Use of phosphorylated proteins in the preparation of products for the diagnosis, prognosis evaluation and treatment of hepatocarcinoma

Abstract

This invention relates to the application of a phosphorylated protein in the preparation of products for the diagnosis, prognostic assessment, and treatment of liver cancer. The phosphorylated protein is selected from one or more combinations of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2. siRNA functional perturbation confirmed that SH3KBP1, SLK, EHD2, EPB41L3, and NEXN are essential for maintaining a large lipid droplet phenotype, while CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2 limit lipid droplet size. Cell migration and invasion experiments demonstrated that interfering with EPB41L3 significantly enhanced the migration and invasion abilities of liver cancer cells while reducing lipid droplets; while interfering with CPD, BET1, UFL1, RRP1B, and OGFR increased lipid droplets while inhibiting cell migration. Clinical data analysis showed that low expression of EPB41L3 was significantly associated with shortened overall survival in HCC patients. Monitoring the expression or phosphorylation level of these phosphorylated proteins can provide important information for predicting the progression of liver cancer, the risk of metastasis, and the development of treatment strategies.

Description

Technical Field

[0001] This invention relates to the field of bioinformatics, and in particular to the application of a phosphorylated protein in the preparation of products for the diagnosis, prognosis assessment and treatment of liver cancer. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide, and metastasis is the most critical factor determining the prognosis of HCC patients. Abnormal lipid metabolism is a prominent feature of HCC, and lipid droplets (LDs), as key organelles for intracellular lipid storage and metabolic regulation, are receiving increasing attention for their role in cancer metastasis. Accumulated lipid droplets can provide energy substrates through β-oxidation, meeting the high energy demands of tumor cell invasion and migration.

[0003] Post-translational modifications of proteins, particularly phosphorylation, play a central role in controlling the function of lipid droplet-related proteins. For example, AMPK phosphorylates PLIN2 / 3 to promote its degradation via chaperone-mediated autophagy, thereby promoting lipolysis. Although individual lipid droplet proteins such as PLIN2 and DGAT1 have been shown to be associated with tumorigenesis, such studies are mostly limited to single molecules and have failed to reveal at the systemic level how phosphorylation acts as a whole network to precisely regulate the dynamic changes of lipid droplets and their function in cancer.

[0004] Phosphoryproteomics offers a powerful strategy for decoding complex signaling networks and post-translational modifications in disease states. However, to date, no studies have performed a global phosphoryproteomic analysis of lipid droplet-related proteins associated with lipid droplet size and abundance in hepatocellular carcinoma.

[0005] Chinese patent CN120965805A discloses a phosphorylated peptide-like compound and its pharmaceutical uses. It provides a novel phosphorylated peptide-like compound; activity studies show that this compound exhibits excellent Cbl-b inhibitory activity, making it an effective Cbl-b inhibitor. Those skilled in the art know that Cbl-b overexpression inhibits T-cell immune responses, and inhibiting Cbl-b can activate the immune system to kill tumor cells. Therefore, the phosphorylated peptide-like compound provided by this invention, or its pharmaceutically acceptable salts or solvates, has the potential to be developed into drugs for treating or alleviating diseases (such as non-small cell lung cancer, breast cancer, prostate cancer, head and neck squamous cell carcinoma, liver cancer, pancreatic cancer, colorectal cancer, and ovarian cancer) that are treated or alleviated by inhibiting Cbl-b. However, this patent does not systematically study the phosphorylation biological regulation of liver cancer lipid droplets, the correlation between this regulation and pathological processes such as the invasion and migration ability of liver cancer cells, or the correlation between phosphorylated proteins and the clinical prognosis of liver cancer. Furthermore, the synthesis process of this phosphorylated peptide-like compound is complex, with cumbersome reaction steps and a low yield.

[0006] Chinese patent CN120998316A discloses a multi-omics-based gene knockout target library for hepatocellular carcinoma (HCC) and its screening method. Through data collection and integration, data screening, and target validation, a gene knockout target library for precision treatment of HCC is ultimately obtained. This invention systematically screens key driver genes for HCC through multi-omics data integration and bioinformatics analysis, revealing the important roles of these genes in the occurrence, development, metastasis, drug resistance, and immune escape of HCC. These genes not only deepen the understanding of the molecular mechanisms of HCC but also provide important theoretical basis and potential intervention targets for the development of targeted therapy and personalized treatment strategies. However, this patent does not systematically study the phosphorylation biological regulation of lipid droplets in HCC, the correlation between this regulation and pathological processes such as the invasion and migration ability of HCC cells, or the correlation between phosphorylated proteins and the clinical prognosis of HCC, and it does not conduct in vitro functional validation.

[0007] Chinese patent CN113564261B discloses lncRNAs related to hepatocellular carcinoma (HCC) and their applications. Analysis of differentially expressed lncRNAs in HCC revealed the high expression of lncRNA-BF368575. This high expression is closely associated with shorter disease-free survival (DFS) in HCC patients; lower lncRNA-BF368575 expression indicates a better prognosis, and its expression level is significantly correlated with the prognosis of HCC patients, suggesting its potential use in predicting HCC prognosis. Functional experiments showed that lncRNA-BF368575 can promote HCC growth by directly binding to phosphorylated proteins in the PI3K / AKT / mTOR signaling pathway, and its pro-cancer effect can be reversed by LY294002. However, this patent does not systematically study the phosphorylation biological regulation of HCC lipid droplets, the correlation between this regulation and pathological processes such as the invasion and migration abilities of HCC cells, and while the patent mentions predicting HCC prognosis, it does not address the correlation between phosphorylated proteins and the clinical prognosis of HCC. Furthermore, the patent does not address core clinical needs such as early screening and diagnosis of HCC.

[0008] Therefore, there is currently a lack of systematic research on key regulatory molecules related to the dynamic changes and metastasis of lipid droplets in liver cancer, based on phosphorylated proteomics technology. Summary of the Invention

[0009] The purpose of this invention is to overcome the problem that existing phosphorylation modifications are mostly limited to single molecules and fail to reveal at the system level how phosphorylation, as a whole network, precisely regulates the dynamic changes of lipid droplets and their functions in cancer, and to provide the application of phosphorylated proteins in the preparation of products for the diagnosis, prognostic assessment and treatment of liver cancer.

[0010] The objective of this invention can be achieved through the following technical solutions: In a first aspect, the present invention provides the application of reagents for detecting the expression or phosphorylation level of phosphorylated proteins in the preparation of products for the diagnosis, prognostic assessment, determination of migration and invasion capabilities, or monitoring of treatment effects of liver cancer, wherein the phosphorylated proteins are selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2.

[0011] Preferably, the product includes a reagent kit.

[0012] Preferably, the product uses tissue or blood samples as test samples.

[0013] Secondly, the present invention provides the use of phosphorylated proteins in the preparation of products that regulate the size of lipid droplets in liver cancer cells, wherein the phosphorylated proteins are selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

[0014] In one embodiment of the present invention, the lipid droplet volume is reduced by knocking down the SH3KBP1, SLK, EHD2, EPB41L3 or NEXN genes by siRNA; and the lipid droplet volume is increased by knocking down the CPD, BET1, UFL1, RRP1B, OGFR or CD2BP2 genes by siRNA. The nucleotide sequence of the sense strand of the siRNA targeting the SH3KBP1 gene is shown in SEQ ID NO.43; The nucleotide sequence of the antisense strand of the siRNA targeting the SH3KBP1 gene is shown in SEQ ID NO.44; The nucleotide sequence of the sense strand of the siRNA targeting the SLK gene is shown in SEQ ID NO.45; The nucleotide sequence of the antisense strand of the siRNA targeting the SLK gene is shown in SEQ ID NO.46; The nucleotide sequence of the sense strand of the siRNA targeting the EHD2 gene is shown in SEQ ID NO.47; The nucleotide sequence of the antisense strand of the siRNA targeting the EHD2 gene is shown in SEQ ID NO.48; The nucleotide sequence of the sense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.49; The nucleotide sequence of the antisense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.50; The nucleotide sequence of the sense strand of the siRNA targeting the NEXN gene is shown in SEQ ID NO.51; The nucleotide sequence of the antisense strand of the siRNA targeting the NEXN gene is shown in SEQ ID NO.52; The nucleotide sequence of the sense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.1; The nucleotide sequence of the antisense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.2; The nucleotide sequence of the sense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.15; The nucleotide sequence of the antisense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.16; The nucleotide sequence of the sense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.29; The nucleotide sequence of the antisense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.30; The nucleotide sequence of the sense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.33; The nucleotide sequence of the antisense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.34; The nucleotide sequence of the sense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.35; The nucleotide sequence of the antisense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.36; The nucleotide sequence of the sense strand of the siRNA targeting the CD2BP2 gene is shown in SEQ ID NO.37; The nucleotide sequence of the antisense strand of the siRNA targeting the CD2BP2 gene is shown in SEQ ID NO.38.

[0015] Thirdly, the present invention provides the use of phosphorylated proteins in the preparation of products that inhibit the migration and invasion of liver cancer, wherein the phosphorylated proteins are selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

[0016] In one embodiment of the present invention, EPB41L3 is knocked down by siRNA to promote cell migration and invasion; Cell migration was inhibited by knocking down CPD, BET1, UFL1, RRP1B and OGFR with siRNA; The nucleotide sequence of the sense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.49; The nucleotide sequence of the antisense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.50; The nucleotide sequence of the sense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.1; The nucleotide sequence of the antisense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.2; The nucleotide sequence of the sense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.15; The nucleotide sequence of the antisense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.16; The nucleotide sequence of the sense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.29; The nucleotide sequence of the antisense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.30; The nucleotide sequence of the sense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.33; The nucleotide sequence of the antisense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.34; The nucleotide sequence of the sense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.35; The nucleotide sequence of the antisense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.36.

[0017] Preferably, the phosphorylated protein is selected from EPB41L3.

[0018] Fourthly, the present invention provides the use of reagents or drugs for regulating the expression or phosphorylation level of phosphorylated proteins in the preparation of products for the treatment of liver cancer, wherein the phosphorylated proteins are selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

[0019] Fifthly, the present invention provides the use of phosphorylated proteins in the preparation of drugs for screening the treatment of liver cancer. The phosphorylated proteins are selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

[0020] This invention first developed a highly efficient and selective phosphorylated proteomics analysis technique. Using this technique, phosphorylated proteomics analysis was performed on various hepatocellular carcinoma cell lines exhibiting different lipid droplet phenotypes (lipid droplet size-dominated and lipid droplet number-dominated) under oleic acid stimulation. By comparing Huh1 and Huh7 cells with significant differences in lipid droplet size, 246 differentially expressed phosphorylated proteins were identified, of which 6 were upregulated in Huh1 cells (large lipid droplets) and 240 were upregulated in Huh7 cells (small lipid droplets). Through siRNA functional perturbation screening, SH3KBP1, SLK, and E were finally confirmed as the most effective phosphorylated proteins. HD2, EPB41L3, and NEXN are essential for maintaining a large lipid droplet phenotype, while CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2 limit lipid droplet size. Further cell migration and invasion experiments demonstrated that interfering with EPB41L3 significantly enhanced the migration and invasion of liver cancer cells while reducing lipid droplets; conversely, interfering with CPD, BET1, UFL1, RRP1B, and OGFR increased lipid droplets while inhibiting cell migration. Clinical data analysis showed that low expression of EPB41L3 was significantly associated with shortened overall survival in HCC patients.

[0021] Compared with the prior art, the present invention has the following beneficial effects: This invention, for the first time, systematically reveals the key role of phosphorylated proteins in regulating the size of lipid droplets in liver cancer cells through efficient phosphorylated proteomics analysis, and directly links lipid droplet dynamics with the migration and invasion capabilities of liver cancer cells.

[0022] This invention has discovered a new set of phosphorylated protein biomarkers associated with hepatocellular carcinoma lipid droplet phenotype and metastatic potential, and in particular, it has identified EPB41L3 as a novel hepatocellular carcinoma metastasis inhibitor.

[0023] Based on these phosphorylated proteins, this invention provides new targets and theoretical basis for the early diagnosis, metastasis risk warning, prognosis assessment, and development of new targeted therapy strategies for liver cancer. Attached Figure Description

[0024] Figure 1 This invention presents the phenotypic analysis of lipid droplets in different HCC cell lines under oleic acid stimulation. Figure a shows confocal microscopy images of lipid droplets in different HCC cell lines under oleic acid stimulation; figure b shows the average volume of each lipid droplet in Huh1 and Huh7 cells under oleic acid stimulation; figure c shows the number of lipid droplets per cell in Huh1 and Huh7 cells under oleic acid stimulation; figure d shows the average volume of each lipid droplet in Tong, SNU354, SNU398, and SNU449 cells under oleic acid stimulation; and figure e shows the number of lipid droplets per cell in Tong, SNU354, SNU398, and SNU449 cells under oleic acid stimulation.

[0025] Figure 2 These are phosphorylated proteomics analysis diagrams of lipid droplet size-dominant (Huh1 / Huh7) and number-dominant (Tong / SNU354 / SNU398 / SNU449) cells of the present invention; where a is a PLS-DA analysis diagram of lipid droplet size-dominant cell populations (Huh1, Huh7), with the x-axis representing the first principal component (Component 1) and the y-axis representing the second principal component (Component 2); b is a heatmap of differentially phosphorylated proteomics in lipid droplet size-dominant cell populations (Huh1, Huh7); c is a volcano plot of differentially phosphorylated sites in lipid droplet size-dominant cell populations (Huh1, Huh7); and d is a PLS-DA analysis diagram of number-dominant cell populations (Tong, SNU354, SNU398, SNU449), with the x-axis representing the first principal component (Component 1) and the y-axis representing the second principal component (Component 2). 2); e is a heatmap of differentially phosphorylated proteomics in the number-dominant cell populations (Tong, SNU354, SNU398, SNU449); f and g are Venn diagrams of differentially upregulated proteins in the number-dominant cell populations.

[0026] Figure 3 This invention uses siRNA transfection technology to knock down target genes and observe the effects on the size and number of lipid droplets in Huh1 and Huh7 cells; where a represents the average volume of each lipid droplet in Huh1 cells after knockdown of STRN, SH3KBP1, SLK, EHD2, EPB41L3, or NEXN genes; b represents the number of lipid droplets per cell in Huh1 cells after knockdown of STRN, SH3KBP1, SLK, EHD2, EPB41L3, or NEXN genes; and c represents the number of lipid droplets per cell in Huh7 cells after knockdown of CPD, PPA2, TRA2B, AFG3L2, EGFR, PLCB3, KPNA2, BET1, CDK12, APLP2, NCL, AGFG1, and NOL9 genes. The average volume of each lipid droplet after knocking down the NUP210, UFL1, G3BP2, RRP1B, OGFR, CD2BP2, or RCC1 genes; d represents the number of lipid droplets per cell in Huh7 cells after knocking down the CPD, PPA2, TRA2B, AFG3L2, EGFR, PLCB3, KPNA2, BET1, CDK12, APLP2, NCL, AGFG1, NOL9, NUP210, UFL1, G3BP2, RRP1B, OGFR, CD2BP2, or RCC1 genes.

[0027] Figure 4This invention describes the effect of siRNA transfection technology on the knockdown of target genes on the size and number of lipid droplets in Tong and SNU354 cells; where a represents the number of lipid droplets per cell in Tong cells after knockdown of BYSL, LIMCH1, JUN, HARS2, FMNL2, APLP2, KRT18, NES, BSG, EGFR, SQSTM1, CAV2, or ARFGAP1 genes; b represents the average volume of each lipid droplet in Tong cells after knockdown of BYSL, LIMCH1, JUN, HARS2, FMNL2, APLP2, KRT18, NES, BSG, EGFR, SQSTM1, CAV2, or ARFGAP1 genes; c represents the number of lipid droplets per cell in SNU354 cells after knockdown of BCKDHA, SYAP1, or PXN genes; and d represents the average volume of each lipid droplet in SNU354 cells after knockdown of BCKDHA, SYAP1, or PXN genes.

[0028] Figure 5 This invention relates to the effects of siRNA transfection technology on the migration and invasion abilities of target genes in Huh1 and Huh7 cells, and to the clinical prognostic analysis of the target genes. Figure a shows a microscopic view of Huh1 cell migration and invasion results measured using a Boyden chamber after knockdown of SH3KBP1, SLK, EHD2, EPB41L3, or NEXN genes; figure b shows a statistical graph of Huh1 cell migration results after knockdown of SH3KBP1, SLK, EHD2, EPB41L3, or NEXN genes; figure c shows a statistical graph of Huh1 cell invasion results after knockdown of SH3KBP1, SLK, EHD2, EPB41L3, or NEXN genes; and figure d shows the effects of siRNA transfection technology on the migration and invasion abilities of target genes in Huh1 and Huh7 cells. Microscopic images of Huh7 cells after knockdown of CPD, BET1, UFL1, G3BP2, RRP1B, OGFR, CD2BP2, or RCC1 genes, measured using Boyden chambers; e is a statistical graph of Huh7 cell migration after knockdown of CPD, BET1, UFL1, G3BP2, RRP1B, OGFR, CD2BP2, or RCC1 genes; f is a statistical graph of Huh7 cell invasion after knockdown of CPD, BET1, UFL1, G3BP2, RRP1B, OGFR, CD2BP2, or RCC1 genes; g is a Kaplan-Meier survival curve of the TCGA-LIHC cohort based on hepatocellular carcinoma RNA-seq data.

[0029] Figure 6The experimental flowcharts are for Examples 1-4. Detailed Implementation

[0030] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments. These embodiments are based on the technical solution of the present invention and provide detailed implementation methods and specific operating procedures. However, the scope of protection of the present invention is not limited to the following embodiments.

[0031] Huh1, Huh7, Tong, and SNU354 were cultured in DMEM / F12 medium. SNU398 and SNU449 were cultured using RPMI-1640 medium.

[0032] DMEM / F12 medium: 3151 mg / L D-glucose, 55 mg / L sodium pyruvate, 365 mg / L L-glutamine, 1.2 g / L sodium bicarbonate, 8.1 mg / L phenol red.

[0033] RPMI-1640 medium: 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L L-glucose and 1500 mg / L sodium bicarbonate, free of protein, lipids and growth factors.

[0034] All culture media were supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin, 1% (v / v) GlutaMAX and 1% (v / v) sodium pyruvate to prepare fresh complete culture media.

[0035] Example 1 Lipid droplet phenotype analysis and phosphorylated proteomics screening of liver cancer cells, the process is as follows: Figure 6 As shown: Six HCC cell lines (Huh1, Huh7, Tong, SNU354, SNU398, and SNU449) were stimulated with 200 μM oleic acid (OA) for 16 hours. Lipid droplet phenotypes were analyzed using high-content imaging. The steps are as follows: Human hepatocellular carcinoma cell lines Huh1 and Huh7 were obtained from the JCRB cell bank in Japan, SNU398, SNU449, and SNU354 from the ATCC cell bank in the United States, and Tong from the Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. All cells were cultured in a humidified incubator at 37°C and 5% CO2. Huh1, Huh7, Tong, and SNU354 were cultured in DMEM / F12 medium (containing 3151 mg / L D-glucose, 55 mg / L sodium pyruvate, 365 mg / L L-glutamine, 1.2 g / L sodium bicarbonate, and 8.1 mg / L phenol red); SNU398 and SNU449 were cultured in RPMI-1640 medium, which contains 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 4500 mg / L D-glucose, and 1500 mg / L sodium bicarbonate, and is free of protein, lipids, and growth factors. All culture media were supplemented with 10% (v / v) fetal bovine serum, 1% (v / v) penicillin-streptomycin, 1% (v / v) GlutaMAX, and 1% (v / v) sodium pyruvate to prepare fresh, complete culture media. When cell confluence reached 80-90%, cells were digested with 0.25% trypsin at a rate of 2 ml per 10 cm dish at 37°C for 2 minutes, passaged, and seeded at a ratio of 1:3.

[0036] Cells were seeded at 100 μL per well in 96-well PhenoPlates and cultured at 37°C and 5% CO2 for 6–8 hours to allow them to adhere. After adhesion, the medium was replaced with fresh complete medium containing different concentrations of oleic acid (OA), including 50, 100, and 200 μM oleic acid, and cultured for another 16 hours. After induction, the medium was aspirated, and the cells were gently washed once with PBS.

[0037] Subsequently, 4% paraformaldehyde (PFA) was added for fixation at room temperature for 20 minutes. After fixation, the cells were washed again with PBS. Staining working solution containing Hoechst 33342 (nuclear dye) and BODIPY 493 / 503 (lipid droplet-specific fluorescent dye) was used, and the cells were incubated at room temperature in the dark for 30 minutes. After staining, the staining solution was aspirated, and the cells were gently washed twice with PBS (200 μL / well). Finally, 100 μL of PBS was added to each well, and the lipid droplets were immediately visualized and quantified using a high-content imaging system.

[0038] The results showed that Huh1 cells exhibited a large lipid droplet phenotype, while Huh7 cells exhibited a small lipid droplet phenotype. Figure 1 b); Tong, SNU354, SNU398, and SNU449 cells showed no difference in lipid droplet volume ( Figure 1d); Tong and SNU354 cells had a higher number of lipid droplets, while SNU398 and SNU449 cells had a lower number of lipid droplets. Figure 1 e); there was no difference in the number of lipid droplets between Huh1 and Huh7 cells (e); Figure 1 c). Based on this, cells were divided into lipid droplet size-dominant (Huh1, Huh7) and number-dominant (Tong, SNU354, SNU398, SNU449) types for subsequent comparative analysis of phosphorylated proteomics.

[0039] Cells were cultured on slides in 6-well plates. When cell confluence reached approximately 50%, 100 μM OA-BSA complex (oleic acid to bovine serum albumin molar ratio of 6:1) was added, and culture continued for 16 hours. After induction, cells were washed twice with pre-chilled PBS, followed by fixation with 4% PFA at room temperature for 30 minutes. Next, cells were permeabilized with 0.1% Triton X-100 at room temperature for 10 minutes, and blocked with 1% BSA at room temperature for 1 hour. Finally, cell nuclei were stained with Hoechst 33342, and lipid droplets were labeled using Bodipy 493 / 503. After washing three times with pre-chilled PBS, the slides were mounted. Lipid droplet images were acquired using a NIKON A1 confocal microscope at 100x oil immersion, and the droplet size was quantitatively analyzed using ImageJ software.

[0040] The results are as follows Figure 1 The results showed that after adding 200 μM OA, lipid droplets accumulated in different liver cancer cells.

[0041] Cells were stimulated with OA and lysed using 500 μL of lysis buffer containing 2% SDS, followed by sonication to ensure complete lysis. 5 μL of 1M DTT (Dithiothreitol) was added to the resulting lysate (approximately 500 μL) to achieve a final DTT concentration of 10 mM, and the mixture was reduced at 60 °C for 1 hour. Subsequently, 12.5 μL of 1M IAA (Iodoacetamide) was added to achieve a final IAA concentration of 25 mM, and the mixture was alkylated at 37 °C in the dark for 30 minutes. Afterward, 2.5 mL of pre-chilled acetone was added, and the mixture was incubated overnight at -20 °C. The protein precipitate was collected by centrifugation and digested overnight at 37 °C using sequencing-grade trypsin (enzyme to substrate ratio of 1:40). Phosphorylated peptides were then enriched using a phosphorylated peptide-specific enrichment probe. The eluted sample was desalted and analyzed by LC-MS / MS.

[0042] The results are as follows Figure 2 As shown in a and d, PLS-DA analysis clearly distinguishes between lipid droplet size-dominated and number-dominated cell populations. Figure 2The heatmap results in b and e further confirmed the differences in phosphorylated protein profiles between the groups, indicating a fundamental difference in the phosphorylated proteins. Importantly, in the size-dominant intracellular comparison (Huh1 vs Huh7), 246 differentially phosphorylated proteins were successfully identified (P < 0.1, FC > 2), of which only 6 were specifically upregulated in Huh1 cells with larger lipid droplets, while 240 were specifically upregulated in Huh7 cells with smaller lipid droplets. Figure 2 b, c). In the lipid droplet count group, compared with SNU398 and SNU449 cells, Tong cells had 13 phosphorylated proteins upregulated, and SNU354 cells had 3 upregulated proteins. Figure 2 (fg), suggesting that these proteins may be potential regulators of lipid droplet number. This result indicates that the regulation of lipid droplet size and number may be closely related to a group of phosphorylated proteins and their mediated signal transduction. This invention is the first to systematically map phosphorylation patterns associated with the lipid droplet phenotype of liver cancer cells, providing a precise target library for subsequent functional studies.

[0043] Example 2 Functional validation of candidate phosphorylated proteins, the procedure is as follows: Figure 6 As shown: siRNA transfection was performed using Lipofectamine 8000 transfection reagent, strictly following the manufacturer's instructions. The simplified steps are as follows: Cells were seeded in 6-well plates and transfected when the cell density reached 60-70%. 100 nM of the corresponding siRNA was transfected into each well. Cells were collected 48 hours after transfection for subsequent analysis. The siRNA sequences used in this study are shown in Table 1.

[0044] Table 1 siRNA sequences Total RNA was extracted from cells using TRIzol reagent, following the manufacturer's instructions. RNA concentration and purity were determined using a Nanodrop One spectrophotometer. cDNA was synthesized using the PrimeScript™ RT kit with 1 μg of total RNA as a template. Quantitative PCR was performed using SYBR Green Mix on a real-time quantitative PCR instrument. The relative expression level of mRNA was calculated using the 2^–ΔΔCt method, with β-actin as an internal control gene. Primer sequences for RT-qPCR are shown in Table 2, and the RT-qPCR amplification system is shown in Table 3.

[0045] Table 2 Primer Sequences Table 3 RT-qPCR amplification system The results are as follows Figure 3 As shown in ab, in Huh1 cells, which originally had large lipid droplets, knocking down five genes—SH3KBP1, SLK, EHD2, EPB41L3, and NEXN—significantly reduced lipid droplet volume, demonstrating that they are essential for maintaining the large lipid droplet phenotype. Knocking down the STRN, SH3KBP1, and NEXN genes also affected the number of lipid droplets in Huh1 cells, increasing their number.

[0046] like Figure 3 As shown in cd, in Huh7 cells, which originally had smaller lipid droplets, knocking down genes such as CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2 significantly increased lipid droplet volume, indicating that their function is to inhibit excessive lipid droplet enlargement. Knockdown of genes such as CPD, PPA2, TRA2B, AFG3L2, EGFR, PCLB3, BET1, CDK12, APLP2, NCL, AGFG1, NOL9, NUP210, and UFL1 also affected the number of lipid droplets in Huh7 cells, increasing their number.

[0047] Conversely, knocking down specific genes in Tong or SNU354 cells did not significantly alter the number of lipid droplets. Figure 4 (ad) This indicates that phosphorylation modification may play a more important role in regulating lipid droplet size than in controlling lipid droplet number in hepatocellular carcinoma. These data confirm that the candidate proteins screened by the phosphorylated proteomics platform of this invention are key effector molecules that directly regulate lipid droplet size in hepatocellular carcinoma cells, achieving a leap from correlation discovery to causal verification.

[0048] Example 3 The effects of candidate phosphorylated proteins on the migration and invasion of liver cancer cells, the process is as follows: Figure 6 As shown: This invention further explores the functions of the aforementioned regulatory factors in tumor metastasis. Cell migration and invasion experiments were performed using a modified 24-well Boyden chamber.

[0049] (1) Transfer experiment: 1×10 5Cells were suspended in 200 μL of serum-free DMEM / F12 medium and seeded in the upper chamber, while fresh complete medium was added to the lower chamber as a chemotactic agent. After culturing at 37°C for 48 hours, unmigrated cells in the upper chamber were wiped off with a cotton swab. Cells that migrated to the lower chamber were fixed, stained, and counted in four randomly selected fields of view under a microscope at 200x magnification.

[0050] (2) Invasion test: The procedure is basically the same as the migration test, except that the upper chamber membrane is pre-coated with Matrigel (Corning) to simulate the extracellular matrix.

[0051] The results are as follows Figure 5 As shown in Figure ac, knockdown of EPB41L3 in Huh1 cells reduced the size of lipid droplets. Simultaneously, the number of migrating and invasive cells increased by approximately 2.3-fold, indicating that EPB41L3 is a potential inhibitor of liver cancer metastasis, suppressing the migration and invasion of liver cancer cells by maintaining larger lipid droplets. Although knockdown of EHD2 also reduced lipid droplet size, it only had a slight inhibitory effect on migration and invasion (approximately 20%). In Huh7 cells, knockdown of CPD, BET1, UFL1, G3BP2, RRP1B, and OGFR increased lipid droplets and inhibited cell migration, but had little effect on invasion. Figure 5 This indicates that the present invention establishes for the first time a functional axis of "phosphorylated protein → lipid droplet size → cell migration / invasion ability", clarifying that these phosphorylated proteins not only regulate cell lipid droplet morphology, but are also key switches affecting liver cancer metastasis, providing a novel target for the development of anti-liver cancer metastasis therapies.

[0052] Example 4 Clinical relevance analysis of candidate phosphorylated proteins, the workflow is as follows: Figure 6 As shown: To investigate the clinical relevance of the identified phosphorylated proteins to hepatocellular carcinoma (HCC) development, patient survival data from the TCGA-LIHC cohort were analyzed. The specific steps were as follows: Kaplan-Meier survival curves were generated using the online tool KMplot (http: / / kmplot.com / analysis / index.php?p=service&cancer=liver_rnaseq) based on HCC RNA-seq data, with all parameters kept at default settings. Differences between groups were assessed using the log-rank test.

[0053] The results are as follows Figure 5As shown in g, low expression of EPB41L3 was significantly associated with shortened overall survival in hepatocellular carcinoma (HCC) patients (P = 0.021), further suggesting its potential role as a metastasis inhibitory protein in HCC. Furthermore, decreased expression levels of BET1 and RRP1B were also associated with poorer prognosis, while the remaining candidate genes did not show significant prognostic value. These findings collectively indicate that phosphorylation-mediated regulation of lipid droplet size (rather than lipid droplet number) is closely related to the invasiveness of HCC cells. Among all identified regulatory factors, EPB41L3, as a novel phosphorylated protein, can regulate both lipid droplet dynamics and metastatic potential. This dual function makes EPB41L3 a highly promising candidate molecule for HCC therapeutic targeting and prognostic assessment.

[0054] All statistical analyses were performed using GraphPad Prism 9.0 software, and data visualizations were generated using Mathematica and Adobe Illustrator. Results are expressed as mean ± standard error of at least three independent trials; see figure captions for specific number of trials. Unpaired two-tailed Student's t-tests were used for comparisons between two groups, and one-way ANOVA combined with Tukey's post-hoc test was used for comparisons of three or more groups. Statistical significance was defined as * P < 0.05, ** P < 0.01, *** P < 0.001, and NS indicates no statistically significant difference (P > 0.05).

[0055] Combining the above research methods and data, we first successfully mapped the phosphorylation regulation of lipid droplet phenotype in liver cancer cells using a phosphorylated proteomics platform. Then, we screened 12 phosphorylated proteins that play a key regulatory role in lipid droplet size phenotype. Among them, SH3KBP1, SLK, EHD2, EPB41L3, and NEXN are essential for maintaining a large lipid droplet phenotype, while CPD, BET1, UFL1, RRP1B, OGFR, and CD2BP2 have the function of inhibiting lipid droplet enlargement. In vitro functional validation revealed that these phosphorylated proteins not only regulate lipid droplet morphology but, more importantly, affect the migration and invasion capabilities of liver cancer cells. EPB41L3 was confirmed as a key factor in inhibiting liver cancer metastasis by regulating lipid droplet size. Clinical data analysis further confirmed that the expression levels of EPB41L3, BET1, and RRP1B are significantly correlated with the prognosis of liver cancer patients. These findings not only reveal a new mechanism by which phosphorylation regulates lipid droplet dynamics and affects liver cancer metastasis but also provide new biomarkers and candidate targets for prognostic assessment and targeted therapy of liver cancer.

[0056] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. The application of reagents for detecting the expression or phosphorylation level of phosphorylated proteins in the preparation of products for the diagnosis, prognostic assessment, determination of metastatic and invasive capabilities, or monitoring of treatment efficacy in liver cancer, characterized in that... The phosphorylated protein is selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

2. The application according to claim 1, wherein the product comprises a reagent kit.

3. The application according to claim 1, characterized in that, The product uses tissue or blood samples as test samples.

4. The application of phosphorylated proteins in the preparation of products that regulate the size of lipid droplets in liver cancer cells, characterized in that, The phosphorylated protein is selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

5. The application according to claim 4, characterized in that, Lipid droplet volume can be reduced by knocking down SH3KBP1, SLK, EHD2, EPB41L3 or NEXN genes with siRNA; lipid droplet volume can be increased by knocking down CPD, BET1, UFL1, RRP1B, OGFR or CD2BP2 genes with siRNA. The nucleotide sequence of the sense strand of the siRNA targeting the SH3KBP1 gene is shown in SEQ ID NO.43; The nucleotide sequence of the antisense strand of the siRNA targeting the SH3KBP1 gene is shown in SEQ ID NO.44; The nucleotide sequence of the sense strand of the siRNA targeting the SLK gene is shown in SEQ ID NO.45; The nucleotide sequence of the antisense strand of the siRNA targeting the SLK gene is shown in SEQ ID NO.46; The nucleotide sequence of the sense strand of the siRNA targeting the EHD2 gene is shown in SEQ ID NO.47; The nucleotide sequence of the antisense strand of the siRNA targeting the EHD2 gene is shown in SEQ ID NO.48; The nucleotide sequence of the sense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.49; The nucleotide sequence of the antisense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.50; The nucleotide sequence of the sense strand of the siRNA targeting the NEXN gene is shown in SEQ ID NO.51; The nucleotide sequence of the antisense strand of the siRNA targeting the NEXN gene is shown in SEQ ID NO.52; The nucleotide sequence of the sense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.1; The nucleotide sequence of the antisense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.2; The nucleotide sequence of the sense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.15; The nucleotide sequence of the antisense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.16; The nucleotide sequence of the sense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.29; The nucleotide sequence of the antisense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.30; The nucleotide sequence of the sense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.33; The nucleotide sequence of the antisense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.34; The nucleotide sequence of the sense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.35; The nucleotide sequence of the antisense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.36; The nucleotide sequence of the sense strand of the siRNA targeting the CD2BP2 gene is shown in SEQ ID NO.37; The nucleotide sequence of the antisense strand of the siRNA targeting the CD2BP2 gene is shown in SEQ ID NO.

38.

6. The application of phosphorylated proteins in the preparation of products that inhibit the migration and invasion of liver cancer, characterized in that, The phosphorylated protein is selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

7. The application according to claim 6, characterized in that, Knocking down EPB41L3 with siRNA promotes cell migration and invasion; Cell migration was inhibited by knocking down CPD, BET1, UFL1, RRP1B and OGFR with siRNA; The nucleotide sequence of the sense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.49; The nucleotide sequence of the antisense strand of the siRNA targeting the EPB41L3 gene is shown in SEQ ID NO.50; The nucleotide sequence of the sense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.1; The nucleotide sequence of the antisense strand of the siRNA targeting the CPD gene is shown in SEQ ID NO.2; The nucleotide sequence of the sense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.15; The nucleotide sequence of the antisense strand of the siRNA targeting the BET1 gene is shown in SEQ ID NO.16; The nucleotide sequence of the sense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.29; The nucleotide sequence of the antisense strand of the siRNA targeting the UFL1 gene is shown in SEQ ID NO.30; The nucleotide sequence of the sense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.33; The nucleotide sequence of the antisense strand of the siRNA targeting the RRP1B gene is shown in SEQ ID NO.34; The nucleotide sequence of the sense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.35; The nucleotide sequence of the antisense strand of the siRNA targeting the OGFR gene is shown in SEQ ID NO.

36.

8. The application according to claim 6, characterized in that, The phosphorylated protein was selected from EPB41L3.

9. The application of reagents or drugs that regulate the expression or phosphorylation level of phosphorylated proteins in the preparation of products for the treatment of liver cancer, characterized in that, The phosphorylated protein is selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

10. The application of phosphorylated proteins in the preparation and screening of drugs for the treatment of liver cancer, characterized in that, The phosphorylated protein is selected from one or a combination of several of SH3KBP1, SLK, EHD2, EPB41L3, NEXN, CPD, BET1, UFL1, RRP1B, OGFR and CD2BP2.

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