Use of a DHCR7 down-regulator in attenuating alcohol-induced liver tumors

Abstract

The present invention provides an application of a DHCR7 down-regulator in attenuating alcohol-induced liver tumors. The present invention reveals that DHCR7 plays a role in regulating liver tumors (alcohol-induced liver tumors), analyzes the important mechanism of action of the cholesterol metabolism pathway regulated by DHCR7 in the development of liver tumors, and provides a drug target and theoretical basis for the development of a new generation of anti-cancer drugs. Taking DHCR7 as a target, drugs for inhibiting (including alleviating or treating) liver tumors can be developed, as well as for diagnosing and prognosticating liver tumors or symptoms.

Description

Technical Field

[0001] The present invention belongs to the fields of biotechnology and pharmacology, and more specifically, the present invention relates to the use of DHCR7 downregulators in attenuating alcohol-induced liver tumors. Background Art

[0002] Liver diseases are the leading cause of death, and a large number of patients die from liver diseases every year worldwide. Excessive alcohol consumption, obesity, and hepatitis (HBV / HCV infection) are the three main causes of liver diseases, which can lead to alcohol-associated liver disease (ALD), non-alcoholic fatty liver disease (NAFLD), and viral hepatitis. The induction mechanisms of liver tumors caused by these three main etiologies are significantly different.

[0003] Although HBV / HCV can induce liver tumors. However, with the introduction of new therapies, the incidence of HBV / HCV-related liver tumors has decreased.

[0004] Liver tumors caused by non-alcoholic steatohepatitis (NASH) and alcohol-associated liver disease (ALD) are rising rapidly, and such liver tumors have completely different induction mechanisms from viral liver tumors. ALD remains the main causative factor for cirrhosis and invasive HCC. Generally, alcohol is metabolized in hepatocytes, leading to the production of toxic metabolites such as acetaldehyde and acetate, the synthesis of fatty acids and cholesterol, and the inhibition of DNA repair, thereby driving somatic mutations that trigger HCC. Closely associated with the global prevalence of NASH, alcohol-induced liver tumors usually occur in patients with a BMI > 25. Long-term excessive alcohol consumption increases the risk of HCC development in NASH patients. Therefore, studying the pathogenesis of alcohol-related liver disease progressing to liver tumors is of great significance for the prevention and treatment of liver tumors, the targeted development of new therapeutic drugs, and the improvement of the health level of the whole population.

[0005] The pathogenesis of liver disease progressing to liver cancer is very complex, including mechanisms such as immune and inflammatory responses, DNA damage, oxidative stress, and autophagy. Increasing evidence indicates that inflammation plays a key role in controlling the progression of liver disease to liver cancer. Chronic inflammation induces immune cells to produce cytokines, transcription factors, activation of nuclear factor-κB (NF-κB), signal transducer and activators of transcription 3 (STAT3), etc., which promotes the survival, proliferation, growth, angiogenesis, and invasion of precancerous cells, and maintains the tumor-associated inflammatory state by inducing the production and attracting new chemokines, ultimately leading to tumor progression and spread. Cytokines, including the IL-1, IL-6, and IL-20 families, can regulate liver inflammation, fibrosis, injury, and repair. Some of these cytokines have been used as targets for treating different types of liver diseases in clinical trials. Hepatocytes can not only produce 80-90% of the circulating proteins related to innate immunity, but also produce some cytokines (such as IL-7, IL-11, and IL-33). These cytokines play an important regulatory role in liver function. Recent studies have shown that Th17 cells in helper T cells respond to toxic or metabolic damage (such as HBV / HCV infection) in the progression of various chronic liver diseases such as NASH, ALD, cholestatic liver disease, and liver cancer. Th17 is a helper T cell differentiated from TH0 cells under the stimulation of IL6 and IL23, mainly secreting pro-inflammatory factors such as cytokines of the IL17 family (IL-14, 17A, IL-17F, IL-17B, IL-17C, and IL-17E), IL22, etc. RORγ is its important transcription factor. The IL-17A homodimer (also known as IL-17) is a cytokine produced in large amounts in Th17 cells and has relatively high biological activity. Its receptors are IL-17RA and IL-17RC.

[0006] The IL-17 signaling pathway plays an important regulatory role in NASH or alcohol-induced liver injury. During injury, neutrophils are rapidly recruited to the liver, promoting hepatocyte injury and macrophage activation. In this process, Th17 cells release IL-17A, inducing neutrophil recruitment to the damaged liver. A novel TNF-TNFRI-dependent caspase-2-S1P-SREBP-DHCR7-mediated cholesterol synthesis pathway that responds to the IL-17 signaling pathway has been discovered in ALD and NASH diseases. Specifically knocking out IL17RA in steatotic hepatocytes (but not normal hepatocytes) significantly reduces the expression of TNFRI, SREBP1 / 2, and DHCR7 proteins, leading to defects in the cholesterol / fatty acid biosynthesis pathway, and ultimately making mice with hepatocyte-specific knockout of IL-17RA resistant to the progression of liver tumors.

[0007] The synthesis of lipids in the liver is mainly regulated by the SREBP1 / 2 pathway of transcription factors in hepatocytes. The activity of SREBP1 / 2 can be regulated through multiple pathways, including the INSIG:SREBP cleavage-activating protein (SCAP) pathway and the non-apoptotic caspase-2-dependent pathway (mediating the SCAP-independent cleavage pathway). Generally, SREBP1 / 2 is transported to the nucleus to activate the transcription of downstream target genes. IL-17RA-deficient hepatocytes exhibit defects in decholesterol / cholesterol and cholesterol synthesis. Summary of the Invention

[0008] The object of the present invention is to provide the application of a DHCR7 downregulator in weakening alcohol-induced liver tumors.

[0009] In the first aspect of the present invention, the application of 7-dehydrocholesterol reductase (DHCR7) is provided, for: being used as a target for inhibiting liver tumors, preparing a composition for inhibiting liver tumors; being used as a target for screening drugs for inhibiting liver tumors; or being used as a marker for diagnosing or prognosticating liver tumors, preparing a diagnostic reagent for diagnosing or prognosticating liver tumors; preferably, the liver tumor is an alcohol-induced liver tumor.

[0010] In another aspect of the present invention, the application of a downregulator of 7-dehydrocholesterol reductase is provided, for preparing a composition for inhibiting liver tumors; preferably, the liver tumor is an alcohol-induced liver tumor.

[0011] In one or more embodiments, the inhibition of liver tumors includes: preventing, alleviating, or treating liver tumors.

[0012] In one or more embodiments, the liver tumor is liver cancer.

[0013] In one or more embodiments, the DHCR7 regulates the liver tumor-related pathways by modulating the cholesterol metabolism pathway.

[0014] In one or more embodiments, the DHCR7 exerts its function by regulating the downstream effector molecule TNF.

[0015] In one or more embodiments, the downregulators of 7-dehydrocholesterol reductase include (but are not limited to): substances that downregulate the activity of 7-dehydrocholesterol reductase or substances that downregulate the expression, stability of 7-dehydrocholesterol reductase or reduce its effective action time.

[0016] In one or more embodiments, the downregulators include (but are not limited to) those selected from: small molecule chemical antagonists or inhibitors against 7-dehydrocholesterol reductase; reagents for knocking out or silencing 7-dehydrocholesterol reductase; binding molecules (such as antibodies or ligands) that specifically bind to 7-dehydrocholesterol reductase; or reagents that interfere with the interaction between 7-dehydrocholesterol reductase and effector molecules.

[0017] In one or more embodiments, the small molecule chemical antagonists or inhibitors against 7-dehydrocholesterol reductase include AY9944.

[0018] In one or more embodiments, the reagents for knocking out or silencing 7-dehydrocholesterol reductase include (but are not limited to): CRISPR gene editing reagents against 7-dehydrocholesterol reductase, interfering molecules that specifically interfere with the expression of the coding gene of 7-dehydrocholesterol reductase, homologous recombination reagents or site-directed mutagenesis reagents against 7-dehydrocholesterol reductase, and the homologous recombination reagents or site-directed mutagenesis reagents introduce loss-of-function mutations to 7-dehydrocholesterol reductase.

[0019] In one or more embodiments, the interfering molecules include siRNA, shRNA, miRNA, antisense nucleic acids, etc., or constructs that can form the siRNA, shRNA, miRNA, antisense nucleic acids, etc.

[0020] In one or more embodiments, the expression constructs (expression vectors) for introducing downregulators such as sgRNA or interfering molecules into cells include: viral vectors, non-viral vectors; preferably, the expression vectors include: adeno-associated virus vectors, lentiviral vectors, adenoviral vectors.

[0021] In another aspect of the present invention, there is provided the use of reagents that specifically recognize or amplify 7-dehydrocholesterol reductase for preparing diagnostic reagents or kits for diagnosing or prognosticating liver tumors; preferably, the liver tumors are alcohol-induced liver tumors.

[0022] In one or more embodiments, the reagents include (but are not limited to): binding molecules (such as antibodies or ligands) that specifically bind to 7-dehydrocholesterol reductase protein; primers that specifically amplify the 7-dehydrocholesterol reductase gene; probes that specifically recognize the 7-dehydrocholesterol reductase gene; or, chips that specifically recognize the 7-dehydrocholesterol reductase gene.

[0023] In another aspect of the present invention, there is provided a pharmaceutical composition or kit for inhibiting liver tumors, comprising: a downregulator of 7-dehydrocholesterol reductase; preferably, the liver tumor is an alcohol-induced liver tumor.

[0024] In one or more embodiments, the liver tumor is a liver tumor cell line, including Hep3B or Huh7.

[0025] In another aspect of the present invention, there is provided a method for screening potential substances for inhibiting liver tumors, the method comprising:

[0026] (1) Treating an expression system expressing 7-dehydrocholesterol reductase with a candidate substance; and,

[0027] (2) Detecting the expression or activity of 7-dehydrocholesterol reductase in the system; if the candidate substance downregulates (significantly downregulates, such as downregulating by more than 10%, 20%, 50%, 80%, etc., or causing it not to be expressed or having no activity) the expression or activity of 7-dehydrocholesterol reductase statistically, then the candidate substance is a potential substance for reducing liver tumors;

[0028] Preferably, the liver tumor is an alcohol-induced liver tumor.

[0029] In one or more embodiments, in step (1), the system is a liver tumor cell (culture) system.

[0030] In one or more embodiments, step (2) further includes: detecting the expression or activity of the downstream molecule TNF regulated by DHCR7 in the system; if the expression or activity of the downstream molecule TNF decreases (significantly decreases, such as decreasing by more than 10%, 20%, 50%, 80%, etc.), then the candidate substance is a potential substance for reducing liver tumors.

[0031] In one or more embodiments, the candidate substances include (but are not limited to): regulatory molecules or their constructs (such as shRNA, siRNA, gene editing agents, expression vectors, recombinant viral or non-viral constructs, etc.) designed against 7-dehydrocholesterol reductase, its fragments or variants, its coding gene or its upstream and downstream molecules or signaling pathways, chemical small molecules (such as specific inhibitors or antagonists), interaction molecules, etc.

[0032] In one or more embodiments, the system is selected from: a cellular system (such as a cell or cell culture expressing 7-dehydrocholesterol reductase), a subcellular (culture) system, a solution system, a tissue system, an organ system, or an animal system.

[0033] In one or more embodiments, the method further includes: performing further cell experiments and / or animal tests on the obtained potential substances to further select and determine substances useful for inhibiting liver tumors from the candidate substances.

[0034] Other aspects of the present invention will be apparent to those skilled in the art from the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Degree of tumor development in DHCR7 heterozygous mutant mice after DEN induction and alcohol plus high-fat diet feeding. A. Representative diagram of liver tumors in DHCR7 heterozygous knockout mice. B. Statistics of tumor formation size and tumor burden in DHCR7 heterozygous knockout mice after different grouped feedings (normal diet vs. high-fat diet vs. alcohol + high-fat diet); compared with other groups, only the tumors in the alcohol and high-fat diet feeding group were significantly smaller and the tumor burden was significantly reduced.

[0036] Figure 2 After feeding with high-fat or combined alcohol diet, the number of lipid accumulation regions in the livers of DHCR7 heterozygous mutant mice was significantly lower than that of wild-type mice. The left picture is a representative diagram of the mouse liver after H&E staining, and the circled area is the tumor area. The right picture is a statistical chart of the number of lipid accumulation regions. Among them, *, P < 0.05; ****, P < 0.0001.

[0037] Figure 3 During the tumor formation induced by alcohol and high-fat diet, inflammation and fibrosis in DHCR7 heterozygous mutant mice were significantly reduced. Immunohistochemical staining and statistics of inflammation and fibrosis marker proteins. Among them, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

[0038] Figure 4 Degree of tumor development in wild-type mice after DEN induction and alcohol plus high-fat diet feeding, combined with treatment with a DHCR7 inhibitor (AY9944). A. Flow chart of mouse induction and feeding; B. Statistics of tumor formation size and tumor burden in wild-type mice after feeding with a DHCR7 inhibitor. After treatment with a DHCR7 inhibitor in wild-type mice fed with alcohol and high-fat diet, the tumors were significantly smaller and the tumor burden was significantly reduced.

[0039] Figure 5 1. The DHCR7 inhibitor AY9944 can inhibit the proliferation of human liver tumor cell lines. The liver tumor cell lines Hep3B and Huh7 were treated with different concentrations of the inhibitor AY9944, and the cell viability decreased as the drug concentration increased.

[0040] Figure 6 2. During the process of alcohol combined with high-fat diet-induced tumor formation, after treatment with the DHCR7 inhibitor (AY9944), the lipid accumulation area in mice decreased significantly, and inflammation and fibrosis were significantly reduced. A. H&E staining confirmed that the inhibitor decreased the lipid accumulation area in the liver, and indicators such as inflammation and fibrosis also decreased. The immunohistochemical staining statistical chart is as shown in A. B. Gene expression analysis showed that the inhibitor could decrease the expression of fibrosis-related marker genes aSMAm and Timp1 in the liver. And the expression of the TNF gene decreased after the activity of DHCR7 was inhibited. Detailed implementation manners

[0041] Through in-depth research, the present inventors have revealed that DHCR7 plays a role in regulating liver tumors (alcohol-induced liver tumors), analyzed the important mechanism of action of the cholesterol metabolism pathway regulated by DHCR7 in the development of liver tumors, and provided a drug target and theoretical basis for the development of a new generation of anti-cancer drugs. Taking DHCR7 as a target, drugs for inhibiting (including alleviating or treating) liver tumors can be developed, as well as for diagnosing and prognosticating liver tumors or symptoms.

[0042] DHCR7

[0043] DHCR7 is an enzyme involved in the last step of cholesterol biosynthesis, which converts 7-dehydrocholesterol (7DHC) into cholesterol. Moreover, the present inventors unexpectedly found that DHCR7 plays a role in regulating liver tumors (alcohol-induced liver tumors), which is unexpected.

[0044] The amino acid sequence of human DHCR7 can be as shown in GenBank accession number 1717; the amino acid sequence of murine DHCR7 can be as shown in GenBank accession number 13360. The present invention may also include DHCR7 homologs from other species and their applications.

[0045] The DHCR7 described in the present invention can be naturally occurring, for example, it can be isolated or purified from mammals. In addition, the DHCR7 can also be artificially prepared, for example, recombinant DHCR7 can be produced according to conventional genetic engineering recombination techniques for use in experiments or clinical applications. When applied, recombinant DHCR7 can be used. The DHCR7 includes full-length DHCR7 or its bioactive fragments. Based on the amino acid sequence of DHCR7, its corresponding nucleotide coding sequence can be easily obtained.

[0046] The amino acid sequence of DHCR7 formed by substitution, deletion, or addition of one or more amino acid residues is also included in the present invention. DHCR7 or its bioactive fragments include alternative sequences of some conserved amino acids, and the amino acid-substituted sequences do not affect their activity or retain part of their activity. Appropriate amino acid substitution is a well-known technique in the art, and this technique can be easily implemented and ensures that the biological activity of the resulting molecule is not changed. These techniques enable those skilled in the art to recognize that generally, changing a single amino acid in a non-essential region of a polypeptide will basically not change its biological activity.

[0047] Any bioactive fragment of DHCR7 can be applied to the present invention. Here, the meaning of the bioactive fragment of DHCR7 refers to a polypeptide that can still maintain all or part of the functions of full-length DHCR7. Generally, the bioactive fragment can maintain at least 50% of the activity of full-length DHCR7. Under more preferred conditions, the active fragment can maintain 60%, 70%, 80%, 90%, 95%, 99%, or 100% of the activity of full-length DHCR7.

[0048] In the research of the present invention, diethylnitrosamine (DEN) was used to induce liver tumors in animals, and combined with alcohol and high-fat feeding to accelerate the development of liver tumors, thereby establishing an in vivo liver tumor model. It was found that knocking out DHCR7 in animals or inhibiting the activity of DHCR7 can weaken the process of liver tumors in animals. Treatment of human liver tumor cell lines with an inhibitor of DHCR7 was found to significantly inhibit the proliferation of liver tumor cells, and the survival rate was inversely proportional to the dose of the drug. This research shows that DHCR7 plays an important role in regulating the development of alcohol-induced liver tumors and can be used as a candidate drug target, providing a basis for the development of new anti-tumor drugs.

[0049] DHCR7 Down-Regulators and Their Applications

[0050] Based on the above new findings of the present inventors, the present invention provides the use of a down-regulator of DHCR7 or its coding gene for preparing a composition for alleviating or inhibiting liver tumors.

[0051] As used herein, the "down-regulator" includes inhibitors, antagonists, blockers, and blockers, etc., and these terms can be used interchangeably.

[0052] As used herein, the terms "inhibit" or "down-regulate" or "weaken" or "reduce", etc., refer to a statistically significant "inhibition" or "down-regulation" or "weakening" or "reduction". Compared with the control group, there is a significant "inhibition" or "down-regulation" or "weakening" or "reduction"; more specifically, for example, an "inhibition" or "down-regulation" or "weakening" or "reduction" of more than 20%, preferably more than 50%, more preferably more than 80%.

[0053] The down-regulator of DHCR7 or its encoding gene refers to any substance that can reduce the activity of DHCR7, reduce the stability of DHCR7 or its encoding gene, down-regulate the expression of DHCR7, reduce the effective action time of DHCR7, or inhibit the transcription and translation of the DHCR7 gene. These substances can all be used in the present invention as substances useful for down-regulating DHCR7, and thus can be used to inhibit liver tumors. For example, the down-regulator is: an interfering RNA molecule or antisense nucleotide that specifically interferes with the expression of the DHCR7 gene; an antibody or ligand that specifically binds to the protein encoded by the DHCR7 gene; and so on.

[0054] As a preferred embodiment of the present invention, the down-regulator is a small molecule compound targeting DHCR7. Those skilled in the art can adopt methods suitable for screening small molecule compounds to screen such small molecule compounds. The screening can rely on various compound libraries existing or to be developed in the art, or establish some new compound libraries by themselves.

[0055] As a preferred embodiment of the present invention, the small molecule compound is AY9944 (Cas: 366-93-8), and its structural formula is:

[0056]

[0057] In the present invention, the small molecule compound of DHCR7 (such as AY9944) can be a compound in a pure form, or a compound with a purity greater than 85% (preferably greater than 90%, for example greater than 95%, 98%, 99%). When the chemical structure is known, the small molecule compound can be obtained by chemical synthesis. The present invention also includes precursors of the compound, and the "precursor" refers to a compound that, when taken by an appropriate method, is metabolized or undergoes a chemical reaction in the patient's body to be converted into the active compound.

[0058] The present invention also includes isomers, solvates of small molecule compounds of DHCR7, or pharmaceutically acceptable salts in other forms thereof (such as forms other than hydrochloride form), as long as they also have the same or substantially the same function as AY9944. The "pharmaceutically acceptable salts" refer to salts formed by the reaction of the compound with inorganic acids, organic acids, alkali metals or alkaline earth metals, etc. These salts include (but are not limited to): (1) salts formed with the following inorganic acids: such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) salts formed with the following organic acids, such as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid, or arginine. Other salts include salts formed with alkali metals or alkaline earth metals (such as sodium, potassium, calcium or magnesium), in the form of esters, carbamates, or other conventional "prodrugs". The compound has one or more asymmetric centers. Therefore, these compounds can exist as racemic mixtures, individual enantiomers, individual diastereoisomers, diastereoisomer mixtures, cis or trans isomers.

[0059] Based on the structure of this lead molecule inhibitor, derivatives can be modified to achieve a better effect of inhibiting liver tumors.

[0060] As a preferred embodiment of the present invention, the downregulator can be a DHCR7-specific interfering RNA molecule (such as siRNA, shRNA, miRNA, etc.). Those skilled in the art can understand that according to the DHCR7 sequence information provided in the present invention, such interfering RNA molecules can be prepared. There is no particular limitation on the preparation method of the interfering RNA molecule, including but not limited to: chemical synthesis method, in vitro transcription method, etc. The interfering RNA can be delivered into cells by using an appropriate transfection reagent, or can also be delivered into cells by using a variety of techniques known in the art.

[0061] In some embodiments, RNAi is used to inhibit DHCR7. RNAi is an evolutionarily conserved cellular defense mechanism for controlling the expression of foreign genes in most eukaryotes including humans. RNAi is usually triggered by double-stranded RNA (dsRNA) and causes sequence-specific mRNA degradation of single-stranded target RNA. The mediator of mRNA degradation is small interfering RNA duplexes (siRNAs), which are usually generated by the intracellular cleavage of long dsRNA. The length of siRNAs is usually about 21 nucleotides (for example, 21-23 nucleotides). After introducing small RNA or RNAi into cells, it is believed that the sequence is delivered to an enzyme complex called RISC (RNA-induced silencing complex). RISC recognizes the target and cleaves it with an endonuclease. It is worth noting that if a larger RNA sequence is delivered into cells, the RNase III enzyme (Dicer) will convert the longer dsRNA into 21-23nt ds-siRNA fragments.

[0062] In a preferred embodiment, RNA interference is carried out using shRNA technology. shRNA is an RNA sequence that can form a tight hairpin and can be used to silence gene expression through RNA interference. shRNA uses a vector introduced into cells and utilizes a promoter (such as U6) to ensure that shRNA is always expressed. This vector is usually passed on to daughter cells, enabling genetic inheritance of gene silencing. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which then binds to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it binds. shRNA is transcribed by RNA polymerase III.

[0063] As an alternative embodiment, an antisense compound that specifically hybridizes to one or more nucleic acids encoding DHCR7 is used to regulate DHCR7 expression. Specific hybridization of the oligomer to its target nucleic acid interferes with the normal function of the nucleic acid. This regulation of the function of the target nucleic acid by a compound that specifically hybridizes to the target nucleic acid is generally referred to as "antisense".

[0064] As a preferred method of the present invention, the CRISPR / Cas (such as Cas9) system can be used for targeted gene editing to knockout the DHCR7 gene in the targeted disease region. Common methods for knocking out the DHCR7 gene include: co-transfecting sgRNA or nucleic acids capable of forming the sgRNA, Cas9 mRNA or nucleic acids capable of forming the Cas9 mRNA into the targeted region or targeted cells. After determining the target site, known methods can be used to introduce sgRNA and Cas9 into the cells. The nucleic acids capable of forming the sgRNA are nucleic acid constructs or expression vectors, or the nucleic acids capable of forming the Cas9 mRNA are nucleic acid constructs or expression vectors. These expression vectors are introduced into the cells to form active sgRNA and Cas9 mRNA in the cells.

[0065] As a preferred method of the present invention, homologous recombination can be used to specifically target DHCR7, resulting in defective expression or lack of expression. The Cre and loxp methods can also be applied to selectively knockout, reduce expression or inactivate related genes in the genomes of animals or cells.

[0066] The above are some preferred ways to downregulate DHCR7. It should be understood that based on this target, after those skilled in the art understand the general scheme of the present invention, other known methods or developing methods in the art can also be used to regulate DHCR7, and these methods are also included in the present invention.

[0067] Applications related to diagnosis and prognostic evaluation

[0068] In the present invention, a target that plays an important regulatory role in the occurrence and development of liver tumors (alcohol-induced liver tumors) is disclosed. Based on this new discovery of the inventors, DHCR7 can be used as a target (or biomarker) for inhibiting liver tumors or screening drugs for inhibiting liver tumors: (i) for tumor typing, differential diagnosis, and / or susceptibility analysis; (ii) for evaluating therapeutic drugs, drug efficacy, prognosis, and selecting appropriate treatment methods for relevant liver tumor populations. For example, populations with abnormal gene expression of DHCR7 can be isolated, enabling more targeted treatment.

[0069] The prognosis of liver tumors in a subject providing the sample to be evaluated can be predicted by determining the expression or activity of DHCR7 in the sample to be evaluated, and appropriate drugs can be selected for treatment. Generally, a threshold for DHCR7 can be specified. When the expression of DHCR7 is higher than the specified threshold, a treatment regimen for inhibiting DHCR7 can be considered. The threshold is easily determined by those skilled in the art. For example, the threshold for abnormal DHCR7 expression can be obtained by comparing the expression of DHCR7 in normal human cells or tissues with that in patient cells or tissues. Depending on different measurement parameters, measurement instruments, etc., the specific value of the threshold can be different.

[0070] Various techniques known in the art can be used to detect the presence and expression of the DHCR7 gene, and these techniques are all included in the present invention. For example, existing techniques such as Southern blotting, Western blotting, DNA sequence analysis, PCR, etc. can be used, and these methods can be used in combination.

[0071] The present invention also provides reagents for detecting the presence and expression of DHCR7 or its coding gene in an analyte. Preferably, when detecting at the gene level, primers for specifically amplifying DHCR7 or a probe for specifically recognizing DHCR7 can be used to determine the presence of the DHCR7 gene; when detecting at the protein level, an antibody or ligand that specifically binds to the protein encoded by DHCR7 can be used to determine the expression of DHCR7.

[0072] The design of specific probes for the DHCR7 gene is a technique well-known to those skilled in the art. For example, a probe can be prepared that can specifically bind to a specific site on the DHCR7 gene and does not specifically bind to other genes outside the DHCR7 gene, and the probe is labeled with a detectable signal.

[0073] The method of using an antibody that specifically binds to DHCR7 to detect the expression of DHCR7 in an analyte is also a technique well-known to those skilled in the art.

[0074] The present invention also provides a kit for detecting the presence or absence and expression of the DHCR7 gene in an analyte, which kit comprises: primers for specifically amplifying the DHCR7 gene; probes for specifically recognizing the DHCR7 gene; or antibodies or ligands that specifically bind to the protein encoded by the DHCR7 gene; or a chip for specifically assaying the DHCR7 gene.

[0075] In addition, the kit may further include various reagents required for DNA extraction, PCR, hybridization, color development, etc., including but not limited to: extraction solutions, amplification solutions, hybridization solutions, enzymes, control solutions, color development solutions, washing solutions, etc.

[0076] In addition, the kit may further include an instruction manual and / or nucleic acid sequence analysis software, etc.

[0077] Drug screening

[0078] After learning the close correlation between DHCR7 and liver tumors, substances that inhibit the expression or activity of DHCR7 or its encoding gene can be screened based on this feature. Drugs that are truly useful for inhibiting liver tumors can be found from the said substances.

[0079] Therefore, the present invention provides a method for screening potential substances (candidate substances or candidate drugs) for inhibiting liver tumors, which method comprises: treating a system expressing DHCR7 with a candidate substance; and detecting the expression or activity of DHCR7 in the said system; if the candidate substance can inhibit the expression or activity of DHCR7, it indicates that the candidate substance is a potential substance for inhibiting liver tumors. The system expressing DHCR7 is preferably a cell (or cell culture) system, and the cells can be cells that endogenously express DHCR7; or can be cells that recombinantly express DHCR7. In addition, the usefulness of the potential substance can also be evaluated by observing the interaction between DHCR7 and its upstream and downstream proteins.

[0080] In a preferred embodiment of the present invention, when performing the screening, in order to more easily observe the change in the expression or activity of DHCR7, a control group (Control) can also be set up, and the control group can be a system expressing DHCR7 without adding the candidate substance. The control group includes but is not limited to: a blank control without adding the candidate substance, a control with an empty plasmid, etc.

[0081] As a preferred embodiment of the present invention, the method further comprises: performing further cell experiments and / or animal tests on the obtained potential substances to further select and determine substances that are truly useful for inhibiting liver tumors.

[0082] On the other hand, the present invention also provides potential substances for inhibiting liver tumors obtained by the screening method described above. These preliminarily screened substances can form a screening library, so that people can ultimately screen out substances useful for inhibiting the expression and activity of DHCR7 and thus inhibiting liver tumors from it.

[0083] Drug composition

[0084] The present invention also provides a drug composition, which contains a down-regulator of the aforementioned DHCR7 or its coding gene in an effective amount (such as 0.000001 - 50 wt%; preferably 0.00001 - 20 wt%; more preferably 0.0001 - 10 wt%), and a pharmaceutically acceptable carrier.

[0085] As a preferred embodiment of the present invention, there is provided a composition for inhibiting liver tumors, which composition contains a down-regulator of DHCR7 or its coding gene in an effective amount, and a pharmaceutically acceptable carrier.

[0086] In the preferred embodiment of the present invention, the down-regulator includes but is not limited to: small chemical molecule antagonists or inhibitors against DHCR7, reagents for knocking out or silencing DHCR7, binding molecules specifically binding to DHCR7 (such as antibodies or ligands), and the like. In a more specific embodiment, the down-regulator includes but is not limited to: small chemical molecule inhibitors, CRISPR gene editing reagents against DHCR7, interfering molecules specifically interfering with the expression of the coding gene of DHCR7, homologous recombination reagents or site-directed mutagenesis reagents against DHCR7, and the homologous recombination reagents or site-directed mutagenesis reagents cause loss-of-function mutations in DHCR7.

[0087] As used herein, the "effective amount" refers to an amount that can produce a function or activity in humans and / or animals and is acceptable to humans and / or animals. The "pharmaceutically acceptable carrier" refers to a carrier for administering a therapeutic agent, including various excipients and diluents. This term refers to such pharmaceutical carriers: they are not necessarily active ingredients themselves and have no excessive toxicity after administration. Suitable carriers are well-known to those of ordinary skill in the art. Pharmaceutically acceptable carriers in the composition may contain liquids, such as water, saline, buffer solutions. Additionally, there may be auxiliary substances in these carriers, such as fillers, lubricants, glidants, wetting agents or emulsifiers, pH buffering substances, etc. The carrier may also contain cell transfection reagents.

[0088] After learning about the use of the down-regulator of DHCR7 or its coding gene, various methods well-known in the art can be used to administer the down-regulator or its coding gene, or its drug composition to mammals or humans.

[0089] Preferably, gene therapy means can be adopted. For example, a down-regulator of DHCR7 can be directly administered to a subject by means such as injection; or, an expression unit (such as an expression vector or a virus, etc., or siRNA) carrying the down-regulator of DHCR7 can be delivered to the target site through a certain route and make the active DHCR7 down-regulator expressed. The specific situation depends on the type of the down-regulator, and these are all well-known to those skilled in the art.

[0090] The effective amount of the down-regulator of DHCR7 or its coding gene according to the present invention may vary with the mode of administration and the severity of the disease to be treated, etc. The selection of the preferred effective amount can be determined by those of ordinary skill in the art according to various factors (such as through clinical trials). The said factors include but are not limited to: the pharmacokinetic parameters of the down-regulator of DHCR7 or its coding gene, such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, etc.

[0091] In the specific embodiments of the present invention, some dosing regimens for animals such as mice are given. It is easy for those skilled in the art to convert the dosing dose for animals such as mice into the dosing dose applicable to humans. For example, it can be calculated according to the Meeh-Rubner formula: Meeh-Rubner formula: A = k × (W 2 / 3 ) / 10,000. In the formula, A is the body surface area, calculated in m 2 ; W is the body weight, calculated in g; K is a constant, which varies with the animal species. Generally speaking, for mice and rats it is 9.1, for guinea pigs it is 9.8, for rabbits it is 10.1, for cats it is 9.9, for dogs it is 11.2, for monkeys it is 11.8, and for humans it is 10.6. It should be understood that according to the differences in the drug and the clinical situation, the conversion of the dosing dose can vary according to the evaluation of an experienced pharmacist.

[0092] The present invention also provides a medicine box containing the said pharmaceutical composition or directly containing the down-regulator of DHCR7 or its coding gene. In addition, the medicine box may further include an instruction manual for explaining the usage method of the drugs in the medicine box.

[0093] Therefore, liver tumors are malignant tumors with relatively high incidence and fatality rates, and lipids and cholesterol provide energy for cancer cells. Studying the impact of its regulatory pathways on the liver tumor process is of great significance. The present invention studies the molecular mechanism of DHCR7 regulating liver tumors, proposes a new molecular target, and analyzes the important mechanism of the cholesterol metabolism pathway regulated by DHCR7 in the development of liver tumors, providing a drug target and theoretical basis for the development of a new generation of anti-cancer drugs.

[0094] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and not to limit the scope of the present invention. For the experimental methods without specific conditions noted in the following embodiments, they are generally carried out under conventional conditions such as those described in "Molecular Cloning: A Laboratory Manual", Third Edition, edited by J. Sambrook et al., published by Science Press, or according to the conditions recommended by the manufacturer.

[0095] Materials and Methods

[0096] 1. Materials and Reagents

[0097] Cell culture medium (DMEM) and fetal bovine serum (FBS) were purchased from Life Technologies.

[0098] AY9944 inhibitor was purchased from Cayman.

[0099] DEN was purchased from Sigma.

[0100] Oligonucleotides were synthesized by Shanghai Sangon Biotech.

[0101] 2. Antibodies

[0102] Rabbit anti-Desmin antibody, rabbit anti-α-SMA antibody, mouse anti-F4 / 80 antibody, and Sirius Red Stain Kit were purchased from Abcam.

[0103] 3. Cell Culture

[0104] Hep 3B and Huh7 (human liver tumor cell lines) were both purchased from ATCC. The single cell layer was cultured at 37°C and 5% CO2, and the cells grew in medium DMEM (Dulbecco's Modified Eagle's Medium containing 100 U / ml penicillin and 100 μg / ml streptomycin) plus 10% FBS.

[0105] 4. Mouse Breeding

[0106] Heterozygous knockout DHCR7 mice and wild-type mice were bred in a specific pathogen-free (SPF) mouse house. When they were two weeks old, male mice were selected and injected intraperitoneally with DEN (25 mg / kg). Then, according to the experimental needs, they were fed different diets and / or injected with DHCR7 inhibitor every two days.

[0107] 5. IHC

[0108] Mouse liver specimens were fixed with 4% neutral formaldehyde, routinely paraffin-embedded, sectioned continuously at 4 μm, and subjected to antigen repair, background blocking, primary antibody incubation, and DAB color development after primary antibody incubation. After sealing the slides, they were quantified under a microscope.

[0109] 6. MTT Assay

[0110] 500 μL of 10,000 liver tumor cells per well (24-well plate) were added. After culturing in an incubator for 24 hours, different concentrations of AY9944 were added. After 48 hours, 10 μL of MTT solution (5 mg / ml) was added to each well, and incubation was continued in the cell incubator for 4 hours. Then, 100 μL of Formazan solubilization solution was added to each well, mixed properly, and incubation was continued in the cell incubator until all Formazan was observed to dissolve under an ordinary optical microscope. Usually, incubation at 37 °C for about 3 - 4 hours will cause all the purple crystals to dissolve. The absorbance was measured at 570 nm.

[0111] 7. Establishment of DHCR7 heterozygous deletion mice (DHCR7 het )

[0112] Mice were purchased from The Jackson Laboratory (Strain#007453).

[0113] Example 1: DHCR7 plays an important role in the growth of liver tumors

[0114] To study the role of DHCR7 in the development of liver tumors in mice, the inventors first injected DEN inducer into 2-week-old male DHCR7 heterozygous deletion mice (DHCR7 het ) to induce the formation of liver tumors.

[0115] When the mice reached 3 months old, they were grouped and raised, with a total of three groups:

[0116] Normal diet feeding group (chow);

[0117] Pair-fed group (pair-fed) (HFD);

[0118] Alcohol and high-fat diet feeding group (HFD + EtOH);

[0119] In each different feeding group, wild-type and DHCR7 heterozygous deletion male mice of the same age were raised together. Approximately 22 weeks after the start of grouped feeding (9 months (9M) for the normal diet group), the mice were analyzed.

[0120] The results showed that the number and size of tumors in DHCR7 heterozygous knockout mice were significantly reduced compared to the normal diet feeding group and the high-fat diet feeding group after DEN induction, and the tumor burden was significantly reduced ( Figure 1 A - B).

[0121] This result indicates that DHCR7 plays an important role in the growth of liver tumors. Taking it as a down-regulation target, the number of liver tumors in DHCR7-deficient animals is significantly reduced.

[0122] Example 2: DHCR7 plays an important regulatory role in the progression of liver tumors

[0123] The inventor further analyzed the phenotypes of tumor mice with heterozygous knockout of DHCR7. The results showed that, compared with the normal diet group and the high-fat diet group, the liver lipid accumulation area in the alcohol and high-fat diet group of mice was significantly reduced in the heterozygous knockout mice ( Figure 2 ).

[0124] After immunohistochemical staining, it was found that during the tumor formation process in mice with heterozygous knockout of DHCR7, indicators such as fibrosis and inflammation were also significantly improved in the alcohol and high-fat diet control groups ( Figure 3 ).

[0125] The above results further confirmed that DHCR7 plays an important positive regulatory role during the progression of the mouse liver to liver tumors, and significant improvement can be obtained by downregulating DHCR7.

[0126] Example 3: Small molecule inhibitors of DHCR7 can inhibit the development of liver tumors in vivo

[0127] Based on the above results, it was confirmed that DHCR7 plays an important regulatory role in alcohol combined with high-fat-induced liver tumors. Then, if the activity of DHCR7 is inhibited by using small molecule inhibitors of DHCR7, can the progression of tumors be delayed or blocked? The inventor verified this.

[0128] A liver tumor animal model was induced with DEN. Approximately 10 weeks after DEN induction, alcohol and high-fat diet were started. Nine weeks later, while feeding alcohol and high-fat diet, AY9944 was intraperitoneally injected 3 times a week at a concentration of 10 mg / kg to intervene in the process of alcohol-induced liver tumor formation.

[0129] Nine weeks after injection, the tumor growth of mice was analyzed. The results showed that by inhibiting the activity of DHCR7 in vivo with AY9944, the size and burden of tumors were also significantly reduced ( Figure 4 A - B).

[0130] These results directly indicated that DHCR7 can resist the development of alcohol-induced liver tumors and plays an important role.

[0131] Example 4: Small molecule inhibitors of DHCR7 can inhibit the growth of human liver tumor cells

[0132] In the foregoing examples, research was conducted on animal models. In this example, the inhibitory effect of DHCR7 inhibitors on human liver tumor cells was studied.

[0133] The present inventors used AY9944 to treat liver tumor cell lines of different individuals, including Hep3B and Huh7, and observed the effect of the inhibitor on cell proliferation.

[0134] The results showed that the inhibitor AY9944 could significantly inhibit cell growth, and as the concentration of the inhibitor increased, the cell survival rate decreased ( Figure 5 ).

[0135] This result further indicates that human liver tumor cell lines are highly sensitive to inhibitors of DHCR7.

[0136] Example 5, TNF is affected by DHCR7 and is its downstream effector molecule

[0137] The staining results showed that during the process of alcohol combined with high-fat diet-induced tumor formation, the lipid accumulation area in mice treated with the DHCR7 inhibitor (AY9944) decreased significantly, and inflammation and fibrosis were significantly reduced ( Figure 6 A).

[0138] Furthermore, the present inventors analyzed the expression of multiple genes before and after treatment with the DHCR7 inhibitor (AY9944). As Figure 6 shown in B, gene expression analysis showed that the inhibitor could decrease the expression of fibrosis-related marker genes aSMA and Timp1 in the liver.

[0139] The present inventors also found that the expression of the TNF gene decreased significantly after the activity of DHCR7 was inhibited. This result suggests that TNF is affected by DHCR7 and is its downstream effector molecule.

[0140] Summary and Discussion

[0141] In the above examples, the animal liver tumor model demonstrated that the deletion of DHCR7 in vivo or the direct inhibition of DHCR7 activity could inhibit the progression of liver tumors, and in vitro cell experiments further demonstrated that DHCR7 could affect the proliferation of human tumor cells. These results suggest that DHCR7 can be used as a candidate target for inhibiting liver tumors, and its inhibitor AY9944 can be used as a candidate compound for anti-liver tumors. Based on the structure of this inhibitor, derivatives can be modified to achieve better anti-cancer effects.

[0142] The above-described embodiments merely represent several implementation manners of the present invention. Their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent for the present invention. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention shall be subject to the appended claims. At the same time, all the documents mentioned in the present invention are cited in this application for reference, just as if each document is separately cited for reference.

Claims

1. Use of a down-regulator of 7-dehydrocholesterol reductase for preparing a composition for inhibiting liver tumors; The liver tumor is an alcohol-induced liver tumor; The down-regulator of 7-dehydrocholesterol reductase is a chemical small molecule antagonist or inhibitor against 7-dehydrocholesterol reductase, and the chemical small molecule antagonist or inhibitor against 7-dehydrocholesterol reductase is AY9944.

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