Silent subfragment for inhibiting K562 cell proliferation as well as inhibition method and application thereof

Abstract

The invention discloses a silent subfragment for inhibiting K562 cell proliferation. The nucleotide sequence of the silent subfragment is as shown in SEQ ID NO: 1-3. The proliferation of the K562 cells can be effectively inhibited by knocking out the three silents. By knocking out three silents Ps1, Ps2 and Ps3, K562 cell proliferation can be efficiently inhibited, and the proliferation speed is an important factor directly influencing leukemia disease development, so that leukemia development can be retarded by inhibiting K562 cell proliferation, and a new strategy is provided for leukemia treatment. The compound can also be used for preparing drugs or products for treating or diagnosing leukemia, and a new method is provided for diagnosis and treatment of leukemia. The obtained K562 cell strain with three silents of Ps1, Ps2 and Ps3 knocked out can be used as a new material for studying leukemia, and a new material and a new research direction are provided for preventing or treating leukemia.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and in particular to a silencer fragment for inhibiting K562 cell proliferation and an inhibition method and application thereof. Background Art

[0002] Leukemia is a malignant clonal disease originating from hematopoietic stem cells. Its complex pathogenesis is associated with multiple factors such as biology, physics, chemistry, genetics and other blood diseases. Among them, the proliferation of leukemia cells in the bone marrow and other hematopoietic tissues is out of control, resulting in the inhibition of normal hematopoiesis and infiltration into other organs. In China, the incidence of leukemia is 3 to 5 per 100,000 people, among which acute myeloid leukemia is the most common, and the incidence populations of acute leukemia and chronic leukemia are different: acute myeloid leukemia is more common in adults, while acute lymphocytic leukemia is more common in children. There are many treatment methods for leukemia, including molecular targeted drug therapy, chemotherapy, radiotherapy, blood or bone marrow transplantation, and adjuvant therapy for various symptoms. The prognosis after treatment varies from person to person. For some patients, long-term survival or cure can be achieved through appropriate treatment, and the disease may relapse or continue to progress in some patients. In terms of prevention, since the specific pathogenesis has not yet been clarified, there are currently no specific preventive measures, and limited prevention can only be carried out by avoiding contact with known risk factors.

[0003] K562 cells (human chronic myeloid leukemia cells) are a leukemia cell line. The speed of their cell proliferation is an important factor that directly affects the development of the disease. Therefore, inhibiting the proliferation of K562 cells can slow down the development of leukemia and provide a new strategy for the treatment and prevention of leukemia.

[0004] Silencers are cis-regulatory elements located in the non-coding region of the genome, which play an important role in regulating gene expression. Silencers generally inhibit gene expression by binding to inhibitory transcription factors (TFs) or changing chromatin structure. At present, research on silencers is mainly focused on the mechanism level, and there are few studies on their biological functions. There are no reports on the research on silencers and K562 cell proliferation or treatment / prevention of leukemia. Summary of the invention

[0005] The purpose of the present invention is to provide a silencer fragment for inhibiting K562 cell proliferation and an inhibition method and application thereof, so as to provide a new idea for leukemia prevention and treatment by inhibiting K562 cell proliferation.

[0006] According to the first aspect of the present invention, a silencer fragment for inhibiting K562 cell proliferation is provided, and the nucleotide sequence of the silencer fragment is shown in SEQ ID NO: 1-3. Thus, by knocking out the three silencer fragments, K562 cell proliferation can be inhibited, or it can be used as a new approach to prevent and treat leukemia.

[0007] According to a second aspect of the present invention, a use of a silencer fragment in inhibiting K562 cell proliferation is provided, and the nucleotide sequence of the silencer fragment is shown in SEQ ID NO: 1-3. Thus, by efficiently inhibiting K562 cell proliferation, new ideas and methods can be provided for the research, diagnosis, prevention and treatment of leukemia.

[0008] According to the third aspect of the present invention, there is provided the use of a silencer fragment in the preparation of a drug for preventing and treating leukemia, thereby providing a new solution for the prevention and treatment of leukemia.

[0009] According to a fourth aspect of the present invention, a method for inhibiting K562 cell proliferation is provided, which is achieved by simultaneously knocking out three silencers in K562 cells, the three silencers being located at 87964523-87964773bp, 87915951-87916201bp, and 87953529-87954017bp of chromosome 10, respectively; the nucleotide sequences of the three silencers are shown in SEQ ID NO: 1-3. Thus, the method can efficiently inhibit K562 cell proliferation, providing a new strategy and method for studying leukemia.

[0010] In certain embodiments, the method comprises the following steps: S1: designing and synthesizing sgRNA sequences for knockout according to the nucleotide sequences of the three silencers; S2: connecting the synthesized sgRNA sequences to the PX459 V2 vector; S3: transfecting K562 cells with the plasmid obtained in step S2 to knockout the three silencers in the K562 cells.

[0011] In certain embodiments, the sgRNA sequence is shown in SEQ ID NO:4-9.

[0012] According to a fifth aspect of the present invention, the use of the above method in inhibiting the proliferation of K562 cells is provided. Thus, the proliferation of K562 cells can be efficiently inhibited by this method, and a new way and method is developed for the prevention, treatment and research of leukemia.

[0013] According to a sixth aspect of the present invention, a method for preparing a K562 cell line with a knockout silencer is provided, wherein the silencer comprises any one or a combination of multiple positions located at 87964523-87964773 bp, 87915951-87916201 bp, and 87953529-87954017 bp of chromosome 10, and the nucleotide sequence of the silencer is as shown in SEQ ID NO: 1-3, and the method comprises the following steps: S1: designing and synthesizing an sgRNA sequence for knockout according to the nucleotide sequence of the silencer, and the sgRNA sequence is as shown in SEQ ID NO: 4-9; S2: connecting the synthesized sgRNA sequence to PX459 V2 vector; S3: transfecting K562 cells with the plasmid obtained in step S2; S4: screening the transfected cells in step S3 using a culture medium containing puromycin; S5: detecting the surviving cells screened in step S4, obtaining positive cells with silencer knockout, and amplifying and culturing the positive cells to obtain K562 cell lines with silencer knockout. Thus, through this method, K562 cell lines with one, two or three silencers knocked out can be efficiently obtained, providing new materials for the study of leukemia.

[0014] According to the seventh aspect of the present invention, a new K562 cell line is provided, the K562 cell line is prepared by the above method, and the three silencers on chromosome 10 are knocked out, and the three silencers are located at 87964523-87964773bp, 87915951-87916201bp, and 87953529-87954017bp of chromosome 10, respectively; the nucleotide sequences of the three silencers are shown in SEQ ID NO: 1-3. Therefore, the new cell line can be used as a new material for studying leukemia, and provide research materials and directions for studying new leukemia targeted drugs.

[0015] According to an eighth aspect of the present invention, there is provided a use of the K562 cell line in the preparation of a product for preventing, treating or diagnosing leukemia, wherein three silencers on chromosome 10 of the K562 cell line are knocked out.

[0016] Beneficial effects of the present invention: The present invention discloses three silencer fragments Ps1, Ps2, and Ps3 that inhibit the proliferation of K562 cells. By knocking out the three silencers, the proliferation of K562 cells can be effectively inhibited. By knocking out the three silencers Ps1, Ps2, and Ps3, the proliferation of K562 cells can be efficiently inhibited, and the speed of its proliferation is an important factor that directly affects the development of leukemia. Therefore, inhibiting the proliferation of K562 cells can slow down the development of leukemia, providing a new strategy for the prevention and treatment of leukemia. It can also be used in the preparation of drugs or products for the treatment or diagnosis of leukemia, providing a new method for the diagnosis, prevention and treatment of leukemia. The obtained K562 cell strain with the three silencers Ps1, Ps2, and Ps3 knocked out can be used as a new material for studying leukemia, providing new materials and research directions for the treatment of leukemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The results of the dual luciferase assay for the activities of the three silencers Ps1, Ps2, and Ps3 are shown in the figure: *** indicates extremely significant differences ( p <0.001); Figure 2 The figure is the result of PCR detection after silencer knockout: the first lane from the left is a marker, the second lane is the electrophoresis band of Ps1 silencer WT wild type, the third lane is the electrophoresis band after Ps1 silencer knockout, the fourth lane is the electrophoresis band of Ps2 silencer WT wild type, the fifth lane is the electrophoresis band after Ps2 silencer knockout, the sixth lane is the electrophoresis band of Ps3 silencer WT wild type, and the seventh lane is the electrophoresis band after Ps3 silencer knockout; Figure 3 The results of the proliferation test of K562 cells and wild-type cells after the three silencers Ps1, Ps2, and Ps3 were knocked out: the left figure WT represents wild-type K562 cells, and the right figure Ps1_Ps2_Ps3-KO represents K562 cells after the three silencers Ps1, Ps2, and Ps3 were knocked out; Figure 4 The results of CCK8 detection of cell proliferation: WT represents the wild-type K562 cell detection results, Silencer KO represents the K562 cell detection results in which the three silencers Ps1, Ps2, and Ps3 were knocked out. ** in the figure indicates that the difference is extremely significant ( p <0.01). DETAILED DESCRIPTION

[0018] The invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0019] Example 1 Screening of silencers The target silencers were screened using the silencer screening system and screening method disclosed in patent CN112538493A. The specific steps are as follows: After culturing K562 cells, collect K562 cells and extract genomic DNA, which is sheared into DNA fragments of about 300 bp, connect the sheared DNA fragments with Illumina adapters, and use primers containing the homology arm sequence of the MAS-seq vector for PCR amplification, purify the PCR products, and then homologously recombine the purified PCR products into the MAS-seq vector to construct a MAS-seq screening plasmid, transfect the MAS-seq screening plasmid into 293T cells, extract RNA, and enrich the marker gene mRNA, reverse transcribe the marker gene mRNA to obtain cDNA, and perform PCR amplification on the cDNA, purify the PCR products, and perform high-throughput sequencing on the purified PCR amplification products (output library); The blank MAS-seq vector without any screening fragment was transfected into 293T cells, and the total RNA was extracted. The marker gene mRNA with a poly (A) tail was separated and purified, and then reverse transcribed into cDNA. The reverse transcription product was treated with RNase A and RNaseH to obtain purified cDNA. The obtained cDNA was amplified by PCR, and the purified PCR product (input library) was subjected to high-throughput sequencing.

[0020] The sequencing data of the input and output libraries were analyzed by CRADLE software, and three silencers were found on chromosome 10, which were recorded as Ps1, Ps2, and Ps3. Their specific locations in the genome (hg38 reference genome) are shown in Table 1: Table 1 Location and sequence of the three silencers Ps1, Ps2 and Ps3 Silent sub-name Genomic location Nucleotide sequence Ps1 Chr10: 87964523-87964773 SEQ ID NO: 1 Ps2 Chr10: 87915951-87916201 SEQ ID NO:2 Ps3 Chr10: 87953529-87954017 As shown in SEQ ID NO:3 The nucleotide sequences of the three silencers Ps1, Ps2 and Ps3 are as follows: Ps1 silencer (SEQ ID NO: 1): CCCTGTCTCAAAAACACACACACACACACACACACACACAAAGAAATACATTGATTTTTCACATAGGTAGTAAGAGAAACATTCTTTTTGAACTCAGCTGTTTGTGAATTGAATTTTGTAATTCAAATGCTATATTATGTAAACTATTGATGACTTTCAATCTGCATTTATTTTGTATAATTATTTAGTTAATATTTGCCACTTATATTCCTTAAAAAATAAAATTGAGGTTGGGCGTGGTGGCTCACACT Ps2 silencer (SEQ ID NO:2): GATGTTTATATATTGAAATTATTGGAAGTAAGGTATGTTTATATTAGAAAGATTTGTAGTCTAGATTATCCAAGTTTTGGGAGTATTACCTCTCTGCTTTTGTTTATCTACTTTTTTAGTCTCTACTTTCCAAGTATCTATAGGCAAATTTTCCCATTTCCCTTTGGAAAGTGCTGTTTTCTTGCTTTTTTTCCGCCTTTCCATTGTGTCAGACTTATAAGGCAATCAGCCAACTGTGGGCATGAAATCCT Ps3 silencer (SEQ ID NO:3): TCGCTTGATCTCAGGAGTTCGAGACCAGCCTGGACAACAAGGTGAAACCCCATCTCTACTAAAAACACAAAAATTAGCTGGGCATAGTGGCACACACCTGTAGTCCCAGCTTCTTGGGAGGC TGAGGCAGGAGAATCGCTTGAATCCAGGAGGCAGAGGTTGCAGTTAGCCGAGATCATGCCACTGCACTGCAGCCTGGACATCGGAGCAAGACTTTGTCTTAGAAATAAATAAATAAATATAA AATAAAATAAATGGGAAGTTGTGTATATAAATTATAAATGCTACATTCAGAAAAGCTTTTGAAGGTTGTCAGACAGTTTCTTAAAGGAAGTTCACCAGTTCTTTATTGAACATTGAAGAAAACATACAGTTTAGACTGGCATTAAAACTGAAAGAAGTGGCCAGACGCAGTGGTAGACGCAGTGGTTCACGCCTGTAATCCCAGCACTTTGGGAGGTCAAGGTGGATGGATCACCTGAGGTCAGG Example 2 Verification of silencer activity First, the homology arm sequences left: AACATTTCTCTGGCCTAACTGGCCG (SEQ ID NO: 16); right: CGTACCTGAGTCTTCCACGGGGTTGG (SEQ ID NO: 17) were added to both ends of the three silencers, and silencer detection sequences with homology arm sequences were artificially synthesized.

[0021] The silencer with homology arm sequence was cloned into the upstream of PGK promoter of vector PGL4.53 (Promega, #E1960) using homologous recombination enzyme (Vazyme, #C117). The constructed plasmid was co-transfected with pRL-CMV Renilla reporter vector (Promega, #E1960) into cells. After 24 hours of transfection, cells were treated with luciferase lysis buffer to prepare cell lysate. The cell lysate was added to a 96-well plate and dual luciferase activity was detected according to the following steps: Firefly luciferase activity assay: Add firefly luciferase substrate and immediately read the luminescence intensity of firefly luciferase.

[0022] Renilla luciferase activity detection: Add Renilla luciferase substrate and read the luminescence intensity of Renilla luciferase.

[0023] The ratio of the firefly luciferase signal to the Renilla luciferase signal was calculated as the relative activity value of the experimental group.

[0024] Test results such as Figure 1 As shown in the figure, compared with the control group (PGL4.53), the detection values ​​of silencers Ps1, Ps2, and Ps3 were significantly reduced ( p <0.001), indicating that Ps1, Ps2, and Ps3 have strong silencer activity and can be used for subsequent research.

[0025] Example 3 Knockout and identification of the three silencers Ps1, Ps2, and Ps3 Design three sgRNAs for silencer knockout, the specific sequences are: Ps1 F sgRNA (SEQ ID NO:4): GGACTGTTAGTGTGTAGTACAGG, Ps1 R sgRNA (SEQ ID NO:5): GGTGACTCACTGCTACTTTACGG; Ps2 F sgRNA (SEQ ID NO:6): GGTCCATAGTGAAGCGTTTGAGG, Ps2 R sgRNA (SEQ ID NO:7): GGAAGCTTATCTGTCAAGCTTGG; Ps3 F sgRNA (SEQ ID NO:8): GGAGGTGGTTACAGGGCTCGTGG, Ps3 R sgRNA (SEQ ID NO:9): GGCAGGCTTTGTAGGCCACAGGG.

[0026] The three silencers Ps1, Ps2, and Ps3 in K562 cells were knocked out by CRISPR / Cas9 technology: the CRISPR / Cas9 expression vector PX459 V2 (Addgene, #118632) was digested with restriction endonuclease BbsI, and then these sgRNAs (Ps1 F sgRNA, Ps1 R sgRNA, Ps2 F sgRNA, Ps2 RsgRNA, Ps3 F sgRNA, Ps3 R sgRNA) were ligated to the linearized vector PX459 V2 using T4 DNA ligase, thus obtaining a PX459 V2 vector containing 6 different sgRNAs. Then, these 6 vector plasmids were mixed together and the concentrations were measured. K562 cells were cultured and then transfected by electroporation. The transfection conditions were: 1 μg plasmid / million cells. 24 hours after transfection, the K562 cell culture medium was replaced with a screening medium containing 1.5 μg / mL puromycin. During the screening process, non-transfected cells will die rapidly due to puromycin inhibiting protein synthesis, usually falling off within 24-72 hours. Successfully transfected cells can survive by resisting the toxicity of puromycin because they carry the PuroR gene.

[0027] After the transfected cells were screened for 3 days with a selection medium containing puromycin, the normal medium (without puromycin) was replaced, and the surviving K562 cells were sorted by flow cytometry to obtain single cells. Then the sorted single cells were placed in a 96-well plate for expansion culture (one cell per well). After the cells were fully grown, they were transferred to a 24-well plate and continued to be cultured until the confluence was above 80%. Half of the digested cells were continued to be cultured for subsequent research, and the other half was used for PCR detection. The principle of PCR detection is: if the silencer is successfully knocked out, then the corresponding primers are designed to amplify the wild-type and knocked-out positive cell lines. The length of the PCR amplification product of the positive cell line will be shorter than that of the wild type. For example, if PS1 is successfully knocked out, the length of the PCR product of the wild-type cell is 620 bp, and the length of the PCR product of the knocked-out positive cell line is 369 bp.

[0028] Each silencer detection primer is: Ps1 primer F (SEQ ID NO:10):TCTCCAGGTCGATTTGACCA, Ps1 primer R (SEQ ID NO:11):GAGTCGTTGCTCACCTTTGC; Ps2 primer F (SEQ ID NO:12): TGACTGAATCGTGTTGACCC, Ps2 primer R (SEQ ID NO:13): TGACTGAATCGTGTTGACCC; Ps3 primer F (SEQ ID NO:14): CTGTGGTGCTGTCATCCTTT, Ps3 primer R (SEQ ID NO:15): ACTCTGCCTCAAGGATGACA.

[0029] After DNA is extracted from each sample cell, the three sets of primers are used for amplification at the same time. When the test results after PCR amplification by the three sets of primers are all positive, the cell is a cell in which all three silencers have been knocked out, and the remaining half of the cell is retained for further amplification and culture. Figure 2 When the cell is shown in the figure, it indicates that the three silencers of the cell have been knocked out, as follows: the PCR amplification product of Ps1 KO is shorter than that of the left control group WT, indicating that the silencer Ps1 has been knocked out; the PCR amplification product of Ps2 KO is also shorter than that of the left control group WT, indicating that the silencer Ps2 has also been knocked out; the PCR amplification product of Ps3 KO is also shorter than that of the left control group WT, indicating that the silencer Ps3 has also been knocked out; therefore, the three silencers Ps1, Ps2, and Ps3 of the cell have all been knocked out. After PCR electrophoresis detection, the K562 cells in which the three silencers Ps1, Ps2, and Ps3 have all been knocked out were expanded and cultured, and some of them were taken for gene sequencing. The sequencing results also further showed that the three silencers Ps1, Ps2, and Ps3 were all knocked out. After amplifying and culturing K562 cells in which all three silencers Ps1, Ps2 and Ps3 have been knocked out, a K562 cell line in which all three silencers Ps1, Ps2 and Ps3 have been knocked out can be obtained, and this cell line can also be used as a new material for leukemia research.

[0030] Example 4 Study on the phenotypic changes of K562 cells after the three silencers Ps1, Ps2 and Ps3 were knocked out The proliferation of K562 cells after the three silencers Ps1, Ps2, and Ps3 were knocked out was detected. First, the same number of K562 cells after the three silencers Ps1, Ps2, and Ps3 were knocked out and WT wild-type cells (WT wild-type cells refer to K562 cells without any treatment) were inoculated in a 100 mm culture dish. The number of cells inoculated was 2×10 6 After 48 hours, the difference in cell mass was observed. Figure 3 As shown in: Figure 3The middle right figure Ps1_Ps2_Ps3-KO represents K562 cells in which the three silencers Ps1, Ps2, and Ps3 are all knocked out. The number of cells is significantly less than that of the WT wild-type cells in the left figure, indicating that the proliferation rate of K562 cells in which the three silencers Ps1, Ps2, and Ps3 are all knocked out is significantly slower than that of the wild-type group, indicating that the proliferation of K562 cells can be effectively inhibited after the three silencers Ps1, Ps2, and Ps3 are all knocked out.

[0031] In addition, the cell proliferation was detected using the CCK8 kit (Biyuntian, C0041). Figure 4 As shown in: Silencers KO indicates K562 cells in which Ps1, Ps2, and Ps3 are all knocked out. The CCK8 OD value is extremely significantly lower than the OD value of WT wild-type cells, indicating that the proliferation rate of K562 cells in which Ps1, Ps2, and Ps3 are all knocked out is significantly slower than that of the wild-type, indicating that the proliferation of K562 cells can be effectively inhibited after the three silencers Ps1, Ps2, and Ps3 are all knocked out.

[0032] In summary, knocking out the three silencers Ps1, Ps2, and Ps3 in K562 cells at the same time can effectively inhibit the proliferation of K562 cells, and the speed of its proliferation is an important factor that directly affects the development of leukemia. Therefore, the proliferation of K562 cells can be inhibited by knocking out the three silencers Ps1, Ps2, and Ps3 in K562 cells, thereby slowing down the development of leukemia. For example, sgRNA (such as SEQ ID NO: 4-9) for knocking out the three silencers Ps1, Ps2, and Ps3 can be matched with a knockout vector (such as a CRISPR / Cas9 expression vector) to prepare a corresponding drug for the treatment of leukemia; or siRNA that can inhibit the function of the three silencers Ps1, Ps2, and Ps3 can be prepared into a corresponding drug, which can also be used for the prevention and treatment of leukemia; or any method that can knock out the three silencers Ps1, Ps2, and Ps3 can be used to inhibit the proliferation of K562 cells, and then used to treat leukemia. The K562 cell line obtained after the three silencers Ps1, Ps2, and Ps3 were knocked out can be used as a new material for leukemia prevention and treatment research, providing a new strategy for the research, prevention, and treatment of leukemia. Therefore, knocking out the three silencers Ps1, Ps2, and Ps3 in K562 cells can effectively inhibit their proliferation, which can provide new ideas and directions for the research and treatment of leukemia.

Claims

1. A silencer fragment that inhibits the proliferation of K562 cells, wherein: The nucleotide sequence of the silencer fragment is shown in SEQ ID NO: 1-3.

2. Use of the silencer fragment described in claim 1 in inhibiting the proliferation of K562 cells.

3. Use of the silencer fragment described in claim 1 in the preparation of drugs for preventing and treating leukemia.

4. A method for inhibiting the proliferation of K562 cells, wherein: The method is achieved by simultaneously knocking out three silencers in K562 cells, wherein the three silencers are located at 87964523-87964773 bp, 87915951-87916201 bp, and 87953529-87954017 bp of chromosome 10, respectively; the nucleotide sequences of the three silencers are shown in SEQ ID NOs: 1-3.

5. The method according to claim 4, wherein: The method comprises the following steps: S1: Design and synthesize the sgRNA sequence for knockout according to the nucleotide sequences of the three silencers; S2: ligating the synthesized sgRNA sequence into the PX459 V2 vector; S3: The plasmid obtained by ligation in step S2 is transfected into K562 cells to knock out the three silencers in the K562 cells.

6. The method according to claim 5, wherein: The sgRNA sequences are shown in SEQ ID NO:4-9.

7. Use of the method according to claim 5 or 6 in inhibiting the proliferation of K562 cells.

8. A method for preparing a K562 cell line with silencer knockout, wherein: The silencer includes any one or a combination of multiple locations located at 87964523-87964773 bp, 87915951-87916201 bp, and 87953529-87954017 bp of chromosome 10, and the nucleotide sequences of the three silencers are shown in SEQ ID NO: 1-3. The method includes the following steps: S1: Designing and synthesizing an sgRNA sequence for knockout according to the nucleotide sequence of the silencer, wherein the sgRNA sequence is shown in SEQ ID NO: 4-9; S2: ligating the synthesized sgRNA sequence into the PX459 V2 vector; S3: Transfect K562 cells with the plasmid obtained by ligation in step S2; S4: screening the cells transfected in step S3 using a culture medium containing puromycin; S5: Detect the surviving cells screened in step S4 to obtain positive cells with silencer knockout, and amplify and culture the positive cells to obtain a silencer knockout K562 cell line.

9. A K562 cell line, wherein: The K562 cell line is prepared by the method described in claim 8.

10. Use of the K562 cell line described in claim 9 in the preparation of products for preventing, treating or diagnosing leukemia.

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