CN113913519A - Application of CUL9 as molecular marker in preparation of drugs for regulating iron death of colorectal cancer - Google Patents

Abstract

The invention provides an application of CUL9 as a molecular marker in preparation of a drug for regulating and controlling iron death of colorectal cancer, which is characterized in that a molecule related to colorectal cancer and iron death is screened by combining whole exon sequencing and database differential analysis, namely CUL9 as the molecular marker plays an important role in inhibiting iron death of colorectal cancer, an MDM2 molecule as an upstream molecule of CUL9 can enhance the inhibition effect of CUL9 on iron death of colorectal cancer, and an in vivo and in vitro experiment verifies that an MDM2 inhibitor can enhance the curative effect of an inducer for inhibiting iron death of colorectal cancer; finally, through the biological experimental methods of the present invention, MDM2 inhibitors can overcome iron death tolerance of colorectal cancer caused by CUL9 and improve the efficacy of iron death inducers; in addition, the iron death inducing agent can also be used for preparing a medicament for treating the middle and advanced stages of colorectal cancer, wherein the medicament comprises a CUL9 molecular marker and an MDM2 inhibitor.

Description

Application of CUL9 as molecular marker in preparation of drugs for regulating iron death of colorectal cancer

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of CUL9 as a molecular marker in preparation of drugs for regulating and controlling iron death of colorectal cancer.

Background

Colorectal cancer is a common malignant tumor of the digestive tract, and the health threat to people in China is not easy to be looked at. According to global epidemiological data (GLOBOCAN 2020) issued by the International agency for research on cancer (IARC) of the world health organization, 193.16 ten thousand new cases and 93.52 ten thousand cases of colorectal cancer were estimated to be located at the third and second sites of all malignant tumors in 2020. In recent decades, the incidence of colorectal cancer has increased with the improvement of people's living standard and the change of dietary structure.

In recent years, with the continuous and deep research on colorectal cancer, new technical means and drug research and development are rapidly developed, and the prognosis of colorectal cancer is on the improvement trend than the past decades. However, for clinical diagnosis and treatment of colorectal cancer, tumor stage is still an important prognostic factor, and there is great heterogeneity in prognosis of early colorectal cancer and middle and late colorectal cancer, so that research on new tumor-targeted therapy and mechanism thereof is critical to improve prognosis of colorectal cancer (especially, prognosis of middle and late colorectal cancer). Iron death is a cell death pattern resulting from iron-dependent lipid peroxidation, and the accumulation of large numbers of reactive oxygen species, and differs from apoptosis, necrosis, and autophagy at both the morphological and biochemical levels. Compared with normal cells, the tumor cells have larger iron demand, obviously higher Reactive Oxygen Species (ROS) level, and are more prone to iron death due to high dependence on iron metabolism. Therefore, induction of iron death is gradually considered as a new therapeutic strategy in tumors. Iron death caused by ROS involvement is ubiquitous in the occurrence and development of tumors, for example, GPX4 inhibition can promote iron death, ACSL4 overexpression can promote iron death and the like. However, there are few reports on the molecules for enhancing the therapeutic effect of iron death inducers for colorectal cancer, and the scheme is not clear. Therefore, the search for a new method for developing a treatment for colorectal cancer that can improve the efficacy of iron death inducers is important.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide the application of CUL9 as a molecular marker in preparation of drugs for regulating and controlling iron death of colorectal cancer.

In order to achieve the above purpose, the solution of the invention is as follows:

in one aspect, the invention aims to screen molecules related to iron death of colorectal cancer by combining whole exon sequencing and database differential analysis, namely provides an application of CUL9 as a molecular marker in preparation of a method for regulating and controlling iron death of colorectal cancer.

As another aspect, the invention aims to experimentally verify the application of CUL9 as a molecular marker in the preparation of drugs for inhibiting iron death of colorectal cancer.

As a further aspect, it is an object of the present invention to provide the use of an MDM2 molecule for the preparation of a medicament for modulating the inhibition of colorectal cancer iron death by CUL 9.

As yet another aspect, it is an object of the present invention to provide in vitro and in vivo experiments demonstrating the use of an MDM2 inhibitor for the preparation of a medicament for enhancing the therapeutic efficacy of an iron death inducer for colorectal cancer.

As a final aspect, it is an object of the present invention to provide the use of a MDM2 molecule as an upstream molecule of CUL9 for the preparation of a medicament for enhancing the inhibition of iron death from colorectal cancer by CUL 9.

Further preferably, the use of an iron death inducer for the manufacture of a medicament for the treatment of mid-to late colorectal cancer.

Further preferably, the medicament comprises a CUL9 molecular marker and a MDM2 inhibitor.

Due to the adoption of the scheme, the invention has the beneficial effects that:

through the biological experimental method, the MDM2 inhibitor can overcome the iron death tolerance of colorectal cancer caused by CUL9 and improve the curative effect of the iron death inducer.

Drawings

FIG. 1 is a schematic diagram of the whole exon sequencing analysis and the mRNA differential expression analysis in example 1 of the present invention.

Fig. 2 is a diagram of how CUL9 was overexpressed (left and upper right panels) and knockout cell system (lower right panel) were constructed in example 2 of the present invention, wherein x < 0.05.

FIG. 3 is a diagram showing the change of phenotype of HCT116 cells treated with Erastin (left) and Ferrostatin-1 (right) in example 2.

FIG. 4 is a graph showing the changes in expression and pathway of iron death-related molecules after the knock-out of CUL9 and the knock-down of HnRNPC, respectively, in example 2 of the present invention.

FIG. 5 is a diagram showing the protein interaction relationship between CUL9 and HnRNPC in example 2 of the present invention (left side); in the RKO system, the CUL9 knockout can up-regulate the HnRNPC protein expression diagram (right side).

FIG. 6 is a graph of the protein interaction of TP53 to inhibit CUL9-HnRNPC (left) and the increase in protein interaction between MDM2 and TP53 after RKO overexpresses CUL9 in example 3 of the invention (right).

FIG. 7 shows that MDM2 is inhibited in example 3 to inhibit the ubiquitination of CUL9-HnRNPC, which is blocked by TP53 mutation (left side); MDM2 blockade increased Erastin-induced iron death of TP53 wild CRC cells (right).

FIG. 8 is a graph showing that the clone formation assay of example 4 of the present invention shows the synergy between an MDM2 inhibitor (Nutlin) and an iron death inducing agent (Erastin) in TP53 wild-type CRC cells (left side); the CTG experiment suggests: at the same time, it was found that Nutlin in RKO CRC cell line enhances the susceptibility profile to Erastin (right).

FIG. 9 is a graph showing the in vivo anticancer activity of Erastin in HCT116 cells overexpressed in the control group and CUL9 in example 4 of the present invention.

Detailed Description

The invention provides application of CUL9 as a molecular marker in preparation of drugs for regulating and controlling iron death of colorectal cancer.

The present invention will be further described with reference to the following examples.

Example 1:

screening for colorectal cancer (CRC) and iron death-related molecules in conjunction with whole exon sequencing and database differential analysis:

1) CRC liver metastasis complete exon sequencing (WES) case selection

The present example was approved by the ethical committee and the specimens were obtained from CRC liver metastasis patients received by the colorectal professional group of the secondary zhongshan hospital of the university of compound denier, both signed with written informed consent. And (3) inclusion standard: a) the age is greater than 18 years; b) no history of other malignant tumors; c) preoperative examination proves that liver metastasis is accompanied and only limited to the liver; d) before operation, no anti-tumor treatment is carried out; e) obtaining a tumor specimen; f) the operation pathology of the colorectal focus is definitely adenocarcinoma, and the operation pathology of the liver focus is definitely metastatic adenocarcinoma; g) no loss of expression of mismatch repair (MMR) protein was observed in immunohistochemical staining of surgical specimens.

2) Sequencing specimen collection

All samples submitted for inspection are fresh samples. And (3) carrying out combined inspection on 10 groups of samples, wherein each group comprises 3 CRC primary foci, CRC liver metastases and normal colon epithelial tissues which are all surgical specimens. Immediately treating all the specimens in vitro with liquid nitrogen, and storing in a refrigerator at-80 ℃; gross cuts were made in a frozen microtome (HM550, Thermo Fisher Microm, Germany) at-50 ℃ prior to presentation.

3) Second generation sequencing (Whole exome sequencing)

Exome capture and pooling was performed by kit (Ion AmpliSeq)^TMExome Kit 4x Duo, Life Technology). Sequencing was also performed as one-way sequencing on an Ion Torrent Proton platform, with a sequencing depth of 100X, and corresponding data analysis was performed by Torrent Suit software.

4) Data analysis and Gene screening

After obtaining the original data, mutation analysis and gene screening were performed according to the following procedures: first, SNV Quality values greater than 20 in this study are considered reliable sites; secondly, screening the SNV of the specific exon aiming at the sample; thirdly, annotating the specific SNV, wherein a reference database is derived from SeattleSeq SNP association (http:// snp.gs. Washington. edu /), and screening out the SNV with influence, including a frame shift code (frameshift), missense (missense), a splice-site variation (splice-acceptor and splice-donor), a stop codon mutation and the like; and fourthly, screening the pathogenic people in the SNV influencing the gene function by utilizing 5 kinds of software such as SIFT, Polyphen-2, Mutation assessor, Condel and FATHMM. It is considered meaningful to meet one of the following criteria: a) SIFT is less than or equal to 0.05; b) PPH2 is more than 0.45; c) FATHMM < 0; d) CONDEL _ LABEL is "Deleterious"; e) impact is "medium" or "high"; and fifthly, performing conversion analysis on the results of SIFT, Polyphen-2 and Mutation assessor in the last step by using transfic software, wherein the result is 'high _ impact' and is considered as a driving gene.

5) TCGA data extraction and analysis

Clinical pathology and mRNA data (http:// www.cbioportal.org) were downloaded from cBioportal in TCGA on 25.6.2020. The expression quantity of mRNA FPKM of the related gene is determined by geoman, and the mRNA expression abundance of the related gene of a matched patient normal intestinal epithelial mucosa sample can also be determined.

As a result:

1) basic clinical information for full exon sequencing patients

In this example, total exon sequencing was performed on samples of CRC primary foci, normal intestinal mucosa and liver metastases from 10 patients with simultaneous liver metastasis. The basic and clinical pathology information of the patients is detailed in table 1. The average sequencing depth of the samples was 89X. Of the 10 patients, 5 male patients had an average age of 59.2 years. All samples were examined by the pathologist as adenocarcinoma.

Table 1: whole exome sequencing cases clinical and pathological conditions

PNI: a nerve insult; VI: cancer emboli can be found in vessels; TDs (time dependent dispersions): cancer nodule

2) Whole exon sequencing and TCGA differential expression analysis

The data analysis flow is detailed in fig. 1. Through analysis, 511 driving genes were detected in the primary focus of CRC, and 638 driving genes were detected in the liver metastasis. Based on the preliminary results of the subject groups, a gene set was obtained by subsequent analysis of gene mutation frequency, enrichment analysis and literature review, as shown in Table 2. By utilizing TCGA mRNA data, selecting iron death important molecules DPP4 and SLC7A11 for mRNA differential expression analysis, combining internal and external analysis, taking a molecule intersection, and finally selecting E3 ligase Cullin9 as a possible key molecule for regulating CRC iron death as a next research target.

Table 2: screening Gene lists for target Capture sequencing

Example 2:

verification that CUL9 can inhibit iron death mechanism of colorectal cancer

1) Cell culture

Human colorectal cancer cell lines HCT116(CCL-247), Caco2(HTB-37), HT29(HTB-38), RKO (FS-0347) cells were cultured in DMEM medium and included 10% fetal bovine serum and 1% penicillin/streptomycin at 37 ℃ with 5% CO₂The wet type constant temperature incubator. HEK293T (CRL 4500) and human colorectal cancer cell line SW1463(CCL-234) Using RPMI-1640 Medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin at 37 deg.C with 5% CO₂The wet type constant temperature incubator.

2) Co-immunoprecipitation

(1) Thawing the protein lysate on ice in a total volume of 500. mu.l, and a total amount of protein of 250. mu.g-1 mg; (2) add 20. mu.l agarose or agarose beads to 500. mu.l protein lysate to eliminate non-specific binding; (3) shaking on an end-to-end mixer at 4 ℃ for 30 min; (4) spinning at 4000rpm for 5s at 4 ℃, and collecting the supernatant; (5) adding an antibody; (6) stirring for at least 2h on an end-to-end stirrer (4 ℃); (7) add 20. mu.l of magnetic beads to the lysate and antibody mixture; (8) stirring in a cold storage room for 2h or overnight; (9) the beads were spun down and washed 3 times with 750 μ l IP buffer for 10min each in a cold room; (10) at least once in a cold room with 750 μ L, 10mmol/L Tris pH 7.4 for at least 10 min; (11) add 20. mu.l loading buffer (containing 7.5% beta-mercaptoethanol); (12) thawing the input aliquot and adding 40% loading buffer containing 7.5% beta-mercaptoethanol; (13) heating all samples (including the input) to 95 ℃ to separate the beads from the antibody/protein complex; (14) place the sample on ice to ensure that the lid does not snap open; (15) the tube was gently shaken to ensure that the beads and proteins were separated.

3) Immunoblot detection

Sample preparation, protein extraction and quantitation

The cell culture medium was aspirated, and the residual medium was washed clean with pre-cooled PBS and discarded. The cells were lysed by adding the appropriate amount of protein lysate containing protein inhibitors and scraping the cells with a cell scraper, shaking overnight at 4 ℃. The resulting mixture was centrifuged at 13000rpm for 30min at 4 ℃ to collect the supernatant, which was then subjected to concentration measurement. Protein concentration was measured using a Bradford (Biorad #5000205) kit. In order to prepare a protein concentration standard curve, 2000 mu g/mL BSA standard protein is diluted, then 5 mu l of the standard BSA protein after gradient dilution is added into 1mL Bradford reagent, the mixture is uniformly mixed and is subjected to a light-shielding reaction at room temperature for 5min, then an enzyme reader is used for measuring the absorbance at 595nm, and the standard curve preparation is carried out according to the absorbance and the concentration value of the standard protein. The method for determining the protein of the sample comprises the following specific steps: taking 1mL of Bradford reagent, adding 2 mu l of sample to be detected, and fully and uniformly mixing; standing at room temperature in dark place for 5min, adding 200 μ l into 96-well plate, and measuring absorbance at 595nm with microplate reader; protein concentration was calculated from the standard curve and dilution factor, and the protein concentration was finally diluted to 2000. mu.g/mL.

(ii) SDS-PAGE

The preparation of 10% SDS-PAGE gel by TGX FastCast Acrylamide Kit (Biorad #1610173) comprises the following steps: (a) cleaning the long glass plate and the short glass plate, drying, placing on a rack, clamping, adjusting the position to be horizontal, and preparing for glue pouring; (b) preparing a lower layer glue according to the instruction, adding a proper amount of 10% Ammonium Persulfate (APS) and N, N, N ', N' -Tetramethylethylenediamine (TEMED) by volume, fully and uniformly mixing, adding 7.5mL of the prepared lower layer glue into each glue plate, and adding 1mL of double distilled water into the upper layer for sealing; (c) after the solidification is carried out for 20-30min at room temperature, a refracted ray between water and glue can be seen, which indicates that the glue at the lower layer is solidified, at the moment, the water at the upper layer is poured out, and the residual water is completely absorbed by absorbent paper; (d) preparing an upper layer adhesive, adding a proper amount of 10% APS and TEMED, fully and uniformly mixing, adding into a glass plate, and inserting a 15-hole comb to prevent bubbles; (e) after the upper layer is solidified, the comb can be pulled out, and SDS-PAGE electrophoresis is carried out.

Protein sample treatment: (a) taking out a protein sample from-80 ℃, adding 40% volume of loading buffer solution containing beta-mercaptoethanol after fusion, namely adding 16 mul of loading buffer solution into 40 mul of protein sample; (b) boiling the protein sample in a water bath for 7 min; (c) the protein sample is centrifuged at 13000rpm for 5min at 4 ℃ to obtain a sample which is used for SDS-PAGE electrophoresis.

SDS-PAGE electrophoresis: (a) adding 3 mul of protein molecular weight markers or 21 mul of processed protein samples into the protein loading holes, wherein the holes without the added samples can be filled with loading buffer solution or waste protein samples for pressing belts; (b) the electrophoresis condition is that after 80V runs for 30min, 120V electrophoresis is changed to the end of buffer solution running gel, or the whole 120V electrophoresis is carried out to the end of buffer solution running gel.

(iii) protein gel transfer membrane: before membrane transfer, a membrane transfer buffer is prepared and placed in a refrigerator for precooling, and a 7.3-8.6cm nitrocellulose membrane (NC membrane) is prepared. The specific steps of the film transfer are as follows: (a) soaking an NC membrane and SDS-PAGE gel which is subjected to electrophoresis in a membrane transfer buffer solution (buffer) for a plurality of minutes, and soaking a clamp, a sponge pad and filter paper for membrane transfer in the membrane transfer buffer; (b) carefully prying the glass plate from one side of the short glass plate, and cutting off the upper separation gel layer and the blue sample buffer solution at the lowest part of the separation gel; (c) carefully peeling the glue from the glass plate, and placing the glue on filter paper to avoid generating bubbles; (d) marking a corner of an NC membrane by using a pencil, then covering a membrane on SDS-PAGE gel, and completely covering filter paper to avoid generating bubbles; (e) covering the sponge cushion, clamping the sponge cushion by a clamp, putting the sponge cushion into a film-rotating groove, adding pre-cooled film-rotating liquid, and rotating the film on ice under the film-rotating condition of 100V for 85min or 80V for 110 min; (f) after the membrane transfer, the NC membrane was taken out and placed in TBST buffer.

(iv) antibody incubation: the membrane was removed from the TBST buffer, stained with 1mL ponceau red stain (Sigma # P71701L) for 2min, then washed slightly with TBST buffer and photographed with a chemiluminescence apparatus (Immobilon Western, Millipore Corporation, Mass.) to see if the membrane was well transferred. After photographing, the primary antibody incubation was started by washing off the residual ponceau with TBST buffer and then blocking with 5% skim milk for 1.5 h. Prior to incubation of the primary antibody, the remaining skim milk was washed off with TBST buffer for 3 times, 5min each. After the primary antibody incubation was completed, the membrane was washed 3 times with TBST buffer for 5min each time, and then the secondary antibody was incubated. The secondary antibody was formulated with 5% skim milk. The secondary antibody was incubated for 1-2h, and the membrane was then washed 5 times with TBST buffer for 5min each. Chemiluminescence autoradiography was performed using Amersham ECL Western Blotting Detection Reagent (GE/Amersham # RPN2134) and Detection was performed using Amersham Imager 600 images (GE/Amersham # 29083461).

4) Cell viability assay

Cell viability assay Using CellTiter-Glo^TMA kit. After the colorectal cancer cell lines to be detected are treated for 24 hours, the cells are digested, centrifuged and resuspended, and the cell density is adjusted to be added into a 96-well plate according to 100 mu l of wells, wherein each group has 3 wells. The CTG detection was performed on regular cultures at 1d, 3d, 5d, 7d and 9d post inoculation, respectively. And during detection, adding 100 mu l/hole of CTG reagent, uniformly mixing for 2min at the speed of 600m/s, standing at room temperature for 10min, detecting a luminous value by using an enzyme-labeling instrument, establishing a standard curve after obtaining the luminous value, and performing corresponding statistical comparison.

As a result:

1) a CUL9 knockout system of HCT116 was constructed by the method of CRIPR-Cas 9. Due to the large open reading frame of CUL9, CUL9 was endogenously overexpressed in the Caco2 cell line by means of a Synergistic Activation Media (SAM) dual vector lentivirus. Western Blotting results showed significant overexpression in Caco2 cells and significant knockdown in HCT116 cells (FIG. 2).

Wherein, in fig. 2, a CUL9 overexpression model of an intestinal cancer cell line Caco2 and a CUL9 knockout model of HCT116 are constructed by a CRISPR gene editing method, the left figure shows the change of the mRNA level of CUL9 after the construction of overexpression, and the right upper figure shows the change of the protein level of CUL9 after the construction of overexpression; the lower right panel shows protein levels of CUL9 after knockout.

2) CUL9 knockout and control HCT116 cells were treated with iron death inducer Erastin and iron death inhibitor Ferrostatin-1. The results showed that cell viability of CUL9 knockout HCT116 cells was inhibited after treatment with different doses of Erastin, with statistical significance (< 0.05) (fig. 3). Correspondingly, after the addition of Ferrostatin-1, the cell viability inhibition caused by the CUL9 knockout is complemented, and the cell viability of the two is not obviously different (P >0.05) on the 5 th day of the experiment.

Among them, as shown in fig. 3, when Erastin, an iron death inducer, was added to the HCT116 cell line, iron death occurred more easily in the CUL9 knockout HCT116 with increasing concentration (left panel); the inhibitory phenotype resulting from the CUL9 knockout can be partially complemented by the iron death inhibitor Ferrostatin1 (right panel).

3) To further confirm that this phenotype is independently present in TP53 wild-type colorectal cancer cell lines, WB experiments were next performed on CUL9-KO, HnRNPC-KD and co-knockout cells, respectively, to detect their downstream pathways. The results showed that after knock-down of HnRNPC, expression of GPX4 was elevated and expression of FTH1 was slightly elevated (fig. 4).

Among them, as shown in FIG. 4, in HCT116 cell line, the deletion of CUL9 and HnRNPC, respectively, resulted in the finding of alterations in the iron death-related markers GPX4, FTH1, COX2 and FACL4 (left panel); corresponding Fe²⁺And changes in MDA (right panel).

4) HnRNPC is probably the downstream molecule for CUL9 to play a role, and the phenotype experiment also verifies the finding. Further, the results of co-immunoprecipitation, Western blot and qRT-PCR show that protein interaction relationships exist between CUL9 and p53, and between CUL9 and HnRNPC, and that p53 negatively regulates HnRNPC (FIG. 5).

As shown in FIG. 5, it can be found through co-immunoprecipitation that protein interaction exists between CUL9 and p53, protein interaction exists between CUL9 and HnRNPC, and protein interaction does not exist between p53 and HnRNPC.

Example 3:

MDM2 can regulate and control the inhibition effect of CUL9 on colorectal cancer iron death

Co-immunoprecipitation of CUL9 with HnRNPC showed a significant decrease in the expression of Ubiquitin (UB) protein after overexpression of TP53 (fig. 6), and thus, it was further found that protein interaction between MDM2 and p53 was increased after RKO overexpresses CUL9 (fig. 7). Next, MDM2 blockade was found to increase Erastin-induced iron death in TP53 wild-type CRC cells (fig. 7), with statistically significant changes in molecular expression associated with iron death.

Wherein, as shown in FIG. 6, inhibition of the interaction of CUL9-HnRNPC protein can be found after p53 is knocked out and overexpressed (left panel); (right panel) overexpression of CUL9 correlated with the p53 upstream molecule MDM 2.

As shown in figure 7, (left panel) CUL9-HnRNPC ubiquitination modification was significantly inhibited using MDM2 inhibitor Nutlin 3; (right panel) the combined use of the MDM2 inhibitor Nutlin and the iron death inducer Erastin can further increase the protein expression of the iron death markers GPX4 and FACL 4.

Example 4:

in vivo and in vitro experiments prove that the inhibitor of MDM2 can enhance the curative effect of the iron death inducer for colorectal cancer

1) Clone formation experiments

Different treated CRC cell lines (500 cells per well) were seeded into 6-well plates and cultured for 3 weeks. Then, the cells were fixed with 4% paraformaldehyde for 10min and stained with crystal violet solution for 45 min. After washing with distilled water, colony images were obtained with a scanner (Microtek, TMA1600III) and counted with ImageJ software (National Institutes of Health, USA). All experiments were performed in three wells and the whole study was repeated three times.

2) Subcutaneous tumor formation of nude mouse and adding medicine

Nude mice with 4-6 weeks age of subcutaneous tumorigenesis were purchased from Shanghai Ling biotechnology company, and were bred for 1 week to adapt to a new environment, and then were prepared for subcutaneous inoculation of tumor cells. Obtaining tumor cells with good growth state in logarithmic phase, performing subcutaneous inoculation, and inoculating 2% pentobarbital to nude miceAfter sodium anesthesia, cells were injected subcutaneously under the lateral proximal axilla of the left forelimb of nude mice, and the cell concentration was adjusted to 5X10⁶Injection at 200. mu.l/injection. The medicine is divided into 4 groups, namely EV + Erastin, CUL9-OE + Erastin, EV + Erastin + Nutlin and CUL9-OE + Erastin + Nutlin, the medicine is added when the sizes of tumors are similar, and 3 medicines in each group are marked. EV + Erastin and CUL9-OE + Erastin groups are treated with 30mg/kg Erastin, EV + Erastin + Nutlin groups and CUL9-OE + Erastin + Nutlin groups are treated with 30mg/kg Erastin +200mg/kg Nutlin. After the Nutlin is prepared, the nude mice are subjected to intragastric administration for 14 days by using an intragastric injection needle continuously every day within 24 hours, and Erastin is subjected to intraperitoneal injection for 1 time every 2 days. The body weight and tumor size of the nude mice were measured every 6 days during the period, and 24 hours after the last administration, the nude mice were killed by breaking the neck, and the tumors were taken out, recorded by photographing, and weighed.

3) Viral packaging and viral infection

(1) And (3) packaging the virus: and (3) carrying out escherichia coli DH5 alpha competence transformation on a target plasmid needing to package the virus, a virus package helper plasmid delta 8.9 and VSVG, picking out monoclonal shake bacteria overnight, extracting and amplifying plasmids, and preparing for virus package. HEK293T cells at 3X 10⁶Spreading the cells/disc to a 10cm cell culture dish, culturing for 48h, and performing lipofectamine 3000 liposome transformation when the cell fusion degree reaches 95%, wherein the specific steps are as follows: (a) prior to lipofection, HEK293T cells were replaced with 5mL of fresh medium; (b) mixing 21 μ l lipofectamine 3000 with 500 μ l Opti-MEM medium; (c) 5. mu.g of delta 8.9, 500ng of VSVG and 5. mu.g of the target plasmid were mixed with 500. mu.l of Opti-MEM medium, and then 21. mu.l of P3000 reagent was added thereto and mixed well; (d) fully mixing the solutions in the steps (b) and (c), and standing for 10min at room temperature; (e) slowly dripping the DNA liposome compound into HEK293T cells, and changing 12mL of fresh culture medium after overnight incubation; (f) collecting culture medium supernatants containing viruses for 24h, 48h and 72h, combining, centrifuging at 5000rpm for 30min, removing residual cell debris, subpackaging according to 1 mL/tube, and freezing at-80 ℃ for later use.

(2) Viral infection: after the cells are plated for 24 hours, virus infection can be carried out when the cells grow to 40 percent of fusion degree, and the specific steps are as follows: (a) the virus was removed from-80 ℃ and thawed and Polybrene (Sigma #107689) was added to a final concentration of 8. mu.g/mL; (b) sucking out the culture medium covering the cells, adding a proper amount of Polybrene virus solution containing 8 μ g/mL, such as 5mL virus solution in a 10cm culture dish, 1mL virus solution in a 6-well plate, and 50 μ l virus solution in a 96-well plate; (c) some fresh culture media can be supplemented when virus liquid is added properly, so that the virus infection effect is enhanced; (d) the virus was infected the next day, the virus fluid was discarded, and then fresh complete medium was added for culture.

As a result:

clonogenic experiments showed that the TP53 enhancer (Nutlin) and the iron death inducer (Erastin) had a synergistic effect in TP53 wild-type CRC cells (fig. 8). The effect of inhibitors of MDM2 (Nutlin) on Erastin sensitivity was studied, and synergy was also observed with CUL9 over-expression in RKO (fig. 8). HCT-116 cells over-expressed in the control group and CUL9 were implanted subcutaneously in nude mice to determine whether the over-expression of CUL9 inhibited Erastin's anti-cancer activity in vivo. Compared with a control group, the over-expression of CUL9 promotes the growth of tumors, inhibits the in vivo activity of Erastin, and reduces MDA and Fe²⁺And (4) horizontal. When Erastin and Nutlin were used in combination, a significant reduction in tumor size, i.e., a significant reduction in subcutaneous tumors in nude mice over-expressed with CUL9, was observed, which was statistically significant, in contrast to MDA and Fe²⁺The levels were significantly elevated (fig. 9).

Wherein, as shown in fig. 8, (left panel) combined application of Erastin and Nutlin can significantly inhibit the clonogenic capacity of the colorectal cancer cell line RKO; (right panel) the Erastin and Nutlin have combined cell viability inhibiting effect as proved by cell viability experiment.

As shown in fig. 9, (upper left panel) animal experiments show that over-expression of CUL9 can significantly inhibit tumor-inhibiting effect of Erastin, and combined application of Nutlin can overcome iron death tolerance caused by over-expression of CUL 9; (upper right panel) change in weight of subcutaneous implants; (lower left panel) expression levels of MDA from different treatment groups; (lower right panel) different treatment groups Fe²⁺The expression level of (3).

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. It will be readily apparent to those skilled in the art that various modifications to these embodiments and the generic principles defined herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments. Those skilled in the art should appreciate that many modifications and variations are possible in light of the above teaching without departing from the scope of the invention.

Claims (8)

1. Use of CUL9 as a molecular marker in the preparation of a medicament for regulating iron death in colorectal cancer.
2. Use of CUL9 as a molecular marker in the preparation of a medicament for inhibiting iron death in colorectal cancer.
3. Use of a MDM2 molecule in the preparation of a medicament for modulating the inhibition of iron death from colorectal cancer by CUL 9.
4. Use of an MDM2 inhibitor for the preparation of an inducer of iron death in colorectal cancer enhancement.
5. Use of a MDM2 molecule as an upstream molecule of CUL9 in the preparation of a medicament for enhancing the inhibition of CUL9 against iron death from colorectal cancer.
6. 6. Use of an iron death inducer in the manufacture of a medicament for the treatment of mid to late colorectal cancer.
7. 7. Use according to claim 6, characterized in that: the drug comprises a CUL9 molecular marker.
8. 8. Use according to claim 6, characterized in that: the medicament also includes an MDM2 inhibitor.

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