Application of MED8 inhibitor in preparation of medicine for reversing drug resistance of sorafenib

Abstract

The invention relates to the field of biological medicine, in particular to application of an MED8 inhibitor in preparation of a product for reversing drug resistance of sorafenib or increasing sensitivity of sorafenib. According to the invention, a sorafenib-resistant HCC cell strain is constructed, and data shows that MED8 is remarkably up-regulated in sorafenib-resistant HCC cells, and the sensitivity of MED8 to sorafenib can be increased by reducing the expression of MED8 in the sorafenib-resistant HCC cells; mED8 interacts with TRIP4 to stabilize TRIP4 protein, so that EMT is induced, and drug resistance of HCC cells to sorafenib is promoted; the drug resistance of sorafenib in the HCC is reversed by targeting an MED8-TRIP4 axis; a new target and a treatment mechanism are provided for developing a novel treatment medicine based on sorafenib, and the treatment effect on HCC patients is improved.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to use of a MED8 inhibitor in the preparation of a drug for reversing sorafenib resistance. Background Art

[0002] Epithelial-mesenchymal transition (EMT) is a process by which epithelial cells acquire mesenchymal traits, and cancer cells can exploit the EMT process to enhance tumorigenesis, invasion, metastasis, stemness, therapeutic resistance, and plasticity. Accumulating evidence has shown that EMT significantly contributes to HCC resistance to sorafenib, emphasizing its role as a key mechanism of HCC therapeutic resistance. However, the mechanisms by which EMT affects sorafenib resistance in HCC remain poorly understood. Thyroid hormone receptor interactor 4 (TRIP4), a component of the tetrameric ASC-1 transcriptional co-integrator complex, is essential for EMT. TRIP4 regulates cellular processes such as proliferation, differentiation, DNA repair, and apoptosis, affecting EMT and contributing to therapeutic resistance in various cancers. However, the role of TRIP4-mediated EMT in HCC sorafenib resistance and its upstream regulatory mechanisms remain unclear.

[0003] Mediator complex subunit 8 (MED8), part of the Mediator complex head module, is essential for RNA polymerase II regulation and mediates interactions with regulatory proteins during mRNA transcription. Multiple studies have shown that MED8 contributes to tumor progression. However, the role and regulatory mechanism of MED8 in HCC drug resistance have not been reported.

[0004] Thyroid hormone receptor interactor 4 (TRIP4) is a component of the tetrameric ASC-1 transcriptional co-integrator complex and is essential for EMT. TRIP4 regulates cellular processes such as proliferation, differentiation, DNA repair, and apoptosis. It affects EMT and contributes to therapeutic resistance in various cancers. However, the role of TRIP4-mediated EMT in sorafenib resistance in HCC and its upstream regulatory mechanisms remain unclear.

[0005] Experimental qRT-PCR and western blot analysis showed that MED8 mRNA and protein levels in HCC tissues were significantly increased compared with adjacent noncancerous tissues and were associated with advanced TNM stage and poor overall survival. Functional assays showed that reduced MED8 expression inhibited HCC cell proliferation and EMT, promoted cell apoptosis, and increased sensitivity to sorafenib. MED8 overexpression increased TRIP4 protein levels, TRIP4 overexpression offset the effects of MED8 knockdown, and TRIP4 inhibition inhibited MED8-driven EMT. Mechanistically, MED8 interacted with TRIP4, reduced its ubiquitination, and stabilized TRIP4 protein levels. Summary of the invention

[0006] In view of the above-mentioned deficiencies in the prior art, the present invention provides an application of MED8 in sorafenib resistance.

[0007] The technical solution adopted by the present invention is:

[0008] Use of MED8 inhibitors in preparing products for reversing sorafenib resistance or increasing sorafenib sensitivity.

[0009] Preferably, a product for reversing sorafenib resistance is provided, comprising the MED8 inhibitor for use.

[0010] Preferably, the MED8 inhibitor includes: one or more substances that inhibit the activity of MED8, and substances that inhibit the expression and stability of MED8, or reduce its effective action time.

[0011] Preferably, the MED8 inhibitor includes: gene editing substances targeting MED8, interfering molecules that specifically interfere with the expression of the coding gene of MED8, or homologous recombination substances or site-directed mutagenesis substances targeting MED8, gene editing substances, homologous recombination substances or site-directed mutagenesis substances. Finally, further screening found that the small molecule compound 7695-0983 can target and inhibit the expression of FAT10, thereby enhancing the sensitivity of drug-resistant cells to sorafenib treatment.

[0012] Preferably, an anti-tumor pharmaceutical composition is provided, comprising an effective dose of the product and sorafenib.

[0013] Preferably, use of a pharmaceutical composition of the product in preparing an anti-tumor product is provided.

[0014] The beneficial effects of the present invention compared with the prior art are as follows:

[0015] The application of MED8 in sorafenib resistance of the present invention is to construct sorafenib-resistant HCC cell lines, and it is found that the expression of MED8 is significantly upregulated therein. Knocking down the expression of MED8 in resistant cells can increase their sensitivity to sorafenib treatment. The results show that silencing MED8 shortens the half-life of TRIP4 protein, while overexpression of MED8 prolongs the half-life. Mechanism studies show that MED8 regulates EMT by interacting with thyroid hormone receptor interacting protein 4 (TRIP4), reducing its ubiquitination and stabilizing TRIP4 protein levels.

[0016] The present invention constructs a sorafenib-resistant HCC cell line, and data show that MED8 is significantly upregulated in sorafenib-resistant HCC cells, and reducing the expression of MED8 in sorafenib-resistant HCC cells can increase their sensitivity to sorafenib.

[0017] The present invention finds that MED8 interacts with TRIP4 to stabilize TRIP4 protein, thereby inducing EMT and promoting the resistance of HCC cells to sorafenib.

[0018] This invention provides a new mechanistic insight into reversing sorafenib resistance in HCC by targeting the MED8-TRIP4 axis, providing the possibility for developing new therapeutic strategies.

[0019] The present invention provides a new target and therapeutic mechanism for the development of a new therapeutic drug based on sorafenib, and is expected to improve the therapeutic effect of HCC patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Expression level of mediator complex subunit 8 (MED8) in hepatocellular carcinoma (HCC) and its relationship with patient prognosis; (A) shows the mRNA expression level of MED8 in hepatocellular carcinoma (LIHC). (B) shows the expression level of MED8 protein in HCC; (C) The difference in MED8 expression between normal and tumor tissues was further quantified by MED8 score, and the expression of MED8 in tumor tissues was significantly higher than that in normal tissues; (D) Immunohistochemistry (IHC) analysis showed the protein expression of MED8 in normal and tumor tissues; (E) The mRNA expression level of MED8 in non-tumor and tumor tissues was compared by IHC analysis; (F) Western blot analysis showed the protein expression level of MED8 in different HCC samples; (G) Kaplan-Meier survival curve showed the relationship between MED8 expression level and patient overall survival (OS); (H) Another set of Kaplan-Meier survival curves further confirmed the relationship between MED8 expression and patient prognosis.

[0021] Figure 2 Expression levels of MED8 in different hepatocellular carcinoma (HCC) cell lines and their effects on sorafenib sensitivity; (A) Western blot analysis showed the protein expression levels of MED8 in different HCC cell lines (L02, HepG2, SMMC7721, MHCC97H, HCCLM03 and Huh7); (B) Quantitative analysis of the relative expression levels of MED8 protein in part A; (C) The relative expression levels of MED8 mRNA in different HCC cell lines were shown; (D) Cell viability assay showed the sensitivity of different HCC cell lines to sorafenib; (E) Western blot analysis of the protein expression levels of MED8 in sorafenib-resistant HCC cell lines (Huh7SR and HCCLM03 / SR); (F) Quantitative analysis of the relative expression levels of MED8 protein in resistant HCC cell lines in part E.

[0022] Figure 3 The role of MED8 in HCC cells, especially its impact on sorafenib resistance; (A) shows that in MHCC97H cells, after knocking down MED8 (shMED8-1# and shMED8-2#) by shRNA technology, the sensitivity of cells to sorafenib increased; (B) shows that knocking down MED8 (shMED8-1# and shMED8-2#) in HCCLM03 cells also increased the sensitivity to sorafenib; (C) After overexpressing MED8 (pcDNA-MED8) in HepG2 cells, the sensitivity of cells to sorafenib decreased, and the IC50 value showed that cells overexpressing MED8 had enhanced resistance to sorafenib; (D) Immunofluorescence analysis showed that after knocking down MED8 in MHCC97H and HCCLM03 cells, E-cadherin ( (E) EdU incorporation assay showed that knockdown of MED8 reduced the proliferation rate of MHCC97H and HCCLM03 cells; (F) Flow cytometry analysis showed that knockdown of MED8 increased the apoptosis rate of MHCC97H and HCCLM03 cells; (G) The results of part D were further confirmed, showing that knockdown of MED8 significantly increased the proportion of apoptotic cells in MHCC97H and HCCLM03 cells; (H) Flow cytometry analysis was used to explore the effect of MED8 on sorafenib resistance in HCC cells; (I) Subcutaneous xenograft model showed that MHCC97H cells with MED8 knockdown responded better to sorafenib treatment in vivo, and tumor growth was significantly inhibited.

[0023] Figure 4The role of MED8 in hepatocellular carcinoma (HCC) cells, especially its impact on epithelial-mesenchymal transition (EMT) and sorafenib resistance; (A) shows the association analysis of MED8 gene expression and gene sets associated with sorafenib resistance in HCC cells; (B) Immunofluorescence analysis showed that in MHCC97H and HCCLM03 cells, E-cadherin (epithelial cell marker) expression increased, while vimentin (mesenchymal cell marker) expression decreased after knockdown of MED8 (shMED8-1#); (C) Western blot analysis showed that in MHCC97H / SR and HCCLM03 / SR cells, E-cadherin and N-cadherin (epithelial cell markers) expression increased, while vimentin expression decreased after knockdown of MED8; (D) Cell viability assay showed that in MHCC97H / SR cells, knockdown of MED8 increased the sensitivity of cells to sorafenib; (E) Western Blot analysis showed that in HCCLM03 / SR cells, the expression of E-cadherin and N-cadherin increased, while the expression of vimentin decreased after knocking down MED8, which was consistent with the results in part C; (F) Cell viability assay showed that in HCCLM03 / SR cells, the sensitivity of cells to sorafenib was also increased after knocking down MED8, and the addition of TGF-β could reverse this effect; (G) Apoptosis analysis showed that in MHCC97H / SR and HCCLM03 / SR cells, the apoptosis rate was increased after knocking down MED8; (H) Flow cytometry analysis showed that in MHCC97H / SR and HCCLM03 / SR cells, the proportion of apoptotic cells increased after knocking down MED8; (I) Subcutaneous xenograft model showed that MHCC97H cells with MED8 knockdown responded to sorafenib treatment in vivo.

[0024] Figure 5Interaction between MED8 and TRIP4 in hepatocellular carcinoma (HCC) and its impact on sorafenib resistance; (A) A protein interaction network is shown, highlighting the potential interaction between MED8 and TRIP4; (B) The figure shows the changes in the expression levels of multiple EMT-related proteins after knockdown of MED8 (shMED8) in MHCC97H / SR cells; (C) Western blot analysis showed that MED8 and TRIP4 were highly expressed in HCC tissues; (D) The relative expression levels of MED8 protein in non-tumor and tumor tissues in part C were quantitatively analyzed; (E) The relative expression levels of TRIP4 protein in non-tumor and tumor tissues were also quantified; (F) The scatter plot shows the positive correlation between the expression levels of MED8 and TRIP4 proteins; (G) Western Blot analysis showed that in MHCC97H cells, the expression level of TRIP4 protein was reduced after knockdown of MED8 (shMED8-1# and shMED8-2#); (H) Similar results were observed in HCCLM03 cells, and TRIP4 protein expression was reduced after knockdown of MED8; (I) Western blot analysis showed that in HepG2 cells, the expression level of TRIP4 protein was increased after overexpression of MED8; (J) In MHCC97H / SR cells, co-transfection experiments showed the co-localization of MED8 and TRIP4 in cells; (K) In HCCLM03 / SR cells, co-transfection experiments also showed the co-localization of MED8 and TRIP4; (L) In HepG2 / SR cells, co-transfection experiments showed the co-localization of MED8 and TRIP4.

[0025] Figure 6The stability of the MED8-NEDD4 complex in liver cancer cells and its relationship with proteasome degradation; (A) qRT-PCR analysis of the changes in the mRNA levels of MED8 and TRIP4 after knockdown of MED8 (shMED8-1# and shMED8-2#) in MHCC97H / SR and HCCLM03 / SR cells; (B) Co-immunoprecipitation (Co-IP) experiments showed the interaction between TRIP4 and MED8 in MHCC97H / SR cells; (C) Immunofluorescence analysis showed the interaction between TRIP4 and MED8 in MHC Co-localization of TRIP4 and MED8 in C97H / SR and HCCLM03 / SR cells; DAPI was used to stain the nucleus, while TRIP4 and MED8 were labeled with green and red fluorescence, respectively; (D) The molecular docking model predicted the interaction interface between TRIP4 and MED8, showing the three-dimensional structure and interaction region of the two proteins; (E) The schematic diagram shows the different domains of TRIP4 protein, including ZF-C2HC5, ZF, ASCH domains, and the key region for interaction with MED8. (F) Western blot analysis of the changes in TRIP4 protein levels after overexpression of MED8 in HepG2 / SR cells.

[0026] Figure 7Effect of MED8 on TRIP4 protein stability and its role in HCC; (A) Western blot analysis showed changes in TRIP4 protein expression levels in MHCC97H / SR cells in the presence of proteasome inhibitor MG132; (B) Similar results were observed in HCCLM03 / SR cells, where MG132 treatment increased TRIP4 protein expression; (C) Western blot analysis showed changes in TRIP4 protein expression levels in HepG2 / SR cells after overexpression of MED8; (D) In ​​MHCC97H / SR cells, TRIP4 protein expression levels were reduced after knockdown of MED8 (shMED8-1#); (E) In HCCLM03 / SR cells, knockdown of MED8 also led to reduced TRIP4 protein expression; (F) Western Blot analysis showed the changes in TRIP4 protein expression levels after overexpression of MED8 in HepG2 / SR cells; (G) The half-life of TRIP4 protein was evaluated by adding the protein synthesis inhibitor cyclohexamide (CHX); (H) Similar results were observed in HCCLM03 / SR cells, where the half-life of TRIP4 protein was shortened after knockdown of MED8; (I) In HepG2 / SR cells, the half-life of TRIP4 protein was prolonged after overexpression of MED8; (J) Cell viability assay showed that in MHCC97H / SR cells, knockdown of (K) In HCCLM03 / SR cells, knockdown of MED8 also reduced the sensitivity of cells to sorafenib; (L) In HepG2 / SR cells, overexpression of MED8 increased the sensitivity of cells to sorafenib; (M) Co-immunoprecipitation (Co-IP) experiments showed the interaction between TRIP4 and MED8 in MHCC97H / SR cells; (N) The interaction between TRIP4 and MED8 was also observed in HCCLM03 / SR cells; (O) Western blot analysis showed the changes in the ubiquitination level of TRIP4 in HepG2 / SR cells after overexpression of MED8.

[0027] Figure 8 Resistance regulation model, describing how MED8 and TRIP4 affect sorafenib resistance in hepatocellular carcinoma (HCC) cells, especially by regulating the process of epithelial-mesenchymal transition (EMT). Left (blue background) - sorafenib-resistant HCC cells; right (pink background) - sorafenib-sensitive HCC cells; molecular structure of sorafenib at the bottom. This conceptual model summarizes the interaction between MED8 and TRIP4 in HCC and their regulatory effects on EMT and sorafenib resistance, providing a theoretical basis for the development of new HCC treatment strategies.

[0028] Fig. 9 In drug-resistant liver cancer cells, mediator complex subunit 8 (MED8) promotes cell resistance by regulating autophagy; (A) Pearson correlation analysis between MED8 expression and autophagy activity score in TCGA-LIHC patients. (B) Immunofluorescence analysis of autophagy activity in MHCC97H / SR and HCCLM03 / SR blank control group, MED8 knockdown group, control group with autophagy activator Rapamycin group, and MED8 knockdown group with Rapamycin added; (C) Electron microscopy analysis of autophagy activity in MHCC97H / SR and HCCLM03 / SR blank control group, MED8 knockdown group, control group with Rapamycin added, and MED8 knockdown group with Rapamycin added. (D) Western Blot experiments were used to detect the expression levels of Beclin1, LC3I, and LC3II in the blank control group, MED8 knockdown group, control group plus Rapamycin group, and MED8 knockdown plus Rapamycin group of MHCC97H / SR and HCCLM03 / SR; (E) Drug sensitivity tests were performed to determine the IC50 values ​​of the blank control group, MED8 knockdown group, control group plus Rapamycin group, and MED8 knockdown plus Rapamycin group of MHCC97H / SR and HCCLM03 / SR. DETAILED DESCRIPTION

[0029] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0030] Attached Figure 2 It is known that mediator complex subunit 8 (MED8) is part of the head module of the large multiprotein complex Mediator, which is essential for RNA polymerase II regulation and mediates interactions with regulatory proteins during mRNA transcription. Preclinical studies of the present invention revealed that increased expression of MED8 in sorafenib-resistant HCC was associated with advanced TNM stage and poor overall survival. Reduced MED8 expression inhibited HCC cell proliferation and epithelial-mesenchymal transition (EMT), promoted cell apoptosis, and increased sensitivity to sorafenib. Overexpression of MED8 increased thyroid receptor interacting protein 4 (TRIP4) protein levels. TRIP4 overexpression offset the effects of MED8 knockdown, while TRIP4 inhibition inhibited MED8-driven EMT. Mechanistically, MED8 interacts with TRIP4, reduces its ubiquitination and stabilizes TRIP4 protein levels. The MED8-TRIP4 axis plays a role in sorafenib resistance in HCC and can be used as a therapeutic target for HCC treatment.

[0031] MED8 is a ubiquitin-like protein. The preclinical study of the present invention revealed that MED8 was significantly upregulated in sorafenib-resistant liver cancer cells, and reducing the expression of MED8 in sorafenib-resistant liver cancer cells can increase their sensitivity to sorafenib, and clarified its specific mechanism: MED8 overexpression significantly inhibits TRIP4 ubiquitination, thereby stabilizing the expression of EMT and affecting the sensitivity of HCC targeted therapy, promoting the resistance of liver cancer cells to sorafenib.

[0032] The first aspect of the present invention provides use of a MED8 inhibitor in preparing a product for reversing sorafenib resistance or increasing sorafenib sensitivity.

[0033] Sorafenib is an oral multi-kinase inhibitor that can target serine and / or threonine and receptor tyrosine kinases in tumor cells and tumor blood vessels, and has the dual effects of inhibiting tumor cell proliferation and angiogenesis. Sorafenib has a certain therapeutic effect on renal cancer, liver cancer, melanoma and non-small cell lung cancer. The FDA has approved sorafenib for the treatment of liver cancer. However, due to the complex progression of liver cancer, patients who respond to sorafenib treatment often respond for a maximum of about 4 months (median PFS and TTP are both 3.7 months), and then develop drug resistance, and disease progression occurs after drug resistance.

[0034] In the use provided by the present invention, the MED8 inhibitor includes: one or more substances that inhibit the activity of MED8, and substances that inhibit the expression and stability of MED8, or reduce its effective action time.

[0035] In the use provided by the present invention, the MED8 inhibitor includes: a gene editing substance targeting MED8, an interfering molecule that specifically interferes with the expression of the gene encoding MED8, or a homologous recombination substance or a site-directed mutagenesis substance targeting MED8, wherein the gene editing substance, the homologous recombination substance or the site-directed mutagenesis substance causes a functional weakening or loss-of-function mutation in MED8.

[0036] In the use provided by the present invention, the product has at least one of the following effects:

[0037] 1) Slow down the tumor growth rate;

[0038] 2) Increase the expression level of apoptosis-related proteins;

[0039] 3) reduce tumor volume;

[0040] 4) reduce tumor weight;

[0041] 5) Increase sensitivity to sorafenib or inhibit sorafenib resistance.

[0042] In the use provided by the present invention, it was found that reduced expression of MED8 inhibited HCC cell proliferation and epithelial-mesenchymal transition (EMT), promoted cell apoptosis, and increased sensitivity to sorafenib. Overexpression of MED8 increased the level of thyroid receptor interacting protein 4 (TRIP4) protein. TRIP4 overexpression offset the effect of MED8 knockdown, while TRIP4 inhibition inhibited MED8-driven EMT. TRIP4 is essential for regulating cell migration, invasion, and EMT. Ubiquitination is an important mechanism for regulating TRIP4 protein expression. MED8 directly interacts with TRIP4 in HCC cells, resulting in degradation of TRIP4 through the ubiquitin-proteasome pathway. MED8 inhibits ubiquitination and degradation of TRIP4, thereby regulating its protein abundance, promoting EMT, and ultimately leading to sorafenib resistance.

[0043] The indication for sorafenib is a tumor; preferably, the tumor is selected from non-melanoma skin cancer, breast cancer, lung cancer, colorectal cancer, prostate cancer, gastric cancer, liver cancer, cervical cancer, esophageal cancer, thyroid cancer, bladder cancer, non-Hodgkin's lymphoma, pancreatic cancer, leukemia, kidney cancer, endometrial cancer, lip / oral cancer, melanoma, ovarian cancer, brain and central nervous system tumors, laryngeal cancer, multiple myeloma, nasopharyngeal cancer, gallbladder cancer, bile duct cancer, oropharyngeal cancer, hypopharyngeal cancer, Hodgkin's lymphoma, testicular cancer, salivary gland cancer, vulvar cancer, penile cancer, Kaposi's sarcoma, mesothelioma or vaginal cancer. In a specific embodiment of the present invention, the tumor refers to liver cancer. More specifically, liver cancer cells can be, for example, HepG2 and Huh7 cells. The present invention has found that MED8 is significantly upregulated in sorafenib-resistant liver cancer cells, and reducing the expression of MED8 in sorafenib-resistant liver cancer cells can increase their sensitivity to sorafenib.

[0044] A second aspect of the present invention provides a product for reversing sorafenib resistance, including the MED8 inhibitor in the use.

[0045] The third aspect of the present invention provides a method for reversing sorafenib resistance, comprising administering a therapeutically effective amount of the aforementioned product to an individual.

[0046] In the method provided by the present invention, the individual is a mammal, such as a rodent, an artiodactyl, a perissodactyl, an immunomodulator, a primate, etc. Primates are, for example, monkeys, apes or Homo sapiens. The individual can benefit from treatment with the aforementioned product.

[0047] In the present invention, "therapeutically effective amount" generally refers to an amount that can achieve the effect of reversing sorafenib resistance after an appropriate administration period; specifically, it is embodied in: increasing the sensitivity of sorafenib-resistant cells to sorafenib by inhibiting autophagy mediated by the NEDD4-PTEN / AKT axis.

[0048] The third aspect of the present invention provides an anti-tumor pharmaceutical composition, comprising an effective dose of the aforementioned product and sorafenib.

[0049] The fourth aspect of the present invention provides an anti-tumor method, comprising administering a therapeutically effective amount of the aforementioned pharmaceutical composition to an individual.

[0050] In the method provided by the present invention, the individual is a mammal, such as a rodent, an artiodactyl, a perissodactyl, a lagomorph, a primate, etc. Primates are, for example, monkeys, apes or Homo sapiens. The individual can benefit from treatment with the aforementioned drug combination.

[0051] In the present invention, the effective dose refers to the dose at which the drug can show its efficacy. Because the drug must have a certain dose to be absorbed by the body before a certain drug concentration can be reached, and the drug effect can only appear when a certain drug concentration is reached. If the dose is too small, the effective concentration cannot be obtained in the body, and the drug cannot play its effective role. However, if the dose is too large and exceeds a certain limit, the effect of the drug may change qualitatively and may produce different degrees of toxicity to the body. Therefore, in order to exert the effective effect of the drug while avoiding its adverse reactions, it is necessary to strictly control the dosage range of the drug.

[0052] The pharmaceutical composition provided by the present invention also includes pharmaceutically acceptable carriers, additives, adjuvants or excipients.

[0053] Pharmaceutically acceptable carriers generally refer to carriers used for the administration of therapeutic agents, which do not induce the production of antibodies harmful to the individual receiving the composition and are not excessively toxic after administration. These carriers are well known to those skilled in the art, for example, Remington's Pharmaceutical Sciences (Mack Pub. Co., NJ 1991) discloses relevant content about pharmaceutically acceptable carriers. Specifically, the carrier may be a combination of one or more of, including but not limited to, saline, buffer, glucose, water, glycerol, ethanol, adjuvants, and the like.

[0054] The pharmaceutical composition provided by the present invention can be adapted to any form of administration, which can be oral or parenteral administration, for example, it can be pulmonary, nasal, rectal and / or intravenous injection, and more specifically, it can be intradermal, subcutaneous, intramuscular, intra-articular, intraperitoneal, pulmonary, oral, sublingual, nasal, transdermal, vaginal, oral or parenteral administration.

[0055] Those skilled in the art can select a suitable formulation form according to the mode of administration. For example, the formulation form suitable for oral administration may include but is not limited to pills, tablets, chewable tablets, capsules, granules, drops or syrups, etc. For another example, the formulation form suitable for parenteral administration may include but is not limited to solutions, suspensions, reconstitutable preparations or sprays, etc. For another example, the formulation form suitable for rectal administration may generally be a suppository.

[0056] In the present invention, "therapeutically effective amount" generally refers to an amount that can achieve an anti-tumor effect after an appropriate administration period.

[0057] In the present invention, "therapeutically effective amount" generally refers to an amount that can achieve an anti-tumor effect after an appropriate administration period.

[0058] The present invention is further described below by way of examples, but the scope of the present invention is not limited thereby.

[0059] Example 1

[0060] Cell culture and reagents

[0061] Human HCC cell lines (MHCC97H, HCCLM03, and HuH7) were obtained from the National Authentication Cell Culture Collection of the Chinese Academy of Sciences. To establish sorafenib-resistant clones, MHCC97H, HCCLM03, and HepG2 cells were exposed to increasing concentrations of sorafenib to induce resistance. Cells were cultured in DMEM containing 10% FBS and 1% P / S at 37°C in a 95% air, 5% CO2 incubator and low-dose sorafenib was added. After an initial 24-hour culture under standard conditions, resistant cell cultures were treated with 10 ng / mL of TGF-β for 48 hours to induce EMT.

[0062] Cell transfection

[0063] shRNA-mediated RNA duplexes targeting MED8 and TRIP4 were synthesized by Sangon Biotech (Shanghai, China). Plasmids of FLAG-MED8, MYC-TRIP4, MYC-TRIP4-zinc finger (ZF), MYC-TRIP4-ΔActivating Signaling Cointegrator 1 complex homology (ASCH), and MYC-TRIP4-ASCH were constructed and provided by Aardman Biotech (Nanchang, China). Plasmid encoding fusion protein wMYC-Ub was purchased from Miaoling Biotechnology (Wuhan, China). HCC cells were transfected with shRNA or plasmids using Lipofectamine 3000. Stable HCC cell lines transfected with sh-NC, shMED8-1, or shMED8-2 plasmids were prepared using puromycin. shRNA targeting MED8 sequence: shMED8-1 #5'-GGGAGUUUCAUUUGCAAGUTT-3' and shMED8-2 #5'-CAAACCUUCUGGAGAAAAUCA-3'

[0064] Clinical HCC specimens

[0065] Fresh HCC tissues and adjacent liver tissues from 40 patients who underwent HCC resection in Jiangxi Cancer Hospital were obtained. Informed consent was obtained from each patient, and the study protocol was approved by the Ethics Committee of Jiangxi Cancer Hospital (Registration No. 2022ky308).

[0066] Liquid chromatography-tandem mass spectrometry (LC-MS / MS) analysis

[0067] MHCC97H / SR cells were transfected with sh-NC or shMED8 and trypsinized and quantified. Approximately 20 μL of 1× electrophoresis buffer was used for this procedure. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE) gels were stained with Coomassie Brilliant Blue for 3 hours and then washed with eluent overnight. The stained gels were then analyzed using LC-MS / MS according to established methods.

[0068] Immunohistochemical analysis

[0069] Immunohistochemistry (IHC) analysis was performed according to established protocols. The primary antibodies used included anti-MED8 from Proteintech (Wuhan, China). Two pathologists from the Department of Pathology, Jiangxi Cancer Hospital, independently evaluated the protein expression intensity in normal paracancerous tissues and HCC tissues. The staining intensity was scored according to previously established criteria.

[0070] Apoptosis analysis

[0071] Apoptosis was assessed by flow cytometry using the Annexin V-FITC / PI apoptosis detection kit following the manufacturer's guidelines. Briefly, 3 × 10^5 cells were seeded into 6-well plates. Cells were harvested after sorafenib treatment, stained with Annexin V-FITC and propidium iodide (PI) for 15 min at room temperature in the dark, and analyzed by flow cytometry. All experiments were performed at least three times.

[0072] Immunofluorescence analysis

[0073] Immunofluorescence analysis followed standard protocols. Briefly, HCC cells were seeded onto coverslips and cultured under different treatment conditions for 24 h. Direct immunostaining of E-cadherin and vimentin was performed on HCC cells. After fixation and permeabilization, cells were incubated with Alexa-Fluor 594 anti-E-cadherin and Alexa-Fluor 488 anti-vimentin antibodies at 4 °C overnight. Nuclei were stained with DAPI, and all antibodies were diluted in PBS containing 1% bovine serum albumin. The fluorescence intensity of the cells was examined using a fluorescence microscope.

[0074] The primer sequences used for qRT-PCR detection are as follows:

[0075] MED8:

[0076] 5'-ATTGGAGACCTTCAGGCAGC-3'(forward,SEQ ID NO:1)

[0077] 5′-ATGCATGGAAGCCGACTTGA-3′

[0078] TRIP4:

[0079] 5'-TGAGAGTGCTGAAGAGATAC-3'

[0080] 5'-CTGTCTGTTTCTCCCTTTCT-3'

[0081] GAPDH:

[0082] 5′-AGAAGGCTGGGGCTCATTTG-3′

[0083] 5′-AGGGGCCATCCACAGTCTTC-3′

[0084] Subcutaneous xenograft experiments

[0085] The tumor-forming ability of MED8 knockdown-stable MHCC97H cells was evaluated in a subcutaneous xenograft mouse model, with or without sorafenib treatment. Six-week-old female BALB / c nude mice were obtained from Beijing Sibeifu Biotechnology Co., Ltd. (Beijing, China). MHCC97H-shNC and MHCC97H-shMED8-1# cell lines were constructed. A total of 1×10^7 cells were suspended in 200μL PBS and injected subcutaneously into the right flank of each mouse. One week later, mice in the treatment group were injected intraperitoneally with sorafenib (30mg / kg) daily. Tumor size was recorded every 5 days with a caliper, and the volume (V) was calculated using the formula (L×W^2) / 2. After the completion of the study, the mice were euthanized, the tumors were excised, and the photos were taken. All animal experiments were performed at Kangtai Medical Laboratory Services Hebei Co., Ltd. (KT2024-06-24-01).

[0086] Proteasome activity assay

[0087] Proteasome activity was assessed using the Abcam Proteasome Activity Assay Kit (ab107921). MHCC97H / SR, HCCLM03 / SR, and HepG2 / SR cells were transfected with specific plasmids and shRNAs, and trypsin digested and quantified. Each 1 mg protein extract was mixed with assay buffer to a final volume of 100 μL and added in triplicate to individual wells of a 96-well black plate. Proteasome activity in each well was assessed by monitoring the change in fluorescence after the introduction of proteasome substrates. Excitation and emission were measured at 350 / 440 nm wavelengths for 25 minutes at 37°C using an Infinite 200PRO microplate reader.

[0088] Other tests

[0089] Western blotting, qRT-PCR, and co-immunoprecipitation assays were performed; cell function assays, such as CCK-8 and EdU staining, were performed19. All primers were synthesized by Sangon Company.

[0090] Statistical analysis

[0091] Statistical analysis was performed using SPSS22.0 or GraphPad Prism 9 software, and all results are expressed as standard deviation (SD). Statistical comparisons between two groups were performed using Student's t-test, while analysis of variance with post hoc tests was applied for multiple group comparisons. Correlations were assessed using Spearman correlation analysis, with statistical significance at p < 0.05.

[0092] Table 1. Association between MED8 expression and clinicopathological characteristics of HCC patients in the training and validation cohorts.

[0093]

[0094]

[0095] Note: P values ​​represent the probability of MED8 expression levels between variable subgroups determined by the test. Abbreviations: AFP, alpha-fetoprotein; HBsAg, hepatitis B surface antigen; TNM, tumor-node-metastasis.

[0096] The table suggests that elevated MED8 expression was significantly associated with sex, tumor number, tumor microsatellites, venous penetration, and advanced TNM stage, but not with age, HBsAg, tumor size, tumor capsule, cirrhosis, or lymph node metastasis.

[0097] Example 2

[0098] Reducing the expression of MED8 in sorafenib-resistant liver cancer cells can enhance sorafenib-induced apoptosis in vitro and in vivo. In order to study whether MED8 in HCC cells is related to sorafenib resistance, the present invention examined the effect of MED8 on the sensitivity of HCC to sorafenib by treating HCC cells with different concentrations of sorafenib. Cell viability assays showed that inhibition of MED8 increased the sensitivity of MHCC97H and HCCLM03 cells to sorafenib ( Figure 3 A, 3B). Increased expression of MED8 reduces the sensitivity of HepG2 cells to sorafenib ( Figure 3 C). EdU and flow cytometry experiments confirmed that MED8 downregulation enhanced the sorafenib-induced inhibitory effect on HCC cell proliferation ( Figure 3 D). Figure 3 D, 3E) and induction of HCC cell apoptosis ( Figure 3 F, 3G) Lar. After 35 days, the tumor volume and weight of the shMED8-1# and sh-NC+sorafenib groups were lower than those of the sh-NC group, and the reduction in the shMED8-1#+sorafenib group was the most significant ( Figure 3 H-3J). These data suggest that MED8 inhibition enhances the sensitivity of HCC cells to sorafenib.

[0099] Example 3

[0100] Knocking down the expression of MED8 inhibits HCC cell proliferation and epithelial-mesenchymal transition (EMT), promotes cell apoptosis, and increases sensitivity to sorafenib. Previous studies have emphasized the important role of autophagy in sorafenib resistance. Therefore, the present invention further explored whether reducing the expression of MED8 in HCC-SR cells increases sensitivity to sorafenib by affecting autophagy. We used RNA-seq data and clinical information of HCC samples obtained from the TCGA database to evaluate this issue. Our GSVA analysis showed that MED8 expression was associated with multiple EMT markers ( Figure 4 A). Immunofluorescence analysis and examination of changes in the expression of EMT markers after MED8 knockout to elucidate the relationship between MED8 and EMT markers showed that decreased MED8 expression increased E-cadherin levels and decreased vimentin levels, implying that downregulation of MED8 may inhibit EMT in HCC cells. ( Figure 4 B-4F) To verify the involvement of MED8 in sorafenib resistance via EMT, we downregulated MED8 in sorafenib-resistant HCC cells and introduced EMT activators to assess the sensitivity of sorafenib changes. Our results showed that reduced MED8 expression increased the sensitivity of resistant HCC cells, but this effect was offset by EMT activators. Fig. 4F. These findings suggest that MED8 regulates EMT and affects the sensitivity of HCC cells to sorafenib.

[0101] Example 4

[0102] Knockdown of MED8 expression promotes sorafenib-induced apoptosis by inhibiting autophagy in HCC-SR cells. Previous studies have emphasized the important role of autophagy in sorafenib resistance. Therefore, the present invention further explores whether reducing the expression of MED8 in HCC-SR cells increases sensitivity to sorafenib by affecting autophagy. First, bioinformatics analysis results showed that the expression of MED8 was positively correlated with autophagy ( Fig. 9 In addition, immunofluorescence analysis was used to detect autophagy ( Fig. 9 B) level. In the two HCC-SR cell lines, the autophagic response level of shMED8 was reduced compared with the control group. The addition of Rapamycin could reverse the low autophagic response level caused by shMED8. Next, we used electron microscopy imaging experiments to detect the number of autophagosomes in the two groups of drug-resistant cell lines ( Fig. 9 C), the results showed that the number of autophagosomes in the shMED8 group decreased compared with the control group. After shMED8 was added with Rapamycin, the number of autophagosomes increased compared with the shMED8 group. This once again verified that MED8 played an important role in regulating autophagy. Western blot analysis showed ( Fig. 9D), the levels of Beclin1 and LC3II, autophagy markers, in the shMED8 group were lower than those in the control group, and the level of LC3I was higher; after adding Rapamycin (autophagy activator), the opposite results were obtained. Finally, we conducted cell resistance experiments on the two cell lines. The experimental results showed that compared with the control group, the shMED8 group was more sensitive to sorafenib; compared with the Rapamycin group, the sensitivity of the Rapamycin+shMED8 group to sorafenib was increased. In summary, the data of the present invention show that MED8 enhances its resistance to sorafenib in HCC-SR cells by regulating autophagy.

[0103] Example 5

[0104] Downregulation of MED8 can promote sorafenib-induced apoptosis in HCC resistant cells by inhibiting MED8-driven EMT through suppressing thyroid receptor interacting protein 4 (TRIP4) protein levels. To elucidate the role of MED8 in regulating EMT and affecting sorafenib sensitivity in HCC cells, we used the STRING interaction network to investigate genes interacting with MED8 and predicted that TRIP4 and MED8 interact with each other ( Figure 5 A). We performed triplicate LC-MS / MS analysis of MED8-silenced and control HCC cells. TRIP4 protein expression was decreased in MED8-silenced HCC cells ( Figure 5 B). We then examined the correlation between MED8 and TRIP4 in HCC tissues. Western blot analysis showed that MED8 and TRIP4 were highly expressed in HCC tissues, indicating that their levels were positively correlated ( Figure 5 C-5F). In addition, it was observed that TRIP4 protein expression decreased when MED8 levels were decreased and increased when MED8 expression was upregulated ( Figure 5 G-5I). To determine whether MED8 regulates EMT through TRIP4, we overexpressed TRIP4 in MED8-silenced sorafenib-resistant HCC cell lines and analyzed EMT changes by western blotting. TRIP4 upregulation counteracted the EMT inhibition caused by MED8 downregulation ( Figure 5 J-5K). Inhibition of TRIP4 expression in sorafenib-resistant HCC cells with stable MED8 overexpression suppressed the MED8-induced EMT process ( Figure 5 L). These findings suggest that MED8 regulates EMT via TRIP4 in sorafenib-resistant HCC cells.

[0105] Example 6

[0106] The interaction between MED8 and TRIP4 affects the resistance of HCC cells to sorafenib. The effect of MED8 on TRIP4 expression was evaluated by measuring TRIP4 mRNA levels in sorafenib-resistant HCC cells with MED8 overexpression or knockdown using qRT-PCR. The analysis showed that MED8 did not affect TRIP4 mRNA levels, which means that MED8 regulates TRIP4 expression post-transcriptionally rather than transcriptionally ( Figure 6 A). Subsequent IP analysis confirmed the interaction between MED8 and TRIP4 ( Figure 6 B). Immunofluorescence experiments confirmed the co-localization of TRIP4 and MED8 in sorafenib-resistant HCC cells, supporting their protein-protein interaction ( Figure 6 C). Docking analysis identified the interaction between TRIP4 and MED8 ( Figure 6 D). A series of MYC-tagged TRIP4 truncated plasmids were constructed to characterize the domains that directly interact with MED8. TRIP4 consists of three main domains: the N-terminal domain, the middle ZF domain responsible for DNA binding, and the C-terminal ASCH domain ( Figure 6 E). Using the structural information, a truncated TRIP4 mutant was constructed to identify the domains that interact with MED8. Localization experiments determined that the N-terminal domain (amino acids 1-150) and the ASCH domain (amino acids 301-544) within the C-terminal region of TRIP4 interact with MED8 ( Figure 6 F) These findings suggest that MED8 and TRIP4 interact in sorafenib-resistant HCC cells.

[0107] Example 7

[0108] In HCC cells, MED8 stabilizes TRIP4 protein expression by inhibiting proteasome degradation. This study investigated the molecular mechanism by which MED8 regulates TRIP4 protein expression. Existing literature suggests that MED8 is involved in the formation of ubiquitin ligase complexes. We hypothesize that MED8 affects TRIP4 degradation and ubiquitination in sorafenib-resistant HCC cells. Our results showed that the proteasome inhibitor MG132 inhibits TRIP4 degradation ( Figure 7 A-7C), indicating that TRIP4 undergoes proteasomal degradation in sorafenib-resistant HCC cells. We investigated whether MED8 affects TRIP4 protein stability by regulating proteasomal degradation. MED8 knockdown significantly reduced TRIP4 levels, while MG132 treatment completely prevented this reduction. ( Figure 7D-7E). In contrast, MG132 treatment blocked MED8-mediated upregulation of TRIP4 expression in HepG2 / SR cells (Figure F). To investigate the effect of MED8 expression on the half-life of TRIP4, sorafenib-resistant HCC cells were treated with CHX. Our results showed that silencing MED8 shortened the half-life of TRIP4 protein, whereas MED8 overexpression prolonged the half-life. ( Figure 7 G-7I). Taken together, these data suggest that MED8 regulates TRIP4 protein expression via the ubiquitin-proteasome system. Given that MED8 is involved in the formation of ubiquitin ligases, this raises the possibility that MED8 may broadly regulate the function of the proteasome degradation machinery, leading to nonspecific effects on TRIP4 expression. To test this hypothesis, we performed an in vitro proteasome activity assay and found that neither upregulation nor downregulation of MED8 affected proteasome function in sorafenib-resistant HCC cells. ( Figure 7 J-7L) These findings suggest that altering MED8 does not alter overall proteasome activity but specifically affects TRIP4 expression. Co-IP analysis showed that MED8 knockdown increased endogenous TRIP4 ubiquitination, whereas MED8 overexpression decreased endogenous TRIP4 ubiquitination ( Figure 7 M-7O). These results suggest that MED8 inhibits the degradation of TRIP4 protein, thereby enhancing its stability.

[0109] The above is only a preferred embodiment of the present invention, and does not limit the structure of the present invention in any form. Any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention are within the scope of the technical solution of the present invention.

Claims

1. Use of MED8 inhibitors in the preparation of drugs for reversing sorafenib resistance.

2. Use of MED8 inhibitors in products that increase sorafenib sensitivity.

3. Use of the MED8 inhibitor according to claim 1 in preparing a product for reversing sorafenib resistance or increasing sorafenib sensitivity, characterized in that: Provided is a product for reversing sorafenib resistance, including the MED8 inhibitor used in the application.

4. The use of the MED8 inhibitor according to claim 1 in the preparation of a drug for reversing sorafenib resistance, characterized in that: MED8 inhibitors include: one or more substances that inhibit the activity of MED8, and substances that inhibit the expression and stability of MED8, or reduce its effective action time.

5. The use of the MED8 inhibitor according to claim 1 in the preparation of a drug for reversing sorafenib resistance, characterized in that: MED8 inhibitors include: gene editing substances targeting MED8, interfering molecules that specifically interfere with the expression of the gene encoding MED8, or homologous recombination substances or site-directed mutagenesis substances targeting MED8, gene editing substances, homologous recombination substances or site-directed mutagenesis substances.

6. Use of the MED8 inhibitor according to claim 1 in the preparation of a drug for reversing sorafenib resistance, characterized in that: Provided is an anti-tumor pharmaceutical composition, comprising an effective dose of the product and sorafenib.

7. Use of the MED8 inhibitor according to claim 1 in the preparation of a drug for reversing sorafenib resistance, characterized in that: Provided is the use of the pharmaceutical composition of the product in preparing anti-tumor products.

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