CN111346229A

CN111346229A - Application of telomere binding protein HP1BP3 in preparation of tumor cell regulating agent

Info

Publication number: CN111346229A
Application number: CN201911082026.4A
Authority: CN
Inventors: 松阳洲; 时光; 黄军就
Original assignee: National Sun Yat Sen University
Current assignee: Sun Yat Sen University; National Sun Yat Sen University
Priority date: 2019-05-17
Filing date: 2019-11-07
Publication date: 2020-06-30
Anticipated expiration: 2039-11-07
Also published as: CN110179985A; CN111346229B

Abstract

The invention discloses an application of telomere binding protein HP1BP3 in preparing a tumor cell regulating agent. The research and analysis of the invention show that telomere related protein HP1BP3 is obviously enriched and positioned on the telomeres of ALT mechanism tumor cells, the genome stability of the ALT mechanism tumor cells is adjusted by adjusting the epigenetic state of telomere chromatin, the activity of the ALT mechanism tumor cells and the length of the telomeres are changed, and the growth and the propagation of telomerase negative tumor cells (ALT) are further adjusted. Therefore, the HP1BP3 has an important telomere regulation function in tumor cells of an ALT mechanism, can develop an effective tumor cell regulation agent and an ALT mechanism tumor resisting medicine aiming at the regulation mechanism of HP1BP3 on the ALT mechanism tumor cells, has a great significance for overcoming cancer, and has a good application prospect.

Description

Application of telomere binding protein HP1BP3 in preparation of tumor cell regulating agent

Technical Field

The invention belongs to the technical field of tumor regulation and control. More particularly, relates to the application of telomere binding protein HP1BP3 in preparing ALT mechanism tumor cell regulator.

Background

Approximately 85% of human tumor cells use telomerase mechanism to extend Telomeres, and 10% -15% of tumor cells use homologous recombination-based telomere extension replacement (ALT) mechanism. Compared with telomerase positive tumor cells, ALT mechanism tumor cells have higher malignancy degree and higher clinical treatment difficulty. Treatment of ALT tumors is a medical problem today. ALT-mechanised tumor cells typically have molecular markers such as telomere length heterogeneity, high frequency of cross-over of sister chromatids in the telomere region (SCE), high levels of extrachromosomal telomeric DNA (C-circles), and telomere-co-localized promyelocytic leukemia nucleosomes (APBs). They reflect to a large extent the activity of ALT cells, and the growth and reproduction of ALT tumor cells are influenced by the activity of ALT cells. Compared with telomerase positive tumor cells, ALT tumor cells have more DNA damage to telomeres. DNA damage is a prerequisite for ALT cell maintenance. ALT cells extend telomeres via a telomere DNA damage-induced Replication mechanism (Break-induced Replication). However, excessive telomeric DNA damage can lead to cell senescence and death. The mechanism of how ALT tumor cells maintain their telomere stability is not well understood. Therefore, the maintenance mechanism for researching the telomere stability of the ALT tumor cells can not only answer basic scientific questions in the telomere research field, but also provide important reference value for ALT tumor prevention and control.

The telomere binding protein HP1BP3 is known as heterochromyin protein 1 binding protein 3, and its amino acid sequence is 554 amino acids in full length, Gene ID at NCBI: 15441. HP1BP3 is similar to histone H1, and HP1BP3 protein contains three H15 domains that recognize nucleosome-associated DNA (linker-DNA) and is capable of binding nucleosome-associated DNA, so HP1BP3 is considered to be an epigenetic regulatory protein similar to histone H1. In the G1-S phase of the epidermal cancer A431 cells, HP1BP3 can regulate the formation of heterochromatin, and the knockout of HP1BP3 can block the cell cycle in the S phase, which shows that HP1BP3 plays an important role in the normal proliferation of cells and the formation of heterochromatin structure. In the process of forming tumors in a hypoxic environment, the HP1BP3 has promotion effects on the formation and proliferation of tumors and the resistance of tumor cells to the environment. It is shown that HP1BP3 may regulate tumor cells through epigenetic mechanism of action.

Disclosure of Invention

The invention aims to solve the technical problems and technical defects of the existing tumor cell regulation and provides application of telomere binding protein HP1BP3 in preparing an ALT mechanism tumor cell regulation agent. The telomere binding protein HP1BP3 has important telomere regulation function in tumor cells of ALT mechanism.

The invention aims to provide application of telomere binding protein HP1BP3 in preparing ALT mechanism tumor cell regulator.

The invention also aims to provide a medicinal preparation for resisting ALT mechanism tumor cells.

The above purpose of the invention is realized by the following technical scheme:

compared with telomerase positive cancer cells, telomerase negative tumor cells extend telomeres independently of telomerase, a mechanism known as ALT. The invention analyzes that HP1BP3 is positioned on the telomere of ALT mechanism tumor cells, and regulates the molecular marker and telomere length of the ALT mechanism tumor cells. HP1BP3 can be located at telomeres of ALT mechanism tumor cells, and interacts with telomere core protein TRF1 and TRF2 through HP1BP 3. In ALT mechanism tumor cells, the expression of HP1BP3 protein is reduced by using an RNA interference technology, and the level of molecular markers and the length of telomeres of the ALT mechanism tumor cells are increased. Therefore, HP1BP3 has an important telomere regulation function in tumor cells of ALT mechanism, and can be used for preparing an effective ALT mechanism tumor cell regulator aiming at the regulation mechanism.

The invention analyzes the telomere stability and cell proliferation of the ALT mechanism tumor cells regulated and controlled by HP1BP 3. The expression of HP1BP3 protein is reduced in ALT mechanism tumor cells by using an RNA interference technology, so that the telomere damage is increased. Knocking down HP1BP3 severely affected tumor cell proliferation by ALT mechanism. Mechanistically, HP1BP3 maintains the stability of ALT-mechanistic tumor cells by stabilizing chromatin assembly.

Therefore, the following applications should be within the scope of the present invention:

the telomere binding protein HP1BP3 is used in preparing ALT mechanism tumor cell regulator.

Specifically, the application is that the telomere binding protein HP1BP3 regulates ALT tumor cell growth in ALT mechanism tumor cells through an epigenetic mechanism.

The application of the inhibitor of telomere binding protein HP1BP3 in preparing medicine for resisting ALT mechanism tumor cells.

The application of the expression inhibitor of telomere binding protein HP1BP3 in preparing medicine for resisting ALT mechanism tumor cells.

In addition, the anti-ALT mechanism tumor medicament comprising an effective amount of the telomere binding protein HP1BP3 inhibitor and/or the expression inhibitor thereof is also within the protection scope of the invention.

Specifically, the medicine also comprises pharmaceutically acceptable auxiliary materials, and is prepared into an injection preparation or an oral preparation.

Preferably, the injection preparation is freeze-dried powder injection, and the oral preparation is powder tablets, capsules or granules.

The invention has the following beneficial effects:

the invention provides application of telomere binding protein HP1BP3 in preparing ALT mechanism tumor cell regulating agent. The invention finds that HP1BP3 in ALT mechanism tumor cells can be positioned on telomeres, and the loss of function of HP1BP3 in the ALT mechanism tumor cells can promote the activity of an ALT mechanism, extend the telomeres to maintain the telomere length, and ensure that the telomeres are unstable and the cell proliferation is hindered. HP1BP3 maintained chromatin stability of ALT-mechanised tumor cells. Therefore, according to the function of HP1BP3, the ALT mechanism tumor cell resisting medicine is developed and applied, has significance for overcoming cancer, and has good application prospect.

Drawings

FIG. 1 shows the co-localization of endogenous HP1BP3 protein with the telomere core protein TRF2 in different cells.

FIG. 2 shows the result of chromatin co-immunoprecipitation by HP1BP3 binding to telomeric DNA.

FIG. 3 shows the results of in vivo co-immunoprecipitation of HP1BP3 with telomere core proteins TRF1 and TRF 2.

FIG. 4 shows the change in the number of molecular markers APB and C-circles of ALT mechanism tumor cells in the control group and cells (U2OS, WI38-V13 and SAOS2) that knocks down the ALT mechanism of HP1BP 3. The A picture is an APB experimental detection picture, the B picture is an APB quantity statistical picture, and the C and D pictures are a C-circles experimental detection picture and a gray level analysis picture.

FIG. 5 is a graph of the change in telomere length in HP1BP 3-knockdown U2OS cell line. A picture is a detection picture of immunoblotting of knocking-down HP1BP3 protein, B picture is a detection picture of telomere length of cells after knocking-down HP1BP3 protein, and C picture is a statistical picture of telomere length.

FIG. 6 shows co-localization of gamma H2A to telomeric DNA in HP1BP 3-knockdown U2OS, WI38-V13 and SAOS2 cell lines. A. Panels C-E are immunofluorescence panels for detection of γ H2A and telomeric DNA, and panels B and F are statistical panels of γ H2A co-localization with telomeric DNA.

FIG. 7 shows the cell proliferation in HP1BP 3-knockdown U2OS, WI38-V13 and SAOS2, Hela and MG63 cell lines. The upper panel shows proliferation of the ALT tumor cell line, and the lower panel shows proliferation of the non-ALT tumor cell line.

FIG. 8 shows the measurement of cell growth metabolism in HP1BP 3-knocked-down U2OS, WI38-V13, SAOS2, Hela and MG63 cell lines. The upper panel shows the growth metabolism of the ALT tumor cell line, and the lower panel shows the growth metabolism of the non-ALT tumor cell line.

FIG. 9 shows the detection of telomere damage, ALT activity and cell growth in HP1BP3 knockout U2OS cells. Panel A shows the expression of HP1BP3 protein in HP1BP3 knock-out cells. B-D are telomere lesion, ALT activity (APB and C-circle) and statistical analysis. And E picture shows the growth metabolism of HP1BP3 knockout U2OS cells.

FIG. 10 shows chromatin packing in HP1BP 3-knockdown U2OS cells.

FIG. 11 shows chromatin packing in HP1BP 3-knocked down WI38-VA13 cells.

FIG. 12 shows chromatin packing in HP1BP3 knock-out U2OS cells.

Detailed Description

The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.

Unless otherwise indicated, reagents and materials used in the following examples are commercially available.

Example 1

1. In order to research the regulation and control function of telomere binding protein HP1BP3 on ALT mechanism tumor cells, the specific experimental design is as follows:

s1, performing immunofluorescence experiments, and detecting the positioning condition of endogenous HP1BP3 protein on telomeres;

s2, chromatin co-immunoprecipitation, namely detecting the binding condition of HP1BP3 on telomere DNA;

s3, co-immunoprecipitation, namely detecting the interaction of HP1BP3 and telomere core proteins TRF1 and TRF 2;

s4, knocking down the expression level of HP1BP3 in U2OS, WI38-VA13 and SAOS2 cells, and researching the relation between the expression level and the activity of an ALT mechanism;

s5, carrying out a telomere length detection experiment, and detecting the change of the telomere length in the cells with the HP1BP3 knocked down;

s6, carrying out telomere dysfunction induced lesion focus experiments, and detecting telomere damage conditions in cells knocked down by HP1BP 3;

s7, cell proliferation experiments and growth metabolism conditions are studied, and the proliferation conditions of 5 cell lines after HP1BP3 is knocked down are studied;

s8, establishing an HP1BP3 gene knockout cell line, and researching the correlation between gene knockout and phenotype;

89. nucleosome digestion experiments were performed to examine the effect of HP1BP3 on chromatin packing of ALT-mechanised tumor cells.

2. The experimental materials were as follows:

reagent: the rabbit polyclonal antibody HP1BP3 endogenous antibody used in the experiment is self-made, and is diluted by 3% Bovine Serum Albumin (BSA) according to a ratio of 1:1000 after being purified; PML antibody (available from Santa Cruz, Inc., product No. sc-966) was used diluted 1:100 with 3% BSA; FLAG antibody (purchased from Sigma, product number F7425) diluted 1:5000 with 3% Bovine Serum Albumin (BSA) at the time of use; GST antibody (purchased from Abmart corporation, product No. M20007) was diluted 1:5000 with 3% Bovine Serum Albumin (BSA) at the time of use; secondary antibody (goat anti-mouse, FITC labeled, purchased from Invitrogen, product No. a11017, goat anti-rabbit, TXRED labeled, purchased from alligator, product No. LK-GAR5492), diluted with 3% BSA at 1:2000 when used; PNA telomere probe (available from Panagene, cat # F1009-5) at a working concentration of 10 nM. HP1BP3 siRNAs, produced by Gima Biotech, at a working concentration of 80 nM.

Tumor cells (Hela), tumor cells (U2OS), and HEK293T cells were purchased from the national academy of sciences cell Bank of Shanghai. Culturing according to a conventional tumor cell culture method, placing cultured tumor cells into a 24-well plate (placing a cover glass into the 24-well plate before use), allowing the cells to grow adherent to the wall on the cover glass, sucking away a culture medium when the cell growth reaches 85% -95% of cell confluency, and washing twice with Phosphate Buffered Saline (PBS); the culture medium comprises the following components: DMEM medium, 10% FBS.

3. Experimental methods

(1) Immunofluorescence: spreading a sterile slide in a 24-well plate, adding 0.1% gelatin for treatment for 30min, and then inoculating the digested cells into the plate; collecting cells after one day, sucking away cell culture medium, washing with PBS 3 times, and fixing with 4% paraformaldehyde ice for 15 min; washing with PBS for 5min for 3 times; adding the permeabilization solution to react for 10min, and washing with PBS for 3 times in the same way; sealing with 5% sheep serum at room temperature for 1 h; adding the prepared primary anti-solution into the holes, and incubating overnight at 4 ℃; washing with blocking solution for 5min for 3 times; incubating the secondary antibody solution for 1h at room temperature; the same with closed liquid washing 3 times; mounting was done for fluorescence microscopy.

(2) Co-immunoprecipitation: collecting cells, lysing the cells with precooled RIPA buffer solution, standing for 30min on ice, and taking supernatant after high-speed centrifugation. Protein A/G agarose particles and antibody were added and incubated for 4h or overnight at 4 deg.C, the particles were washed 2 times with RIPA buffer after low speed centrifugation, then washed 3 times with PBS, the particles were suspended in 40. mu.l sample buffer, boiled at 100 deg.C for 5min, centrifuged at high speed for 15s in a short time, and the supernatant was extracted for SDS-PAGE electrophoresis.

(3) Western blot analysis: the protein samples of each cell were subjected to SDS-PAGE gel separation, and the proteins were then electroporated onto Hybond-P membranes. The blot was blocked with 5% skimmed milk powder at room temperature for 1h and incubated overnight at 4 ℃. Washing the primary antibody on the blotting membrane, then incubating with a secondary antibody with a far infrared light label, washing the secondary antibody on the blotting membrane, and detecting a fluorescence luminescence signal.

(4) Chromatin immunoprecipitation (ChIP) technique cells were fixed with 1% formaldehyde at room temperature (about 1 × 10)⁷) After 10min, the crosslinking was terminated with glycine to a final concentration of 0.125M. Cells were harvested by low speed centrifugation and washed 2 times with PBS. Cells were lysed in a buffer containing 50mM Tris pH 8.0, 10mM EDTA, 1% SDS. Carrying out ultrasonic disruption treatment to break the chromatin DNA into fragments with the size of 200-800 bp. The lysate supernatant was centrifuged at high speed and the antibody, pretreated magnetic beads and 20. mu.g chromatin were incubated overnight at 4 ℃. Washed 1 time each in low salt buffer, high salt buffer, lithium chloride buffer and TE buffer. The chromatin and DNA complexes are then de-crosslinked, and the enriched DNA is recovered and purified on a column and used for quantitative PCR or ChIP-seq library construction. If telomere DNA is detected, the enriched DNA is loaded on membrane, and treated by ultraviolet cross-linking and isotope P is used³²Tagged telomeres (CCCTAA)₃And (4) probe hybridization.

(5) Fluorescent Quantitative in situ hybridization (Q-FISH): nocoazole treatment cells for 2-4 h before cell collection, resuspending the cells with 0.075M KCl preheated at 37 ℃, standing for 30min, and then centrifuging at 1200rpm for 5 min; fixing the cells with ice-cold fixative (methanol: glacial acetic acid ═ 3:1) at room temperature for 30min, centrifuging at 1200rpm for 5min, repeating the fixing and centrifuging steps 3 times; the cell pellet was resuspended in 0.5ml ice-cold fixative and aerial-dripped onto an ice-cold slide to obtain a dispersed karyotype. Fixing with 4% formaldehyde for 2min, washing with PBS for 2 times, 5min each time, and gradient dehydrating with 70% -90% -100% ethanol for 5min each time. After the slide is dried, hybridization solution containing the probe is added, denaturation treatment is carried out for 5min at 85 ℃, hybridization treatment is carried out for 2h at 37 ℃, and TBST is washed for 3 times, 5min each time. And performing gradient dehydration by using 70-90-100% ethanol for 5min each time, drying the slide, sealing the slide, and keeping out of the sun for fluorescence microscopy.

(6) Telomere Dysfunction-induced lesion foci (telomee dye function induced foci, TIF): firstly, a glass sheet is paved in a 12-hole plate, the glass sheet is treated for 30min by 1% gelatin, and cells with certain density are inoculated in the 12 holes. Collecting cells after one day, sucking away cell culture medium, washing with PBS 3 times, and fixing with 4% paraformaldehyde ice for 15 min; washing with PBS for 5min for 3 times; adding the permeabilization solution to react for 10min, and washing with PBS for 3 times in the same way; blocking with 5% sheep serum for 1h at room temperature. Incubating overnight at 4 ℃ with primary anti-TRF 2 and primary anti-gamma H2A; washing with blocking solution for 5min for 3 times; incubating the secondary antibody solution for 1h at room temperature; the same with closed liquid washing 3 times; mounting was done for fluorescence microscopy.

(7) C-circles detection experiment: extracting genome DNA and enzyme digestion treatment. 3 gradients of cleaved DNA were used for each sample to perform amplification reactions, 50ng, 100ng, 200ng, respectively, for C-circles using Phi29 DNA polymerase. Amplification was performed according to the following procedure: after incubation at 30 ℃ for 8h, polymerase was inactivated at 65 ℃ for 20 min. The C-circles amplified sample is loaded on the membrane, treated by UV cross-linking, and hybridized with an isotope-labeled telomere (CCCTAA)3 probe. Control groups were hybridized using Alu repeat probes. Finally, Totallab was used for grey scale quantification.

(8) Nucleosome microspherical enzyme digestion experiment: after collection of cells, they were washed 1 time with PBS and resuspended and washed 3 times with 1ml of solution A (100mM NaCl, 10mM Tris pH 7.5, 3mM MgCl2, 1mM CaCl2, 0.5mM PMSF, protease inhibitor). The cells were then lysed with 1ml of solution A (plus NP40 to 0.7% concentration). After gentle mixing, incubate for 3min on ice and resuspend with 450. mu.l of solution A (without NP 40). Each sample was dispensed into 4 PCR tubes, 100. mu.l each. Adding 1U of Micrococcus enzyme for digestion for 4 gradient times, 0, 2, 4, 8min or 0, 5, 10, 20 min. After the reaction time had elapsed, the addition of 1-fold volume (100. mu.l) of TES-proK solution per tube was stopped and incubated at 37 ℃ for 2h or overnight. DNA was extracted with phenol chloroform and then examined with 1.2% strength TAE agarose gel, and 20. mu.l of each lane was electrophoresed at a constant pressure of 50V for about 10 hours. And staining the Gel with Gel Red, photographing to record the position of a DNAmarker, and performing Southern detection.

(9) CRISPR/Cas9 gene knockout technology and establishment of HP1BP3 gene knockout line: a gRNA targeting HP1BP3 gene was designed, and primers were synthesized to clone the DNA sequence of the gRNA into a pX330 vector. Transferring the gene into ALT tumor cells by a transfection method, collecting the cells after 48h, extracting genomic DNA, amplifying a target region by a PCR mode, and detecting the activity of gRNA by T7E1 assay or sequencing. Establishing a resistance (Puromycin) cell line stably expressed by Cas9, then constructing effective gRNA into a pLenti-gRNA virus vector of a BSD antibody again, infecting the cell line after packaging viruses, and carrying out drug screening of resistance. Gene status was detected by T7E1 assay or sequencing, and the protein knock-out effect was detected by immunoblotting with HP1BP3 endogenous antibody.

4. The experimental results are shown in FIGS. 1-10, respectively:

FIG. 1 shows the co-localization of endogenous HP1BP3 protein with the telomere core protein TRF2 in different cells. The red color shows the TRF2 protein signal, and the green color shows the HP1BP3 protein signal. The result shows that the HP1BP3 is obviously co-localized with TRF2 protein in ALT mechanism tumor cells.

FIG. 2 shows the result of chromatin co-immunoprecipitation by HP1BP3 binding to telomeric DNA. The results indicated that HP1BP3 bound to telomeric DNA.

FIG. 3 shows the results of in vivo co-immunoprecipitation of HP1BP3 with telomere core proteins TRF1 and TRF 2. The results show that HP1BP3 interacts with TRF1 and TRF 2.

FIG. 4 shows the change in the number of molecular markers APB and C-circles of ALT mechanism tumor cells in the control group and in the cells U2OS, WI38-V13 and SAOS2 that knocks down the ALT mechanism of HP1BP 3. The results show that the knocking-down of the HP1BP3 protein increases the number of APB and C-circles in ALT mechanism tumor cells.

FIG. 5 is a graph of the change in telomere length in HP1BP 3-knockdown U2OS cell line. The result shows that the knocking-down HP1BP3 protein increases the length of telomere in ALT mechanism tumor cells.

FIG. 6 shows co-localization of gamma H2A to telomeric DNA in HP1BP 3-knockdown U2OS, WI38-V13 and SAOS2 cell lines. The result shows that the knocking-down HP1BP3 protein increases the co-localization of gamma H2A and telomere DNA in ALT mechanism tumor cells.

FIG. 7 shows the cell proliferation in HP1BP 3-knockdown U2OS, WI38-V13 and SAOS2, Hela and MG63 cell lines. The result shows that the knocking-down of the HP1BP3 protein seriously reduces the ALT mechanism tumor cell expansion.

FIG. 8 shows the measurement of cell growth metabolism in HP1BP 3-knocked-down U2OS, WI38-V13, SAOS2, Hela and MG63 cell lines. The result shows that the knocking-down of the HP1BP3 protein seriously reduces the growth and metabolism of ALT mechanism tumor cells.

FIG. 9 shows the detection of telomere damage, ALT activity and cell growth in HP1BP3 knockout U2OS cells. The result shows that the HP1BP3 gene knockout increases telomere damage, ALT cell activity and reduces cell growth metabolism.

FIGS. 10-12 are measurements of telomere chromatin packing in HP1BP3 knockdown and knocked-out cells. FIG. 10 shows chromatin packing in HP1BP 3-knockdown U2OS cells. FIG. 11 shows chromatin packing in HP1BP 3-knocked down WI38-VA13 cells. FIG. 12 shows chromatin packing in HP1BP3 knock-out U2OS cells.

The results indicate that ALT cell telomere chromatin became loose after knockdown and knockdown of HP1BP 3.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. Application of telomere binding protein HP1BP3 in preparing tumor cell regulator.

2. The telomere binding protein HP1BP3 is used in preparing ALT mechanism tumor cell regulator.

3. The use according to claim 1 or 2, wherein the use is the telomere binding protein HP1BP3 in regulating ALT tumor cell growth in ALT-mechanistic tumor cells.

4. The application of the inhibitor of telomere binding protein HP1BP3 in preparing medicine for resisting ALT mechanism tumor cells.

5. The application of the expression inhibitor of telomere binding protein HP1BP3 in preparing medicine for resisting ALT mechanism tumor cells.

6. A medicine for resisting ALT mechanism tumor, which is characterized by comprising an effective amount of telomere binding protein HP1BP3 inhibitor.

7. A medicine for resisting ALT mechanism tumor, which is characterized by comprising an effective amount of an expression inhibitor of telomere binding protein HP1BP 3.

8. The medicine according to claim 6 or 7, which is characterized by further comprising pharmaceutically acceptable auxiliary materials and is prepared into an injection preparation or an oral preparation.

9. The medicine of claim 8, wherein the injection preparation is a freeze-dried powder injection, and the oral preparation is a powder tablet, a capsule or a granule.