A hepatocellular carcinoma biomarker - solute carrier family 37 member 3 and application thereof

Abstract

The application provides a hepatocellular carcinoma biomarker-solute carrier family 37 member 3 (SLC37A3) and application thereof, and belongs to the technical field of biological medicine. The application finds that the expression level of SLC37A3 in a hepatocellular carcinoma tissue is higher than that in a normal hepatocyte tissue; high expression of SLC37A3 predicts poor prognosis of a HCC patient; the expression level of SLC37A3 is related to immune cell infiltration in HCC, and knockdown of SLC37A3 can inhibit proliferation and metastasis of hepatoma cells and promote apoptosis of hepatoma cells; SLC37A3 is involved in the regulation of glucose homeostasis. The SLC37A3 provided by the application can be a prospective diagnostic and prognostic biomarker of HCC with a sugar metabolism regulation function, and provides a basis for further research on accurate diagnosis and treatment targets of HCC.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a hepatocellular carcinoma biomarker - a member of the solute carrier family 37 and its application. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common type of liver cancer, accounting for over 90% of all liver cancer types. The high mortality rate of HCC is usually due to frequent recurrence and metastasis. As the largest metabolic organ in the body, the liver contains various types of cytokines, adhesion molecules, and cell-cell interactions, forming a unique metabolic regulatory network. Hepatic metabolic disorders are a major risk factor for HCC progression. Due to the insidious onset, high recurrence and metastasis rates, and poor prognosis of HCC, early diagnosis is relatively difficult, and more than 50% of patients miss the optimal surgical period. Furthermore, first-line treatments such as sorafenib exhibit strong resistance, making HCC treatment still challenging, especially for advanced HCC. Therefore, identifying accurate diagnostic and prognostic biomarkers may be a prospective strategy for the diagnosis and treatment of HCC.

[0003] The solute carrier (SLC) superfamily is an indispensable family of membrane transport proteins. SLC transporters participate in the transmembrane transport of substrates, serving as channels for the transport of key nutrients, metabolites, and drugs within cells. SLC family members are specifically expressed in various metabolic organs, including the liver, kidneys, and heart, which are involved in nutrient metabolism, signal transduction, and other important physiological activities. The SLC37 family is a transmembrane sugar transporter located on the endoplasmic reticulum membrane, primarily involved in the exchange of sugars and phosphates. Currently, four members have been identified: SLC37A1, SLC37A2, SLC37A3, and SLC37A4. The various biological functions of SLC37A3 have been gradually confirmed; for example, as a carrier for transporting nitrogenous bisphosphates, SLC37A3 may be involved in the treatment of osteoporosis; mutations in SLC37A3 are associated with congenital hyperpancreatic islet dysfunction. Recent studies suggest that SLC37A3 is a potential biomarker for intracranial aneurysms and retinal diseases. However, the role of SLC37A3 in cancer development, especially in HCC, remains unknown. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a biomarker for hepatocellular carcinoma, namely SLC37A3, which is a prospective diagnostic and prognostic biomarker for HCC with glucose metabolism regulation function, providing a basis for the accurate diagnosis and therapeutic target of HCC.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0006] This invention provides a biomarker for hepatocellular carcinoma, wherein the biomarker is SLC37A3.

[0007] Preferably, the SLC37A3 is expressed at a higher level in hepatocellular carcinoma tissue than in normal hepatocellular tissue.

[0008] Preferably, SLC37A3 knockdown inhibits the proliferation and metastasis of liver cancer cells, and SLC37A3 knockdown promotes apoptosis of liver cancer cells.

[0009] Preferably, the SLC37A3 participates in the regulation of glucose homeostasis.

[0010] Preferably, high expression of SLC37A3 predicts poor prognosis in HCC patients.

[0011] This invention also provides the application of the above-mentioned biomarkers in the preparation of prospective diagnostic kits for hepatocellular carcinoma.

[0012] This invention also provides the application of the above-mentioned biomarkers in the preparation of a hepatocellular carcinoma prognostic diagnostic kit.

[0013] Preferably, the kit includes a negative control, which is normal hepatocyte tissue.

[0014] This invention also provides the application of the above-mentioned biomarkers in constructing a prognostic prediction model for hepatocellular carcinoma.

[0015] The beneficial effects of this invention are:

[0016] This invention demonstrates through bioinformatics analysis that the overall expression level of SLC37A3 in hepatocellular carcinoma (HCC) cells, across different stages and clinical or pathological features, is significantly higher than that in normal tissues. This similar trend in SLC37A3 expression levels was also observed in HCC cells and IHC experiments.

[0017] Survival analysis showed that patients with high SLC37A3 expression had significantly lower overall survival, 1-year survival, 3-year survival, and 5-year survival rates than patients with low SLC37A3 expression.

[0018] This invention demonstrates through cell function experiments that knocking down SLC37A3 can inhibit the proliferation and metastasis of liver cancer cells and promote apoptosis of liver cancer cells. Xenograft tumor experiments also show that knocking down SLC37A3 can significantly inhibit the occurrence of HCC tumors in vivo.

[0019] This invention demonstrates, through functional interaction network analysis and in vitro validation experiments, that SLC37A3 can form a unique functional interaction network with several related genes. Immune infiltration analysis showed that SLC37A3 expression levels are associated with immune cell infiltration in HCC. Furthermore, RNA-seq analysis of SLC37A3 knockdown HCC cells revealed significant alterations in TIDM-related signaling pathways. Significant changes were also observed in the expression levels of insulin secretion-related and glycolysis / gluconeogenesis-related genes, suggesting that SLC37A3 may be involved in the regulation of glucose homeostasis. Attached Figure Description

[0020] Figure 1 Overall and different expression levels of SLC37A3 in HCC tissues;

[0021] Figure 2 Expression levels of SLC37A3 in various clinical and pathological features of primary HCC tissue;

[0022] Figure 3 The effect of SLC37A3 expression on HCC-related genes;

[0023] Figure 4 The effect of SLC37A3 expression on immune infiltrating cell subsets;

[0024] Figure 5 The relationship between SLC37A3 expression and HCC prognosis;

[0025] Figure 6 The function of SLC37A3 in the proliferation, metastasis and apoptosis of HCC cells in vitro;

[0026] Figure 7 Effects of SLC37A3 expression on glucose metabolism-related signaling pathways in HCC cells;

[0027] Figure 8 The effect of SLC37A3 expression on the occurrence of HCC tumors in vivo;

[0028] Figure 9 mRNA and protein expression levels of si-SLC37A31-3 after SLC37A3 knockout. Detailed Implementation

[0029] This invention provides a biomarker for hepatocellular carcinoma, namely SLC37A3. This invention uses a combination of bioinformatics analysis and in vitro / in vivo experiments to investigate the role of SLC37A3 in HCC progression.

[0030] This invention first analyzed the expression levels of SLC37 family genes in hepatocellular carcinoma (HCC). The overall and different stages of SLC37A3 expression levels in HCC tissues were significantly higher than those in normal tissues. Figure 1 Further analysis showed that SLC37A3 expression levels were significantly higher in primary HCC tissues with various clinical and pathological features compared to normal tissues, including patient sex, ethnicity, TP53 mutation, weight, lymph node metastasis, tumor grade, histological subtype, and age. This invention detected the expression levels of SLC37A3 in the normal human hepatocyte cell line HL7702, and the hepatocellular carcinoma cell lines HepG2 and HuH-7. Compared to HL7702 cells, both the mRNA and protein expression levels of SLC37A3 were significantly increased in HepG2 and HuH-7 cells. The IHC staining results of SLC37A3 in primary human HCC tumor tissues and adjacent normal control tissues were also consistent with the results of bioinformatics analysis and cell experiments. Figure 2 The above results indicate that SLC37A3 may be a novel diagnostic biomarker for HCC, with higher expression levels in hepatocellular carcinoma tissues than in normal hepatocyte tissues.

[0031] This invention combines bioinformatics analysis and in vitro validation experiments, showing that the expression levels of CALU, YEATS2, and TRRAP are positively correlated with the expression of SLC37A3 in HCC; however, OCEL1, SELENBP1, and ECHS1 are negatively correlated with the expression of SLC37A3. Figure 3 SLC37A3 may contribute to the occurrence and development of HCC through interactions with the aforementioned related genes.

[0032] This invention investigated the prognostic value of SLC37A3 in hepatocellular carcinoma (HCC). Survival analysis showed that patients with high SLC37A3 expression had significantly lower overall, 1-year, 3-year, and 5-year survival rates compared to patients with low SLC37A3 expression. Cox regression analysis confirmed that SLC37A3 is an independent risk factor for HCC. Figure 5 The above results preliminarily demonstrate that high expression of SLC37A3 may predict poor prognosis in HCC patients.

[0033] This invention further elucidates the role of SLC37A3 in HCC progression and conducted related functional experiments to verify the function of SLC37A3 in the proliferation, metastasis, and apoptosis of HCC cells in vitro. The results showed that knockdown of SLC37A3 inhibited the proliferation and metastasis of liver cancer cells, but promoted apoptosis of liver cancer cells. Figure 6 Similarly, results from xenograft tumor experiments also showed that knocking down SLC37A3 significantly inhibited HCC tumorigenesis in vivo, accompanied by decreased Ki-67 proliferation markers and increased TUNEL apoptosis markers. Figure 8 Therefore, the above results suggest that SLC37A3 may also be a prospective prognostic biomarker for HCC.

[0034] In the tumor microenvironment of HCC, this invention found that SLC37A3 expression was significantly positively correlated with six immune infiltrating cell subsets, including B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells. Changes in SLC37A3 copy number were significantly correlated with the infiltration levels of B cells, CD8+ T cells, and CD4+ T cells. Furthermore, SLC37A3 expression was significantly positively correlated with the expression levels of representative immune checkpoints, including CD274, PDCD1, CTLA4, and TIGIT. Figure 4 The RNA-seq results from SLC37A3 knockdown also showed that, after inhibiting SLC37A3 expression, several immune-related KEGG pathways were significantly altered, including primary immunodeficiency, Th1 and Th2 cell differentiation, PD-L1 expression and the PD-1 checkpoint pathway in cancer, the T cell receptor signaling pathway, and Th17 cell differentiation. Figure 7 A). The above results indicate that SLC37A3 expression may be involved in the regulation of immune homeostasis in the HCC tumor microenvironment.

[0035] This invention selected HuH-7 cells transfected with si-NC- and si-SLC37A3- and further investigated the novel biological functions of SLC37A3 through RNA sequence analysis. The results showed that after inhibiting SLC37A3 expression, T1DM-related signaling pathways were manifested in the top 20 most important KEGG pathways, with significant alterations in these pathways. The expression levels of insulin secretion-related (IGFBP1, KCNN4) and glycolysis / gluconeogenesis-related (TPI1, ALDH1B1) genes also changed significantly, indicating that SLC37A3 may be involved in the regulation of glucose homeostasis. Figure 7 ).

[0036] This invention also provides the application of the biomarker SLC37A3 in the preparation of a prospective diagnostic kit for hepatocellular carcinoma and in the preparation of a prognostic diagnostic kit for hepatocellular carcinoma. The kit includes a negative control, which is normal hepatocyte tissue.

[0037] This invention also provides the application of the biomarker SLC37A3 in constructing a prognostic prediction model for hepatocellular carcinoma.

[0038] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0039] Unless otherwise specified, the following embodiments are all conventional methods.

[0040] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0041] Example 1

[0042] Cell Culture: Human normal hepatocyte cell line HL7702 and hepatocellular carcinoma cell line HuH-7 were obtained from the Cell Biology Institute, Chinese Academy of Sciences (Shanghai, China). Human hepatocellular carcinoma cell line HepG2™ was obtained from ATCC (HB-8065) ​​(Virginia, USA). HL7702 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum (FBS, Gibco, NY, USA), 100 mg / mL streptomycin (Solarbio, Beijing, China), and 100 U / mL penicillin (Solarbio, China). HepG2 and HuH-7 cells were cultured in DMEM medium (Gibco, NY, USA) containing 10% FBS, 1% non-essential amino acids (Cell Technologies, Beijing, China), 100 mg / mL streptomycin, and 100 U / mL penicillin. All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0043] RNA isolation and quantitative real-time PCR (qRT-PCR): Total RNA was isolated from HL7702, HepG2, and HuH-7 cells using TRIzol reagent (Invitrogen, CA, USA) according to the manufacturer's instructions. cDNA synthesis was performed using a cDNA synthesis kit (Takara, Shiga, Japan). mRNA expression levels were quantified by qRT-PCR using SGExcel Fast SYBR Green (Sangon Biotech, Shanghai, China). Relative mRNA expression levels were normalized to β-actin expression levels and measured by comparison using the Ct method. Primer sequences are listed in Table 1.

[0044] Table 1. Relevant primer sequences

[0045]

[0046]

[0047]

[0048] Western blot: Cells were lysed using RIPA lysis buffer (Solarbio, Beijing, China) and a protease inhibitor (Solarbio, China). Lysate was collected and centrifuged at 10,000 g for 15 min at 4 °C. The supernatant was collected to determine total protein concentration using a BCA protein assay kit (Elabscience, Wuhan, China). Equal volumes of protein extract (50 μg / well) were loaded into an 8–12% sodium dodecyl sulfate polyacrylamide gel electrophoresis apparatus and then transferred to a polyvinylidene fluoride membrane (Millipore, MA, USA). The membrane was blocked with 5% bovine serum albumin blocking buffer for 2 h at room temperature. The membrane was then coated with primary antibodies β-TUBULIN (1:2000, Sungene Biotech, Tianjin, China), SLC37A3 (1:500, #PA5-42500, Invitrogen, CA, USA), and CYCLIN. D1 (1:1000, #A2708, Abclonal, Wuhan, China), PCNA (1:1000, #10205-2-AP, Proteintech, Wuhan, China), N-CADHERIN (1:1000; #A0433, Abclonal, Wuhan, China), VIMENTIN (1:1000, #10366-1-AP, Proteintech, Wuhan, Wuhan), BAX (1:1000, #A7626, Abclonal, Wuhan, China), BCL-2 (1:1000, #12789-1-AP, Proteintech, Wuhan, China), CASPASE3 (1:500, #A0214, Abclonal, Wuhan, China), IGFBP1 (1:500, #DF7130, Affinity Biosciences, OH, United States), KCNN4 (1:500, #DF4132, Affinity Biosciences, OH, United States) TPI1 (1:1000, #DF12023, Affinity Biosciences, OH, United States) and ALDH1B1 (1:1000, #DF3755, Affinity Biosciencies, OH, United States) were incubated overnight at 4°C. The membrane was then incubated with goat anti-rabbit IgG secondary antibody HRP (1:3000, #AS014, ABclonal, Wuhan, China) at room temperature for 1 hour. Protein bands were detected using an enhanced chemiluminescence assay kit (Advansta, Menlo Park, USA). The relative expression levels of target proteins were normalized to β-tubulin expression levels.

[0049] RNA interference: Following the manufacturer's protocol, SLC37A3 siRNA and negative control (NC) siRNA (GenePharma, Shanghai, China) were transfected with Lipofectamine 2000 (Invitrogen, CA, USA) for 6 hours. Cells were then harvested after 24, 48, or 72 hours, depending on experimental requirements. Considering the higher transfection stability of shRNA, SLC37A3 shRNA and negative control shRNA (GenePharma, Shanghai, China) were transfected in xenograft tumor models. The sequences of siRNA and shRNA (purchased from GenePharma) are listed in Table 2.

[0050] Table 2 Sequences of siRNA and shRNA

[0051]

[0052]

[0053] In Table 2, the last two T bases of the sequences SEQ ID NO: 45 to SEQ ID NO: 52 are TT overhangs to facilitate unwinding.

[0054] Statistical analysis: Data are expressed as mean ± standard deviation of repeated trials. Statistical analysis was performed using GraphPad Prism 8.0 software (GraphPad Inc., California, USA). t-tests were used for comparisons between two groups. One-way ANOVA was used for comparisons among the three groups. Furthermore, Pearson correlation tests were used to analyze the correlation between SLC37A3 expression and its positively or negatively correlated genes or representative immune checkpoints. Spearman correlation coefficient and Wilcoxon rank-sum test were used to calculate the correlation between SLC37A3 expression or gene copy number variation and six infiltrating immune cell types. Log-rank tests were used for survival analysis. P < 0.05 was generally considered significant (*); highly significant (**); and extremely significant (***).

[0055] 1. Analysis of SLC37 family gene expression levels

[0056] UALCAN (http: / / ualcan.path.uab.edu) is a database containing TCGA RNA sequencing (RNA-Seq) results. UALCAN was used to analyze the expression levels of SLC37 family genes in HCC, including the expression of SLC37A3 in different clinicopathological features of HCC. SLC37 family gene expression data were downloaded from the following TCGA databases: SLC37A1 (http: / / 985.so / 5bth), SLC37A2 (http: / / 985.so / 5bt3), SLC37A3 (http: / / 985.so / sc5h), and SLC37A4 (http: / / 985.so / 5b08).

[0057] The Gene Expression Interaction Analysis (GEPIA) database was used to analyze the expression levels of SLC37 family genes at different stages of hepatocellular carcinoma. Data were downloaded from the following website: http: / / gepia.cancer-pku.cn / detail.php?gene=&clicktag=stageplot.

[0058] The expression levels of SLC37A1-4 in HCC were analyzed using the UALCAN database. The results showed that the mRNA expression levels of SLC37A1 and SLC37A3 were significantly increased in primary liver cancer tissues compared to normal tissues. However, there was no significant difference in the expression levels of SLC37A2 and SLC37A4 between the two tissue types. Figure 1 A).

[0059] The expression levels of SLC37A1-4 at different stages of HCC were analyzed using the GEPIA database. The results showed that the expression levels of SLC37A1 and SLC37A3 increased significantly with the progression of HCC, while the expression levels of SLC37A2 and SLC37A4 did not change significantly. Furthermore, during HCC progression, the expression level of SLC37A3 (p = 0.0107) increased more significantly than that of SLC37A1 (p = 0.0491). Figure 1 B).

[0060] The expression level of SLC37A3 increased more significantly than that of SLC37A1 during the progression of HCC, so SLC37A3 was selected as the target gene for subsequent research.

[0061] Further investigation was conducted into the effects of patient gender, patient race, TP53 mutation, patient weight, nodal metastasis, tumor grade, histological subtypes, and patient age on SLC37A3 expression levels. The results showed that, compared to normal tissues, the expression level of SLC37A3 in primary HCC tissues with the aforementioned clinical and pathological characteristics was significantly higher than that in normal tissues. Figure 2 A).

[0062] The expression levels of SLC37A3 in the human normal hepatocyte cell line HL7702, and HCC cell lines HepG2 and HuH-7 were measured. The results showed that the mRNA expression level of SLC37A3 was significantly increased in HepG2 and HuH-7 cells compared to HL7702 cells. Figure 2 B). The trend of protein expression changes is consistent with that of mRNA expression. Figure 2 C).

[0063] SLC37A3 expression was detected in primary human hepatocellular carcinoma (HCC) tissue and adjacent normal tissue as controls. IHC examination showed that SLC37A3 staining was stronger in HCC proto-carcinoma tissue, while staining was relatively weaker in adjacent normal tumor tissue. Figure 2 D). In summary, SLC37A3 expression was significantly enhanced in HCC cells and tumor tissues.

[0064] 2. Functional Network Analysis

[0065] The GeneMANIA database (http: / / GeneMANIA.org / ) establishes an interaction network among genes with similar functions, and gene prioritization is determined based on genomics and proteomics data. This study used the GeneMANIA database to query and construct a functional network among SLC37A3-related genes in hepatocellular carcinoma (HCC). The data can be downloaded from: http: / / genemania.org / search / homo-sapiens / SLC37A3. LinkedOmics (http: / / www.linkedomics.org / ) is a clinical database containing data from multiple cancer groups, used to analyze the expression levels of SLC37A3-related genes in HCC. The data ID is 106768.

[0066] A functional interaction network of SLC37A3 and its related genes was constructed. SLC37A3 can physically interact with all-trans retinoic acid-induced differentiation factor (ATRAID), ADP ribosylation factors such as GTPase 15 (ARL15), SPRY domain-containing gene 7 (SPRYD7), solute carrier family 5 member 3 (SLC5A3), and zinc finger protein 177 (ZNF177), and can also genetically interact with SERTA domain-containing gene 4 (SERTAD4) and exoprotein glycosyltransferase 2 (EXT2). SLC37A3 is co-expressed with ARL15, KDEL endoplasmic reticulum protein retention receptor 2 (KDELR2), transmembrane 9 superfamily member 3 (TM9SF3), zinc metallopeptidase STE24 (ZMPSTE24), and SERTAD4. SLC37A3 also co-localizes with TRAF3 interacting protein 2 (TRAF3IP2), solute carrier family 16 member 6 (SLC16A6), KDELR2, ZMPSTE24, and SERTAD4. Furthermore, SLC37A3 shares protein domains with solute carrier family 16 member 6 (SLC16A6), solute carrier family 16 member 7 (SLC17A7), solute carrier family 37 member 1 (SLC37A1), solute carrier family 37 member 2 (SLC37A2), and solute carrier family 37 member 4 (SLC37A4). SLC37A3 forms a functional interaction network with its related genes, primarily playing biological roles in phosphate ion transport, anion detransport activity, and transmembrane transport activity. Figure 3 A).

[0067] Further analysis of SLC37A3-related genes in HCC was conducted using the LinkedOmic database. The expression levels of 13,371 genes were positively correlated with SLC37A3 expression, while the expression levels of 6,551 genes were negatively correlated with SLC37A3 expression. The heatmap shows the top 50 significantly positive and negative SLC37A3-related genes. Figure 3 B).

[0068] Pearson correlation analysis showed that SLC37A3 was positively correlated with caloprotein (CALU), protein containing two YEATS domains (YEATS2), and transformation / transcription domain-associated protein (TRRAP), and negatively correlated with blocking protein / ELL domain containing one OCEL1 (SELENBP1), selenium-binding protein 1 (SELENBA1), and enol-coenzyme A hydratase 1 (ECHS1). Figure 3 C).

[0069] The mRNA expression levels of the above-mentioned genes were detected in HL7702, HepG2, and HuH-7 cells. Compared with HL7702 cells, the expression levels of CALU, YEATS2, and TRRAP were significantly higher in HepG2 and HuH-7 cells, and were positively correlated with SLC37A3 expression. Conversely, the expression levels of OCEL1, SELNEBP1, and ECHS1 were significantly lower in HepG2 and HuH-7 cells, and were negatively correlated with SLC37A3 expression. Figure 3 D). The results are consistent with those obtained from bioinformatics.

[0070] SLC37A3-specific siRNA and negative control (NC) siRNA were transfected into HepG2 and HuH-7 cells to inhibit SLC37A3 expression. The knockout efficiency of SLC37A3 siRNA was measured, and subsequent studies selected si-SLC37A3-2 (which exhibited the highest knockout efficiency). Figure 9 The mRNA expression levels of representative genes positively or negatively correlated with SLC37A3 expression were measured in HepG2 and HuH-7 cells. Results showed that compared to the si-NC group, the expression levels of CALU, YEATS2, and TRRAP were significantly decreased in the si-SLC37A3 group, and were positively correlated with SLC37A3 expression. The expression levels of CALU, YEATS2, and TRRAP in the si-SLC37A3 group were significantly higher than those in the si-NC group, and were negatively correlated with SLC37A3 expression. Figure 3 E). In summary, the results indicate that SLC37A3 can interact with multiple genes to form a functional network and play a unique biological role in liver cancer.

[0071] 3. Immunosmotic analysis

[0072] The Tumor Immunological Assessment Resource (TIMER) database (https: / / cistrome.shinyapps.io / timer / ) is used for comprehensive research on the relationship between tumors and infiltrating immune cells.

[0073] The correlation between SLC37A3 expression and six infiltrating immune cell types was analyzed, including B cells, CD4+ T cells, CD8+ T cells, neutrophils, macrophages, and dendritic cells. The TIMER database was also used to analyze the relationship between gene copy number variation and infiltrating immune cell abundance.

[0074] The correlation between SLC37A3 expression and representative immune checkpoints was analyzed using the GEPIA database, including CD274, programmed cell death 1 (PDCD1), cytotoxic T lymphocyte-associated protein 4 (CTLA4), and T cell immune receptor with Ig and ITIM domains (TIGIT).

[0075] Tumor-infiltrating immune cells are an indispensable component of the tumor microenvironment, participating in the occurrence and development of various cancers. The results of immune infiltration analysis showed that SLC37A3 expression was significantly positively correlated with the number of B cells, CD8+ T cells, CD4+ T cells, macrophages, neutrophils, and dendritic cells infiltrating HCC, and negatively correlated with tumor purity. Figure 4 A). Changes in SLC37A3 copy number were significantly correlated with the infiltration levels of B cells, CD8+ T cells, and CD4+ T cells. Figure 4 B).

[0076] Further analysis of the relationship between SLC37A3 expression and representative immune checkpoints using the GEPIA database revealed a significant positive correlation between SLC37A3 expression and the expression levels of CD274, PDCD1, CTLA4, and TIGIT. Figure 4 C).

[0077] The results indicate that SLC37A3 may be involved in the regulation of immune homeostasis in the HCC tumor microenvironment.

[0078] 4. Survival analysis and Cox regression analysis

[0079] The Kaplan-Meier plotter (http: / / kmplot.com / analysis) is an online database used to analyze the impact of specific gene expression on tumor survival. This database was selected to determine the prognostic value of SLC37A3 in HCC. Data ID number 84255.

[0080] Cox regression analysis was used to comprehensively assess the impact of multiple factors on overall survival in HCC. Data were downloaded from the TCGA database (https: / / portal.gdc.cancer.gov / ). HCC RNA sequence data in level 3 HTSeq FPKM format were obtained and converted to TPM format and log2 transformation. Statistical analysis and visualization were performed using R packages.

[0081] This study investigated the impact of SLC37A3 expression on the prognosis of hepatocellular carcinoma (HCC). The overall survival rate was significantly lower in the high SLC37A3 expression group than in the low SLC37A3 expression group. The median survival time in the low SLC37A3 expression group (82.9 months) was longer than that in the high SLC37A3 expression group (45.7 months). The 1-year, 3-year, and 5-year survival rates in the high SLC37A3 expression group were all significantly lower than those in the low SLC37A3 expression group, suggesting that high SLC37A3 expression may predict a poor prognosis for HCC. Figure 5 A).

[0082] Univariate and multivariate Cox regression analyses were performed to examine whether SLC37A3 was an independent prognostic factor for HCC. Univariate Cox regression analysis showed that SLC37A3, tumor T3 and T4 stages, and pathological stages III and IV were significantly associated with overall survival in HCC. Multivariate Cox regression analysis demonstrated that SLC37A3 may be an independent risk factor for HCC prognosis among the aforementioned factors. Figure 5 B).

[0083] The results showed that high SLC37A3 expression, as an independent risk factor, can predict poor prognosis in HCC patients.

[0084] 5. Analyze the function of SLC37A3 in the proliferation, metastasis and apoptosis of HCC cells in vitro.

[0085] Cell viability assay: Cell viability was determined using the Cell Counting Kit-8 (CCK-8, Beyotime, Beijing, China) according to the manufacturer's instructions. HepG2 and HuH-7 cells (5000 cells / well) were seeded in 96-well plates and transfected with si-SLC37A3 and si-NC as previously described. CCK-8 solution was added to the plates during the last 4 hours of the experiment. Absorbance at 450 nm was measured using a microplate reader at 24, 48, and 72 hours post-transfection. Cell viability was calculated based on the absorbance at 450 nm.

[0086] Cell migration assay: Cell migration was measured using a wound healing assay. HepG2 and HuH-7 cells were seeded in 24-well plates and transfected with si-SLC37A3 and si-NC as previously described. Transfected cells were grown to approximately 100% confluence, and wounds were created using a sterile pipette tip. Cells were then cultured in medium containing 1% FBS to eliminate the influence of cell proliferation. The interstitial region was captured by optical microscopy at 0 and 48 hours post-transfection. Cell migration rate was calculated using ImageJ software.

[0087] Apoptosis assays: HepG2 and HuH-7 cells were seeded in 6-well plates and transfected with si-SLC37A3 and si-NC as previously described. Forty-eight hours post-transfection, 1 × 10⁵ cells / assay were harvested and washed with cold phosphate-buffered saline (PBS). Apoptosis staining was performed using the Annexin V / Propidium Iodide (PI) Apoptosis Detection Kit (Tianjin Sunshine Biotechnology Co., Ltd., China) according to the manufacturer's protocol. Fluorescence intensity was measured by flow cytometry and analyzed using FlowJo software. In HCC xenograft models, apoptosis in tumor tissues was detected using the terminal deoxynucleotidyl transferase dUTP nick-end marker (TUNEL) apoptosis detection kit (Beyotime, Beijing, China) according to the manufacturer's instructions. Images were captured by fluorescence microscopy.

[0088] This study investigated the effects of SLC37A3 on the proliferation, metastasis, and apoptosis of HCC cells in vitro. HepG2 and HuH-7 cells were transfected with si-SLC37A3 and si-NC to inhibit SLC37A3 expression. Compared with the si-NC group, the cell viability of the si-SLC37A3 group was significantly reduced at 24, 48, and 72 hours after transfection into HepG2 and HuH-7 cells. Figure 6 A). In HepG2 and HuH-7 cells, the cell migration rate of the si-SLC37A3 group was also significantly lower than that of the si-NC group ( Figure 6 B). Conversely, si-SLC37A3 transfection of HepG2 and HuH-7 cells significantly increased the percentage of apoptosis (B). Figure 6 C).

[0089] The protein expression levels of representative markers of proliferation, metastasis, and apoptosis were detected after SLC37A3 silencing. Compared with the si-NC group, the protein expression levels of proliferation (CYCLIN D1, PCNA) and metastasis (N-CADHERIN, VIMENTIN) markers were significantly decreased in the si-SLC37A3 group, but the protein expression level of the apoptosis marker (BAX / BCL2, Cleaved-CASPASE3) was significantly increased in HepG2 and HuH-7 cells. Figure 6 (D, E). Western blot results were consistent with cell viability, migration, and apoptosis assays.

[0090] The results showed that silencing SLC37A3 could inhibit the proliferation and metastasis of HCC cells in vitro and promote their apoptosis.

[0091] 6. Analyze the relationship between SLC37A3 and signaling pathways related to glucose metabolism in HCC cells.

[0092] RNA Sequencing and Data Analysis: HuH-7 cells were seeded in 6-well plates and transfected with si-SLC37A3 and si-NC as previously described. Forty-eight hours post-transfection, high-quality total RNA was extracted from these cells using TRIzol reagent. RNA-Seq was performed with the assistance of Applied Protein Technology Co., Ltd. (Shanghai, China). The RNA-Seq library was prepared by... RNA Library Preparation Kit (Massachusetts, USA) Follow the manufacturer's instructions. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis was performed using the R package clusterProfiler. Differential expression analysis was performed using the DESeq2R package based on gene read counts. A p-value < 0.05 was considered a differentially expressed gene (DEG).

[0093] si-SLC37A3 and si-NC were transfected into HuH-7 cells to suppress SLC37A3 expression. KEGG pathway enrichment analysis showed that several signaling pathways were significantly altered after SLC37A3 expression was suppressed. Figure 7 A shows the top 20 most important signaling pathways, including natural killer cell-mediated cytotoxicity, cytokine-cytokine receptor interactions, allogeneic transplantation regeneration, graft-versus-host disease, autoimmune thyroid disease, and type 1 diabetes mellitus (T1DM). Figure 7 A). Compared with the si-NC group, the si-SLC37A3 group had 822 significantly upregulated genes and 962 significantly downregulated genes. Figure 7 B).

[0094] The SLC37 family of genes encodes transmembrane glucose transporters. As a glucose-phosphate exchanger, SLC37A3 mutations are associated with congenital hyperinsulinemia. RNA-seq results showed that after inhibiting SLC37A3 expression, type 1 diabetes mellitus (T1DM)-related signaling pathways were also observed in the top 20 most prominent KEGG pathways. T1DM is primarily caused by pancreatic β-cell destruction, leading to insulin deficiency and disordered glucose metabolism.

[0095] Further cluster analysis was conducted on glucose metabolism-related genes. For example... Figure 7As shown in Figure C, SLC37A3 transfection significantly altered insulin secretion and glycolysis / gluconeogenesis signaling pathways. In the insulin secretion signaling pathway, the expression levels of insulin-like growth factor binding protein-1 (IGFBP1) and insulin-like growth factor binding protein-4 (IGFBP4) were significantly higher in the si-SLC37A3 group than in the si-NC group. Conversely, in the si-SLC37A3 group, the expression levels of potassium inward rectifier channel subfamily J member 11 (KCNJ11), potassium-calcium activated channel subfamily N member 4 (KCNN4), potassium-calcium activated channel subfamily N member 1 (KCNN1), and pancreatic and duodenal homology box 1 (PDX1) were significantly lower than in the si-NC group. In the glycolysis / gluconeogenesis signaling pathway, the expression levels of alcohol dehydrogenase 4 (ADH4) and phosphoenolpyruvate carboxykinase 2 (PCK2) were significantly higher in the si-SLC37A3 group than in the si-NC group. However, compared to the si-NC group, the expression levels of phosphoglycerate kinase 1 (PGK1), acyl-CoA synthase family member 1 (ACSS1), acyl-CoA synthase family member 2 (ACSS2), glucose-6-phosphate isomerase (GPI), triphosphate isomerase 1 (TPI1), and aldehyde dehydrogenase family member B1 (ALDH1B1) were significantly decreased in the si-SLC37A3 group. Figure 7 C).

[0096] To further validate these results, specific si-SLC37A3 and si-NC were transfected into HCC cells to further inhibit SLC37A3 expression. The mRNA expression levels of IGFBP1, KCNN4, TPI1, and ALDH1B1 were consistent with the RNA sequence results in HepG2 and HuH-7 cells after SLC37A3 expression inhibition. Figure 7 D). Further analysis of the protein expression levels of IGFBP1, KCNN4, TPI1, and ALDH1B1 in the si-NC and si-SLC37A3 groups revealed that these levels were consistent with their mRNA expression levels. Figure 7 In summary, these results elucidate that inhibition of SLC37A3 expression primarily affects glucose metabolism-related insulin secretion and glycolysis / gluconeogenesis signaling pathways in HCC cells.

[0097] (3) Relationship between SLC37A3 and the occurrence of HCC in vivo

[0098] Xenograft Tumor Model: Male BALB / c nude mice aged 4 to 6 weeks were purchased from HFK Bioscience (Beijing, China). All mice were housed under specific pathogen-free (SPF) conditions with ample water and food. Logarithmically growing HuH-7 cells were transfected with sh-SLC37A3 and sh-NC, as previously described. To induce the HCC xenograft tumor model, 5 × 10⁶ transfected HuH-7 cells were suspended in 150 μL of Matrigel (1:1, BD Biosciences, CA, United States) and subcutaneously injected into the right axilla of the BALB / c mice. Tumor volume was measured every two days for approximately 7 days post-injection. Tumor volume was measured as follows: length × width 2 / 2. Mice were sacrificed approximately 21 days post-injection for subsequent experiments. All mouse experimental procedures were performed according to the guidelines approved by the Laboratory Animal Ethics Committee of Tianjin Medical University Zhu Xianyi Memorial Hospital and Tianjin Institute of Endocrinology.

[0099] Hematoxylin-eosin (H&E) and Immunohistochemical (IHC) Staining: For H&E staining, mouse tumor tissue was collected, fixed with 4% paraformaldehyde, and embedded in paraffin. The embedded paraffin blocks were cut into 5 μm sections and stained with H&E. For IHC staining, paraffin sections of human and mouse tissues were dewaxed with xylene containing ethanol and water. Antigen recovery and nonspecific site blocking were then performed before incubation overnight at 4°C with primary antibodies SLC37A3 (1:100, #PA5-42500, Invitrogen, CA, United States) and Ki-67 (1:100, #AF0198, Affinity Biosciences, OH, United States). After washing three times with PBS, sections were incubated with anti-rabbit IgG secondary antibody and visualized using the DAB Immunohistochemical Chromogenic Kit (Sangon Biotech, Shanghai, China). Images were captured by optical microscopy.

[0100] An in vivo SLC37A3 knockout HCC xenograft model was constructed. The knockout efficiency of SLC37A3 shRNA was examined, and sh-SLC37A3-3, which had the highest knockout efficiency, was selected for xenograft experiments. Figure 8 A, B). HuH-7 cells transfected with sh-SLC37A3 or sh-NC were then subcutaneously injected into the right axilla of male BALB / c nude mice. Mice implanted with HuH-7 cells transfected with sh-NC showed smaller tumors compared to mice implanted with HuH-7 cells transfected with sh-SLC37A3. Figure 8(C, D). Compared with the sh-NC group, the mean tumor weight of the sh-SLC37A3 group was significantly reduced, which is consistent with the tumor growth curve. Figure 8 E).

[0101] The expression level of the tumor proliferation marker Ki-67 in paraffin-embedded tumor tissues was detected by in vitro chromogenic chromatography (IHC). IHC results showed that Ki-67 staining was stronger in sh-NC transfected tumor tissues, while it was relatively weaker in sh-SLC37A3 transfected tumor tissues. Figure 8 F). TUNEL staining was used to detect the degree of apoptosis in paraffin-embedded tumor tissue. Compared with the sh-NC group, the number of TUNEL-positive cells in the sh-SLC37A3 group was significantly higher (F). Figure 8 G).

[0102] The combined results show that knocking down SLC37A3 can significantly inhibit the proliferation of HuH-7 cells and promote their apoptosis, thereby further inhibiting the occurrence of HCC tumors in vivo.

[0103] The findings of this invention provide valuable insights into SLC37A3 as a prospective diagnostic and prognostic biomarker for HCC with glucose metabolism regulation function, and lay the foundation for further research on precise diagnostic and therapeutic targets for HCC.

[0104] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of reagents for detecting the hepatocellular carcinoma biomarker SLC37A3 in the preparation of prospective diagnostic kits for hepatocellular carcinoma.

2. Application of reagents for detecting the hepatocellular carcinoma biomarker SLC37A3 in the preparation of hepatocellular carcinoma prognostic diagnostic kits.

3. The application according to claim 1 or 2, characterized in that, The kit includes a negative control, which is normal hepatocyte tissue.

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