Application of heat shock protein 27 in malignant lymphoma proliferation and vinetulas drug resistance
By targeting HSP27 with inhibitors, the proliferation and drug resistance of malignant lymphoma cells are mitigated, providing a therapeutic strategy for improved lymphoma treatment.
Patent Information
- Application Number
- CN202411416974.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-10-11
- Publication Date
- 2024-12-27
Smart Images
Figure CN119199120A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to application of protein heat shock protein 27 in malignant lymphoma proliferation and venetola resistance, belonging to the technical field of biopharmaceuticals. Background Art
[0002] Malignant lymphoma is a blood system malignancy that has received widespread attention and importance. It is mainly composed of Hodgkin's lymphoma and non-Hodgkin's lymphoma. Lymphoma can invade any part of the body and has various clinical manifestations, usually characterized by swollen lymph nodes, fever, weight loss, night sweats, etc. At present, the treatment of lymphoma mainly relies on chemotherapy and radiotherapy, and the specific regimen depends on the type, stage and prognostic factors of the lymphoma. Although systemic chemotherapy, radiotherapy and other advanced treatments, including rituximab or immune checkpoint inhibitors, have improved the prognosis of patients with malignant lymphoma, there are still some patients who are insensitive to treatment or even relapse, and have a poor prognosis and a high mortality rate, which has attracted widespread attention from the public.
[0003] Heat shock protein 27 (HSP27) is encoded by the HSPB1 gene, which is upregulated during cellular stress and is associated with protein misfolding. Heat shock protein 27 (HSP27), a member of the low molecular weight heat shock protein family, is an important protein involved in drug resistance, cell growth, apoptosis, tumor occurrence and metastasis. These functions mediated by HSP27 may be related to its effects on other proteins. Especially in malignant tumor cells, heat shock protein 27 can protect tumor cells from apoptosis or other types of cell death, thereby promoting tumor cell growth and metastasis, and causing tumor cells to develop multidrug resistance to a variety of anti-tumor drugs. However, the current research on the role and function of heat shock protein 27 in the occurrence, development and multidrug resistance of malignant tumors is still very limited. Summary of the invention
[0004] The purpose of the present invention is to provide a targeted medical use of heat shock protein 27, and specifically to an application of protein heat shock protein 27 in malignant lymphoma proliferation and venetoclax resistance.
[0005] In the first aspect of the present invention, there is provided the use of protein heat shock protein 27 in the preparation of products that promote the growth and / or proliferation of malignant lymphoma cells and the use of protein heat shock protein 27 as a target in the preparation of products that prevent and / or treat the emergence of resistance to the Bcl-2 inhibitor venetoclax in malignant tumor cells.
[0006] In a preferred example, the cell is a malignant tumor cell, and the malignant tumor cell is a malignant lymphoma cell.
[0007] In another aspect of the present invention, there is provided a use of protein heat shock protein 27 in the preparation of a product for promoting distal metastasis of malignant tumor cells.
[0008] In another aspect of the present invention, the use of protein heat shock protein 27 as a target in the preparation of a product for inhibiting the distal metastasis of malignant tumor cells is provided. In another aspect of the present invention, there is provided a use of protein heat shock protein 27 in the preparation of a product for preventing and / or treating malignant lymphoma.
[0009] In another preferred embodiment, the malignant tumor cells are malignant lymphoma cells.
[0010] In another aspect of the present invention, there is provided the use of an inhibitor of protein heat shock protein 27 in the preparation of a product for inhibiting cell growth and proliferation, inhibiting cell invasion and / or migration, inhibiting the distal metastasis of malignant tumor cells, inhibiting the metastasis of malignant tumor cells, and treating malignant tumors.
[0011] In another aspect of the present invention, there is provided a use of an inhibitor of protein heat shock protein 27 in the preparation of a product for reversing resistance to the Bcl-2 inhibitor venetoclax.
[0012] In another preferred embodiment, the inhibitor is an sgRNA that can interfere with the transcription and / or expression of protein heat shock protein 27.
[0013] In another preferred embodiment, the cell is a malignant tumor cell, specifically a malignant lymphoma cell; and the malignant tumor is a malignant lymphoma.
[0014] In another aspect of the present invention, an experimental method for the application of heat shock protein 27 in the proliferation of malignant lymphoma and venetoclax resistance is provided, comprising the following steps: detecting the expression of HSP27 in different malignant lymphoma cell lines by Western blotting; constructing a malignant lymphoma cell line overexpressing HSP27; constructing a malignant lymphoma cell line knocked out of HSP27; the proliferation changes of tumor cells in JeKo-1 cells overexpressing HSP27; the proliferation changes of tumor cells in JeKo-1 cells knocked out of HSP27; the expression of HSP27 in cell lines Rec-1 and Rec-Ven-R that are sensitive and resistant to the Bcl-2 inhibitor venetoclax.
[0015] The present invention studies the application of heat shock protein 27 in the proliferation of malignant lymphoma and venetola resistance, and proves that the protein heat shock protein 27 (HSP27) is abnormally highly expressed in malignant lymphoma tissues, and HSP27 can promote the growth and proliferation of malignant lymphomas. At the same time, high expression of HSP27 can cause malignant lymphoma cells to become resistant to the Bcl-2 inhibitor venetola, while interfering with HSP27 can inhibit the growth and proliferation of malignant lymphoma cells and increase the sensitivity of lymphoma cells to the drug venetola. Thus, it is proved that heat shock protein 27 can be used as a target for the treatment of lymphoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0016] Figure 1 The expression of protein heat shock protein 27 in different malignant lymphoma cell lines.
[0017] Figure 2 To overexpress HSP27 in the malignant lymphoma cell line JeKo-1.
[0018] Figure 3 To knock out HSP27 in the malignant lymphoma cell line JeKo-1.
[0019] Figure 4 Figure 2 shows the changes in tumor cell proliferation in JeKo-1 cells overexpressing HSP27.
[0020] Figure 5 The figure shows the changes in tumor cell proliferation in HSP27 knockout JeKo-1 cells.
[0021] Figure 6 The expression of HSP27 in the cell lines Rec-1 and Rec-Ven-R that are sensitive and resistant to the Bcl-2 inhibitor venetoclax.
[0022] Figure 7 The figure shows the changes in tumor cell apoptosis after the addition of the Bcl-2 inhibitor venetoclax in HSP27 knockout JeKo-1 cells. DETAILED DESCRIPTION
[0023] After extensive and in-depth research, the inventors have proved that heat shock protein 27 can be used as a target for the treatment of lymphoma. Using it as a target, drugs that inhibit tumor resistance can be developed, as well as diagnosis and prognosis evaluation of related diseases or symptoms. The specific experimental method includes the following steps: 1. Western blotting was used to detect the expression of HSP27 in different malignant lymphoma cell lines. Specifically, the expression level of HSP27 protein was detected by Western blotting using rabbit anti-HSP27 antibody, while GAPDH was used as an internal reference and rabbit anti-GAPDH was used as a primary antibody for detection.
[0024] Experimental results: as attached Figure 1 As shown, at the protein level, HSP27 is highly expressed in malignant lymphoma cell lines.
[0025] 2. Construction of malignant lymphoma cell lines overexpressing HSP27; The specific steps are as follows: the GFP gene in the plasmid pLenti CMV GFP Puro is replaced with the sequence of the HSP27 gene, and then the lentiviral particles are transfected into HEK293T cells using the packaging plasmids pMDLg / pRRE, pRSV-Rev and pMD2.G. After 48 hours of transfection, the viral supernatant is collected and added to the lymphoma cell line JeKo-1 for infection. After 24 hours of infection, 1 mg / ml puromycin is added to screen the infected cells, and then the overexpression of HSP27 protein in the lymphoma cell line JeKo-1 is verified by Western blotting.
[0026] Experimental results: as attached Figure 2 As shown, HSP27 overexpression was successfully constructed in the malignant lymphoma cell line JeKo-1.
[0027] 3. Construction of HSP27 knockout malignant lymphoma cell lines; Specifically: Design the guide RNA sequence (guide RNA) for knocking out HSP27: 5'-TGAGTTGCCGGCTGAGCGCGCGG-3' 5'-TGGTCGAAGAGGCGGCTATGCGG-3' Experimental method: The packaging plasmids pMDLg / pRRE, pRSV-Rev and pMD2.G were used to transfect lentiviral particles into HEK293T cells. After 48 hours of transfection, the viral supernatant was collected and added to the lymphoma cell line JeKo-1 for infection. After 24 hours of infection, 1 mg / ml puromycin was added to screen the infected cells, and the knockout of HSP27 protein in the lymphoma cell line JeKo-1 was verified by Western blotting.
[0028] Experimental results: as attached Figure 3 As shown, HSP27 knockout was successfully constructed in the malignant lymphoma cell line JeKo-1.
[0029] Among them, pLenti CMV GFP Puro was purchased from Addgene, catalog number 17448; pMDLg / pRRE was purchased from Addgene, catalog number 12251; pRSV-Rev was purchased from Addgene, catalog number 12253; pMD2.G was purchased from Addgene, catalog number 12259; lentiCRISPRv2 was purchased from Addgene.
[0030] 4. Changes in tumor cell proliferation in JeKo-1 cells with overexpression of HSP27; The specific method includes: press 2 x 10 5 Normal JeKo-1 cells or JeKo-1 cells overexpressing HSP27 were seeded in 24-well plates at a cell density of 100 cells / well, and the proliferation intensity of the cells at different times was calculated using trypan blue every 24, 48, and 72 hours.
[0031] Experimental results: as attached Figure 4 As shown, at the two time points of 48h and 72h, the proliferation intensity of JeKo-1 cells overexpressing HSP27 was significantly higher than that of normal JeKo-1 cells, and the difference was statistically significant.
[0032] 5. Changes in tumor cell proliferation in HSP27 knockout JeKo-1 cells; The specific method is: press 2 x 10 5 Malignant lymphoma cells JeKo-1 or HSP27 knockout JeKo-1 were seeded in 24-well plates at a cell density of 100 cells / well, and the proliferation intensity of the cells at different times was calculated using trypan blue every 24, 48, and 72 hours.
[0033] Experimental results: as attached Figure 5 As shown, at the three time points of 24h, 48h and 72h, the proliferation intensity of JeKo-1 cells with HSP27 knockout was significantly lower than that of JeKo-1 cells, and the difference was statistically significant.
[0034] 6. Expression of HSP27 in cell lines Rec-1 and Rec-Ven-R that are sensitive and resistant to the Bcl-2 inhibitor venetoclax; The method includes: inducing the construction of a cell line Rec-Ven-R that is resistant to the Bcl-2 inhibitor venetoclax in a malignant lymphoma cell line Rec-1 that is sensitive to Bcl-2, inducing the construction of a cell line Rec-Ven-R that is resistant to the Bcl-2 inhibitor venetoclax by gradually increasing the dose of the Bcl-2 inhibitor venetoclax (up to 1000nM) in the malignant lymphoma cell line Rec-1, and then using a rabbit anti-HSP27 antibody to perform Western blotting to detect the expression level of HSP27 protein, while using ß-actin as an internal reference and mouse anti-ß-actin as a primary antibody for detection.
[0035] Experimental results: as attached Figure 6 As shown, the expression of HSP27 in the cell line Rec-Ven-R, which is resistant to the Bcl-2 inhibitor venetoclax, was significantly higher than that in the cell line Rec-1, which is sensitive to the Bcl-2 inhibitor venetoclax.
[0036] 7. Flow cytometry was used to detect the effect of Bcl-2 inhibitor venetoclax on inducing apoptosis of malignant lymphoma cells after inhibiting HSP27; The specific experimental method includes the following steps: a.) Take normal JeKo-1 and HSP27 knockout JeKo-1 cells in logarithmic growth phase and culture them at 5 x 10 5 The cells were seeded at a density of 100 cells / well in a 24-well plate, and 1 μM of the Bcl-2 inhibitor venetoclax was added. The culture was continued for 24 h. Figure 7 (a) indicates, and 48h, 7 (b) indicates. After 24h and 48h of culture, the cell suspension was collected, washed twice with pre-cooled PBS after centrifugation, and resuspended with 100μL of 1 x Annexin-V after centrifugation, and then 2μL of PI was added to each 100μL of cell suspension, gently mixed, incubated at room temperature in the dark for 15min, and detected on a flow cytometer within 30min; b.) Data processing: FlowJo was used to analyze and plot the flow cytometry data of cell apoptosis; GraphPad Prism 8.0 software was used to record the data for statistical analysis and plotting.
[0037] Experimental results: as attached Figure 7 As shown in (a) and (b), the apoptosis rate of JeKo-1 cells with HSP27 knockout after addition of Bcl-2 inhibitor venetoclax was significantly increased at 24h and 48h compared with that of normal JeKo-1 cells, and the difference was statistically significant.
[0038] Among them, Annexin V was purchased from BD Biosciences; Propidium iodide (PI) was purchased from Invitrogen; Venetola was purchased from MedChemExpress.
[0039] This method proves that the protein heat shock protein 27 (HSP27) is abnormally highly expressed in malignant lymphoma tissues, and HSP27 can promote the growth and proliferation of malignant lymphoma. At the same time, high expression of HSP27 can cause malignant lymphoma cells to become resistant to the Bcl-2 inhibitor venetoclax. Interfering with HSP27 can inhibit the growth and proliferation of malignant lymphoma cells and increase the sensitivity of lymphoma cells to the drug venetoclax, thus proving that heat shock protein 27 can be used as a target for the treatment of lymphoma.
Claims
1. Application of heat shock protein 27 in malignant lymphoma proliferation and venetola resistance, characterized in that: The application includes the use of protein heat shock protein 27 in the preparation of products that promote the growth and / or proliferation of malignant lymphoma cells and the use of protein heat shock protein 27 as a target in the preparation of products that prevent and / or treat the emergence of resistance to the Bcl-2 inhibitor venetoclax in malignant tumor cells.
2. The use according to claim 1, characterized in that: The cells are malignant tumor cells, and the malignant tumor cells are malignant lymphoma cells.
3. Application of protein heat shock protein 27 in the preparation of products that promote distal metastasis of malignant tumor cells.
4. Application of protein heat shock protein 27 as a target in the preparation of products for inhibiting the distal metastasis of malignant tumor cells.
5. Use of protein heat shock protein 27 in the preparation of products for preventing and / or treating malignant lymphoma.
6. The use according to claim 3, 4 or 5, characterized in that: The malignant tumor cells are malignant lymphoma cells.
7. Use of inhibitors of protein heat shock protein 27 in the preparation of products for inhibiting cell growth and proliferation, inhibiting cell invasion and / or migration, inhibiting the distal metastasis of malignant tumor cells, inhibiting the metastasis of malignant tumor cells, and treating malignant tumors.
8. Application of inhibitors of protein heat shock protein 27 in the preparation of products for reversing resistance to the Bcl-2 inhibitor venetoclax.
9. The use according to claim 7 or 8, characterized in that: The inhibitor is an sgRNA that can interfere with the transcription and / or expression of protein heat shock protein 27.
10. The use according to claim 7 or 8, characterized in that: The cells are malignant tumor cells, specifically malignant lymphoma cells; and the malignant tumor is malignant lymphoma.
11. An experimental method for the application of heat shock protein 27 in the proliferation of malignant lymphoma and venetola resistance, characterized in that: The following steps are involved: Western blotting was used to detect the expression of HSP27 in different malignant lymphoma cell lines; Construction of malignant lymphoma cell lines overexpressing HSP27; Construction of HSP27 knockout malignant lymphoma cell line; Changes in tumor cell proliferation in JeKo-1 cells with overexpression of HSP27; Changes in tumor cell proliferation in HSP27 knockout JeKo-1 cells; Expression of HSP27 in the cell lines Rec-1 and Rec-Ven-R that are sensitive and resistant to the Bcl-2 inhibitor venetoclax.