Application of FOSL1 gene in preparation of medicine for inducing tumor cell copper death

Abstract

The invention is applicable to the technical field of biological medicine, and provides application of an FOSL1 gene in preparation of a medicine for inducing tumor cell copper death, and the tumor is non-small cell lung cancer. The mitochondrial injury can be specifically induced by regulating the expression of the FOSL1 gene in a targeted manner, so that copper death is triggered. On the basis of differential expression and regulation effects of FOSL1 in radiation-resistant NSCLC cells, a core mechanism of FOSL1 for mediating mitochondrial injury, triggering copper death and sensitizing radiotherapy is defined, a medicine capable of inducing copper death of the radiation-resistant NSCLC cells is provided, and an accurate, safe and efficient tumor cell radiation resistance reversal scheme is provided.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to an application of a FOSL1 gene in the preparation of a drug that induces copper death in tumor cells. Background Art

[0002] Lung cancer leads the world in mortality among human malignancies, with non-small cell lung cancer (NSCLC) accounting for approximately 80% of all lung cancer cases. Radiotherapy or concurrent chemoradiotherapy has become a core treatment approach for NSCLC patients. However, during treatment, tumor cells can acquire radiation resistance, leading to failure of local control and compromising treatment efficacy. The factors contributing to radiation resistance are complex and include hypoxia, abnormalities in signaling pathways associated with radiation resistance, and aberrant expression of genes involved in DNA damage and repair responses.

[0003] Copper death is a 2+ Copper cell death, a novel type of programmed cell death triggered by mitochondrial overload, may offer new insights into reversing radiation resistance. The mechanism of copper cell death involves copper ions specifically binding to acylated proteins in the mitochondrial tricarboxylic acid (TCA) cycle, inducing oligomerization of toxic proteins and collapse of the mitochondrial respiratory chain, ultimately leading to cell death. Copper cell death is independent of traditional apoptosis / necrosis pathways and could theoretically circumvent common apoptosis resistance mechanisms in tumor cells.

[0004] Although progress has been made in research on the mechanisms of NSCLC radiation resistance, current technologies still face the following key bottlenecks: existing copper death-inducing strategies (such as copper-loaded drugs) lack specific molecular targets for radiation-resistant cells, resulting in unclear mechanisms of action and uncontrollable efficacy; traditional copper-loaded drugs induce death through exogenous copper ion overload, which can easily disrupt the body's copper homeostasis and induce systemic adverse reactions such as liver / neurotoxicity, limiting their clinical applicability; single intervention in pathways such as hypoxia or DNA repair is difficult to overcome the multi-mechanistic synergistic effects of radiation resistance and cannot circumvent the apoptosis / necrosis resistance mechanism of tumor cells.

[0005] Therefore, it is urgent to find a target and new drug that can induce copper death in radiation-resistant NSCLC cells to improve the prognosis of patients with radiation-resistant lung cancer and provide new alternative strategies for the treatment of clinically refractory NSCLC. Summary of the Invention

[0006] The purpose of the present invention is to provide the use of FOSL1 gene in the preparation of a drug for inducing copper death of tumor cells, aiming to solve the problems raised in the background technology.

[0007] In order to solve the above problems, the present invention is implemented by providing a use of the FOSL1 gene in the preparation of a drug that induces copper cell death in tumor cells.

[0008] Furthermore, the tumor is lung cancer.

[0009] Furthermore, the tumor is non-small cell lung cancer.

[0010] Furthermore, by targeting and regulating FOSL1 gene expression, mitochondrial damage was specifically induced, thereby triggering copper death.

[0011] Another object of the present invention is to provide a use of the FOSL1 gene in the preparation of a drug for reversing the radiation resistance of tumor cells.

[0012] Another object of the present invention is to provide a drug for inducing copper cell death in tumor cells, comprising a pharmaceutically acceptable carrier and a FOSL1 overexpression plasmid.

[0013] Furthermore, the preparation method of the drug comprises the following steps: adding an aqueous NaOH solution to a mixed solution of ethanol, oleic acid, and n-hexane and mixing the mixture to obtain a microemulsion system; Cu(NO3)2·3H2O and 1,3,5-benzenetricarboxylic acid were added to the microemulsion system to react and obtain Cu-MOF; Cu-MOF was modified with DSPE PEG2000 to obtain nanoparticles; The nanoparticles are co-incubated with a FOSL1 overexpression plasmid to obtain the drug.

[0014] Based on the differential expression and regulatory role of FOSL1 in radiation-resistant NSCLC cells, the present invention clarifies the core mechanism of FOSL1 in mediating mitochondrial damage, triggering copper death, and sensitizing radiotherapy, and provides a drug that can induce copper death in radiation-resistant NSCLC cells, as well as a precise, safe, and efficient solution for reversing tumor cell radiation resistance. Among them, by targeted regulation of FOSL1 gene expression, the introduction of exogenous copper ions can be circumvented, eliminating the risk of copper metabolism disorders from the root; by activating copper death independent of traditional death pathways, the mitochondrial function and DNA repair ability of radiation-resistant cells are synchronously destroyed, playing an important guiding role in the clinical treatment of radiation-resistant lung cancer. The drug provided by the present invention effectively improves the prognosis of patients with radiation-resistant lung cancer through the above-mentioned mechanism, and provides a new alternative strategy for the treatment of clinically refractory NSCLC. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Figure 2 is the result of differential gene enrichment analysis of cells in different treatment groups (A549-RR group and A549 group).

[0016] Figure 2 Figure 2 shows the results of differential gene enrichment analysis in cells of different treatment groups (A549-RR group and A549-RR+ES group).

[0017] Figure 3 This is a graph showing gene expression enriched in copper-related death pathways.

[0018] Figure 4 The figures are comparisons of differentially expressed genes in cells of different treatment groups; in the figure, A is a comparison of differentially expressed genes between the A549-RR group and the A549 group; B is a comparison of differentially expressed genes between the A549-RR group and the A549-RR+ES group.

[0019] Figure 5 Figure 3 shows the results of copper ion concentration (A) and cell death rate after trypan blue staining (B) under different treatment conditions (n=3, *P<0.05, **P<0.01).

[0020] Figure 6 Schematic diagram of the synthesis process of the nanomedicine provided in an embodiment of the present invention.

[0021] Figure 7 Graphs showing the characterization results of the nanomedicine provided in an embodiment of the present invention; in the graph, a is a transmission electron microscope image of Cu-MOF; b is a mapping of each element in the transmission electron microscope of CMDP; c and d are transmission electron microscope images of Cu-MOF showing the characteristic peak distribution of each element in Cu-MOF by XRD and XPS, respectively; e is the Fourier transform infrared spectroscopy (FTIR) result of CMDP; f and g are diagrams showing the hydrated particle size distribution and potential change of CMDP, respectively; h is a transmission electron microscope image of CMDP degraded at different pH values; i is the copper ion release of CMDP at different pH values ​​detected by ICP (inductively coupled plasma analyzer); j is the characteristic peak of ·OH in CMDP displayed by ESR; k and l are the results and quantitative diagrams of GSH consumption detected by UV spectrophotometer.

[0022] Figure 8 Figures 2 and 3 are the results of cell experiments in different groups; in the figure, a is the detection result of CMDP on the clone-forming ability of A549-RR; b is the live-dead staining after A549-RR is treated with CMDP; c is the flow cytometry detection result; d is the ROS level after A549-RR is treated with CMDP; e is the mitochondrial ROS generation; f is the mitochondrial membrane potential level; g is the change in mitochondrial calcium ion concentration.

[0023] Figure 9Figures 2 and 3 are the results of in vivo animal experiments in different groups; in the figure, a is a photo of A549-RR cell xenograft tumors in SCID mice treated with different groups; b is a comparison of weight changes in different groups of A549-RR tumor-bearing SCID mice; c is a comparison of tumor volumes in different groups of A549-RR cell tumor-bearing SCID mice; d is a comparison of H&E staining, immunohistochemistry, FOSL1, and Ki67 protein changes in A549-RR cell xenograft tumor tissue in SCID mice after treatment with different groups. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0025] Based on the "apoptosis / necrosis resistance" phenotype exhibited by radiation-resistant NSCLC cells, the present invention demonstrates for the first time that the transcription factor FOSL1 (FOS Like 1) exhibits significant differential expression in radiation-resistant model cells under the influence of a copper death inducer. FOSL1 is a key target for regulating the copper death process in radiation-resistant NSCLC cells; targeted regulation of FOSL1 can specifically induce mitochondrial damage (including membrane potential collapse and respiratory chain complex dysfunction), thereby triggering copper death. Based on these findings, the present invention proposes a novel therapeutic strategy: by designing drugs that can target and regulate FOSL1, it is possible to specifically induce copper death in radiation-resistant NSCLC cells.

[0026] Specifically, one embodiment of the present invention provides the use of the FOSL1 gene in the preparation of a drug that induces copper apoptosis in tumor cells. By targeting and regulating FOSL1 gene expression, mitochondrial damage is specifically induced, thereby triggering copper apoptosis in tumor cells such as NSCLC. This mechanism can reverse the radiation resistance of tumor cells such as NSCLC.

[0027] In another embodiment of the present invention, a drug for inducing copper cell death and reversing tumor cell radiation resistance is provided, comprising a pharmaceutically acceptable carrier and a FOSL1 overexpression plasmid.

[0028] Specifically, the preparation method of the above-mentioned medicine comprises the following steps: S1. Add 0.5-1 mL of a 0.1-0.15 M NaOH aqueous solution to a mixed solution of 1-1.5 mL of ethanol, 0.2-0.4 mL of oleic acid, and 0.15-0.2 mL of n-hexane, and stir the mixture at 45-55° C. to obtain a constant-temperature microemulsion system.

[0029] S2. Add 8-12 mg of Cu(NO3)2·3H2O and 8-12 mg of 1,3,5-benzenetricarboxylic acid to the above microemulsion system, heat to 65-75°C for reaction, and then centrifuge and wash to obtain Cu-MOF; S3. Modify the Cu-MOF with DSPE PEG2000 to obtain nanoparticles. Specifically, disperse DSPE-PEG2000 in sterile enzyme-free water, disperse 10-15 mg of the obtained Cu-MOF in 2 mL of DSPE-PEG2000 solution with a concentration of 0.8-1.2 mg / mL, and vigorously stir overnight for modification.

[0030] S4. Co-incubate the above-mentioned nanoparticles with the FOSL1 overexpression plasmid for 10-15 hours to obtain the above-mentioned drug.

[0031] The preparation method of the FOSL1 overexpression plasmid is as follows: the purchased commercial FOSL1 plasmid (HumanpLenti-HA-FOSL1-puro) is amplified in large quantities; first, positive clones are picked, and single colonies are picked from the transformed LB plate and inoculated into 3-5 mL of LB liquid medium (with Amp 100 μg / mL); then, the culture is shaken at 37°C and 200-250 rpm for 12-16 hours (until OD600 ≈ 2.0 and the culture medium is turbid); the harvested bacterial liquid is centrifuged (4000 rpm, 10 minutes, 4°C) to collect the bacteria and discard the supernatant; and a commercially available plasmid extraction kit is used to purify the plasmid according to the specific process details in the instruction manual to obtain the FOSL1 overexpression plasmid, and the concentration (Nanodrop) and purity (A260 / A280 ≈ 1.8) are measured.

[0032] Example 1: To establish a radiation-resistant model for lung cancer A549 cells, parental A549 cells in the logarithmic growth phase were irradiated with 6 Gy (1.02 Gy / min) and then cultured. The cells were passaged when they reached the end of their logarithmic growth phase. The next day, they were irradiated with the same dose. This process was repeated five times for a total dose of 30 Gy. Surviving cells were then isolated and isolated to obtain a single clone, designated A549-RR.

[0033] The copper death inducer elesclomol (ES) was added to A549-RR cells, and the A549 (untreated A549 cells), A549-RR (irradiated A549-RR cells), and A549-RR+ES (A549-RR cells treated with the copper death inducer) groups were set up. High-throughput transcriptome sequencing and differentially expressed gene enrichment analysis revealed that the differentially expressed genes in A549-RR compared with A549 cells were enriched in pathways closely related to copper death. After the copper death inducer ES was added to radiation-resistant cells (A549-RR+ES), the expression of genes enriched in the copper death mechanism was reversed (such as Figure 1 and Figure 2 Compared with the parental A549 group and the A549-RR group, the expression of genes enriched in the copper death-related pathway was significantly different. After the addition of copper death inducers, the gene expression was reversed, including the FOSL1 gene (as shown in Figure 2). Figure 3 shown).

[0034] Example 2: High-throughput sequencing was performed on the A549, A549-RR, and A549-RR+ES cells of Example 1 above by qPCR, and the top nine differentially expressed genes in the sequencing results were verified to confirm the reliability of the sequencing results. The target gene FOSL1 (such as Figure 4 In addition, FOSL1 siRNA fragments were synthesized according to existing technologies, and the changes in the degree of copper death after knocking down FOSL1 in A549 and A549-RR+ES cells were detected by copper ion concentration determination and trypan blue staining. The results are shown in FIG. Figure 5 As shown in A and B; the results showed that compared with normal A549-RR+ES cells (ES group) and A549 cells (control group), when FOSL1 was knocked down in A549-RR+ES cells (siFOSL1+ES group), the copper ion concentration decreased and the cell death rate decreased, confirming that FOSL1 is a key target of copper death in A549 and A549-RR.

[0035] Example 3: Figure 6 As shown, based on the fact that FOSL1 is a key gene that induces copper cell death in cells, this embodiment provides a nanomedicine for inducing copper cell death in tumor cells and reversing the radiation resistance of tumor cells, and the preparation method thereof comprises the following steps: S1. Add 0.8 mL of a 0.125 M NaOH aqueous solution to a mixed solution of 1.2 mL of ethanol, 0.3 mL of oleic acid, and 0.17 mL of n-hexane, and stir the mixture at 50° C. to obtain a constant-temperature microemulsion system.

[0036] S2. Add 10 mg of Cu(NO3)2·3H2O (dissolved in 0.2 mL of deionized water before adding) and 10 mg of 1,3,5-benzenetricarboxylic acid (dissolved in a mixed solution of 0.13 mL of deionized water and 0.17 mL of ethanol before adding) to the above microemulsion system, heat to 70°C, and keep the reaction for 2 hours under continuous stirring. Then collect the product by centrifugation and wash it three times with a mixture of cyclohexane and anhydrous ethanol to obtain Cu-MOF.

[0037] S3. Modify the Cu-MOF with DSPE PEG2000 to obtain nanoparticles CMD; specifically, disperse DSPE-PEG2000 in sterile enzyme-free water, disperse 12 mg of the obtained Cu-MOF in 2 mL of 1 mg / mL DSPE-PEG2000 solution, and vigorously stir overnight for modification.

[0038] S4. The nanoparticles CMD and the FOSL1 overexpression plasmid are co-incubated for 12 hours to obtain the nanodrug CMDP.

[0039] The structure and performance of the nano drug CMDP prepared above were characterized. Figure 7 Among them, the transmission electron microscope image shows that the Cu-MOF nanoparticles have a round shape (such as Figure 7 The X-ray diffraction pattern (XRD) shows multiple diffraction peaks (such as Figure 7 (c) shows that the obtained nanomedicine is a crystalline material rather than a completely amorphous material; X-ray photoelectron spectroscopy (XPS) analysis confirmed the presence of Cu, C, N, P and O elements in CMDP nanomedicine (such as Figure 7 d). Fourier transform infrared spectroscopy (FT-IR) results show that after DSPE-PEG2000 modification, the -1 and 1100cm -1 Significant new peaks appeared at 1240 cm, corresponding to ν(CH) and ν(COC) of the PEG chain, confirming its successful encapsulation. -1 The ν(P=O) peak further verifies the presence of DSPE phospholipids. The characteristic carboxylic acid peaks of Cu-MOF are 1602 / 1398 cm -1 The intensity decreases, suggesting that the PEG layer partially shields the MOF surface. After FOSL1 plasmid loading, 1220 cm -1 The peak at 1650cm is enhanced, confirming the presence of the DNA phosphate backbone. -1 The weak peak may be attributed to the vibration of nucleic acid bases, and the Cu-O vibration peak shifts to a low wave number, indicating that the plasmid is connected to the MOF surface Cu through the phosphate group. 2+In addition, the ν(COC) peak of PEG is broadened, suggesting that the plasmid is bound to the PEG chain through hydrogen bonds (e.g. Figure 7 When the hydrated particle size and ζ potential of the CMDP nanoparticles were measured at each stage of synthesis, their diameter changed from 184 nm to 220 nm, and the ζ potential value changed from negative to positive, further demonstrating that DSPE-PEG2000 had successfully coated the nanoparticle surface and the FOSL1 overexpression plasmid had been successfully loaded (as shown in Figure 5). Figure 7 f and g). When the CMDP nanomedicine was exposed to solutions with pH values ​​of 7.4 and 6.4, the CMDP nanomedicine still maintained its intact material morphology. However, when exposed to a solution with a pH value of 5.4 for the same period of time, the material degraded and lost its original morphology. This phenomenon is attributed to the inherent instability of the internal coordination bonds of the CMDP nanomedicine (e.g. Figure 7 In addition, under GSH and hydrogen peroxide conditions, CMDP nanomedicine showed more intense GSH consumption, which may be due to the Cu+ / Cu 2+ Can react with GSH and hydrogen peroxide, resulting in rapid consumption of GSH (such as Figure 7 of k and l), and generate reactive oxygen species (such as Figure 7 As shown in j), the above results show that the embodiment of the present invention successfully synthesized the nano drug CMDP with copper death inducing effect.

[0040] Example 4: Cell experiments were performed according to the following groups: -IR was the non-irradiation group, +IR was the irradiation group; Con group was the control group without any treatment; FOSL1 OE The group is the FOSL1 gene overexpression group, i.e., the commercial FOSL1 overexpression plasmid; the CMD group is the empty copper-based nanomaterial without the FOSL1 overexpression plasmid, i.e., the nanoparticle CMD prepared in Example 3; the CMDP group is the nanodrug loaded with the FOSL1 overexpression plasmid, i.e., the nanodrug CMDP prepared in Example 3. Figure 8 shown.

[0041] The clone formation experiment showed that -IR (Con group, FOSL1 OE group, CMD group, CMDP group) and +IR (Con group, FOSL1 OE group, CMD group, CMDP group). Under the same irradiation dose, the cell colony formation in the CMD and CMDP groups was significantly reduced (e.g. Figure 8 In addition, PI staining experiments showed that the number of cell death in the +IR CMDP group was the highest compared with other groups (as shown in a). Figure 8Flow cytometry was used to detect cell apoptosis, and it was found that the +IR CMDP group had the most cell death, further confirming the killing effect of CMDP on tumor cells (as shown in Figure 2). Figure 8 c).

[0042] DCFH-DA was used as a fluorescent probe to detect the ability of CMDP to generate ROS in tumor cells. Compared with other groups, the +IR CMDP group produced the most ROS (e.g. Figure 8 The accumulation of mitochondrial ROS in cells was further confirmed by the mitochondrial specific probe MitoSOX Red (as shown in d). Figure 8 The results are consistent with the above results, proving that CMDP can induce the production of a large amount of reactive oxygen species and cause mitochondrial damage after entering tumor cells and irradiation.

[0043] The present invention uses JC-1 to detect mitochondrial membrane potential and finds that compared with other groups, the membrane potential of the +IRCMDP group decreases the most, confirming that the +IRCMDP group causes severe mitochondrial damage (such as Figure 8 At the same time, Hoechst 33342 and Rhod-2 AM were used to detect the fluorescence of mitochondrial calcium ions, and it was found that the Rhod-2 AM fluorescence intensity increased in the +IR CMDP nanoparticles group (as shown in f). Figure 8 The above results all prove that under radiation induction, CMDP can induce mitochondrial damage and affect mitochondrial function, and its tumor killing effect is significantly better than that of the -IR group and the radiotherapy alone group.

[0044] Example 5: The tumor killing effect of the nanomedicine CMDP prepared in Example 3 was verified by in vivo experiments. Specifically, 6-week-old female SCID mice were raised in an isolator. The standard feed was sterilized by cobalt source irradiation, and the bedding and drinking water were sterilized by high temperature and high pressure. As in Example 4, the experiment was divided into 8 groups: -IR (Con group, FOSL1 OE group, CMD group, CMDP group) and +IR (Con group, FOSL1 OE The FOSL1 stable cell model was established by inoculating cells from different treatment groups subcutaneously on the outside of the right leg of mice. The tumor growth was observed and recorded every day. 3 At 3 pm, 20 Gy local irradiation was given at a dose rate of 2.0 Gy / min. After 14 days of continuous observation, the subcutaneous tumor was removed for subsequent functional experiments (e.g. Figure 9 The results showed that the body weight of mice did not change significantly after treatment in different groups (as shown in a). Figure 9The subcutaneous tumors were measured and photographed, and it was found that the tumor volume of mice in the +IR CMDP group was the smallest after irradiation compared with the other treatment groups (as shown in b). Figure 9 HE staining of tumor tissue and immunohistochemical staining showed that compared with the other treatment groups, the tumor cell nuclear volume in the +IR CMDP group was reduced, the chromatin was densely stained, and the nuclear chromatin was fragmented. Immunohistochemical staining showed that Ki-67 gradually decreased, and the expression of FOSL1 in the +IR CMDP group was still high (as shown in Figure 4). Figure 9 The above mouse experimental results demonstrate the tumor-killing effect of the nanomedicine CMDP.

[0045] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be determined by the appended claims.

Claims

1. Application of FOSL1 gene in the preparation of drugs that induce copper-induced death of tumor cells.

2. The use according to claim 1, characterized in that The tumor is lung cancer.

3. The use according to claim 2, characterized in that The tumor is non-small cell lung cancer.

4. The use according to claim 1, 2 or 3, characterized in that: By targeting and regulating FOSL1 gene expression, mitochondrial damage is specifically induced, thereby triggering copper death.

5. Application of FOSL1 gene in the preparation of drugs for reversing radiation resistance of tumor cells.

6. The use according to claim 5, characterized in that The tumor is lung cancer.

7. The use according to claim 6, characterized in that The tumor is non-small cell lung cancer.

8. A drug for inducing copper cell death in tumor cells, comprising a pharmaceutically acceptable carrier, characterized in that: A FOSL1 overexpression plasmid is also included.

9. The drug for inducing copper cell death in tumor cells according to claim 8, characterized in that The tumor is non-small cell lung cancer.

10. The drug for inducing copper cell death in tumor cells according to claim 8 or 9, characterized in that The preparation method of the medicine comprises the following steps: adding an aqueous NaOH solution to a mixed solution of ethanol, oleic acid, and n-hexane and mixing the mixture to obtain a microemulsion system; Cu(NO3)2·3H2O and 1,3,5-benzenetricarboxylic acid were added to the microemulsion system to react and obtain Cu-MOF; Cu-MOF was modified with DSPE PEG2000 to obtain nanoparticles; The nanoparticles are co-incubated with a FOSL1 overexpression plasmid to obtain the drug.

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