.
And adding an equal amount of AGO2 into the miR-181a group and the NC group, combining the AGO2 with miRNA, detecting PPFIA1 mRNA combined with the miR-181a in 12h, 24h and 48h respectively, and performing qPCR experimental detection. The experimental result is shown in figure 5, the PPFIA1 is enriched, and the experiment proves that miR-181a targets the PPFIA1.
1.5 flag-PPFIA1 fusion protein IP product mass spectrometry results KEGG integration signal transduction
The lentiviral vector with the flag tag expressing the flag-PPFIA1 fusion protein infects cells. For the study of the expression of flag-PPFIA1 fusion protein IP products for electrophoresis, SDS-PAGE gels were placed in Coomassie blue staining solution and stained well with shaking. Protein mass spectrometry was performed on SDS-PAGE gels after the staining. The interaction protein was detected by mass spectrometry of the flag-PPFIA1 IP product, and the integration analysis was performed using the KEGG database, the results of which are shown in FIG. 6.
The integration results of mass spectrometry showed a signal transduction pathway associated with PPFIA1. From the results of this integrated assay we can speculate that the effects on PPFIA1 are linked to the PTK signaling pathway. Abnormal expression of PTK is often associated with the development of malignant tumors, metastasis and chemotherapy resistance. The degree of phosphorylation of protein Tyr is an important factor in directly regulating signals in numerous signaling pathways inside the body. The phosphorylation of Tyr is a reversible dynamic process, controlled mainly by the PTK and PTP families of enzymes. The PTK activity of CML is abnormally high, which can cause a variety of signaling abnormalities.
As can be seen from the above results, one of the target genes of miR-181a is PPFIA1.KEGG integrated signal transduction revealed that PPFIA1 is involved in signal transduction pathways such as PTK.
Example 2
Inhibition of BCR-ABL1 independent CML resistant cell lines by PPFIA1-siRNA
2.1 Drug resistance detection of CML drug-resistant strain
K562-IMR and KCL22-IMR CML drug-resistant cell lines are established in an incremental mode of drug concentration.
The K562 cells are derived from Shanghai cell bank of China academy of sciences; KCL22 cells were donated by Muschen professor of los angeles children hospital;
different concentrations of imatinib solution were prepared and relative viability of cells of the sensitive strain and the drug-resistant strain was detected by CCK8 for 48 h.
As shown in fig. 7, the sensitive strain was more sensitive to imatinib than the drug resistant strain under the action of imatinib.
IC50 of K562, K562-IMR, KCL22-IMR were 0.1285. Mu.M, 3.369. Mu.M, 0.2005. Mu.M, 4.026. Mu.M, respectively. The drug resistance of K562-IMR to Imatinib drug was 26.22 times and the drug resistance of KCL22-IMR to Imatinib drug was 20.08 times.
And simultaneously carrying out SNP sequencing on the K562-IMR and the KCL22-IMR. The 6 more frequent mutation sites in the BCR-ABL-dependent drug resistance were selected, and the base sequencing of the more frequent mutation sites in the two drug-resistant cell lines was examined, and no mutation was found (FIG. 8). K562-IMR and KCL22-IMR were demonstrated to be Imatinib-resistant cell lines independent of BCR-ABL 1.
2.2 PPFIA1-siRNA reduces the viability of K562-IMR and KCL22-IMR cells
The effect of PPFIA1-siRNA on the viability of K562-IMR, KCL22-IMR cells was examined by lipo2000 transfection.
K562-IMR, KCL22-IMR cells were treated with 100nM PPFIA1-siRNA for 24h, 48h and 72h. After culturing for a predetermined period of time, the relative activities of K562-IMR and KCL22-IMR cells were measured by the method of CCK-8. According to the instruction, the optimal acting time of the PPFIA1-siRNA is 24-96h. The experimental results are shown in FIG. 9, and the PPFIA1-siRNA has strong inhibition effect on K562-IMR and KCL22-IMR cells at 48h and 72h. At 48h the relative viability of the K562-IMR and NC was reduced by 13.88% and 16.13% respectively, and the KCL22-IMR was reduced by 18.00% and 19.17% respectively.
The specific procedures for transfection were:
1) CML resistant cells with good growth conditions were taken and counted by a cell counting plate. Adding RPMI-1640 culture medium without serum and antibiotics into centrifuge tube after calculation, mixing, diluting, and mixing with 3×10 5 The number of cells/well was seeded in 6-well plates;
2) Setting groups BK (blank control group), NC (negative control), PPFIA1-siRNA group and miR-181a mic group, wherein each group is provided with 3 compound holes, and the concentration of transfected NC/siRNA/miRNA mic is 100nM per hole;
3) Taking a sterile EP tube, and sucking Lipofectamine at a rate of 5. Mu.L/well TM 2000, opti-MEM was added at 250. Mu.L/well for dilution and slowly and thoroughly mixed. Suction is carried out on EP pipes according to groupsTaking 10 mu L/hole of 20 mu M nucleic acid of each group, adding Opti-MEM into the mixture according to 250 mu L/hole for dilution, and standing the mixture in an ultra-clean workbench for 5min at room temperature;
4) According to dilution of Lipofectamine TM 2000 and diluted nucleic acid volumes 1:1, gently and fully mixing, and incubating for 20min at room temperature;
5) After incubation, 500. Mu.L Lipofectamine per well was pipetted TM 2000-RNA complexes were carefully added slowly to the plated cells;
6) The 6-well plate was placed in a cell incubator at 37℃and a volume fraction of 5%, and allowed to stand. Transfection was completed after 6h days.
2.3 Influence of PPFIA1-siRNA on human CML bone marrow specimen cell viability
As shown in FIG. 10, PPFIA1-siRNA transfected (100 nM) with human CML bone marrow specimen cells (from the second people's hospital, guangdong province) for 72h, the average viability of the PPFIA1-siRNA group was found to be 84.8% after the comparison of the human CML bone marrow specimen cells with the NC group. Compared with NC group, PPFIA1-siRNA can inhibit proliferation of human CML marrow specimen cells to a certain extent.
2.4 Influence of PPFIA1-siRNA on the clonogenic Capacity of CML-resistant cells after treatment
Experimental set up groups were each: blank (Blank), 100nM NC (positive control), 100nM PPFIA1-siRNA, 3 wells per group.
1) After counting, diluting the cells by using serum-free double-antibody-free RPMI-1640 medium, and inoculating the cells into a 96-well plate at 5000 cells/50 mu L per well;
2) Lipofectamine was pipetted in an amount of 0.25. Mu.L/well TM 2000, diluted with 25. Mu.L/well of the reduced serum medium Opti-MEM, was slowly and thoroughly mixed. Each group of 20. Mu.M nucleic acid was pipetted at 0.5. Mu.L/well per group, diluted by addition of Opti-MEM at 25. Mu.L/well, and incubated at room temperature for 5min, respectively;
3) According to dilution of Lipofectamine TM 2000 and diluted nucleic acid volumes 1:1, gently and fully mixing, and incubating for 20min at room temperature; 50. Mu.L Lipofectamine per well was pipetted TM Slowly dropping 2000-RNA complex into the cell suspension;
4) The 96-well plate was placed in a cell incubator at 37℃and a volume fraction of 5%, and allowed to stand. Transfection was completed after 6 h;
5) A sterile centrifuge tube was taken, 25mL of 1.2% soft agarose gel solution (incubated) was poured into the tube, 25mL of 20% FBS 1640 medium (incubated) was added into the tube, and the mixture was thoroughly mixed. Adding 1mL of mixture (containing 0.6% soft agarose gel solution and 10% FBS 1640 culture medium) into each hole of a 6-hole plate, cooling and solidifying to obtain lower layer gel;
6) A sterile centrifuge tube was taken, 25mL of 0.7% soft agarose gel solution (incubated) was poured into the tube, 25mL of 20% FBS 1640 medium (incubated) was added into the tube, and the mixture was thoroughly mixed. After 6h of transfection of the small nucleic acids, the cell suspension was collected. Each well of cells was mixed with 1mL of an upper layer gel mixture (containing 0.4% soft agarose gel solution and 10% FBS 1640 medium), and after mixing, the mixture was added dropwise to the lower layer gel and shaken well. Cooling and solidifying to obtain cell-containing upper gel, and placing the gel in a carbon dioxide incubator for culturing;
7) After the appearance of clones was observed, the PBS solution was slowly flowed through the glue plane 3 times and was aspirated. Adding 500 mu L of 4% paraformaldehyde solution into each hole, fixing for 15-30 min, and cleaning a glue plane for 3 times by PBS;
8) 1mL of 0.005% crystal violet dye solution is added into each hole, and the mixture is stood until the clone groups are dyed;
9) Sucking out 0.005% crystal violet dye liquor, adding PBS, and repeatedly washing until the dye liquor is washed off; photographing and storing, and counting the number of clones in each group.
As shown in FIG. 11, the transfection of PPFIA1-siRNA reduced the clone of K562-IMR cells by 37.83%, and the transfection of PPFIA1-siRNA reduced the clone of KCL22-IMR cells by 35.88%;
the results of clone formation showed that the number of clones knocked down with PPFIA1 was significantly smaller than that of the random control NC group, and the size of clones knocked down with PPFIA1 was also smaller than that of the NC group. The results show that the knockdown of PPFIA1 significantly inhibited the clonogenic capacity of CML-resistant cells.
2.5 B-NDG mice were treated with PPFIA1-siRNA after onset by intravenous injection of K562-IMR/luciferase cells
2.5.1 construction of K562-IMR/luciferase cells
K562-IMR cells are screened by stably transferring a luciferase reporter gene vector (Beijing Biocytogen) to the K562-IMR cells, and the K562-IMR cells are cultured for 1 week by puromycin, so that the K562-IMR/luciferase cells are obtained. K562-IMR/luciferase cells were diluted with complete medium (RPMI-1640 medium plus 10% foetal calf serum, 1% double antibody) and grown in 96 Kong Quanbai opaque plates with different numbers of K562-IMR/luciferase cells, incubated for 30min after addition of 200 Xluciferase substrate. After incubation, the sample is detected by a multifunctional enzyme-labeled instrument, and the experimental result is shown in fig. 12A.
The sample was adjusted to plate mode in a small animal living body luminescence imaging system, and different numbers of K562-IMR/luciferase cells were planted in 96 Kong Quanbai opaque plates, and the test results were shown in FIG. 12B. The experimental result shows that the more the number of the K562-IMR/luciferase cells is, the stronger the fluorescence intensity detected by the instrument is, which shows that the K562-IMR/luciferase cells can react with a luciferase substrate and emit fluorescence, and can be used for tracing the K562-IMR/luciferase cells in the follow-up body of the B-NDG mice.
2.5.2 PPFIA1-siRNA treatment inhibits proliferation of K562-IMR/luciferas cells in B-NDG mice
4 week old B-NDG (NOD.CB17-Prkdc) scid IL2rg tm1 Bcgen) mice were purchased from Beijing Bai-ao Session, B-NDG mice with NOD-scid as genetic background, in which the Il2rg gene was knocked out, and the breed of mice lacked mature T, B and NK cells.
After two weeks of quarantine, each B-NDG mouse was injected 1X 10 by tail vein 6 K562-IMR/luciferase cells; on the day after injection (day 0), each B-NDG mouse was subjected to in vivo imaging test to confirm that K562-IMR/luciferas cells were injected into the B-NDG mouse via tail vein. After B-NDG onset, the hair is dull and dull, the body is emaciated, appetite is lost, and the bow back phenomenon occurs (figure 13A).
Starting from the 10 th day after injection, carrying out grouping treatment on mice (3 mice in each group), respectively treating the mice with medicaments every other day, carrying out in-vivo imaging detection after treatment, and observing proliferation and metastasis conditions of K562-IMR/luciferase cells in the mice; the treatment groups were as follows:
(1) IM group: each B-NDG mouse was given 200. Mu.L of imatinib solution, 15mg/kg.
(2) NC group: each B-NDG mouse was given 200. Mu.L of 10nmol NC solution.
(3) PPFIA1-siRNA: 200. Mu.L of 10nmol PPFIA1-siRNA solution was given to each B-NDG mouse;
(4) miR-181a: each B-NDG mouse was given 200. Mu.L of 10nmol miR-181a solution.
The death time of the B-NDG mice was recorded, the weight of each group of B-NDG mice was weighed, the changes were recorded, and survival curves of the B-NDG mice after drug treatment in the different groups were made.
Compared with NC group, B-NDG mice after treatment have slower weight drop after disease treatment, bioluminescence intensity representing disease degree is reduced, and survival time of mice in PPFIA1-siRNA treatment group is longer, and experimental results are shown in figure 13.
Treatment with PPFIA1-siRNA was able to inhibit the growth of K562-IMR/luciferase cells in B-NDG mouse models in which human cells K562-IMR/luciferase were transplanted, and the PPFIA1-siRNA group was able to prolong the survival time of B-NDG mice by about 2 days;
the experimental result can show that the CML drug resistance independent of BCR-ABL1 can be overcome to a certain extent through the PPFIA1-siRNA through in vivo experiments of B-NDG mice.
The above results show that PPFIA1-siRNA can effectively inhibit CML drug-resistant cell growth independent of BCR-ABL1 to a certain extent.
Example 3
PPFIA1-siRNA overcomes dependence on BCR-ABL1 resistance by inhibiting leukemia stem cell renewal and promoting apoptosis
3.1 detection of reduced c-kit protein phosphorylation levels by phosphorylated protein chips
In order to understand the mechanism of action of PPFIA1 in transferring invasiveness and cloning, the phosphorylated protein chip analysis is carried out after the detection of transfected PPFIA1-siRNA, and specifically comprises the following steps:
1) Protein extraction was performed after transfection of K562-IMR with PPFIA 1-siRNA.
2) Protein samples (50 mg each) were labeled with biotin and hybridized to a pre-phosphorylation array (full-month biosystems, CA, usa) using an antibody array kit (full-month biosystems, usa) to detect specific cancer signaling phosphorylated antibody profiles.
3) Finally, the fluorescence intensity was scanned with GenePix4000B (axon instruments, houston, tex., USA) using GenePixPro6.0.
The calculation method of the phosphorylation ratio is as follows: reduced phosphorylation ratio = phosphorylation value/non-phosphorylation value.
The phosphorylated signal proteins were compared to a control using an antibody chip system. Transfection of PPFIA1-siRNA reduced the phosphorylation level of the protein site as shown in FIG. 14, about 60 protein sites had reduced phosphorylation levels, with the reduction in phosphorylation levels of c-kit protein, FKHR protein, etc. being more pronounced, PPFIA1-siRNA/NC was about 0.25. The experiment suggests that miR-181a/PPFIA1 can inhibit drug resistance by inhibiting self-renewal of leukemia stem cells.
3.2 detection of the ratio of c-kit+ in CML sensitive and resistant strains by flow cytometry
The c-kit is a hematopoietic stem cell growth factor receptor, and is related to leukemia stem cells, and the proportion of c-kit+ in K562-IMR and KCL22-IMR resistant strains and K562 and KCL22 sensitive strains is detected by a flow cytometer.
K562-IMR and KCL22-IMR resistant strains and K562 and KCL22 sensitive strains were incubated with the flow antibody c-kit, respectively, and the experimental results were shown in FIG. 15, in which the ratio of c-kit+ in K562 was 1.63%, the ratio of c-kit+ in K562-IMR was 3.68% (increase of 2.05%), the ratio of c-kit+ in KCL22 was 0.13%, and the ratio of c-kit+ in KCL22-IMR was 3.69% (increase of 3.56%). The ratio of c-kit+ detected by a flow cytometer can be known that the ratio of c-kit positive in K562-IMR and KCL22-IMR CML resistant strains is higher than that in K562 and KCL22 sensitive strains.
3.3 flow cytometer detection of the ratio of c-kit+ after PPFIA1-siRNA treatment
Treatment of K562-IMR cells and KCL22-IMR cells with PPFIA1-siRNA (100 nM) (2.5X10 per well) 5 Individual cells) 48h later, c-kit+ was detected by flow cytometryThe ratio and experimental results are shown in FIG. 16. The effect on c-kit positive cells after PPFIA1-siRNA treatment was detected by detecting the proportion of marker c-kit+.
In K562-IMR cells, the c-kit+ ratio of NC group was 6.42% and the ratio after PPFIA1-siRNA treatment was 4.04%.
In KCL22-IMR cells, the c-kit+ ratio of NC group was 4.70%, and the ratio after PPFIA1-siRNA treatment was 3.35%.
The ratio of PPFIA1-siRNA to c-kit+ cells was reduced.
3.4 expression of c-kit after PPFIA1-siRNA treatment in Western Blot experiments
To verify whether PPFIA1 has an effect on the c-kit protein, western Blot detection of the expression level of the c-kit protein in K562-IMR cells and KCL22-IMR cells was performed on cells of the drug-resistant strain transfected with PPFIA1-siRNA (obtained in step 2.2 of example 2), as shown in FIG. 17, the transfected PPFIA1-siRNA was able to reduce the expression level of the c-kit protein.
3.5 detection of CD34+ proportion of leukemia Stem cell index in CML sensitive Strain and drug resistant Strain by flow cytometry
Drug resistant cells are likely to be resistant to therapeutic drugs because of their ability to self-renew in Leukemia Stem Cells (LSCs). Leukemia stem cells have stem cell characteristics that can be the root cause of relapse. And meanwhile, the common marker CD34 of leukemia stem cells is detected. The proportion of CD34+ in resistant strains K562-IMR and KCL22-IMR and sensitive strains K562 and KCL22 was detected by flow cytometry. Drug resistant strains K562-IMR and KCL22-IMR and sensitive strains K562 and KCL22 were incubated with the flow antibody CD34, respectively.
As shown in FIG. 18, the ratio of CD 34-positive cells in K562 was 0.28%, the ratio of CD 34-positive cells in K562-IMR was 2.01% (1.73% increase), the ratio of CD 34-positive cells in KCL22 was 0.27%, and the ratio of CD 34-positive cells in KCL22-IMR was 1.72% (1.45% increase). As can be seen from the ratio of CD34 positive cells detected by flow cytometry, the ratios of K562-IMR and KCL22-IMR were higher than those of LSC in K562 and KCL 22.
3.6 flow cytometer detecting the proportion of CD34+ after PPFIA1-siRNA treatment
In most CML patients, TKI can induce molecular remission, but CML is hindered from curing in part because of the sustained presence of CML stem cells. CD34 is a surface marker of Leukemia Stem Cells (LSC), and the ratio of CD34+ was detected by flow cytometry after treating K562-IMR cells and KCL22-IMR cells with miR-181a and PPFIA1-siRNA for 48h, and the experimental results are shown in FIG. 19.
The effect on LSC following PPFIA1-siRNA treatment was detected by detecting the proportion of marker CD34 positive.
In K562-IMR cells, the CD34+ fraction in NC group was 2.94% and the PPFIA 1-siRNA-treated fraction was 1.20%.
In KCL22-IMR cells, the CD34 positive ratio of NC group was 4.00%, and the ratio after PPFIA1-siRNA treatment was 1.40%.
Experimental results show that the PPFIA1-siRNA can inhibit proliferation of K562-IMR cells and KCL22-IMR cells by inhibiting CD34 positive cells.
3.7 detection of apoptosis increase by flow cytometry after PPFIA1-siRNA treatment
In order to study whether PPFIA1-siRNA has influence on apoptosis of cells, the proportion of early apoptosis in K562-IMR cells and KCL22-IMR cells is detected by a flow cytometer through transfection of PPFIA1-siRNA, and experimental verification shows that the proportion of early apoptosis in K562-IMR cells can be increased by 0.70% after treatment of PPFIA1-siRNA and the proportion of early apoptosis in KCL22-IMR cells is increased by 0.32% after treatment of PPFIA 1-siRNA;
transfection of PPFIA1-siRNA can increase the early apoptosis rate of cells. The restoration of the apoptotic pathway by drugs of the apoptotic pathway constitutes a promising approach to anticancer therapy, which may overcome the resistance to therapeutic drugs by stimulating exogenous pathways to induce apoptosis.
The results show that after PPFIA1-siRNA treatment, the proportion of CD34+ and c-kit+ of CML drug-resistant strains is reduced, and the PPFIA1-siRNA can reduce leukemia stem cells to overcome drug resistance independent of BCR-ABL. Meanwhile, the proportion of early apoptosis is reduced after PPFIA1-siRNA treatment, and the PPFIA1-siRNA can induce apoptosis to overcome the drug resistance independent of BCR-ABL.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (4)
1. The application of an agent for inhibiting PPFIA1 gene expression in preparing a medicament for treating drug-resistant chronic granulocytic leukemia independent of BCR-ABL1 is characterized in that: the drug-resistant chronic granulocytic leukemia independent of BCR-ABL1 refers to an imatinib-resistant cell strain without any mutation: T315I, E255K, E255V, G250E, Q252H, Y253H;
the imatinib-resistant cell strain refers to K562-IMR and KCL22-IMR;
the agent for inhibiting the expression of the PPFIA1 gene is siRNA (PPFIA 1-siRNA) targeting the PPFIA1 gene or an expression vector containing the siRNA;
the sequence of the PPFIA1-siRNA is as follows:
sense:5’-CCACAAAGCUCUGGAUGAAdTdT-3’
antisense:5’-UUCAUCCAGAGCUUUGUGGdTdT-3’。
2. the use according to claim 1, characterized in that: the medicine also contains other active ingredients and auxiliary materials.
3. The use according to claim 2, characterized in that: the auxiliary materials are sustained release agent, filler, adhesive, wetting agent, disintegrating agent, absorption promoter, adsorption carrier, surfactant or lubricant.
4. Use according to claim 1 or 2, characterized in that: the dosage forms of the medicine are aerosol, tablet, capsule, pill, powder, suspension, emulsion, granule, lipid agent, buccal agent and suppository.
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