Uses of TRPM2 inhibitors

Abstract

This application discloses the use of a TRPM2 inhibitor. This application establishes for the first time the mechanism by which TRPM2 mediates liver cancer cell proliferation and illustrates how TRPM2 regulates cell proliferation through the Ca2+-CaM-CaMKII signaling pathway. This provides a new target for the development of drugs to treat liver cancer and a new intervention strategy for the clinical intervention and treatment of liver cancer.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the use of TRPM2 inhibitors. Background Art

[0002] Hepatocellular carcinoma (HCC) is a common and highly prevalent malignant tumor. my country, a high-incidence region for liver cancer, accounts for approximately half of the world's cases, with the third and second highest incidence and mortality rates in China, respectively. However, current clinical treatment options for liver cancer are limited, with poor long-term efficacy and a five-year survival rate of only 12%. Chemotherapy, a common treatment, is highly damaging to normal cells. Therefore, researchers are dedicated to identifying better treatment strategies based on the pathogenesis of liver cancer.

[0003] Transient receptor potential melastatin 2 (TRPM2) channel is an oxidative stress-sensitive cation channel that plays a vital role in maintaining intracellular ion homeostasis, especially Ca2+ homeostasis. It has been reported to be involved in various pathophysiological processes such as proliferation, survival, apoptosis, cell cycle regulation, oxidative stress response, etc. in various tumors such as neuroblastoma and gastric cancer. Studies have shown that TRPM2 channels may play a carcinogenic or tumor suppressor role in different tumors, and the mode of action and potential mechanisms of TRPM2 channels in regulating the growth and proliferation of different tumors are different. The role of TRPM2 channels in liver cancer is still unclear. Therefore, clarifying the role of TRPM2 in the occurrence and development of liver cancer and its molecular mechanism is very important for explaining Ca 2+ The impact of homeostasis regulation on abnormal proliferation of liver cancer is of great significance for developing new targets for the treatment of liver cancer. Summary of the Invention

[0004] The object of the present invention is to provide a use of a TRPM2 inhibitor.

[0005] Another object of the present invention is to provide a polynucleotide.

[0006] Another object of the present invention is to provide a method for preventing and / or treating liver cancer.

[0007] To solve the above technical problems, the first aspect of the present invention provides the use of a TRPM2 inhibitor for:

[0008] (i) preparing a medicament for preventing and / or treating liver cancer;

[0009] (ii) prevention or treatment of liver cancer;

[0010] (iii) inhibiting the binding of calcium ions to calmodulin;

[0011] (iv) inhibiting calmodulin expression;

[0012] (v) inhibiting the expression of CaMKII;

[0013] (vi) inhibiting the phosphorylation of CaMKII; or

[0014] (vii) Induces G1 / S phase cell cycle arrest.

[0015] In some preferred embodiments, the TRPM2 inhibitor is selected from the group consisting of: antibodies, polypeptides, sh-RNA, dsRNA, miRNA, antisense oligonucleotides, compounds, or combinations thereof.

[0016] In some preferred embodiments, the TRPM2 inhibitor is sh-RNA.

[0017] In some preferred embodiments, the sh-RNA is selected from any one of the following:

[0018] (a) a polynucleotide having the sequence shown in SEQ ID NO.1;

[0019] (b) a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO. 1;

[0020] (c) a polynucleotide having a sequence complementary to the polynucleotide described in (a) or (b);

[0021] (d) a polynucleotide having the sequence shown in SEQ ID NO. 2;

[0022] (e) a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO. 2; and

[0023] (f) A polynucleotide having a complementary nucleotide sequence to that described in (d) or (e).

[0024] In some preferred embodiments, the TRPM2 inhibitor is ACA.

[0025] In some preferred embodiments, the TRPM2 inhibitor is ACA and a compound represented by formula (I);

[0026]

[0027] Among them, R 1 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro;

[0028] R 2 For hydrogen, halogen, C 1-4alkyl, methoxy or nitro;

[0029] R 3 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro; and / or

[0030] R 4 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro.

[0031] In some preferred embodiments, the TRPM2 inhibitor is a compound represented by formula (I);

[0032]

[0033] Among them, R 1 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro;

[0034] R 2 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro;

[0035] R 3 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro; and / or

[0036] R 4 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro.

[0037] In some preferred embodiments, in formula (I), the halogen is preferably fluorine, chlorine, bromine or iodine;

[0038] In some preferred embodiments, in formula (I), the C 1-4 The alkyl group is preferably a methyl group or an ethyl group, more preferably a methyl group.

[0039] In some preferred embodiments, in formula (I), R 1 is hydrogen, methyl or halogen;

[0040] R 2 is hydrogen, methyl or halogen;

[0041] R 3 is hydrogen or halogen; and

[0042] R 4 For hydrogen, halogen, C 1-4 alkyl, methoxy or nitro.

[0043] In some preferred embodiments, in formula (I), R 1 is hydrogen;

[0044] R 2 For bromine;

[0045] R 3 is hydrogen; and

[0046] R 4 It is a methyl group.

[0047] The second aspect of the present invention provides an isolated polynucleotide, which is selected from any one of the following:

[0048] (a) a polynucleotide having the sequence shown in SEQ ID NO.1;

[0049] (b) a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO. 1;

[0050] (c) a polynucleotide having a sequence complementary to the polynucleotide described in (a) or (b);

[0051] (d) a polynucleotide having the sequence shown in SEQ ID NO. 2;

[0052] (e) a polynucleotide having a homology greater than 95% with the sequence shown in SEQ ID NO. 2; and

[0053] (f) A polynucleotide having a complementary nucleotide sequence to that described in (d) or (e).

[0054] The third aspect of the present invention provides a vector comprising the polynucleotide according to the second aspect of the present invention. In some preferred embodiments, the vector is constructed by lentivirus.

[0055] The fourth aspect of the present invention provides a host cell, wherein the host cell comprises the vector described in the third aspect of the present invention.

[0056] The information of the above SEQ ID NO: 1 and SEQ ID NO: 2 is shown in the following table.

[0057] serial number Sequence information SEQ ID NO: 1 5'-GCGTGATCTACCACCTCATGA-3' SEQ ID NO:2 5'-GGCCAAGGACATGAAGTTTGT-3'

[0058] A fifth aspect of the present invention provides a method for preventing and / or treating liver cancer, comprising the steps of:

[0059] administering a TRPM2 inhibitor or a pharmaceutical composition containing a TRPM2 inhibitor, preferably the polynucleotide, ACA and / or the compound represented by formula (I), more preferably the polynucleotide or the compound represented by formula (I) or shRNA, to the subject; or

[0060] administering a CaM inhibitor to the subject; or

[0061] A CaMKII inhibitor is administered to the subject.

[0062] Compared with the prior art, the present invention has at least the following advantages:

[0063] (1) This invention establishes for the first time the mechanism by which TRPM2 mediates liver cancer cell proliferation and illustrates that TRPM2 can mediate the cell proliferation regulation process through the Ca2+-CaM-CaMKII signaling pathway, providing a new target for the development of drugs to treat liver cancer and a new intervention strategy for the clinical intervention and treatment of liver cancer.

[0064] (2) The present invention found that TRPM2 inhibitors have a good inhibitory effect on liver cancer not only at the cellular level but also at the animal level, and are very good drugs for treating liver cancer;

[0065] (3) The present invention designs and develops a new TRPM2 inhibitor, which not only has a better ability to inhibit liver cancer cells than the commonly used ACA on the market, but also has better selectivity, less impact on normal liver cells, and fewer toxic side effects.

[0066] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] One or more embodiments are exemplarily described by the figures in the corresponding drawings, and these exemplified descriptions do not constitute limitations on the embodiments.

[0068] Figure 1 Representative Western blot images of TRPM2 protein expression levels in human normal liver cell lines (MIHA, HL-7702) and liver cancer cell lines (HCC-LM3, Hep3B, SNU387, SNU449, HepG2, Huh-7) according to the embodiments of the present invention;

[0069] Figure 2 are the results of Western blot quantitative analysis of TRPM2 protein expression levels in human normal liver cell lines (MIHA, HL-7702) and liver cancer cell lines (HCC-LM3, Hep3B, SNU387, SNU449, HepG2, Huh-7) in various liver cells and liver cancer cells according to the examples of the present invention;

[0070] Figure 33 is a graph showing the results of qPCR quantitative analysis of TRPM2 mRNA expression levels in human normal liver cell lines (MIHA, HL-7702) and liver cancer cell lines (HCC-LM3, Hep3B, SNU387, SNU449, HepG2, Huh-7) according to an embodiment of the present invention;

[0071] Figure 4 This is a representative confocol diagram of the in situ RNA expression of TRPM2 in Huh-7 cells detected using RNAscope-specific TRPM2-Cy5 probe according to an embodiment of the present invention;

[0072] Figure 5 This is a confocol representative diagram of the in situ RNA expression of TRPM2 detected by RNAscope in HepG2 cells according to an embodiment of the present invention;

[0073] Figure 6 Representative graphs and IV curves of the time-dependent changes in TRPM2 currents in Huh-7 cells activated by 500 μM ADPR at a voltage of -80 mV during whole-cell electrophysiological recording according to an embodiment of the present invention (n=4, *P<0.05);

[0074] Figure 7 Representative graphs and IV curves of the time-dependent changes in TRPM2 current in HepG2 cells under the action of 30 μM A10 according to an embodiment of the present invention (n=6, *P<0.05);

[0075] Figure 8 Representative images and IV curves of the time-dependent changes in TRPM2 current in HL-7702 cells under the action of 30 μM A10 according to an embodiment of the present invention (n=4);

[0076] Figure 9 Representative images and IV curves of TRPM2 current changes over time in MIHA cells under the action of 30 μM A10 according to an embodiment of the present invention (n=3);

[0077] Figure 10 is a graph showing the change in relative fluorescence intensity of Fluo-4 in Huh-7 cells over time according to an embodiment of the present invention;

[0078] Figure 11 is the quantitative statistical result of the relative fluorescence intensity peak value of single Huh-7 cells F / F0 according to the embodiment of the present invention (31-48 cells per group), ****P<0.0001;

[0079] Figure 12 is a representative graph showing the change in relative fluorescence intensity (F / F0) of Fluo-4 in HepG2 cells over time according to an embodiment of the present invention;

[0080] Figure 13 is the quantitative statistical result of the relative fluorescence intensity peak value of F / F0 of single HepG2 cells according to the embodiment of the present invention (19-42 cells per group), ****P<0.0001;

[0081] Figure 14 Graph showing the effect of TRPM2 inhibitor treatment on HCC cell growth according to an embodiment of the present invention;

[0082] Figure 15 Graph showing the effect of TRPM2 inhibitor treatment on HCC cell clone formation ability according to an embodiment of the present invention;

[0083] Figure 16 Graph showing the effect of treatment with TRPM2 inhibitor A10 or ACA on the growth of two normal hepatocytes according to an embodiment of the present invention;

[0084] Figure 17 Graph showing the effect of treatment with TRPM2 inhibitor A10 or ACA on the clone-forming ability of two normal hepatocytes according to an embodiment of the present invention;

[0085] Figure 18 is a graph showing the expression levels of TRPM2 proteins in Huh-7 and HepG2 cells after shRNA knockdown of TRPM2 according to an embodiment of the present invention;

[0086] Figure 19 is a graph showing mRNA expression after TRPM2 knockdown in Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0087] Figure 20 2 is a graph showing the effect of TRPM2 knockdown on in situ RNA expression in Huh-7 cells according to an embodiment of the present invention;

[0088] Figure 21 2 is a graph showing the effect of TRPM2 knockdown on in situ RNA expression in HepG2 cells according to an embodiment of the present invention;

[0089] Figure 22 Graph showing the effect of TRPM2 knockdown on Huh-7 and HepG2 cell growth according to an embodiment of the present invention;

[0090] Figure 23 Graph showing the effect of TRPM2 knockdown on the clone-forming ability of Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0091] Figure 24 1 is a graph showing the knockdown efficiency of TRPM2 shRNA in normal hepatocytes according to an embodiment of the present invention;

[0092] Figure 25is the mRNA expression graph after TRPM2 knockdown in HL-7702 and MIHA cells according to an embodiment of the present invention;

[0093] Figure 26 Graph showing the effect of TRPM2 knockdown on the growth of HL-7702 and MIHA cells according to an embodiment of the present invention;

[0094] Figure 27 Graph showing the effect of TRPM2 knockdown on the clone-forming ability of HL-7702 and MIHA cells according to an embodiment of the present invention;

[0095] Figure 28 Graph showing the effect of TRPM2 inhibitors or TRPM2 knockdown on cell proliferation according to an embodiment of the present invention

[0096] Figure 29 The effect of TRPM2 knockdown on the proliferation of Huh-7 and HepG2 cells according to the embodiment of the present invention is shown in FIG.

[0097] Figure 30 is a cell cycle distribution diagram of Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0098] Figure 31 Graph showing the effect of TRPM2 knockdown on Huh-7 and HepG2 cell cycle distribution according to an embodiment of the present invention;

[0099] Figure 32 2 is a graph showing the effect of TRPM2 knockdown on the expression levels of key proteins regulating the cell cycle in Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0100] Figure 33 24h treatment or TRPM2 knockdown on the expression levels of key proteins regulating the cell cycle in Huh-7 and HepG2 cells according to the embodiments of the present invention;

[0101] Figure 34 Schematic diagram of the effect of A10 on the cell cycle distribution of normal liver cell lines HL-7702 and MIHA according to an embodiment of the present invention;

[0102] Figure 35 Schematic diagram of the effect of TRPM2 knockdown on cell cycle distribution of normal hepatic cell lines HL-7702 and MIHA according to an embodiment of the present invention;

[0103] Figure 36 2. Graph showing transcriptome data analysis results of liver cancer tissue and adjacent non-cancerous tissue according to an embodiment of the present invention;

[0104] Figure 37GSEA enrichment analysis results of GO gene Cell cycle according to an embodiment of the present invention;

[0105] Figure 38 GSEA enrichment analysis result diagram of KEGG gene Cell cycle according to an embodiment of the present invention;

[0106] Figure 39 Graph showing relative mRNA expression levels of cell cycle regulatory proteins in normal liver tissue (n=50), TRPM2 low-expressing liver cancer tissue (n=187), and TRPM2 high-expressing liver cancer tissue (n=187) according to an embodiment of the present invention;

[0107] Figure 40 This is an H&E staining image of liver tissue sections from patients with liver cancer according to an embodiment of the present invention, and a TRPM2 and Ki67 staining image;

[0108] Figure 41 Schematic diagram of H&E staining of liver tissue sections from patients with liver cancer and immunohistochemical staining of TRPM2 and Ki67 according to an embodiment of the present invention;

[0109] Figure 42 1 is a representative Western blot diagram of the expression levels of proteins such as TRPM2, PCNA, Cyclin D1, Cyclin E1 and Cyclin A2 in tumor tissue (T) and paired non-tumor tissue (N) of liver cancer patients according to an embodiment of the present invention;

[0110] Figure 43 Graph showing the effects of Ca2+ chelators, CaM inhibitors, Zn2+ chelators, or PARP-1 inhibitors alone or in combination with TRPM2 inhibitor A10 on the clonogenicity of Huh-7 cells according to an embodiment of the present invention;

[0111] Figure 44 Graph showing the effects of Ca2+ chelators, CaM inhibitors, Zn2+ chelators, or PARP-1 inhibitors alone or in combination with TRPM2 inhibitor A10 on the clonogenicity of HepG2 cells according to an embodiment of the present invention;

[0112] Figure 45 Graph showing the effect of treatment with a TRPM2 inhibitor, a Ca2+ chelator, or a CaM inhibitor on the proliferation ability of Huh-7 cells according to an embodiment of the present invention;

[0113] Figure 46 Graph showing the effect of TRPM2 inhibitors, Ca2+ chelators or CaM inhibitors on the proliferation of HepG2 cells according to an embodiment of the present invention;

[0114] Figure 47Graph showing the effects of treatment with a TRPM2 inhibitor, a Ca2+ chelator, or a CaM inhibitor on Huh-7 cell viability according to an embodiment of the present invention;

[0115] Figure 48 Graph showing the effects of TRPM2 inhibitors, Ca2+ chelators, or CaM inhibitors on HepG2 cell viability according to an embodiment of the present invention;

[0116] Figure 49 According to the TRPM2 inhibitor, Ca 2+ Figure 2 Effects of chelating agents and CaM inhibitors on cell cycle distribution of Huh-7 and HepG2 cells;

[0117] Figure 50 According to the TRPM2 inhibitor, Ca 2+ The changes of Huh-7 cell cycle distribution over time after treatment with chelators and CaM inhibitors;

[0118] Figure 51 According to the TRPM2 inhibitor, Ca 2+ The changes of HepG2 cell cycle distribution over time after treatment with chelating agents and CaM inhibitors;

[0119] Figure 52 Graph showing the effects of treatment with a TRPM2 inhibitor, a Ca2+ chelator, and a CaM inhibitor on the expression levels of TRPM2, CaM, p-CaMKII (Thr286), CaMKII, Cyclin D1, p-CDK4 (Thr172), CDK4, p21, p27, Cyclin E1, p-CDK2 (Thr160), CDK2, p-Rb (Ser807 / 811), p-Rb (Ser795), Rb, and Cyclin A2 proteins in Huh-7 and HepG2 cells according to the embodiments of the present invention;

[0120] Figure 53 Graph showing the effects of TRPM2 knockdown combined with CaM inhibitor W-7 treatment on the expression levels of TRPM2, CaM, Cyclin D1, p-CDK4 (Thr172), CDK4, p21, p27, Cyclin E1, p-CDK2 (Thr160), CDK2, p-Rb (Ser807 / 811), p-Rb (Ser795), Rb, and Cyclin A2 proteins in Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0121] Figure 54: is a quantitative statistical result diagram of the effects of TRPM2 inhibitors, Ca2+ chelators, and CaM inhibitors on protein expression levels in Huh-7 and HepG2 cells according to the embodiments of the present invention;

[0122] Figure 55 : is a quantitative statistical result diagram of the effect of TRPM2 knockdown combined with CaM inhibitor W-7 treatment on protein expression levels in Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0123] Figure 56 is a representative Western blot diagram of CaM protein expression levels in tumor tissue (T) and paired non-tumor tissue (N) of liver cancer patients according to an embodiment of the present invention;

[0124] Figure 57 is the quantitative statistical analysis result of CaM protein Western blot according to the embodiment of the present invention;

[0125] Figure 58 Graph showing the effect of CaMKII inhibitor KN-93 treatment on the proliferation capacity of Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0126] Figure 59 Graph showing the effect of CaMKII inhibitor treatment on cell cycle distribution of Huh-7 and HepG2 cells according to an embodiment of the present invention;

[0127] Figure 60 The effect of the CaMKII inhibitor KN-93 treatment on the protein expression levels of Huh-7 and HepG2 cells according to the examples of the present invention;

[0128] Figure 61 This is a quantitative statistical result diagram showing the effect of CaMKII inhibitor KN-93 treatment on the expression of different proteins in Huh-7 cells according to an embodiment of the present invention;

[0129] Figure 62 This is a quantitative statistical result diagram showing the effect of the CaMKII inhibitor KN-93 treatment on the expression of different proteins in HepG2 cells according to an embodiment of the present invention;

[0130] Figure 63 1. is a diagram of the PDX transplant tumor modeling process according to an embodiment of the present invention;

[0131] Figure 64 1 is a graph showing the expression level of TRPM2 protein in tumor tissue of a patient used to construct a PDX nude mouse xenograft tumor according to an embodiment of the present invention;

[0132] Figure 65This is a diagram showing the in vivo evaluation of the therapeutic effect of TRPM2 inhibitors on liver cancer using a PDX transplant tumor model according to an embodiment of the present invention;

[0133] Figure 66 This is a comparison of the sizes of PDX transplanted tumors peeled off according to an embodiment of the present invention;

[0134] Figure 67 This is a graph showing statistical results of PDX transplanted tumor weight according to an embodiment of the present invention;

[0135] Figure 68 is a graph showing the growth curve of the PDX transplanted tumor volume according to an embodiment of the present invention;

[0136] Figure 69 This is a diagram for evaluating the effect of TRPM2 knockdown on the in vivo growth of Huh-7 cells in a Huh-7 transplant tumor model according to an embodiment of the present invention;

[0137] Figure 70 2 is a graph showing the statistical analysis results of tumor weight of Huh-7 transplanted tumors in the shNC and shTRPM2 groups according to an embodiment of the present invention;

[0138] Figure 71 : These are representative images of H&E staining of PDX transplanted tumor and Huh-7 transplanted tumor tissue sections and TRPM2 and Ki67 immunohistochemical staining according to the examples of the present invention (scale bar: 100 μm);

[0139] Figure 72 1 is a representative Western blot graph of the expression levels of proteins such as TRPM2, Cyclin D1, Cyclin E1 and Cyclin A2 in PDX transplanted tumor tissues of the Control group and the A10-treated group according to an embodiment of the present invention;

[0140] Figure 73 1 is a graph showing quantitative statistical results of Western blot according to an embodiment of the present invention. DETAILED DESCRIPTION

[0141] Liver cancer is a type of malignant tumor, and there is currently no effective treatment with minimal side effects. TRPM2 channels, as an important target for various diseases, have long been the focus of researchers. In the prior art, the relationship between TRPM2 channels and various cancers has been studied. The mode of action and potential mechanisms of TRPM2 channels in regulating the growth and proliferation of different tumors vary, but no one has yet established a connection between TRPM2 channels and liver cancer, that is, the role of TRPM2 channels in liver cancer is still unclear. Through research, the inventors have established for the first time the mechanism by which TRPM2 mediates the proliferation of liver cancer cells, elucidated that TRPM2 can mediate the cell proliferation regulation process through the Ca2+-CaM-CaMKII signaling pathway, and designed and developed TRPM2 inhibitors, including shRNA and small molecule inhibitors. The inventors found that these two inhibitors not only have a better inhibitory effect on liver cancer cells, but can also selectively kill liver cancer cells without affecting normal liver cells, with less toxic side effects. The selective killing ability of TRPM2 inhibitors has not been found in other cancer cells. Therefore, the inventors believe that drugs that target and inhibit TRPM2 are likely to have good clinical application effects.

[0142] As used herein, the term "TRPM2" refers to transient receptor potential M2. Transient receptor potential (TRP) channels are an important superfamily of cation channels located on the cell membrane. Based on the homology of amino acid sequences, 28 TRP channel proteins have been identified and divided into six subfamilies: TRPC (canonical), TRPM (melastatin), TRPA (ankyrin), TRPP (polycystin), TRPML (mucolipin) and TRPV (vanilloid). Transient receptor potential M2 (TRPM2) channels belong to the TRPM family. TRPM2 channels can be activated by ADP-ribose (ADPR), Ca 2+ It has been shown that TRPM2 acts as an oxidative stress sensor, mediating the intracellular Ca 2+ Its concentration increases and it participates in the physiological and pathological processes of various cells.

[0143] As used herein, the term "CaM" refers to calmodulin, the most common 2+ Binding protein, which not only binds Ca 2+ , but also as Ca 2+ Receptors that sense intracellular Ca 2+ Instantaneous changes in concentration.

[0144] As used herein, the term "CaMKII" is a protein kinase that can bind to the Ca2+ / CaM complex and be activated by its phosphorylation. It is an important downstream kinase in the Ca2+ / CaM signaling pathway.

[0145] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which specific conditions are not specified, are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight. The experimental materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

[0146] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which this application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of this application.

[0147] Example 1: TRPM2 channel protein is functionally expressed in liver cancer cells

[0148] This example demonstrates that TRPM2 channel protein is positively expressed in liver cancer cells.

[0149] (1) Analysis of protein and mRNA expression levels of TRPM2 channels

[0150] Two normal human liver cell lines (HL-7702 and MIHA) and six human hepatocellular carcinoma cell lines (HCC-LM3, Hep3B, SNU387, SNU449, HepG2, and Huh-7) were obtained from the Cell Bank of the Chinese Academy of Sciences in Shanghai and the First Affiliated Hospital of Zhejiang University School of Medicine, respectively. The protein expression levels and mRNA expression levels of the TRPM2 channel were compared and analyzed. The results are shown in Table 1. Figure 1 、 Figure 2 and Figure 3 .

[0151] Figure 1 β-actin was used as an internal reference protein.

[0152] Figure 2 The results were normalized with MIHA cells. *P<0.05, ****P<0.0001.

[0153] Figure 3 The results were normalized with MIHA cells, **P<0.01, ****P<0.0001.

[0154] Depend on Figure 1、 2 As shown in Figures 3 and 4, TRPM2 protein is expressed to varying degrees in normal hepatocytes and hepatoma cell lines;

[0155] The TRPM2 protein expression level and mRNA expression level in the two liver cancer cell lines Huh-7 and HepG2 were significantly higher than those in the normal liver cell lines HL-7702 and MIHA, which is consistent with the phenotypic trend that TRPM2 expression in tumor tissues of liver cancer patients is significantly higher than that in normal liver tissues.

[0156] (2) Verification of TRPM2 in situ RNA expression levels in Huh-7 and HepG2 cells

[0157] Using RNAscope technology, the in situ RNA expression level of TRPM2 in Huh-7 and HepG2 cells was verified using a human-specific TRPM2-Cy5 probe. The results are shown in Figure 4 and Figure 5 .

[0158] Figure 4 In the middle, BF stands for bright field, TRPM2 was labeled with red fluorescent Cy5, DAPI was used to label the cell nucleus (blue), and the DapB gene was used as a negative control. Scale bar: 20 μm.

[0159] Figure 5 Middle, scale bar: 20 μm

[0160] Depend on Figure 4 and Figure 5 It can be seen that TRPM2 in situ RNA is stably expressed in both types of liver cancer cells.

[0161] Example 2: TRPM2 channel protein has ion channel function in liver cancer cells

[0162] This example demonstrates that the TRPM2 channel protein functions as an ion channel in liver cancer cells. The TRPM2 inhibitors used in this example were A10 and ACA, where ACA represents N-(p-amylcinnamyl)anthranilic acid and A10 represents 6-Bromo-8-methyl-2-(3-phenyl-1H-pyrazol-4-yl)-2,3-dihydroquinazolin-4(1H)-one.

[0163] (1) Electrophysiological recording of TRPM2 characteristic currents in liver cancer cells and normal liver cells

[0164] Whole-cell electrophysiology was performed on Huh-7 and HepG2 cells; the physiological recording results are shown in Figures 6 to 9 .

[0165] Depend on Figures 6 to 9It can be seen that the TRPM2-specific intracellular agonist ADPR (500 μM) can quickly induce liver cancer cells to produce TRPM2 currents, and this current can be completely inhibited by TRPM2 inhibitors (A10 or ACA), and presents a characteristic TRPM2I-V curve, indicating that TRPM2 channel protein has ion channel function in liver cancer cells.

[0166] When activated by the same concentration of ADPR, almost no obvious TRPM2 current was recorded, indicating that TRPM2 has only weak channel function in normal hepatocytes.

[0167] (2) Calcium imaging experiments

[0168] Select specific Ca 2+ Fluorescent probe Fluo-4 AM was used to perform live cell calcium imaging experiments on two types of liver cancer cells. After Fluo-4 AM loading, the cells were pre-incubated with solvent control (0.1% DMSO), A10 (30 μM) or ACA (30 μM) for 30 minutes. The results are shown in Figures 10 to 13 .

[0169] Depend on Figures 10 to 13 It can be seen that activating TRPM2 channels with 3 mM H2O2 can significantly increase the Ca 2+ concentration, so that intracellular Ca 2+ The fluorescence intensity increased significantly; TRPM2 inhibitors (A10 or ACA) pre-incubated for 30 minutes could effectively inhibit the Ca2+-induced 2+ influx, indicating that H2O2-mediated Ca 2+ Influx is directly linked to TRPM2 channels.

[0170] Example 3: Inhibiting the function and expression of TRPM2 channels can inhibit the growth of liver cancer cells

[0171] This example demonstrates that inhibiting both the function and expression of TRPM2 channels can inhibit the growth of liver cancer cells, and that TRPM2 inhibitors have selective inhibitory effects on the growth of liver cancer cells and normal liver cells.

[0172] (1) CCK-8 assay to detect the effect of TRPM2 inhibitor treatment on the growth of HCC cells and normal stem cells

[0173] Huh-7 and HepG2 cells were treated with solvent control (0.1% DMSO), 30 μM A10 or ACA for 24, 48 and 72 hours, and then their growth activity was detected by CCK-8. Each experiment was repeated at least three times independently. The data were expressed as mean ± standard error. Two-way ANOVA was used for statistical analysis, and the Bonferroni method was used for pairwise comparison. The results are shown in Figure 14.

[0174] Normal hepatocytes were treated in the same manner as above, and the results are shown in Figure 15 .

[0175] Figure 14 and Figure 16 In the table, **** indicates A10 vs. Control, P < 0.0001; #### indicates ACA vs. Control, P < 0.0001.

[0176] (2) Effects of TRPM2 inhibitor treatment on the clonal formation ability of HCC cells and normal stem cells

[0177] Huh-7 and HepG2 cells were seeded in 6-well plates and treated with Control (0.1% DMSO), A10, or ACA (30 μM) for 10-14 days, fixed with 4% PFA, and stained with 0.1% crystal violet aqueous solution. Figure 15 .

[0178] Normal hepatocytes were treated in the same manner as above, and the results are shown in Figure 16 .

[0179] Depend on Figures 14 to 17 Treatment with the TRPM2 inhibitors A10 and ACA significantly reduced the viability of Huh-7 and HepG2 cells compared to the control group, and also significantly decreased their ability to form colonies. Testing of the normal liver cell lines HL-7702 and MIHA cells revealed that treatment with A10 and ACA had little effect on their viability and colony-forming ability.

[0180] Example 4: Inhibiting the function and expression of TRPM2 channels can inhibit the growth of liver cancer cells

[0181] This example demonstrates that knocking down TRPM2 can inhibit the function and expression of TRPM2 channels and inhibit the growth of liver cancer cells while having little effect on normal liver cells.

[0182] (1) TRPM2 knockdown can significantly inhibit the growth of liver cancer cells

[0183] TRPM2 was specifically knocked down by lentiviral-encapsulated shRNA. Huh-7 and HepG2 cells were transfected with lentiviral-encapsulated negative control shRNA (shNC), TRPM2 shRNA-#1 (shTRPM2), or TRPM2 shRNA-#2 (shTRPM2#2) for 72 hours, and the whole cell lysates were collected. The knockdown efficiency of TRPM2 was detected by Western blot. The results are shown in Figure 18Compared with the negative control shRNA (shNC), both specific TRPM2 shRNAs (shTRPM2 and shTRPM2#2) were able to significantly reduce the expression level of TRPM2 protein in Huh-7 and HepG2 cells, and shTRPM2 had a higher knockdown efficiency.

[0184] Negative Control (shNC) 5'-TTCTCCGAACGTGTCACGT-3' shTRPM2: 5'-GCGTGATCTACCACCTCATGA-3' shTRPM2#2 5'-GGCCAAGGACATGAAGTTTGT-3'

[0185] qPCR was used to detect the mRNA expression of TRPM2 in Huh-7 and HepG2 cells after knockdown. Each experiment was repeated at least three times independently. The data are expressed as mean ± standard error and statistically analyzed by paired sample t test; **P < 0.01, ****P < 0.0001. Figure 19 .

[0186] RNAscope was used to detect the effect of TRPM2 knockdown on the in situ RNA expression level of TRPM2 in Huh-7 and HepG2 cells. The results are shown in the figure. Figure 20 and Figure 21 .

[0187] The effects of TRPM2 knockdown on the growth of Huh-7 and HepG2 cells were detected by CCK-8. Each experiment was repeated at least three times independently. The data are expressed as mean ± standard error. Two-way ANOVA was used for statistical analysis, and the Bonferroni method was used for pairwise comparison. *P < 0.05, ***P < 0.001, ****P < 0.0001, the results are shown in the table. Figure 22 .

[0188] TRPM2 knockdown Huh-7 and HepG2 cells were seeded in 6-well plates, fixed with 4% PFA, and then stained with 0.1% crystal violet solution. The effect of TRPM2 knockdown on the colony formation ability of Huh-7 and HepG2 cells was determined. The results are shown in Figure 23 .

[0189] Depend on Figures 18 to 23 It can be seen that compared with shNC, shTRPM2 can indeed significantly reduce the mRNA expression level of TRPM2, and the results of RNAscope-specific TRPM2 probe detection also observed that after TRPM2 knockdown, the in situ RNA expression level of TRPM2 in Huh-7 and HepG2 cells in the shTRPM2 group was significantly reduced compared with shNC. After TRPM2 knockdown, the growth viability and clone formation ability of Huh-7 and HepG2 cells were significantly reduced compared with shNC. This result is similar to the results of TRPM2 inhibitor treatment, indicating that TRPM2 knockdown can significantly inhibit the growth of liver cancer cells.

[0190] (2) TRPM2 knockdown has little effect on normal hepatocytes

[0191] TRPM2 knockdown experiments were performed using normal hepatocytes, following the same procedures as above. Figures 24 to 27 .

[0192] Depend on Figure 24 and 27 It can be seen that inhibiting TRPM2 channels has little effect on normal hepatocytes.

[0193] Example 5: Inhibition of TRPM2 channel function and expression can induce G1 / S cell cycle arrest

[0194] This example demonstrates that inhibiting the function and expression of TRPM2 channels can induce G1 / S phase cell cycle arrest and inhibit cell proliferation.

[0195] (1) TRPM2 is involved in regulating the proliferation of liver cancer cells

[0196] To clarify the potential mechanism by which inhibition of TRPM2 channel function and expression induces growth inhibition of liver cancer cells, the inventors investigated whether TRPM2 is involved in regulating the proliferation of liver cancer cells. First, 5-ethynyl-2'-deoxyuridine (EdU) was used to determine the effects of TRPM2 inhibitors or TRPM2 knockdown on the proliferation of Huh-7 and HepG2 cells. Figure 28 . Figure 28 In the middle, Control is the solvent control group with 0.1% DMSO. The left figure is a representative image of EdU staining, where BF represents bright field, EdU is labeled with Alexa Fluor 555 red fluorescence, and Hoechst 33342 (Hoechst) is used to label the cell nucleus (blue). Merge is the merged image of EdU and Hoechst. Scale bar: 100 μm.

[0197] Depend on Figure 28 It can be seen that compared with the control group (0.1% DMSO), the proportion of EdU-positive cells in Huh-7 and HepG2 cells was significantly decreased after 24 hours of treatment with 30 μM A10.

[0198] EdU staining was used to quantitatively analyze the effect of TRPM2 knockdown on the proliferation of Huh-7 and HepG2 cells. *P < 0.05, ****P < 0.0001. Each experiment was repeated at least three times independently. The data are expressed as mean ± standard error. Figure 29 . Figure 29The results also showed that the proliferation activity of liver cancer cells in the TRPM2 knockdown group (shTRPM2) was significantly reduced compared with the negative control group (shNC). These results fully confirm that inhibiting the function and expression of TRPM2 channels can significantly inhibit the proliferation of liver cancer cells.

[0199] (2) Inhibiting the function and expression of TRPM2 channels can induce G1 / S cell cycle arrest

[0200] To explore the mechanism of action of TRPM2 in regulating the growth and proliferation of liver cancer cells, the inventors used flow cytometry to explore the role of TRPM2 in cell cycle progression.

[0201] Huh-7 and HepG2 cells were treated with control group (0.1% DMSO) and A10 (30 μM) for 24 h, and then PI staining was used to determine the cell cycle distribution. Figure 30 . Figure 30 Middle, the left figure is a representative image of cell cycle distribution, and the right figure is the statistical results of the percentage of cells in each phase of G1, S, and G2 (two-way ANOVA for significance analysis, followed by Bonferroni method for pairwise comparison, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0202] Depend on Figure 30 It can be seen that after 24 hours of A10 treatment, the proportion of cells in the G1 phase of Huh-7 and HepG2 cells increased significantly, while the proportion of cells in the S phase decreased significantly.

[0203] The same method was used to detect the effect of TRPM2 knockdown on the cell cycle distribution of Huh-7 and HepG2 cells. The results are shown in Figure 31 .

[0204] Western blot was used to detect the expression levels of key proteins regulating the cell cycle. The effects of 24h treatment with TRPM2 inhibitor A10 (30μM) or TRPM2 knockdown on the expression levels of TRPM2, Cyclin D1, p-CDK4 (Thr172), Cyclin E1, CyclinA2, p-CDK2 (Thr160), p-JNK (Thr183 / Tyr185), JNK, Cyclin B1 and p-CDC2 (Tyr15) in Huh-7 cells were analyzed. β-actin was used as an internal reference protein. Each group of experiments was repeated at least three times independently, and the data are expressed as mean ± standard error. Results are shown in Figure 32 .

[0205] Similarly, the effects of 24h treatment with TRPM2 inhibitor A10 (30 μM) or TRPM2 knockdown on the expression levels of TRPM2, CyclinD1, p-CDK4 (Thr172), Cyclin E1, Cyclin A2, p-CDK2 (Thr160), p-JNK (Thr183 / Tyr185), JNK, Cyclin B1 and p-CDC2 (Tyr15) proteins in HepG2 cells were studied. β-actin was used as an internal reference protein. Each group of experiments was repeated at least three times independently. The data are expressed as mean ± standard error. Figure 33 .

[0206] Depend on Figures 30 to 33 It can be seen that inhibiting or knocking down TRPM2 can significantly downregulate the expression and phosphorylation levels of key G1 / S phase cell cycle regulatory proteins such as Cyclin D1, p-CDK4 (Thr172), Cyclin E1, Cyclin A2 and p-CDK2 (Thr160), but has no obvious effect on the expression and phosphorylation levels of p-JNK (Thr183 / Tyr185), JNK and Cyclin B1 and p-CDC2 (Tyr15) which are closely related to G2 / M phase cell cycle regulation, which is consistent with the results of cell cycle detection, further confirming that inhibiting the function and expression of TRPM2 can induce the occurrence of G1 / S phase cell cycle arrest, while having little effect on the G2 / M phase.

[0207] (3) Inhibiting the function and expression of TRPM2 channels can induce G1 / S cell cycle arrest

[0208] The cell cycle distribution of two normal hepatocytes was detected. The normal hepatocyte cell lines HL-7702 and MIHA cells were treated with TRPM2 inhibitor A10 (30μM) for 24 hours to determine the effect of A10 on the cell cycle distribution of normal hepatocyte cell lines HL-7702 and MIHA. Figure 34 The same method was used to determine the effect of TRPM2 knockdown on the cell cycle distribution of normal hepatocyte cell lines HL-7702 and MIHA. Figure 35 .

[0209] Depend on Figure 34 and Figure 35 The results showed that the proportion of cells in the G1, S, and G2 phases in HL-7702 and MIHA cells did not differ significantly from the control group, whether treated with the TPRM2 inhibitor A10 or specifically knocked down TRPM2. This suggests that inhibiting the function and expression of the TRPM2 channel has little effect on the cell cycle of normal hepatocytes. This also further confirms that cell intervention targeting TRPM2 has significant selectivity for normal hepatocytes and hepatocellular carcinoma cells.

[0210] (4) TRPM2 channel expression is positively correlated with cell cycle regulation

[0211] Metascape enrichment analysis software was used to perform differential analysis on the transcriptome data of 35 paired liver cancer tissues and adjacent non-cancerous tissues in the GSE124535 dataset in the GEO database (differential analysis method: Limma package, padj < 0.05, |log2FC| > 1), and enrichment analysis of the upregulated genes was performed. Figure 36 to.

[0212] Figure 36 The top 20 signaling pathways are displayed, and the top-ranked signaling pathway is the cell cycle pathway, which shows the core role of cell cycle regulation in the progression of liver cancer.

[0213] Gene Set Enrichment Analysis (GSEA) was performed on the differentially expressed genes in the expression matrix between liver cancer tissues and adjacent non-cancerous tissues based on the TCGA-LIHC database using clusterProfiler (version: 3.18.1). The differential analysis method used was the Limma package, with padj < 0.05 and |log2FC| > 1. Cell cycle pathway enrichment analysis was also performed on the GO (Gene Ontology) gene set and the KEGG (Kyoto Encyclopedia of Genes and Genomes) gene set. The results are shown in Figure 2. Figure 37 and Figure 38 .

[0214] Depend on Figure 37 and Figure 38 It can be seen that the Cell cycle pathways of both GO and KEGG gene sets were significantly upregulated, suggesting that the cell cycle pathway does play a key positive regulatory role in the occurrence of liver cancer.

[0215] The relative mRNA expression levels of cell cycle regulatory proteins Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E1, Cyclin E2, Cyclin A1, Cyclin A2, and Cyclin B1 in normal liver tissues (n=50), TRPM2 low-expressing liver cancer tissues (n=187), and TRPM2 high-expressing liver cancer tissues (n=187) in the TCGA-LIHC database were compared and analyzed. One-way ANOVA was used for significance analysis, followed by Tukey's post hoc analysis for pairwise comparisons. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. The results are shown in Figure 39.

[0216] Depend on Figure 39 It can be seen that compared with normal liver tissue (Normal), in liver cancer tissues with high TRPM2 expression, the expression of Cyclin D1, Cyclin D2, Cyclin D3, Cyclin E1, Cyclin E2, Cyclin A1 and Cyclin A2, which are closely related to the regulation of G1 and S phase cell cycle, were significantly upregulated; and compared with liver cancer tissues with low TRPM2 expression, in liver cancer tissues with high TRPM2 expression, except for Cyclin A1, the mRNA expression levels of other G1 / S phase regulatory proteins were also significantly increased, suggesting that the expression level of TRPM2 is positively correlated with the expression of G1 / S phase cell cycle regulatory proteins. In addition, we also found that Cyclin B1 is closely related to the regulation of the G2 / M phase cell cycle. Although the expression of Cyclin B1 in normal liver tissue is significantly lower than that in tumor tissue, there is no significant difference in its expression level between liver cancer tissues with low TRPM2 expression and high TRPM2 expression, which also suggests that TRPM2 expression may not be directly related to the G2 / M phase cell cycle regulation of liver cancer.

[0217] In summary, it can be shown that TRPM2 channels play an important role in the cell cycle regulation process. Inhibiting the function and expression of TRPM2 channels can induce G1 / S phase cell cycle arrest, inhibit cell proliferation, and thus slow the growth of liver cancer cells. It is worth mentioning that inhibiting the function and expression of TRPM2 channels has almost no effect on the cell cycle regulation and cell proliferation of normal liver cells. This is consistent with the phenomenon that inhibiting or knocking down TRPM2 has little effect on the growth of normal liver cells in the aforementioned results. It also further shows that the cell intervention method targeting TRPM2 has obvious selectivity in normal liver cells and liver cancer cells with high TRPM2 expression.

[0218] Example 6: In liver cancer tissues with high TRPM2 expression, cell proliferation levels were significantly increased

[0219] (1) Immunohistochemical staining

[0220] Liver tissue sections from patients with liver cancer were stained with H&E. The results of TRPM2 and Ki67 immunohistochemical staining are shown in Figure 40 .

[0221] Depend on Figure 40 It can be seen that the results of immunohistochemical staining showed that in liver cancer tissues with significantly upregulated TRPM2 expression, the proportion of Ki67-positive cells also increased significantly, suggesting that liver cancer tissues with high TRPM2 expression have stronger cell proliferation ability.

[0222] (2) Western blot detection of the expression of proteins related to cell proliferation and cell cycle regulation

[0223] Western blot was used to detect the expression levels of TRPM2, PCNA, Cyclin D1, Cyclin E1, and Cyclin A2 proteins in tumor tissues (T) and paired non-tumor tissues (N) of patients with liver cancer. The results are shown in Figure 42 , β-actin was used as an internal reference protein.

[0224] Depend on Figure 42 It can be seen that compared with normal liver tissue, the expression of PCNA, a key protein for cell proliferation, was significantly increased in liver cancer tissues where TRPM2 expression was significantly upregulated. Correspondingly, the expression levels of Cyclin D1, Cyclin E1, and Cyclin A2, key proteins for cell cycle regulation in the G1 / S phase, were also significantly increased, which was consistent with the immunohistochemistry results.

[0225] The above results indicate that the cell proliferation level is significantly increased in liver cancer tissues with high TRPM2 expression.

[0226] Example 7: Inhibition of TRPM2 can induce G1 / S cell cycle arrest through the Ca2+ / CaM signaling pathway and inhibit cell proliferation

[0227] (1)Ca 2+ and Zn 2+ Chelation research

[0228] Specific Ca2+ that can permeate the cell membrane 2+ Chelating agent: BAPTA-AM;

[0229] Specific Zn that can penetrate cell membranes 2+ Chelating agent: TPEN.

[0230] Ca 2+ Chelating agents, CaM inhibitors, Zn2+ chelators or PARP-1 inhibitors were treated alone or in combination with TRPM2 inhibitor A10 to detect their effects on cell clone formation ability. Huh-7 or HepG2 cells were seeded in 6-well plates and treated with Control (0.1% DMSO), BAPTA-AM (10μM), W-7 (40μM), TPEN (5μM), PJ-34 (20μM) alone or in combination with A10 (30μM) for 10-14 days. After fixation with 4% PFA, the cells were stained with 0.1% crystal violet aqueous solution. The results are shown in Figure 3. Figure 43 and 44 .

[0231] Root Cause Figure 43 and 44 It can be seen that BAPTA-AM alone can chelate intracellular Ca2+ It can significantly inhibit the clone formation ability of Huh-7 and HepG2 cells, and the treatment with BAPTA-AM combined with TRPM2 inhibitor A10 does not further increase the inhibitory effect on cell clone formation ability; however, the use of TPEN alone to chelate intracellular Zn 2+ However, there was no sign of weakening of the clone-forming ability of Huh-7 and HepG2 cells.

[0232] EdU staining was used to quantitatively analyze the effects of TRPM2 inhibitors, Ca2+ chelators, or CaM inhibitors on the proliferation of Huh-7 and HepG2 cells. EdU staining was performed after 24 hours of treatment with control (0.1% DMSO), A10 (30 μM), BAPTA-AM (10 μM), or W-7 (40 μM). Figure 45 and 46 .

[0233] Figure 45 and 46 Middle, left: representative images of EdU staining, scale bar: 100 μm; right: quantitative statistical results of the ratio of EdU-positive cells to Hoechst-positive cells, significance analysis was performed by one-way ANOVA, followed by pairwise comparisons by Dunnett's method, ****P < 0.0001.

[0234] The effects of TRPM2 inhibitors, Ca2+ chelators, or CaM inhibitors alone or in combination on the viability of Huh-7 and HepG2 cells were detected by CCK-8. Control (0.1% DMSO), A10 (30 μM), BAPTA-AM (10 μM), A10+BAPTA-AM, W-7 (40 μM), and A10+W-7 were treated for 24 hours before CCK-8 assay. ****P<0.0001. Each experiment was repeated at least three times independently, and the data are expressed as mean ± standard error. Results are shown in Figure 47 and Figure 48 .

[0235] Depend on Figures 45 to 48 It is known that chelating intracellular Ca 2+ It can significantly reduce the proportion of EdU-positive cells in Huh-7 and HepG2 cells, and also significantly inhibit cell viability. Moreover, BAPTA-AM combined with A10 treatment does not further induce a decrease in cell viability. The above results indicate that the phenomenon of inhibiting TRPM2-mediated liver cancer cell proliferation inhibition is mainly through Ca 2+ Implementation, not Zn 2+ .

[0236] (2) The Ca2+ / CaM signaling pathway plays a role in TRPM2-mediated cell proliferation regulation

[0237] The experimental results in (1) show that 40 μM W-7 treatment can significantly inhibit the cloning ability of Huh-7 and HepG2 cells, reduce the proportion of EdU-positive proliferation cells, and induce a decrease in cell viability; and W-7 combined with A10 treatment does not further inhibit the cell cloning ability, nor does it further increase the decrease in cell viability, which is similar to the results of BAPTA-AM treatment, suggesting that Ca 2+ The TRPM2 / CaM signaling pathway plays an important role in the regulation of TRPM2-mediated cell proliferation.

[0238] Based on the above results, it can be proved that inhibition of TRPM2 can 2+ / CaM signaling pathway plays a role in inhibiting cell proliferation and growth.

[0239] Example 6: Inhibition of TRPM2 through Ca 2+ / CaM signaling pathway induces G1 / S cell cycle arrest

[0240] The cells were treated with Control (0.1% DMSO), A10 (30 μM), BAPTA-AM (10 μM), and W-7 (40 μM) for 24 hours, and then PI staining was used to determine the cell cycle distribution. Figure 49 .

[0241] Figure 49 In the figure, the left side shows the representative image of cell cycle distribution, and the right side shows the statistical results of the percentage of cells in each phase of G1, S, and G2 in different treatment groups. Two-way ANOVA was used for significance analysis, followed by pairwise comparison using the Bonferroni method, *P < 0.05, **P < 0.01.

[0242] Thymidine (TdR) double blockade was used to induce cell cycle synchronization in Huh-7 and HepG2 hepatocellular carcinoma cells, synchronizing them at the G1 / S phase junction. The cells were then released from the cell cycle for 0, 2, 4, 6, 8, and 12 hours with fresh medium containing 0.1% DMSO (Control), A10 (30 μM), BAPTA-AM (10 μM), or W-7 (40 μM), respectively. The effects of different treatments on cell cycle transitions were observed. Specifically, Huh-7 (D) and HepG2 (E) cells were synchronized using TdR (thymidine) double blockade. The cells were then released from the cell cycle for 0-12 hours with fresh medium containing 0.1% DMSO (Control), A10 (30 μM), BAPTA-AM (10 μM), or W-7 (40 μM), respectively. Propidium Iodide staining was used to determine cell cycle distribution at each time point. Each experiment was repeated at least three times independently, and the data were expressed as mean ± standard error. Figure 50 and 51 .

[0243] The percentage of cells entering the S phase after A10 treatment was significantly reduced compared with the control group; similar results were also seen after BAPTA-AM and W-7 treatment, further clarifying the important role of TRPM2 and Ca2+ / CaM signaling pathways in the G1 / S cell cycle regulation of liver cancer cells.

[0244] Depend on Figure 49 It can be seen that both BAPTA-AM and W-7 treatments can significantly increase the proportion of cells in the G1 phase, while the proportions of cells in the S phase and G2 phase decrease to varying degrees.

[0245] Depend on Figure 50 and 51 It can be seen that the percentage of cells entering the S phase after A10 treatment was significantly reduced compared with the control group; similar results were also seen after BAPTA-AM and W-7 treatment, further clarifying the role of TRPM2 and Ca 2+ The important role of the CaM / CaM signaling pathway in the G1 / S cell cycle regulation of liver cancer cells.

[0246] Method 1: Huh-7 and HepG2 cells were treated with TRPM2 inhibitors, Ca2+ chelators, and CaM inhibitors;

[0247] Method 2: TRPM2 knockdown combined with CaM inhibitor W-7 treatment of Huh-7 and HepG2 cells;

[0248] Method 3: TRPM2 inhibitors, Ca 2+ Chelating agents and CaM inhibitors were used to treat Huh-7 and HepG2 cells;

[0249] Method 4: TRPM2 knockdown combined with CaM inhibitor W-7 was used to treat Huh-7 and HepG2 cells.

[0250] Western blot was used to detect its effects on the expression levels of TRPM2, CaM, p-CaMKII (Thr286), CaMKII, Cyclin D1, p-CDK4 (Thr172), CDK4, p21, p27, Cyclin E1, p-CDK2 (Thr160), CDK2, p-Rb (Ser807 / 811), p-Rb (Ser795), Rb and Cyclin A2 proteins, with β-actin as the internal control protein. Figure 52 The results were quantitatively analyzed and the significance was analyzed by one-way analysis of variance. Then, Dunnett's method was used for pairwise comparison with the control group. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Figures 52 to 55 .

[0251] Depend on Figures 52 to 55 It can be seen that after 24 hours of treatment with BAPTA-AM (10μM) and W-7 (40μM), the expression level of TRPM2 protein did not change significantly, while the expression level of CaM was significantly reduced (similar to the result after treatment with 30μM A10 alone); correspondingly, the protein expression levels of key G1 / S cell cycle regulatory proteins Cyclin D1, p-CDK4 (Thr172), Cyclin E1, p-CDK2 (Thr160) and Cyclin A2 were significantly reduced compared with the control group cells;

[0252] Sequestering intracellular Ca 2+ Or inhibition of CaM can significantly inhibit the phosphorylation of Rb and upregulate the expression of p21 and p27.

[0253] TRPM2 knockdown itself can also significantly downregulate the protein expression or phosphorylation levels of CaM, Cyclin D1, p-CDK4 (Thr172), CyclinE1, p-CDK2 (Thr160), Cyclin A2, p-Rb (Ser807 / 811) and p-Rb (Ser795), and upregulate the expression levels of p21 and p27, which is basically consistent with the results of treatment with TRPM2 inhibitor A10.

[0254] TRPM2 knockdown combined with W-7 ​​treatment did not further increase the changes in the expression levels of the above proteins, suggesting that TRPM2 is likely to directly2+ / CaM signaling pathway plays a role in cell cycle regulation.

[0255] (3) Detection of CaM protein expression levels in tissue samples from patients with liver cancer

[0256] The protein expression level of CaM in liver cancer tissue samples was detected. Figure 56 and Figure 57 Compared with normal liver tissue, a significant increase in CaM protein expression level was also observed in liver cancer tissue with high TRPM2 expression, suggesting a positive correlation between the expression levels of TRPM2 and CaM.

[0257] Based on the above results, it can be fully demonstrated that inhibition of TRPM2 can 2+ / CaM signaling pathway, inhibiting the expression and phosphorylation activation of downstream G1 / S phase cell cycle regulatory proteins, inducing G1 / S phase cell cycle arrest, and thus inhibiting the proliferation of liver cancer cells.

[0258] Example 7: Inhibition of CaMKII can induce G1 / S cell cycle arrest and significantly inhibit the proliferation of liver cancer cells

[0259] KN-93 is a cell-permeable competitive CaMKII inhibitor.

[0260] The cells were treated with the inhibitor KN-93, and EdU staining was used to quantitatively analyze the effect of CaMKII inhibitor KN-93 treatment on the proliferation capacity of Huh-7 and HepG2 cells. EdU staining was performed after 24 hours of treatment with control (0.1% DMSO) and KN-93 (5μM). Figure 58 . Figure 58 Middle, the left panel is a representative image of EdU staining, scale bar: 100 μm; the right panel is the quantitative statistical results of the ratio of EdU-positive cells to Hoechst-positive cells, two independent sample t-test, ****P < 0.0001.

[0261] Huh-7 and HepG2 cells were treated with CaMKII inhibitors: Control (0.1% DMSO) and KN-93 (5 μM) were used to treat the cells for 24 hours, and then PI staining was used to determine the cell cycle distribution. Figure 59 . Figure 59 In the middle, the left figure is a representative image of cell cycle distribution, and the right figure is the statistical results of the percentage of cells in each phase of G1, S and G2 in different treatment groups. Two-way ANOVA was used for significance analysis, followed by pairwise comparison using the Bonferroni method, *P < 0.05, ***P < 0.001.

[0262] Effects of CaMKII inhibitor treatment on the expression levels of TRPM2, CaM, p-CaMKII (Thr286), CaMKII, Cyclin D1, p-CDK4 (Thr172), CDK4, p-Rb (Ser807 / 811), p-Rb (Ser795), Rb, p21, p27, Cyclin E1, p-CDK2 (Thr160), CDK2 and Cyclin A2 proteins in Huh-7 and HepG2 cells. The results are shown in Figure 60 , quantitative statistical results are shown in Figures 61 to 62 .

[0263] Example 8: Inhibition or knockdown of TRPM2 channels can significantly slow the growth of PDX and Huh-7 transplanted tumors

[0264] (1) Construction of liver cancer disease model

[0265] Patient-derived xenografts (PDX xenografts) and Huh-7 cell-derived nude mouse xenografts (Huh-7 xenografts) were constructed for in vivo animal studies. Figure 63 , liver cancer tissue removed from liver cancer patients during surgery was inoculated subcutaneously into BALB / c nude mice. After subcutaneous tumor formation, it was amplified and passaged, and drug intervention and treatment were performed after the P3 generation. Western blot results confirmed that TRPM2 expression was significantly upregulated in the tumor tissue of liver cancer patients used for modeling ( Figure 64 ).

[0266] (2) Verification of the inhibitory effects of the commercially available TRPM2 inhibitor ACA and the TRPM2 inhibitor A10 designed by the inventors on PDX transplanted tumors and Huh-7 transplanted tumors.

[0267] TRPM2 inhibitors ACA and A10 were used to conduct drug intervention experiments on PDX transplanted tumor nude mice. The solvent control group (Control, n=5), ACA treatment group (n=5) and A10 treatment group (n=5) were given continuous intraperitoneal injection at a dose of 30 mg / kg / day. After 28 days, the mice were killed by cervical dislocation. The red circle is the transplanted tumor. After 28 days of continuous intraperitoneal injection, it was found that the transplanted tumors of nude mice treated with TRPM2 inhibitors (ACA treatment group or A10 treatment group) were significantly smaller than those of nude mice in the control group (Control group) (see Figure 65 ), and the tumor weight and tumor growth curve of PDX transplanted tumors were significantly decreased compared with the control group ( Figure 66 and Figure 67), but had no significant effect on the body weight of nude mice (body weight of nude mice, the long diameter (a) and short diameter (b) of transplanted tumors were measured every 2 days, and the tumor volume was calculated using the formula: V = 1 / 2 × a × b2, *P < 0.05; G. PDX nude mouse body weight change over time, measured once a day) ( Figure 68 ), suggesting that inhibiting TRPM2 can significantly slow the growth of PDX transplanted tumors with relatively minor side effects on the organism.

[0268] Example 9: Inhibition or knockdown of TRPM2 channels can significantly slow the growth of PDX and Huh-7 transplanted tumors

[0269] The inventors constructed a batch of Huh-7 transplant tumors, and inoculated the same number of Huh-7 cells that were successfully transfected with shTRPM2 or shNC into BALB / c Nude mice subcutaneously for tumor formation. Huh-7 cells were transfected with lentivirus-coated shNC and shTRPM2, respectively. Subsequently, Huh-7shNC (n=9) and Huh-7shTRPM2 (n=9) cells were subcutaneously inoculated at a ratio of 3×105 cells / nude mouse. The mice were observed for 35 consecutive days. When the transplant tumor volume was approximately 1000mm3, the nude mice were killed by cervical dislocation, and the transplant tumors were peeled and collected (the red part was the transplant tumor). The results also observed that the tumor size and tumor weight of the Huh-7 transplant tumors in the TRPM2 knockdown group (shTRPM2 group) were significantly lower than those in the shNC control group ( Figure 69 and Figure 70 ).

[0270] The results of the above two in vivo transplant tumor models fully demonstrated that inhibiting or knocking down the TRPM2 channel can significantly slow down the growth of PDX transplant tumors and Huh-7 transplant tumors.

[0271] Example 10: Inhibition or knockdown of TRPM2 can significantly inhibit cell proliferation and expression of cell cycle-related proteins in PDX and Huh-7 transplanted tumors

[0272] The inventors detected the cell proliferation and G1 / S phase cell cycle regulation key proteins expression in PDX transplanted tumors and Huh-7 transplanted tumor tissues. Figure 71 .

[0273] Depend on Figure 71 It can be seen that in PDX transplanted tumors, ACA and A10 treatment had no significant effect on the protein expression level of TRPM2, but could significantly downregulate the expression level of Ki67, a key proliferation protein.

[0274] Depend on Figures 71 to 73It can be seen that compared with the control group, A10 treatment can significantly reduce the protein expression levels of Cyclin D1, Cyclin E1, and Cyclin A2, indicating that inhibition of TRPM2 can significantly inhibit the cell proliferation level of PDX transplanted tumors and the expression level of G1 / S phase cell cycle regulatory proteins. In Huh-7 transplanted tumors, compared with the shNC control group, the immunohistochemical staining level of TRPM2 in the transplanted tumor tissue of the shTRPM2 group was significantly reduced, proving that TRPM2 was successfully knocked down. The results of Ki67 staining also confirmed that after TRPM2 knockdown, the cell proliferation level of Huh-7 transplanted tumor tissue was significantly reduced.

[0275] The above results fully demonstrate that inhibiting or knocking down TRPM2 can significantly inhibit the cell proliferation level of PDX transplanted tumors and Huh-7 transplanted tumors, and downregulate the expression of G1 / S phase cell cycle regulation-related proteins.

[0276] Example 11: TRPM2 is highly expressed in human liver cancer tissue and negatively correlated with patient prognosis

[0277] We used bioinformatics methods to analyze the GSE124535 dataset from The Cancer Genome Atlas (TCGA) and GEO databases. mRNA expression data from 49 normal liver tissues (non-tumor) and 361 liver cancer tissues (tumor) were extracted from the TCGA database. RNA sequencing data from 35 paired liver cancer and normal liver tissues were obtained from the GSE124535 cohort. Comparative analysis of TRPM2 mRNA expression levels in normal and liver cancer tissues from the two databases revealed that TRPM2 mRNA expression in liver cancer tissues was significantly higher than in normal liver tissues. Subsequently, we divided the 361 liver cancer patients from the TCGA database into high and low TRPM2 expression groups based on TRPM2 mRNA expression. Kaplan-Meier survival analysis was performed using the "Survival Analysis" module in GEPIA2. The results showed that patients in the high TRPM2 expression group had significantly worse survival than those in the low TRPM2 expression group (P = 0.041, HR = 1.4). These results suggest that high TRPM2 expression is closely related to poor prognosis in patients with liver cancer.

[0278] To validate the results of our bioinformatics analysis, we collaborated with the Department of Hepatobiliary Surgery at the First Affiliated Hospital, Zhejiang University School of Medicine, and obtained frozen surgically resected tissue samples from 104 paired clinical HCC patients and 87 paired paraffin-embedded tissue samples. We first analyzed TRPM2 protein expression by Western blot and found that TRPM2 protein expression was higher in HCC tissue than in normal liver tissue in 88 of the 104 patients, representing 84.6% of the total cases. Quantitative statistical analysis further confirmed that TRPM2 protein expression was significantly upregulated in HCC tissue. Subsequently, H&E staining and TRPM2 immunohistochemical staining were performed on paraffin-embedded normal and HCC tissue samples. Similarly, TRPM2 staining intensity was significantly higher in HCC tissue than in normal liver tissue in 53 of the 87 patients.

[0279] We also conducted regular follow-up on 104 patients and verified the correlation between TRPM2 expression levels and overall survival. We also divided patients into high-TRPM2 expression and low-TRPM2 expression groups based on the median relative TRPM2 expression levels detected by Western blot. Kaplan-Meier overall survival comparisons revealed that patients in the high-TRPM2 expression group had significantly shorter postoperative overall survival than those in the low-TRPM2 expression group (HR = 2.044, P = 0.0285), consistent with the results of bioinformatics analysis. In summary, it is clear that TRPM2 is highly expressed in HCC tissues and negatively correlated with patient prognosis.

[0280] Example 12: Drug Screening to Obtain TRPM2 Inhibitors

[0281] The inventors designed the following 20 molecules based on the structure of A10. The molecular structures of A10 and the following 20 TRPM2 inhibitors are shown in Table 1. The ability to inhibit PDX and Huh-7 transplanted tumors was verified at the animal level using the same method as Example 8.

[0282] Table 1

[0283]

[0284] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and that in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention. Sequence Listing <110> Zhejiang University <120> Uses of TRPM2 inhibitors <130> P220158-1CNCNB5 <140> 202210209735X <141> 2022-03-03 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> twenty one <212> DNA <213> artificial sequence <400> 1 gcgtgatcta ccacctcatg a 21 <210> 2 <211> twenty one <212> DNA <213> artificial sequence <400> 2 ggccaaggac atgaagtttg t 21

Claims

1. TRPM2 inhibitors are used to prepare drugs for preventing and / or treating liver cancer; wherein, The TRPM2 inhibitor is sh-RNA, and the sh-RNA is a polynucleotide with a sequence as shown in SEQ ID NO.1 or a polynucleotide with a sequence as shown in SEQ ID NO.2; Alternatively, the TRPM2 inhibitor is ACA and / or A10.

2. An isolated polynucleotide, characterized in that The polynucleotide is a polynucleotide having a sequence as shown in SEQ ID NO.1; or a polynucleotide having a sequence as shown in SEQ ID NO.

2.

3. A carrier, characterized in that The vector comprises the polynucleotide according to claim 2.

4. The carrier according to claim 3, wherein The vector is a lentiviral vector.

5. A host cell, characterized in that The host cell comprises the vector according to claim 3 or 4.

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