Application of SUCO gene in preparation of tumor drugs

Abstract

The invention provides application of an SUCO gene in preparation of tumor drugs, and belongs to the technical field of biological medicines. A brand new gene target SUCO is found, knock-down of the gene can remarkably prolong the survival time of a tumor subject with tumors, and the improvement effect does not aim at the tumors and avoids the characteristics of tumor heterogeneity, which means that the target has a wide prospect of being applicable to treatment of different tumors. The problem that in the prior art, a tumor treatment scheme is poor in universality is solved, and the method has important significance on tumor prevention and treatment.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly to SUCO the application of a gene in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is the third leading cause of death globally. According to the statistics of the WHO, the incidence of cancer is increasing at a rate of 3% per year. How to treat cancer and extend the survival period of patients has become one of the major problems that modern biomedicine urgently hopes to solve.

[0003] Currently, the mainstream treatment methods for cancer include surgical resection, radiotherapy, and chemotherapy. The method of surgical resection is mainly applicable to patients in the early and middle stages of solid tumors; radiotherapy inhibits the proliferation of tumor cells by destroying the DNA structure of tumor cells and is applicable to tumor cells sensitive to radiation, and requires the patient to be in good health; compared with the previous two treatment methods, chemotherapy drugs are applicable to a wider range of tumor types, but have greater side effects. In addition, immunotherapy and targeted therapy have become the "stars" in the field of tumor treatment in recent years due to their high specificity and low side effects. However, due to their high specificity, their applications are only limited to some populations sensitive to them, and issues such as acquired drug resistance and complications after treatment need to be considered.

[0004] In the prior art, no treatment method can be generally applicable to all tumors, which is closely related to the high heterogeneity of tumors. Due to the instability of the tumor cell genome, cells will mutate at a rate much higher than that of normal cells during tumor proliferation. Therefore, there will be differences in tumor cells within the same patient; in addition, there are also differences from genes to phenotypes among the same type of tumors in different patients. Such high heterogeneity makes existing tumor therapies often unable to take all factors into consideration, resulting in large differences in curative effects. Therefore, it is very difficult to find a treatment plan that targets the main body of tumors and is generally applicable to various types of tumors. Summary of the Invention

[0005] The purpose of the present invention is to provide SUCO the application of a gene in the preparation of anti-tumor drugs. The knockdown of this gene can significantly extend the survival time of tumor-bearing subjects. This effect does not directly affect the tumor itself, avoiding the interference of tumor heterogeneity, and has broad prospects for the treatment of different tumors.

[0006] To achieve the above-mentioned invention purpose, the present invention provides the following technical solutions: The present invention provides suco-1 or SUCO the application as a target in the development or screening or preparation of drugs for preventing and / or treating tumors.

[0007] In the present invention, suco-1It is a gene that is widely expressed in the cytoplasm and cell membrane of the model organism Caenorhabditis elegans and is an ortholog of human SUCO (SUN domain containing osteogenic factor). This gene is also known as et-503, CELE_R12E2.2, and the accession number is R12E2.2.

[0008] In the present invention SUCO The gene accession number is C404748 。

[0009] The present invention also provides suco-1 or SUCO The application as a target in the preparation of a drug screening model for preventing and / or treating tumors.

[0010] Preferably, the drug can reduce suco-1 or SUCO The molecular expression level of.

[0011] Preferably, the drug can inhibit suco-1 or SUCO The protein activity of.

[0012] Preferably, the drug includes interfering RNA, gene knockout reagents, antibodies or chemical inhibitors.

[0013] The present invention also provides a drug composition for preventing and / or treating tumors, and the drug composition can inhibit suco-1 or SUCO The expression of.

[0014] Preferably, the tumors include one or more of liver cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, prostate cancer, glioblastoma or pancreatic cancer.

[0015] The present invention also provides a drug for prolonging the survival period of a tumor-bearing subject, and the drug can block or reduce suco-1 or SUCO The expression of.

[0016] Preferably, the drug specifically binds to suco-1 or SUCO Specifically bind.

[0017] The beneficial effects of the present invention: The present invention discovers a brand-new gene target: SUCO gene, knockdown of this gene can significantly prolong the survival time of tumor-bearing subjects. Since tumors are essentially a consumptive disease, their occurrence and development impose a great burden on tumor-bearing subjects, compressing organs, consuming nutrients, and exhausting the lives of tumor-bearing subjects. Therefore, the long-term survival rate is often used clinically to measure the therapeutic effect of tumors. Without changing the size of the tumor, the therapeutic target provided by the present invention can well achieve the effect of prolonging the survival period of tumor-bearing subjects, avoiding the characteristics of tumor heterogeneity, which means that this target has broad prospects for the treatment of different tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Survival curve of nematodes after knockdown of suco-1 gene. Among them, in the figure, A: Tumor nematodes glp-1 （ar202） after knockdown of suco-1 gene, survival results; B: Results of reducing the expression of glp-1 (ar202) intestinal suco-1 gene in tumor nematodes; C: Results of reducing the expression of glp-1 (ar202) epidermal suco-1 gene in tumor nematodes; D: Results of reducing the expression of glp-1 (ar202) muscle suco-1 gene in tumor nematodes; E: Results of reducing the expression of glp-1 (ar202) gonad suco-1 gene in tumor nematodes; F: Results of reducing the expression of glp-1 (ar202) nervous system suco-1 gene in tumor nematodes; Figure 2 Survival curve of nematodes after knockdown of suco-1 gene on the gonad of nematodes. Among them, A: Gonad phenotypes of tumor nematodes glp-1 (ar202) at different time points under the culture environment of 25 °C; B: Results of germ cell counting of the gonad of tumor nematodes glp-1 (ar202) at different time points under the culture environment of 25 °C.

[0019] Figure 3 Results of gene expression differences between the control group and the suco-1 knockdown group. Among them, A: GO analysis diagram of differential genes; B: Interaction analysis diagram of differential genes mainly expressed in the intestine; C: Expression levels of differential genes involved in the intracellular membrane transport system; D: Interaction diagram of differential genes involved in the intracellular membrane transport system; Figure 4 Results of gene expression quantity differences in the intracellular membrane transport system. DETAILED DESCRIPTION OF THE INVENTION

[0020] The technical solutions provided by the present invention will be described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0021] Example 1 glp-1 (ar202) is a temperature-sensitive tumor model nematode. Its tumorigenesis mechanism is that under the environmental stimulation at 25°C, a gain-of-function mutation occurs in the NOTCH signaling pathway. After the enhancement of NOTCH signal function, it stimulates glp-1 (ar202) the proliferation of germ cells in the gonad, and they always remain in the mitotic state and cannot enter meiosis. glp-1 (ar202) The gonad of glp-1 (ar202) proliferates infinitely, ultimately leading to the death of the tumor nematode glp-1 (ar202) due to excessive proliferation of the gonad. The NOTCH pathway is highly conserved among different species. Currently, NOTCH signal mutations have been found in various types of tumors, and NOTCH signal mutations have been proven to play a key role in the occurrence and development of various human cancers (Zhou et al., 2022). Therefore, using the nematode NOTCH pathway mutant glp-1 (ar202) is for research can reveal clues for the treatment of human tumors. The specific implementation method is to use the tumor nematode

[0022] Nematode synchronization Rely on culturing at 15°C to obtain NGM plates containing a large number of tumor nematodes glp-1 (ar202) at the early adult stage (oocytes can be seen under microscopic examination). Inject M9 into the plate with a pipette, gently shake to suspend the nematodes, and then carefully pour them into a 15 mL centrifuge tube; repeat the above steps to collect the nematodes. Centrifuge at 3000g for 30 seconds to deposit the nematodes at the bottom of the tube. Connect a high-pressure sterilized glass pipette to a vacuum pump and carefully suck out the upper layer of M9. Add 4 mL of M9 and 2 mL of lysis solution (prepare nematode lysis solution: bleach, sodium hydroxide solution, M9 buffer, mixed and shaken well in a volume ratio of 3:3:4) to the centrifuge tube, cover it, and shake vigorously at least 50 times. After centrifuging at 3000g for 30 seconds, use a vacuum pump to suck out the upper layer of liquid, then add 4 mL of M9 and 2 mL of lysis solution, cover it, and shake vigorously. Stop immediately until no adult tissues or larval individuals can be seen under the microscope. Centrifuge at 3000g for 30 seconds, and a little white precipitate should be seen. Use a vacuum pump to suck out the upper layer of liquid, then add 10 mL of M9, cover it, and shake to suspend the precipitate. Repeat this step three times. Add 5 - 7 mL of M9, cover it, and shake to suspend the precipitate. At this time, a large number of eggs can be seen under microscopic examination. Place the centrifuge tube at 20°C, shake or rotate to keep the solution with dissolved oxygen. After 1 - 2 days, the larvae hatch. After shaking evenly, take three drops of 5 µL, 10 µL, and 15 µL solutions respectively and place them on NGM plates. Count the larvae in each drop under the microscope to calculate the average density of nematodes in the solution. Take the corresponding volume of liquid drops and add them to the gene interference group and the control group for the survival experiment.

[0023] The experimental methods are as follows: RNA interference of genes: Prepare a solid standard plate for nematode RNA interference (hereinafter referred to as the RNAi plate). Prepare 1 L of nematode culture medium (20 g of agar, 2.3 g of bacterial peptone, add ddH2O to 975 mL), and autoclave it. Add 1 M CaCl 2 solution, 5 mg / mL cholesterol solution, 1 mL of 1 M MgSO 4 solution, 25 mL of buffer A (KH 2 PO 4 108.3 g, K 2 HPO 4 35.6 g, NaCl 112.8 g, add ddH 2 O to 1 L) in sequence, and mix well each time. Add 200 μL of nystatin suspension, add ampicillin antibiotic to a final concentration of 50 μg / mL, add IPTG to a final concentration of 5 mM, and mix well. Dispense the medium into petri dishes and let it stand until the medium solidifies.

[0024] Use a sterile pipette tip or other object to scrape a little RNAi bacteria and streak it on the streaking plate. Incubate at 37 °C overnight. Add carbenicillin (final concentration 50 μg / mL) to the LB liquid medium, inoculate the grown bacteria with an inoculation loop, and incubate at 37 °C on a shaker for 6 - 10 hours. Pipette 100 μL of the bacterial solution onto each 3.5 cm RNAi feeding plate and let it air dry in a sterile fume hood for about 1 hour. Invert the RNAi plate with the inoculated bacteria at room temperature for at least overnight. Then store it at 4 °C for later use. Place the synchronized L1 nematodes on the feeding plate to start the gene interference process.

[0025] Survival period experiment: Prepare the feeding plates for the experiment, and the steps are the same as those for RNAi. Add 100 μL of Escherichia coli HT115 to the 3.5 cm RNAi plate for the control group, and add 100 μL of RNAi Escherichia coli to the 3.5 cm RNAi plate for the experimental group and the control group. Place the RNAi plate with the inoculated bacteria at room temperature overnight.

[0026] Add the synchronized L1-stage nematodes to the RNAi plate and place it in an incubator at 25 °C. The day when the nematodes develop to the L4 stage is recorded as the day before the start of the lifespan experiment, i.e., day 0, and then day 1, day 2, day 3... Record the number of dead nematodes every day. Use SPSS software to statistically analyze the lifespan of the nematodes. The results are as Figure 1 shown in Table A and the whole body group in Table 1.

[0027] The nematode has five major systems from the outside to the inside: the epidermis, muscle, intestine, reproductive, and nervous systems. In order to explore the knockdown suco-1The actual functional sites of the genes. According to the body structure of the nematodes, in the present invention, the suco- 1 genes in different parts were respectively knocked down to observe the survival performance of the tumor nematodes. The present invention constructed gene interference-sensitive glp-1 （ar202） epidermal tissues, glp-1 (ar202) muscle tissues, glp-1 （ar202） intestinal systems, glp-1 (ar202) gonads, glp-1 (ar202) and nervous systems. Interference was performed on these tissue-sensitive tumor nematodes respectively to observe the changes in survival period. suco-1 Interference was performed on these tissue-sensitive tumor nematodes respectively to observe the changes in survival period.

[0028] Construction of gene interference tissue-sensitive nematodes: First, male induction is required. After synchronizing the nematodes for two days, they are placed on the middle plate and cultured until the late L4 stage (this period is defined as 6 - 9 hours after the appearance of the first L4). Wash the nematodes with M9, transfer them to a centrifuge tube and wash twice with M9. Add 5 mL of 10% ethanol solution to each tube, rotate on a mixer for 30 minutes. Wash three times with M9. Centrifuge the nematodes to the bottom, aspirate the precipitate with a pipette, and place it on a sterile position on the middle plate. After 1 hour, pick the nematodes feeding on the bacterial lawn to a new middle plate. Place 6 nematodes per plate, and pick at least 3 plates. Culture on the experimental bench for three days. There should be at least a few male individuals in each plate of the offspring. Pick them out and place them on a small plate. Pick 1 hermaphrodite larva into each of 2 small plates, and let them mate at 15°C. After two days, ensure that mating is observed, and then pick out or kill all the male worms. Observe the sex ratio of the offspring. If there are about half male worms, it indicates successful mating.

[0029] Male induction was performed on the nervous system gene interference-sensitive nematode TU3401, the intestinal system gene interference-sensitive nematode JM45, the epidermal tissue gene interference-sensitive nematode JM43, and the muscle tissue gene interference-sensitive nematode JM44. After successful male induction, they were respectively mated with glp-1 (ar202) hermaphrodite nematodes, and the ratio of male worms to hermaphrodite nematodes was 2 - 4:1. Observe that the proportion of male worms in F1 is about half, indicating successful mating. Pick the hermaphrodite nematodes of the F1 generation to a new nematode solid culture plate, 1 per plate, and wait for egg laying. Pick the hermaphrodite nematodes of the F2 generation to a new nematode solid culture plate, 1 per plate, at least 16 plates, and wait for egg laying. Wait for F3 to hatch, and observe the tumor phenotype of the F3 generation nematodes (they cannot lay eggs under the 25°C environment) and the gene interference tissue-sensitive label (manifested as red fluorescence in the pharynx). If the F3 generation simultaneously has these two phenotypes, it indicates successful construction.

[0030] The gonad gene interference-sensitive nematodes were used with NL2098 andglp-1 (ar202) Obtained by mating. The F3 phenotype needs to be identified by tumor phenotype (inability to lay eggs at 25°C) and PCR.

[0031] After obtaining tissue-sensitive tumor nematodes, the lifespan experiment with gene interference was carried out. The steps were the same as above, and the changes in survival period were observed. The results are as Figure 1 B-F and Table 1 for different tissues.

[0032] The results are as Figure 1 and shown in Table 1 below: Table 1 Results of lifespan experiment data suco-1 The gene is widely expressed in the cell membrane and cytoplasm of nematodes and has a homologous gene SUCO in the human genome. The results show that when only the intestinal suco-1 gene is knocked down, glp-1 (ar202) the survival period is extended by about 30%. When only the nervous system suco-1 is knocked down, glp-1 (ar202) the survival period is extended by about 10%. However, when the expression of the suco-1 gene is inhibited alone in the epidermis, muscle, and gonad, glp-1 (ar202) the survival period of suco-1 is not significantly affected. Therefore, the intestine is the main site where the suco-1 gene exerts its function, playing 3 / 4 of the role, and the nervous system plays 1 / 4 of the role. When knocking down the glp-1 (ar202) gene of nematodes, the survival period of tumor nematodes

[0033] can be extended by 45%. suco-1 To confirm the effect of the suco-1 gene on gonad tumors, in this invention, after knocking down the

[0034] gene, the gonads of nematodes were dissected and stained to observe the changes in the gonads. Nematode gonad dissection: Pipette a drop of PBSL (PBS + 0.2 mM Levamisole) onto a glass slide, then pick the nematodes to be dissected into it and let stand for 1 minute. Cut along the posterior edge of the nematode pharynx (or where the tail width begins to contract) with a sharp scalpel. The liquid pressure will push at least one gonad out of the body. Place 3 mL of PBST (PBS + 0.1% Tween 20) in a glass test tube. Use a glass pipette to aspirate a small amount of PBST, carefully aspirate the dissected worms into it, and transfer them into the test tube. Place at least 20 nematodes with intact gonad structures in this test tube. Let stand for 5 minutes to allow the nematodes to sediment to the bottom of the tube, then carefully remove as much supernatant as possible. Add 3 mL of PBS, and then add formaldehyde solution until the final concentration of formaldehyde is 3%. After standing at room temperature for 2 hours, aspirate as much supernatant as possible. After washing with 5 mL of PBS, let stand until the nematodes sediment to the bottom of the tube, then carefully remove as much supernatant as possible. Add 5 mL of ice-cold methanol, and place the test tube in a -20°C refrigerator for at least 5 minutes. Take out, aspirate the supernatant. Wash once with PBST. Use a glass pipette to aspirate the nematodes from the bottom of the test tube, and then place them on a new glass slide (pre-prepared agarose pad). Carefully remove as much excess liquid as possible, or wait for most of the liquid to air dry. Add the DAPI reaction solution to the glass slide according to the kit instructions. Gently place a coverslip on it. Place the prepared glass slide at 4°C for 1 hour to overnight until there is no visibly obvious liquid on the glass slide. Seal the coverslip along the edge with colorless nail polish. After the nail polish solidifies, it can be viewed under a microscope. First take bright-field photos, and then take fluorescence photos.

[0035] The results are as Figure 2 shown.

[0036] The results show that suco-1 gene knockdown did not reverse the state of excessive proliferation of gonad cells. That is to say, suco-1 the gene does not act on the gonad to extend the host survival period by inhibiting the excessive proliferation of the gonad.

[0037] To explore suco-1 the mechanism by which gene inactivation extends the survival period of tumor nematodes, the present invention uses RNA sequencing technology to compare the mRNA expression levels of the control group and suco-1 the knockdown group.

[0038] The experimental method is as follows: RNA sequencing Prepare worms and calculate the number of worms. N = 2000 - 3000 worms per large plate * 2 * (C + 1) * n. N is the total number of nematodes, C is the number of accessory pores, n is the number of experimental groups, and 2 large plates are prepared for each copy. On average, each worm can receive 50 - 80 eggs. Prepare nematode culture plates. Add antibiotics to the LB solution at a ratio of LB solution: Car (carbenicillin) = 1:1000. Inoculate the strain into the LB solution and shake the bacteria at 37 °C and 220 rpm for a shaking time of ≤10 h. Concentrate the bacterial solution by 10 times, add 1 ml to a 90 mm nematode culture plate, let it dry, place it at room temperature, and use it after overnight incubation. Seed the synchronized L1-stage larvae on the culture plate. When the nematodes grow to the young adult stage, collect the nematodes into a 15 ml centrifuge tube with PBS; there are 3 replicates for each experimental group, and 1 replicate is in 1 tube. Wash with PBS 2 - 3 times, and finally transfer to a 1.5 ml EP tube. Before transfer, moisten the pipette tip with PBST. After collection into the EP tube, centrifuge again and discard as much supernatant as possible, then seal with a sealing film. Keep the tube bottom down and quickly freeze in liquid nitrogen. Then place the sample in dry ice and send it for sequencing.

[0039] The results are as Figure 3 shown, and the genes with relatively large differences in expression levels mainly converge on genes related to Rab family proteins and Golgi vesicles. Rab family proteins are members of the small G protein Ras superfamily and are involved in regulating many steps of membrane trafficking, including vesicle formation, vesicle movement along the actin and tubulin networks, and membrane fusion. Golgi vesicles are an important form for the Golgi apparatus to function, and the Golgi apparatus transports the processed proteins through vesicles. These results suggest suco-1 that the gene may play a role by affecting the cell's membrane trafficking system.

[0040] To confirm suco-1 that the gene plays a role by affecting the endomembrane trafficking system, the present invention detected the expression levels of genes asm-3 , gba-4 , cav-2 , cup-4 , sid-2 and vps-45 in the suco-1 gene knockdown group and the control group.

[0041] The experimental method is as follows: Extract total nematode RNA: Culture about 1000 nematodes as required (three plates for each strain), collect them into a 15 ml centrifuge tube with PBS. Centrifuge at 3000 g for 30 seconds and discard as much supernatant as possible, then transfer to a 1.5 mL Ep tube for grinding. Wash with 500 uL ddH2O 2 - 3 times. Freeze and thaw repeatedly in liquid nitrogen 3 times. If it does not melt during the 3rd time, place it on ice and grind for 10 - 15 seconds with an electric grinder. Subsequently, use the Aowei Biology RNT412 kit to extract total nematode RNA.

[0042] qPCR: Total RNA of nematodes was reverse-transcribed using Yeasen's 11141ES60 kit. qPCR detection was performed using Yeasen's 11184ES08 kit. The system and procedures were all in accordance with the reagent instruction manual.

[0043] The primers used in the experiment are shown in Table 2 below: Table 2 Primer sequences The Ct values obtained by qPCR were finally calculated using ΔΔCt. The formula is: fold change expression = 2 -（ΔΔCt） ΔΔCt = ΔCt (sample) - ΔCt (control average) The results are as Figure 4 shown, cav-2 , cup-4 , sid-2 and vps-45 showed upregulated expression levels in the experimental group, asm-3 , gba-4 showed downregulated expression levels in the experimental group, and the differences in expression levels were all statistically significant. This implies that suco-1 gene inactivation prolongs the survival of the tumor model by affecting the intracellular membrane trafficking system glp-1 (ar202) .

[0044] The membrane trafficking system is an important pathway for intracellular material transport and communication, participates in various physiological processes of organisms, and is a highly conserved program. Among these genes involved in the intracellular membrane trafficking system of nematodes, asm-3 , gba-4, cav-2 , cup-4 and vps-45 all have corresponding human homologous genes, indicating that the pathway that ultimately leads to the prolonged survival after knocking down glp-1 (ar202) genes in the nematode tumor model suco-1 is conserved.

[0045] Currently, regardless of the treatment method adopted, the treatment goal of clinical tumors is to increase the long-term survival rate. suco-1 The gene target can significantly prolong the survival of the tumor-bearing host. And suco-1 the gene mechanism of action does not directly act on tumor cells, avoiding the unstable efficacy caused by tumor heterogeneity. Therefore, suco-1 the gene target has the advantage of universality in the application of tumor treatment and has very high clinical translation value. In the process of developing a protocol to extend the survival of human tumor-bearing patients, it can be achieved by knocking down the genes in the cells of tumor patients that are related to suco-1The human homologous gene SUCO corresponding to the gene is used to determine the corresponding treatment plan.

[0046] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. suco-1 or SUCO as a target in the development, screening or preparation of drugs for preventing and / or treating tumors.

2. suco-1 or SUCO as a target in the application of preparing a drug screening model for preventing and / or treating tumors.

3. The application according to claim 1 or 2, characterized in that, The drug can reduce suco-1 or SUCO the molecular expression level.

4. The application according to claim 1 or 2, characterized in that, The drug can inhibit suco-1 or SUCO protein activity.

5. The application according to claim 1 or 2, characterized in that, the drug comprises interfering RNA, gene knockout reagent, antibody or chemical inhibitor.

6. A pharmaceutical composition for preventing and / or treating tumors, characterized in that, The pharmaceutical composition can inhibit suco-1 or SUCO expression.

7. The pharmaceutical composition according to claim 6, characterized in that, the tumor comprises one or more of liver cancer, lung cancer, breast cancer, colorectal cancer, ovarian cancer, prostate cancer, glioblastoma or pancreatic cancer.

8. A drug for prolonging the survival period of a tumor-bearing subject, characterized in that, The drug can block or reduce suco- 1 or SUCO expression.

9. The drug according to claim 8, characterized in that, The drug binds specifically to suco-1 or SUCO specifically binds.

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