Application of phosphatidylinositol phosphatase SAC1 in prognosis and treatment of liver cancer

Abstract

The invention discloses an application of phosphatidylinositol phosphatase SAC1 in prognosis and treatment of liver cancer. The research shows that the phosphatidylinositol phosphatase SAC1 can obviously inhibit the metastasis of the hepatocellular carcinoma, and a new hepatocellular carcinoma treatment strategy is provided from the perspective of phospholipid metabolism. Therefore, the SAC1 as a novel potential target spot for anti-hepatocellular carcinoma metastasis treatment has a good application prospect.

Description

Technical Field

[0001] The invention relates to the field of biomedicine and pharmaceutical technology, and in particular to application of phosphatidylinositol phosphatase SAC1 in the prognosis and treatment of liver cancer. Background Art

[0002] Primary liver cancer is the sixth most common cancer and the third leading cause of cancer death in the world. Primary liver cancer can be divided into hepatocellular carcinoma, cholangiocarcinoma and mixed liver cancer according to cell type; among them, hepatocellular carcinoma (HCC) accounts for 75%-85% of all primary liver cancers. HCC is not easy to detect and diagnose in the early stage, while most patients in the middle and late stages have metastasized, making it difficult to treat and having a very poor prognosis. Recurrence and metastasis after treatment are the main reasons for the intractability of liver cancer. More and more evidence shows that the metastasis of hepatocellular carcinoma is closely related to lipid metabolism in hepatocytes. Phosphatidylinositol lipids play an important role in lipid signal transduction, tumor progression, cell autophagy, pathogen invasion and related vesicle transport. However, there is currently no relevant literature on whether phosphatidylinositol can regulate the metastasis of hepatocellular carcinoma.

[0003] Phosphatidylinositol phosphatases (PIPases) SAC1 is an important phosphatase in the intracellular phosphatidylinositol phosphate (PIPs) metabolic pathway. It can dephosphorylate phosphatidylinositol-4-phosphate and participate in the regulation of a series of cellular functions such as inositol metabolism, actin cytoskeleton rearrangement, and ATP transport. Previous studies have shown that SAC1 loss is closely related to tumor metastasis. For example, a decrease in SAC1 levels has been observed in invasive breast cancer and prostate cancer. SAC1 loss or inhibition of SAC1 catalytic activity promotes PI4P-dependent secretion or increases the transport of cell surface proteins that regulate cell adhesion and motility. So far, it is unclear whether SAC1 regulates the occurrence and metastasis of hepatocellular carcinoma.

[0004] Hepatitis B is a type of hepatitis caused by infection with the hepatitis B virus. Some patients may develop chronic hepatitis. If not treated in time, it may gradually develop into hepatitis, cirrhosis, liver cancer, etc. Hepatitis B does not necessarily develop into liver cancer. Viral hepatitis is one of the causes of primary liver cancer, but it is not the only one. Risk factors for primary liver cancer also include cirrhosis, alcoholic liver disease, non-alcoholic fatty liver disease, etc. These factors are common causes of clinical primary liver cancer. Chinese patent CN115998876B (Announcement No.) discloses the use of SAC1 as a therapeutic target for hepatitis B, but there is no relevant research report on whether SAC1 can be used as an intervention or treatment target for primary liver cancer. Summary of the invention

[0005] The present invention has found that the loss of SAC1 is associated with shorter overall survival, recurrence-free survival, progression-free survival and disease-specific survival in patients with liver cancer. SAC1 silencing significantly promotes the migration and invasion ability of HCC cells, while SAC1 overexpression produces the opposite effect. Based on this, the purpose of this application is to provide a new application of phosphatidylinositol phosphatase SAC1 in the prognosis and treatment of liver cancer.

[0006] The technical solution adopted by the present invention to achieve its purpose is as follows:

[0007] The first aspect of the present invention provides the use of a phosphatidylinositol phosphatase SAC1 detection reagent in the preparation of a product for liver cancer prognosis judgment.

[0008] In the above-mentioned application technology scheme, the prognosis of liver cancer is evaluated by detecting the expression level of SAC1.

[0009] In the above-mentioned application technology scheme, down-regulation of SAC1 expression level indicates liver cancer metastasis and poor prognosis.

[0010] The second aspect of the present invention provides the use of phosphatidylinositol phosphatase SAC1 as a target in the preparation or screening of drugs for treating liver cancer or inhibiting the migration, invasion and metastasis of liver cancer cells or inhibiting the metastasis of liver cancer.

[0011] In the above-mentioned application technology solution, the drug promotes the expression of SAC1.

[0012] The third aspect of the present invention provides the use of SAC1 protein or an agent that promotes the expression of phosphatidylinositol phosphatase SAC1 in the preparation of a drug for treating liver cancer or inhibiting the migration, invasion and metastasis of liver cancer cells or inhibiting the metastasis of liver cancer.

[0013] In the above-mentioned application technology scheme, the reagents for promoting the expression of phosphatidylinositol phosphatase SAC1 include compounds, proteins, nucleic acids, cells, and plasmids.

[0014] In the above application technology scheme, the reagent for promoting the expression of phosphatidylinositol phosphatase SAC1 is a recombinant plasmid expressing SAC1 protein. Preferably, the recombinant plasmid is obtained by cloning the gene SAC1-Myc into the vector pcDNA3.1(-) / Myc-HisA, and the cloning sites are: BamHI, HindIII.

[0015] In any of the above-mentioned application technical solutions, the liver cancer is hepatocellular carcinoma, preferably metastatic hepatocellular carcinoma.

[0016] The fourth aspect of the present invention provides a drug for treating metastatic liver cancer, wherein the active ingredient of the drug is SAC1 protein or an agent that promotes the expression of phosphatidylinositol phosphatase SAC1; preferably, the liver cancer is hepatocellular carcinoma; preferably, the drug also includes a pharmaceutically acceptable carrier.

[0017] The beneficial effects of the present invention are:

[0018] The present study found that the loss of SAC1 was associated with shorter overall survival, recurrence-free survival, progression-free survival and disease-specific survival, indicating that SAC1 has a tumor suppressor effect and prognostic value in HCC.

[0019] The present invention found that SAC1 silencing significantly promoted the migration and invasion of hepatocellular carcinoma cells, while SAC1 overexpression produced the opposite effect. In the nude mouse tail vein injection lung metastasis model, SAC1 overexpression inhibited HCC distant metastasis. The present invention showed that SAC1 overexpression significantly inhibited HCC metastasis. SAC1 has good application prospects as a new potential target for anti-HCC metastasis treatment, and provides a new anti-HCC metastasis strategy from the perspective of phospholipid metabolism. SAC1 can be used as a new inhibitor of HCC metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The results of public database analysis of SAC1 expression in liver cancer tissues of HCC patients and its prognostic correlation, including: (A) SAC1 mRNA levels in HCC tissues and non-tumor liver tissues from the TCGA dataset; (B) SAC1 mRNA levels in liver cancer tissues of HCC patients with or without metastasis from the GSE364 dataset; (C-D) Overall survival and recurrence survival of HCC patients based on the Kaplan-Meierplotter dataset; *p<0.05; **p<0.01; ns, no statistical difference.

[0021] Figure 2The experiment of the effect of SAC1 on the proliferation, migration and invasion of HCC cells in MHCC97H cells, wherein Figures AB are transfected with 40nM siRNA against SAC1 (siSAC1) or control siRNA (siNC), or pcDNA3.1 or Myc-SAC1 plasmids, (A, C) CCK8 assay shows the effect of SAC1 on cell proliferation; (B, D) EdU assay shows the effect of SAC1 on cell proliferation, scale, 50μm; (EF) scratch assay shows cell migration after 24 hours; (GH) 24 hours after transfection, cells were collected for transwell assay to show the effect of SAC1 on cell migration and invasion, scale, 100μm; (IJ) 48 hours after transfection, Western blotting experiment shows the effect of SAC1 on the expression of EMT marker proteins; *p<0.05; **p<0.01; ns, no statistical difference.

[0022] Figure 3 The experiment of the effect of SAC1 on the proliferation, migration and invasion of HCC cells in PLC / PRF / 5 cells, wherein Figures AB are transfected with 40nM siRNA targeting SAC1 (siSAC1) or control siRNA (siNC), or pcDNA3.1 or Myc-SAC1 plasmids, (A, C) CCK8 assay to determine the effect of SAC1 on cell proliferation; (B, D) EdU assay to show the effect of SAC1 on cell proliferation, scale, 50μm; (EF) scratch assay to determine the cell migration after 48 hours; (GH) 24 hours after transfection, cells were collected for transwell assay to determine the effect of SAC1 on cell migration and invasion, scale, 100μm; (IJ) 48 hours after transfection, Western blotting assay to show the effect of SAC1 on the expression of EMT marker proteins; *p<0.05; **p<0.01; ns, no statistical difference.

[0023] Figure 4These are the experimental results of overexpressing SAC1 in the nude mouse tail vein injection lung metastasis model. In MHCC97H cells stably overexpressing SAC1, (A) the overexpression efficiency of SAC1 was determined by Western blotting experiment; (B) the effect of SAC1 overexpression on cell migration was determined by scratch assay; (C) the effect of SAC1 overexpression on cell migration and invasion was determined by transwell assay, scale bar, 100 μm; (D) diagram of nude mouse tail vein lung metastasis model; (E) lung metastasis probability table; (F) MHCC97H cells with LV-NC or LV-SAC1 were injected into the tail vein of nude mice; 4 weeks after injection, lung tissues were obtained for H&E staining, and metastatic nodules were counted, scale bar, 100 μm (left) or 25 μm (right); *p<0.05; **p<0.01; ns, no statistical difference. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0025] The experimental methods in the following examples are all conventional methods unless otherwise specified; the reagents and materials used are all conventional reagents and materials in the art, unless otherwise specified, and are all commercially available.

[0026] Example 1

[0027] 1 Experimental Materials and Reagents

[0028] 1.1 Experimental Materials

[0029] The liver cancer cells MHCC97H and PLC / PRF / 5 were both preserved and used for a long time by the inventor's laboratory.

[0030] Myc-SAC1 plasmid (SAC1 overexpression plasmid): commissioned to General Biosystems (Anhui) Co., Ltd. to construct, which was obtained by cloning the gene SAC1 phosphatidylinositol phosphatase (SACM1L / SAC1) (Genbank ID: 22908)-Myc into the vector pcDNA3.1(-) / Myc-HisA, cloning site: BamHI (GGATCC)-HindIII (AAGCTT).

[0031] 1.2 Main experimental reagents

[0032] Antibody Name Production Company Part Number Mouseanti-β-actinmonoclonalantibody proteintech 66009-1-Ig Rabbitanti-Claudin-1monoclonalantibody ZENBIO 343203 Rabbitanti-E-Cadherinmonoclonalantibody Abmart TA0131 Rabbitanti-Myc-tagmonoclonalantibody proteintech 16286-1-AP Rabbitanti-N-Cadherinmonoclonalantibody Abmart T55015 Rabbitanti-SACM1Lmonoclonalantibody proteintech 13033-1-AP

[0033] 1.3 siRNA sequences

[0034]

[0035] 2 Experimental methods

[0036] 2.1 Cell transfection

[0037] After inoculating liver cancer cells MHCC97H and PLC / PRF / 5 for overnight culture, transfection can be performed when the density reaches about 80%.

[0038] 2.1.1 SAC1 knockdown

[0039] The transfection complex was prepared using Opti-MEM according to the Lipo3000 instruction manual, and the concentration of siRNA targeting SAC1 was 40 nM.

[0040] 2.1.2 SAC1 overexpression

[0041] According to the instructions of Lipo 3000, Opti-MEM was used to prepare the transfection complex, and the amount of Myc-SAC1 plasmid used was 1 μg (taking a 12-well plate as an example).

[0042] 2.2 Cell proliferation assay

[0043] 2.2.1 CCK8 experiment

[0044] 24 hours after transfection, the cells were digested and counted and then plated into 96-well plates. 100 μl of culture medium and 10 μl of CCK8 mixture were added to each well in the form of liquid replacement at 0, 24, 48, 72, and 96 hours after cell attachment. The plates were incubated in an incubator for 2 hours, and the absorbance at 450 nm was detected using a microplate reader.

[0045] 2.2.2 EdU experiment

[0046] Digest and count the cells that grow well after transfection, place the slide in a new six-well plate and moisten it with collagen, take an appropriate amount of cells and spread them into the six-well plate, shake well, place in an incubator overnight or grow to a suitable density, and perform EdU staining according to the instructions.

[0047] 2.3 Scratch test

[0048] (1) Select cells with a density of more than 90% and good growth status after transfection for scratching. Use a 10 μl plastic pipette tip with a ruler to make scratches perpendicular to the bottom of the well plate;

[0049] (2) Rinse the cells several times with PBS to rinse off the scratched cells, add serum-free or low-serum culture medium, and return to the incubator to continue culturing;

[0050] (3) Take photos at different time points such as 0, 24, and 48 hours to obtain images of migration at different time points.

[0051] 2.4 Transwell experiment

[0052] 2.4.1 Transwell migration assay

[0053] When performing a transwell experiment without laying matrix gel, it is a transwell migration experiment, and the other steps are the same as below.

[0054] 2.4.2 Transwell invasion assay

[0055] (1) The night before, transfer the Matrigel from a -20°C refrigerator to a 4°C refrigerator and thaw it in an ice bath. The next day, dilute the thawed Matrigel with serum-free basal medium DMEM at a ratio of 1:9. Gently pipette to mix well and then add 100 μl to each transwell chamber. After irradiating with ultraviolet light for 1 hour, place the cells in a 37°C incubator and incubate until the Matrigel solidifies.

[0056] (2) Digest cells that have grown well after transfection, digest and centrifuge each group of cells, resuspend the cells in serum-free basal medium and count them, determine the number of experimental cells based on cell characteristics, and calculate the required volume of cell suspension and basal medium;

[0057] (3) Add 700 μl of complete culture medium containing 10% serum to the lower chamber of the 24-well plate, add the cell suspension resuspended in basal culture medium to the upper chamber, and place the transwell plate in an incubator;

[0058] (4) After 24-48 hours, remove the transwell chamber, discard the liquid in the upper and lower layers of the chamber, wash with PBS 1-2 times, and fix the chamber with 4% paraformaldehyde for 30 minutes;

[0059] (5) Take out the fixed chamber and place it in crystal violet staining solution for 30 minutes;

[0060] (6) Place the chamber in ddH2O to wash away excess dye, and gently wipe excess cells on the upper layer of the chamber with a moistened cotton swab. After washing, dry it in a ventilated place and take pictures with a microscope.

[0061] 2.5 Western blotting experiments

[0062] Cells were collected 48 hours after transfection, and the wells were washed with PBS, then lysis buffer was added and shaken on ice for 30 minutes. The protein concentration was detected and adjusted according to the instructions of the BCA kit, and the steps of loading-electrophoresis-transfer-blocking-primary antibody-secondary antibody were performed. The Bio-Rad gel imaging system was used for exposure and data preservation.

[0063] 2.6 Nude mouse tail vein injection lung metastasis model

[0064] (1) Preparation of experimental animals: All animal experiments were approved by the Ethics Committee of Chongqing Medical University (approval number 20240193). This study was conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals and in accordance with the regulations of the People's Republic of China;

[0065] (2) Grouping: First, the mice were randomly divided into 2 groups: LV-NC control group and LV-SAC1 experimental group;

[0066] (3) Tail vein injection: The constructed SAC1 overexpression or control MHCC97H cells were diluted with PBS and 200 μl of the dilution (containing 1×10 6 The lung metastasis model was established by tail vein injection in nude mice (MHCC97H cells with SAC1 overexpression or control group);

[0067] (4) Sample preservation: The mice were killed about 4 weeks after the injection and the lung tissue was removed to observe the formation of nodules on the lung surface and take pictures. In addition, the lung tissue samples were preserved with 4% tissue cell fixative (HE staining was used to detect the type and number of nodules). The mouse carcasses were placed in a dedicated freezer at the animal center for harmless disposal.

[0068] 3 Results

[0069] 3.1 Downregulation of SAC1 levels in liver cancer is associated with metastasis and poor prognosis

[0070] To evaluate the role of SAC1 in HCC development and progression, we analyzed the mRNA levels of SAC1 based on The Cancer Genome Atlas (TCGA) dataset. We found that SAC1 levels were comparable between HCC and normal liver tissues ( Figure 1 A). Considering that previous studies have reported that SAC1 inhibits the metastasis of certain cancers (such as breast cancer and prostate cancer), we investigated whether SAC1 is involved in HCC metastasis. Bioinformatics analysis of the GSE364 dataset showed that SAC1 expression was reduced in metastatic HCC tissues compared with non-metastatic HCC tissues, indicating that SAC1 may be involved in HCC metastasis rather than its occurrence ( Figure 1 B). In addition, to determine the clinical relevance of SAC1 in HCC, we analyzed the correlation between SAC1 expression and patient survival in the Kaplan-Meierplotter dataset. Loss of SAC1 was associated with shorter overall survival, recurrence-free survival, progression-free survival, and disease-specific survival, indicating that SAC1 has a tumor suppressive role and prognostic value in HCC ( Figure 1 CD).

[0071] 3.2 SAC1 inhibits HCC cell migration and invasion in vitro, but has little effect on cell proliferation

[0072] To determine the function of SAC1 in HCC metastasis, we knocked down SAC1 by specific siSAC1 in MHCC97H cells and PLC / PRF / 5 cells ( Figure 2 A; Figure 3 A). Silencing SAC1 was shown to significantly promote scratch healing, migration and invasion ( Figure 2 E, G; Figure 3 In addition, Myc-tagged SAC1 was used to determine the effects of overexpressing SAC1 ( Figure 2 C; Figure 3 C). However, overexpression of SAC1 showed the opposite effect ( Figure 2 F,H; Figure 3 F, H). Given that EMT is one of the key steps in the increased cell mobility and metastasis, we tested the expression of three different EMT markers. Silencing SAC1 significantly increased the levels of mesenchymal markers (N-cadherin) and decreased the levels of epithelial markers (E-cadherin and Claudin-1) in MHCC97H cells and PLC / PRF / 5 cells ( Figure 2 I; Figure 3 I), whereas the opposite result was observed in cells overexpressing SAC1 ( Figure 2 J; Figure 3 J) Taken together, these results indicate that silencing SAC1 induces EMT and promotes metastasis of HCC cells.

[0073] To study the effect of SAC1 on cell proliferation, CCK8 and EdU assays were performed. The results showed that SAC1 expression had little effect on HCC cell proliferation ( Figure 2 AD; Figure 3 AD), which is consistent with the analysis of TCGA datasets, which showed that the levels of SAC1 in HCC tumor tissues and normal liver tissues were comparable. The above data suggest that SAC1 is mainly involved in HCC metastasis rather than its occurrence.

[0074] 3.3 SAC1 overexpression inhibits HCC metastasis in vivo

[0075] To evaluate the inhibitory effect of SAC1 on HCC metastasis in vivo, we injected MHCC97H cells stably overexpressing SAC1 into BALB / c nude mice via tail vein to inhibit HCC metastasis. The overexpression efficiency of SAC1 was determined by Western blotting assay ( Figure 4 A). SAC1 overexpression significantly impeded the wound healing, migration and invasion abilities of MHCC97H ( Figure 4 BC). Four weeks after injection, lung samples were collected from mice for H&E staining to identify metastatic nodules ( Figure 4Compared with the control group, SAC1 overexpression resulted in a decrease in lung tumor nodules and a lower probability of lung metastasis ( Figure 4 EF). Collectively, the above results verified that SAC1 inhibits HCC metastasis in vivo.

[0076] 4 Analysis

[0077] SAC1 is currently the only phosphatase that can effectively reduce PI4P levels in certain vesicles. However, whether SAC1 regulates the metastatic process of HCC remains unclear. We investigated the effect of SAC1 on HCC metastasis and found that SAC1 silencing significantly promoted the migration and invasion of HCC cells, while SAC1 overexpression produced the opposite effect. Our in vitro cell and in vivo mouse experimental data showed that the phosphatase SAC1 can significantly inhibit HCC metastasis.

[0078] Metastasis is one of the major difficulties in the poor prognosis of HCC patients. There is an urgent need to explore the underlying molecular mechanisms of hepatocellular carcinoma metastasis to develop new treatment strategies and improve patient prognosis. In this study, we identified SAC1 as a potential predictor of metastasis and prognosis in HCC patients for the first time, and SAC1 loss predicted a poor prognosis in HCC patients. These data all suggest that SAC1 may play an inhibitory role in HCC metastasis.

[0079] Golgi PI4P binding to mutant binding proteins results in defective vesicle trafficking, which increases secretion and ultimately leads to more aggressive cancers. Genetic mutations such as increased expression of PI4P kinases or associated with Golgi-targeted PI4P binding proteins have been implicated in anterograde protein trafficking in tumor-invasive angiogenesis. However, it is unclear whether other PI4P pools have similar functions. SAC1 is the only hydrolase in eukaryotic cells that can hydrolyze PI4P to PI. Reduced SAC1 expression affects a range of pro-cancer cell processes, such as SAC1 depletion induces mislocalization of intercellular adhesion proteins E-cadherin and β-catenin and perturbations of the peripheral actin cytoskeleton, a hallmark of the early stages of EMT. This process heralds the initiation of amplified cell motility, tissue invasion, and ultimately metastasis. Although reduced SAC1 levels have been reported in invasive breast and prostate cancers, its role in other tumors has not been fully elucidated. In this study, we determined the role of SAC1 in suppressing HCC cell migration, invasion, and metastasis by gain- and loss-of-function experiments in HCC cells. In addition, the lung metastasis model injected into the tail vein of nude mice further verified that SAC1 had a significant inhibitory effect on HCC lung metastasis.

[0080] In summary, we report that SAC1 is a novel inhibitor of HCC metastasis. Our experimental data reveal the key role of SAC1 in HCC metastasis and provide a new potential target for the development of phospholipid metabolism-related HCC therapies.

Claims

1. Application of phosphatidylinositol phosphatase SAC1 detection reagent in the preparation of products for liver cancer prognosis judgment.

2. The use according to claim 1, characterized in that: The prognosis of liver cancer was evaluated by detecting the expression level of SAC1.

3. The use according to claim 2, characterized in that: Down-regulation of SAC1 expression level indicates liver cancer metastasis and poor prognosis.

4. Use of phosphatidylinositol phosphatase SAC1 as a target in the preparation or screening of drugs for treating liver cancer or inhibiting the migration, invasion and metastasis of liver cancer cells or inhibiting the metastasis of liver cancer.

5. The use according to claim 4, characterized in that: The drug promotes the expression of SAC1.

6. Use of SAC1 protein or an agent that promotes the expression of phosphatidylinositol phosphatase SAC1 in the preparation of a drug for treating liver cancer or inhibiting the migration, invasion and metastasis of liver cancer cells or inhibiting the metastasis of liver cancer.

7. The use according to claim 6, characterized in that: The reagents for promoting the expression of phosphatidylinositol phosphatase SAC1 include compounds, proteins, nucleic acids, cells and plasmids.

8. The use according to claim 6, characterized in that: The reagent for promoting the expression of phosphatidylinositol phosphatase SAC1 is a recombinant plasmid expressing SAC1 protein. Preferably, the recombinant plasmid is obtained by cloning the gene SAC1-Myc into the vector pcDNA3.1(-) / Myc-HisA, and the cloning sites are: BamHI, HindIII.

9. The use according to any one of claims 1 to 8, characterized in that: The liver cancer is hepatocellular carcinoma, preferably metastatic hepatocellular carcinoma.

10. A drug for treating metastatic liver cancer, characterized in that: The active ingredient of the drug is SAC1 protein or an agent that promotes the expression of phosphatidylinositol phosphatase SAC1; preferably, the liver cancer is hepatocellular carcinoma; preferably, the drug also includes a pharmaceutically acceptable carrier.

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