New target for overcoming radiotherapy resistance and application thereof
By knocking out or inhibiting Piezo2 gene expression, the low sensitivity and resistance problems of malignant tumors in radiotherapy are solved, and the anti-tumor effect of radiotherapy is significantly improved.
Patent Information
- Application Number
- CN202311161320.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-09-11
- Publication Date
- 2025-03-11
AI Technical Summary
During the treatment of malignant tumors, many tumors have problems such as low radiotherapy sensitivity, radiotherapy resistance, and radiotherapy-induced adverse reactions, which limits the widespread clinical use of radiotherapy.
Drugs for preventing and/or treating radiotherapy to resist colon cancer and/or melanoma are prepared by knocking out or inhibiting Piezo2 gene expression using CRISPR-Cas9 gene editing system, RNAi molecules, or small molecule compounds that inhibit Piezo2 expression.
Deletion of Piezo2 can enhance the anti-tumor effect of radiotherapy, improve the radiotherapy sensitivity of colon cancer and/or melanoma, and reduce radiotherapy resistance.
Smart Images
Figure CN119587696A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the field of biomedicine and relates to a new target for overcoming radiotherapy resistance and its application. Background Art
[0002] Radiotherapy is one of the first-line treatments for malignant tumors. About 60% of newly diagnosed tumor patients receive radiotherapy. [1] Radiotherapy is widely used in the treatment of many cancers, such as lung cancer, esophageal cancer, colorectal cancer, and cervical cancer. However, in the treatment of many malignant tumors, there are many problems such as low radiotherapy sensitivity, radiotherapy resistance, and radiotherapy-induced adverse reactions, which largely limit the widespread clinical use of radiotherapy. [2] . Exploring and discovering effective targets to overcome radiotherapy resistance has become an urgent issue to be addressed.
[0003] In 2010, Ardem Patapoutia's laboratory first identified the Piezo family of cation channels that sense mechanical forces in mammals and discovered their important roles in touch, proprioception, etc. [3] The Piezo family has two members, Piezo1 and Piezo2. Piezo1 is widely expressed in vascular endothelial cells, red blood cells, and myocardial cells, and plays a very important role. Piezo2 mediates the perception of touch and plays an important role in human physiological processes such as mechanical pain, urination, and breathing. [4 -7]. However, how Piezo uses its mechanical sensitivity and ion signals to regulate related physiological and pathological functions still needs further research.
[0004] 1. Jaffray, DA, Image-guided radiotherapy: from current concept to future perspectives. Nat Rev Clin Oncol, 2012.9(12):p.688-99.
[0005] 2.Citrin,DE,Recent Developments in Radiotherapy.N Engl J Med,2017.377(11):p.1065-1075.
[0006] 3.Coste,B.,et al.,Piezo1 and Piezo2 Are Essential Components ofDistinct Mechanically Activated Cation Channels.Science,2010.330(6000):p.55-60.
[0007] 4. Ranade, SS, et al., Piezo1, a mechanically activated ion channel, is required for vascular development in mice. Proc Natl Acad Sci USA, 2014.111(28):p.10347-52.
[0008] 5.Retailleau,K.,et al.,Piezo1 in Smooth Muscle Cells Is Involved inHypertension-Dependent Arterial Remodeling.Cell Rep,2015.13(6):p.1161-1171.
[0009] 6.Woo,SH,et al.,Piezo2 is required for Merkel-cellmechanotransduction.Nature,2014.509(7502):p.622-6.
[0010] 7. Ranade, SS, et al., Piezo2 is the major transducer of mechanical forces for touch sensation in mice. Nature, 2014.516(7529): p.121-5. Summary of the invention
[0011] The object of the present invention is to address the above-mentioned deficiencies in the prior art and to provide a substance for knocking out the Piezo2 gene or a substance for knocking down or inhibiting the expression of Piezo2 for use in the preparation of a drug for preventing and / or treating radiotherapy-resistant colon cancer and / or melanoma.
[0012] The purpose of the present invention can be achieved through the following technical solutions:
[0013] Use of a substance for knocking out the Piezo2 gene or a substance for knocking down or inhibiting the expression of Piezo2 in the preparation of a drug for preventing and / or treating tumor radiotherapy resistance.
[0014] As a preferred embodiment of the present invention, the tumor is colon cancer and / or melanoma.
[0015] As a further preferred embodiment of the present invention, the drug for preventing and / or treating tumor radiotherapy resistance includes products that inhibit the proliferation of radiotherapy-resistant tumor cells and / or enhance tumor radiotherapy sensitivity.
[0016] As a preferred embodiment of the present invention, the substance for knocking out the Piezo2 gene is the CRISPR-Cas9 gene editing system of the Piezo2 gene.
[0017] As a preferred embodiment of the present invention, the substance for knocking down the expression of Piezo2 is an RNAi molecule of the Piezo2 gene.
[0018] As a preferred embodiment of the present invention, the substance that inhibits the expression of Piezo2 is a small molecule compound that inhibits the expression of Piezo2.
[0019] Beneficial effects:
[0020] The present invention uses two tumor cell lines, the radiosensitive MC38 mouse colon cancer cell line and the radiosensitive B16 mouse melanoma cell line. We first used CRISPR-Cas9 technology to knock out Piezo2 in the tumor cell lines MC38 and B16F1. At the same time, an empty vector was used to establish a stable control cell line. After the target gene knockout stable line was amplified, flow cytometry or western-blot technology was used to verify whether the target gene was knocked out. After the stable knockout cell line was established, 1-2×10 6 MC38-Ctrl and MC38-Piezo2 KO, or 1-2 × 10 6 B16F1-Ctrl and B16F1-Piezo2 KO were subcutaneously inoculated into the back of 8-week-old C57BL / 6 mice. When the tumor volume reached 80-120 mm 3 The tumor was given a local irradiation with a dose of 18Gy. The experimental results showed that the loss of Piezo2 would enhance the anti-tumor effect of radiotherapy and enhance the radiosensitivity of colon cancer and / or melanoma. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 :Verification of the effect of Piezo2 knockout
[0022] Figure 2 :Piezo2 knockout can enhance the anti-tumor effect of radiotherapy DETAILED DESCRIPTION
[0023] Example 1
[0024] In order to make the experimental conclusions more solid and reliable, we will use two tumor cell lines, the radiotherapy-sensitive MC38 mouse colon cancer cell line and the radiotherapy-insensitive B16 mouse melanoma cell line. We first used CRISPR-Cas9 technology to knock out Piezo2 in the tumor cell lines MC38 and B16F1, respectively. The three designed guide RNAs targeting the mouse Piezo2 gene were connected to the plasmid lentiCRISPRv2, and the corresponding lentivirus was obtained to make the tumor cells stably express Cas9. Then, the resistance gene puromycin was used for screening for one week to establish stable cell strains with each guide RNA knockout. At the same time, empty vectors were used to establish control cell stable strains. After the target gene knockout stable strain was amplified, flow cytometry or western-blot technology was used to verify whether the target gene was knocked out. Figure 1 It can be seen that MC38 and B16F1 with Piezo2 gene knockout were successfully constructed.
[0025] Example 2
[0026] In order to study whether Piezo2 in tumor cells regulates the anti-tumor effect of radiotherapy, after the stable knockout cell line was established, 1-2×10 6 MC38-Ctrl and MC38-Piezo2 KO, or 1-2 × 10 6 B16F1-Ctrl and B16F1-Piezo2KO were subcutaneously inoculated into the back of 8-week-old C57BL / 6 mice when the tumor volume reached 80-120 mm. 3 The tumor was irradiated once with a dose of 18 Gy. The tumor growth curve was followed up. Tumor volume = (length × width × height) / 2 ( Figure 2 ).according to Figure 2 The tumor growth curves shown show that whether it is a radiotherapy-sensitive colon cancer tumor (left) or a radiotherapy-insensitive melanoma tumor (right), the tumor cells lack Piezo2, and the anti-tumor effect induced by radiotherapy is better, indicating that after radiotherapy, the upregulation of Piezo2 in tumor cells inhibits the anti-tumor effect of radiotherapy and causes radiotherapy resistance.
Claims
1. Use of a substance that knocks out the Piezo2 gene or a substance that knocks down or inhibits the expression of Piezo2 in the preparation of a drug for preventing and / or treating tumor radiotherapy resistance.
2. The use according to claim 1, characterized in that The tumor is colon cancer and / or melanoma.
3. The use according to claim 2, characterized in that The drugs for preventing and / or treating tumor radiotherapy resistance include products that inhibit the proliferation of radiotherapy-resistant tumor cells and / or enhance the sensitivity of tumor radiotherapy.
4. The use according to any one of claims 1 to 3, characterized in that The substance for knocking out the Piezo2 gene is the CRISPR-Cas9 gene editing system of the Piezo2 gene.
5. The use according to any one of claims 1 to 3, characterized in that The substance for knocking down the expression of Piezo2 is an RNAi molecule of the Piezo2 gene.
6. The use according to any one of claims 1 to 3, characterized in that The substance that inhibits Piezo2 expression is a small molecule compound that inhibits Piezo2 expression.