Application of up-regulation LZTS3 gene in preparation of tumor radiotherapy sensitization drug and tumor radiotherapy sensitization drug

Abstract

The invention relates to the field of promoting tumor radiotherapy sensitization by targeted induction of gene expression, in particular to novel application of cancer suppressor protein LZTS3, namely application of up-regulated LZTS3 gene in preparation of lung cancer radiotherapy sensitizer drugs. According to the invention, lung cancer cells H1299 and A549 are used as examples for research, and the influence of the LZTS3 gene on the lung cancer cells is researched through RNA interference (RNAi) silent LZTS3 gene. Researches find that expression of LZTS3 is knocked down in H1299 and A549 cells, the multiplication capacity of lung cancer cells is remarkably enhanced, the number of gamma-H2AX focuses induced by ionizing radiation (IR) is reduced, the number of RAD51 focuses induced by IR is increased, and then the application of the up-regulation LZTS3 gene in preparation of tumor radiotherapy sensitizing drugs is provided. The purpose of up-regulating the expression level of the LZTS3 gene is achieved by knocking out beta-Trcp through lipidosome-mediated small interfering RNA, so that the sensitivity of tumor cells to radiotherapy is enhanced.

Description

Technical Field

[0001] The present invention relates to the field of targeted induction of gene expression to promote tumor radiotherapy sensitization, and in particular to an application of up-regulating LZTS3 gene in the preparation of tumor radiotherapy sensitization drugs and the tumor radiotherapy sensitization drugs. Background Art

[0002] Lung cancer is one of the tumors with the highest cancer mortality rate in the world. In China, the incidence and mortality of lung cancer are both the highest. Treatments for lung cancer include surgery, radiotherapy, chemotherapy, targeted therapy and immunotherapy, but the overall survival rate of lung cancer is still low, with a 5-year survival rate of less than 15%. Radiotherapy is suitable for all stages of lung cancer and can significantly improve the quality of life of lung cancer patients and prolong their survival time. However, some patients still experience tumor recurrence and distant metastasis after radiotherapy, resulting in reduced radiotherapy efficacy. One of the main causes of radiotherapy resistance is that radiotherapy-induced DNA damage triggers a series of DNA damage repair responses, including DNA damage sensing activation, early signal transduction pathway activation, cell cycle arrest and DNA repair, etc. These mechanisms can lead to cell resistance to radiotherapy. Therefore, radiotherapy resistance has become a major challenge in the clinical treatment of lung cancer, and elucidating the regulatory mechanism of radiotherapy resistance in lung cancer cells will help develop effective radiosensitizers.

[0003] In the prior art, there are few reports on the LZTS3 gene, which is currently considered to be a potential tumor suppressor protein that can regulate the growth of tumor cells.

[0004] Gu Xinpei et al. showed through Transwell, actin staining and in vitro transfection experiments that LZTS3 can inhibit tumor cell proliferation and migration. Gene set enrichment analysis (GSEA) showed that LZTS3 was closely related to the Notch signaling pathway, and downregulation of LZTS3 increased the expression of genes such as Notch1, Hes1 and C-myc. In in vivo experiments, a nude mouse human tumor model was constructed and it was found that LZTS3 overexpression could significantly inhibit colorectal adenocarcinoma tumor growth. In addition, RNA-seq and other analyses determined that LZTS3 regulates colorectal adenocarcinoma progression through the downstream interacting protein TAGLN (see: Gu X, Chen S, Wang Z, Bu Q, An S. LZTS3 / TAGLN Suppresses Cancer Progression in Human Colorectal Adenocarcinoma Through Regulating Cell Proliferation, Migration, and Actin Cytoskeleton. Arch Med Res. 2023;54(7):102894.)

[0005] Feng Junpeng et al. found that lncRNA PGM5P4-AS1 was lowly expressed in lung cancer tissues. Overexpression of PGM5P4-AS1 inhibited the proliferation, migration and invasion activity of lung cancer cells through cell cycle assay, wound healing assay and transwell assay. In addition, luciferase reporter gene assay showed that PGM5P4-AS1 had a binding site for miR-1275 and could positively regulate the expression of LZTS3 through sponge miR-1275, suggesting that PGM5P4-AS1 promoted the expression of anti-cancer protein LZTS3 by negatively regulating miR-1275, thereby inhibiting the growth, proliferation, migration and invasion of lung cancer cells. (See: Feng J, Li J, Qie P, Li Z, Xu Y, Tian Z. Long non-coding RNA (lncRNA) PGM5P4-AS1 inhibits lung cancer progression by up-regulating leucine zipper tumorsuppressor (LZTS3) through sponging microRNA miR-1275. Bioengineered. 2021;12(1):196-207.)

[0006] However, the research on the preparation of lung cancer radiosensitizers that upregulate LZTS3 gene expression has not been reported at home and abroad. Summary of the invention

[0007] The purpose of the present invention is to provide a new application of tumor suppressor protein LZTS3, namely, application of up-regulating LZTS3 gene in preparing radiosensitizer drugs for lung cancer.

[0008] Another object of the present invention is to provide a tumor radiotherapy sensitizing drug to increase the sensitivity of tumor cells to radiotherapy.

[0009] The embodiments of the present invention are implemented by the following technical solutions:

[0010] An application of up-regulating LZTS3 gene in preparing tumor radiotherapy sensitization drugs.

[0011] Preferably, the method for up-regulating the LZTS3 gene comprises: adding an inhibitory component that inhibits the expression of the β-Trcp gene to the drug.

[0012] Preferably, the inhibitory component includes: small interfering RNA carrying the β-Trcp gene.

[0013] Preferably, the small interfering RNA carrying the β-Trcp gene is mediated by liposomes.

[0014] A tumor radiotherapy sensitization drug, the chemical components of which include: small interfering RNA carrying beta-Trcp gene.

[0015] The present invention has at least the following beneficial effects:

[0016] The present invention uses lung cancer cells H1299 and A549 as examples for research, and studies the effect of silencing the LZTS3 gene on lung cancer cells by RNA interference (RNAi). The study found that knocking down the expression of LZTS3 in H1299 and A549 cells significantly enhanced the growth ability of lung cancer cells, reduced the number of γ-H2AX foci induced by ionizing radiation (IR), and increased the number of RAD51 foci induced by IR, thereby providing an application of up-regulating the LZTS3 gene in the preparation of tumor radiosensitization drugs.

[0017] The present invention achieves the purpose of up-regulating the expression level of LZTS3 gene by knocking out beta-Trcp through liposome-mediated small interfering RNA, so as to enhance the sensitivity of tumor cells to radiotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0019] Figure 1 is the expression level of LZTS3 protein in H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA;

[0020] Figure 2 The survival curves of H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA;

[0021] Figure 3 is the clonal survival rate of H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA;

[0022] Figure 4 To image the number of γ-H2AX foci induced by IR in H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA;

[0023] Figure 5 To image the number of RAD51 foci induced by IR in H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA;

[0024] Figure 6 The expression level of LZTS3 in H1299 and A549 cells after transfection of β-Trcp gene small interfering RNA. DETAILED DESCRIPTION

[0025] In order to make the purpose, method scheme and advantages of the embodiments of the present invention clearer, the method scheme in the embodiments of the present invention is clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0026] Example 1: Application of up-regulating LZTS3 gene in preparing tumor radiotherapy sensitization drugs.

[0027] Example 2: Based on Example 1, a method for up-regulating the LZTS3 gene is provided, comprising: adding an inhibitory component that inhibits the expression of the β-Trcp gene to the drug.

[0028] Example 3: Based on Example 2, an inhibitory component is provided, including: small interfering RNA carrying the β-Trcp gene.

[0029] Example 4: Based on Example 3, a delivery method of the inhibitory component is provided, wherein the small interfering RNA carrying the β-Trcp gene is mediated by liposomes.

[0030] Example 5: This example provides a tumor radiotherapy sensitization drug, the chemical components of which include: small interfering RNA carrying the β-Trcp gene.

[0031] In the above embodiments, the liposome-mediated small interfering RNA construction method is a prior art, see: Teimoori-Toolabi L. Accurate and efficient siRNA design: the key to competent gene silencing. Cancer Gene 2016 23; 4 (73): 82-10.)

[0032] In the above embodiments, the tumor cells include but are not limited to lung cancer cells.

[0033] Application verification experiment 1: Verify the effect of LZTS3 expression level on tumor cell growth, proliferation and radiotherapy sensitivity.

[0034] I. Materials: Human lung adenocarcinoma cell lines H1299 and A549 were purchased from the American Type Culture Collection (ATCC) and cultured in our laboratory; RPMI1640 medium was purchased from Zhejiang Senrui Biotechnology Co., Ltd., fetal bovine serum was purchased from Novazon Biotechnology Co., Ltd., trypsin and phosphate-buffered saline were purchased from Zhejiang Senrui Biotechnology Co., Ltd.; electrophoresis buffer, RIPA protein lysis buffer and electroporation concentrate were purchased from Hangzhou Huaan Biotechnology Co., Ltd.; LZTS3 antibody was purchased from Sigma, and β-Trcp antibody was purchased from Cell Signaling Technology.

[0035] 2. Application Process

[0036] (1) Cell culture: H1299 and A549 were cultured in RPMI1640 medium containing 10% FBS, and the cell suspension was transferred to a culture dish. The culture dish was gently shaken and placed in an incubator at 37°C and 5% CO2 for culture. The cell status was observed in time, and the cell status was selected for medium replacement, subculturing, freezing, plating, etc. according to the cell status.

[0037] (2) Cell transfection: Inoculate an appropriate amount of cells into a six-well plate. Mix the cells evenly when plating them so that the cell density reaches 50%-70% on the next day.

[0038] Preparation of transfection solution: dilute siRNA with 250ul Opti-Medium (the initial storage concentration of siRNA is 20uM, and the final concentration of cell transfection is 100nM), and gently blow and mix. Gently invert to mix the transfection reagent, then add 250ul Opti-Medium to dilute 6ul Lipofectamine 3000, blow and mix, and let stand at room temperature for 5min. Mix the siRNA dilution and transfection reagent together, blow and mix, and let stand at room temperature for 20min. Add the transfection complex to the six-well cell plate and gently shake the cell plate to mix. Place the cell culture plate in an incubator at 37℃ and 5% CO2, and change to fresh culture medium 4-6h after transfection.

[0039] The method of liposome transfection was used: the above-mentioned small interfering RNA of LZTS3 gene was transfected into H1299 and A549 cells respectively, and siRNA without specific target gene sequence was transfected as negative control. The Western blot electrophoresis of H1299 and A549 cells after transfection of small interfering RNA of LZTS3 gene is shown in the figure below. Figure 1 The results showed that the expression of LZTS3 gene in H1299 and A549 cells was significantly lower than that in cells transfected with control small interfering RNA.

[0040] (3) The above Western blot includes the following steps: After the culture medium in the culture dish is aspirated, the cells are washed twice with PBS. After the PBS is discarded, a pre-prepared lysis buffer mixed with RIPA and PMSF (100:1) is added. After lysis on ice for half an hour, the cells are quickly collected and placed in a pre-cooled high-speed centrifuge, and centrifuged at 14400 rpm for 15 min at 4°C. According to the instructions of the Thermo Fisher BCA protein quantification kit, the absorbance of the sample at 562 nm is measured using an enzyme marker to obtain the protein concentration of the experimental sample. 5× Loading Buffer is mixed with the protein sample at a ratio of 1:4, and the mixture is placed in a pre-opened metal bath heater at 100°C for 5 min to denature the protein. Then, an appropriate amount of 1× Loading Buffer is added to each group of samples to adjust the protein concentration of each group to the same. The Biorad electrophoresis instrument is used for constant voltage electrophoresis at 160V for 45 min. Cut out a PVDF membrane of suitable size, cut off a corner as a mark, put it in methanol to wake up the membrane for 30s, install the clips in the order of black side (-), sponge, filter paper, glue, membrane, filter paper, filter paper, sponge, and white side (+) from bottom to top in the recovered transfer solution, put it in the transfer tank, and transfer it in the pre-cooled transfer solution for 30-120 minutes at a constant flow (the transfer time depends on the specific protein molecular weight). Use TBST to prepare 5% milk, put the transferred membrane into the milk and place it on a shaker at room temperature for 2h. Put the protein band into the corresponding primary antibody dilution solution and put it in a 4℃ refrigerator shaker overnight. The next day, put the protein band into the antibody incubation box and wash it with TBST 3 times, 10min each time. Then use TBST to prepare an appropriate amount of secondary antibody dilution solution at a concentration of 1:3000, incubate the protein band at room temperature on a shaker for 2 hours, and then wash it with TBST 3 times, 10min each time. Prepare the developer at a ratio of 1:1, and note that the developer is stored in a dark box. The washed strips are immersed in the developer, and then the strips are developed and analyzed using the Image Lab software to obtain the protein expression trend.

[0041] (4) Observe and record experimental results:

[0042] 1) Survival curves of H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA

[0043] The cells transfected with LZTS3 small interfering RNA were digested and centrifuged to obtain a cell suspension. 20 μL of the cell suspension was added to a cell counting plate. The cell concentration was measured in a cell counter. 1.5 mL of complete medium was added to each well of the six-well plate. The initial cell number of each group was set to 5*10 4 , that is, the number of cells to be inoculated per well is 5*10 4. In order to reduce experimental errors, 3 replicate wells were taken from each group. The six-well plate was mixed and placed steadily in the incubator for continued culture. After 24 hours of culture, the cells in each well of the first group were digested and counted, and the average value of the three replicate wells was taken. After that, a group of cells was digested and counted every 24 hours, and a total of 4 groups were measured. A cell growth curve was drawn with the growth time as the horizontal axis and the number of cells as the vertical axis. Figure 2 As shown, the cell growth and proliferation in the LZTS3 gene knockdown group was greater than that in the blank control group, indicating that the LZTS3 gene has a significant inhibitory effect on the growth of lung cancer cells.

[0044] 2) Figure 3 The cloning survival rate of H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA. From the results in the figure, it can be seen that the cell cloning rate of the LZTS3 gene knockdown group is greater than that of the control group, and the number of clones formed between the knockdown group and the control group is statistically different (p<0.05), indicating that the LZTS3 gene has a significant effect on the cloning of lung cancer cells.

[0045] 3) Number of γ-H2AX foci induced by IR in H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA

[0046] H1299 and A549 cells were treated with different methods (Scramble, Si-LZTS3-1, Si-LZTS3-2), and then induced with 8Gy radiation, and the results of γ-H2AX immunofluorescence staining and quantitative analysis were performed. Figure 4 As shown in the figure, in H1299 and A549 cells, after knocking down the LZTS3 gene, the number of γ-H2AX foci formed in the cells decreased, indicating that inhibition of LZTS3 leads to a decrease in the degree of DNA damage in lung cancer cells.

[0047] 4) Imaging of the number of RAD51 foci induced by IR in H1299 and A549 cells after transfection with LZTS3 gene small interfering RNA

[0048] H1299 and A549 cells were treated with different methods (Scramble, Si-LZTS3-1, Si-LZTS3-2) and induced with 8Gy radiation to perform immunofluorescence staining and quantitative analysis of RAD51. Figure 4 As shown, after LZTS3 gene inhibition in H1299 and A549 cells, the number of RAD51 foci formed in the cells increased, indicating that inhibition of LZTS3 led to enhanced DNA repair ability of lung cancer cells, suggesting that silencing of LZTS3 led to cell resistance to radiotherapy.

[0049] The above irradiation conditions are: using Elekta medical electron linear accelerator to irradiate cells, the irradiation parameters are set to 6MV-X-ray, source-skin distance 100cm, irradiation field 40cm×40cm. The machine jump number MU is set according to the actual required irradiation dose D.

[0050] Application verification experiment 2: Effect of β-Trcp gene small interfering RNA on LZTS3 expression

[0051] Expression of LZTS3 in H1299 and A549 cells after transfection of liposome-mediated β-Trcp gene small interfering RNA.

[0052] H1299 and A549 cells transfected with liposome-mediated β-Trcp gene small interfering RNA were collected and samples were prepared, and the expression of LZTS3 was detected by Western blot. Figure 6 As shown, knockout of β-Trcp significantly increased the expression of endogenous LZTS3 in H1299 and A549 cells ( Figure 6 ).

[0053] Through liposome-mediated siRNA transfection technology, the expression of β-Trcp gene was successfully inhibited in non-small cell lung cancer A549 and H522 cell lines, which led to the increase of LZTS3 protein expression level. The increase of LZTS3 protein effectively slowed down the growth and proliferation rate of lung cancer cells and increased the sensitivity of lung cancer cells to radiotherapy.

[0054] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An application of up-regulating LZTS3 gene in the preparation of tumor radiosensitization drugs.

2. The use according to claim 1, characterized in that: The method for up-regulating the LZTS3 gene comprises: adding an inhibitory component for inhibiting the expression of the β-Trcp gene to the drug.

3. The use according to claim 2, characterized in that: The inhibitory component includes: small interfering RNA carrying β-Trcp gene.

4. The use according to claim 3, characterized in that: The small interfering RNA carrying the β-Trcp gene is mediated by liposomes.

5. A tumor radiotherapy sensitizing drug, characterized in that: The chemical components of the drug include: small interfering RNA carrying the β-Trcp gene.

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