Application of substance for inhibiting FHL3 gene expression in preparation of medicine for treating hepatocellular carcinoma

Abstract

The invention provides application of a substance for inhibiting FHL3 gene expression in preparation of a medicine for treating hepatocellular carcinoma. The application finds that the substance for inhibiting the expression of FHL3 significantly inhibits the proliferation and metastasis of hepatocellular carcinoma cells, and effectively reduces the volume of tumors in vivo and reduces pulmonary metastasis. Therefore, the substance for inhibiting the expression of the FHL3 gene can be used for preparing the medicine for preventing, relieving and / or treating the hepatocellular carcinoma.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, in particular to the application of a substance for inhibiting FHL3 gene expression in the preparation of a drug for preventing, alleviating and / or treating hepatocellular carcinoma. BACKGROUND

[0002] Hepatocellular carcinoma is one of primary liver cancer, which mainly includes hepatocellular carcinoma, cholangiocellular carcinoma and mixed hepatocellular carcinoma. Hepatocellular carcinoma is derived from the damage of hepatocytes, which is caused by various factors such as hepatitis B virus and alcohol abuse, and the chronic inflammatory damage causes the hepatocytes to become malignant and form malignant tumors. Cholangiocellular carcinoma is derived from cholangiocellular carcinoma, and mixed hepatocellular carcinoma is derived from both.

[0003] Therefore, it is necessary to develop a target for screening a drug for preventing, alleviating and / or treating hepatocellular carcinoma and the drug. SUMMARY

[0004] The present application provides the application of a substance for inhibiting FHL3 gene expression in the preparation of a drug for preventing, alleviating and / or treating hepatocellular carcinoma. The present application has found that the expression of FHL3 in the serum of patients diagnosed as patients is significantly higher than that of healthy people (P<0.01), and the decrease of FHL3 level can inhibit the proliferation or metastasis of hepatocellular carcinoma, so FHL3 can be used as a target for screening a drug for preventing, alleviating and / or treating hepatocellular carcinoma.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: In the first aspect of the present application, FHL3 gene (Gene ID: 2275) is provided as a target gene for screening a drug for preventing, alleviating and / or treating hepatocellular carcinoma, and the screening method includes screening a substance capable of inhibiting FHL3 gene expression.

[0006] In the second aspect of the present application, a substance for inhibiting FHL3 gene expression is provided for the preparation of a drug for preventing, alleviating and / or treating hepatocellular carcinoma.

[0007] Further, the substance for inhibiting FHL3 gene expression includes shRNA for knockdown.

[0008] Further, the sequence of the shRNA is shown in SEQ ID NO. 1-2.

[0009] Further, the drug prevents, alleviates and / or treats hepatocellular carcinoma by at least one of the following effects: inhibiting the proliferation or metastasis of hepatocellular carcinoma.

[0010] In a third aspect of the present application, a medicine for preventing, alleviating and / or treating hepatocellular carcinoma is provided, characterized in that the medicine comprises a substance for inhibiting expression of FHL3 gene.

[0011] Further, the medicine further comprises a pharmaceutically acceptable excipient.

[0012] Further, the excipient is selected from one of a filler, a disintegrant, a binder, a diluent, a lubricant, a sweetener or a colorant.

[0013] Further, the dosage form of the medicine comprises at least one of a granule, a tablet, a pill, a capsule, an injection and a dispersion.

[0014] In a fourth aspect of the present application, FHL3 is provided for use as a molecular marker in the preparation of a product for diagnosing hepatocellular carcinoma.

[0015] In a fourth aspect of the present application, a detection reagent of FHL3 is provided for use in the preparation of a product for diagnosing hepatocellular carcinoma.

[0016] Further, the detection reagent of FLNC comprises one of an ELISA detection kit, a real-time fluorescent quantitative PCR kit and an immunohistochemical detection kit. Alternatively, the detection reagent comprises an antibody for detecting FHL3 protein or a primer (SEQ ID NO. 3-SEQ ID NO. 4) for detecting FHL3 mRNA.

[0017] The one or more technical solutions in the embodiments of the present application have at least the following technical effects or advantages: 1. The present application provides a substance for inhibiting expression of FHL3 gene for use in the preparation of a medicine for preventing, alleviating and / or treating hepatocellular carcinoma. The present application first discovers that FHL3 is highly expressed in liver cancer tissues, and the 5-year survival rate of patients with high expression is reduced by 40%. In combination with YAP detection, the AUC reaches 0.91. Therefore, a detection reagent of FHL3 can be used for preparing a product or kit for diagnosing hepatocellular carcinoma.

[0018] 2. In the present application, shRNA (SEQ ID NO. 1-2) targeting FHL3 significantly inhibits liver cancer proliferation (inhibition rate >65%, *P<0.001) and metastasis (83% reduction in lung metastasis, *P<0.001), indicating that a substance for inhibiting expression of FHL3 gene can be used for preparing a medicine for preventing, alleviating and / or treating hepatocellular carcinoma. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings needed in the following embodiment description will be briefly introduced. Obviously, the drawings in the following description are some embodiments of the present application, and other drawings can also be obtained by those skilled in the art without any creative effort.

[0020] Figure 1 Experimental data for FHL3 as an independent prognostic marker in Example 1; wherein, Figure 1 A: Spontaneous tumor model in mice induced by tumor driver genes. Figure 1 B: Representative liver images (top panel) scale bar: 1 cm, and H&E staining images of the spontaneous tumor model in mice. Figure 1 C: Heatmap showing the transcriptomic differences between the three model tissues. Figure 1 D: Using four gene sets to screen the most relevant differential genes to YAP. Figure 1 E: Detection of YAP, c-MYC, p-AKT and FHL3 expression in the liver tissues of animal models by WB experiment. Figure 1 F: Expression levels of FHL3 in cancer tissues and adjacent tissues in the LIHC expression matrix of the TCGA database. Figure 1 G: Kaplan-Meier curve of overall survival of the FHL3 differential expression group in the LIHC expression matrix of the TCGA database. Figure 1 H: Protein differential expression of FHL3 in 50 pairs of cancer tissues and adjacent tissues. Figure 1 I: qPCR detection of FHL3 mRNA expression in 50 pairs of cancer tissues and adjacent tissues. Figure 1 J: Detection of FHL3 expression in cancer tissues and adjacent tissues by immunohistochemical staining. Figure 1 K: Representative images of FHL3 expression levels detected by immunohistochemical staining. Figure 1 L: Kaplan-Meier curve of overall survival of the FHL3 differential expression group in the Shanghai cohort. Figure 1 M: Forest plot of multivariate Cox proportional hazards model of FHL3 on overall survival.

[0021] Figure 2 FHL3 overexpression or shRNA regulation of hepatocellular carcinoma cell proliferation and invasion in Example 2. Figure 2 A: Expression levels of FHL3 protein in various common HCC cell lines. Figure 2 B: Construction of FHL3 stably overexpressing cell lines in Hep3B (left) and Huh7 (right) cell lines. Figure 2 C: Construction of FHL3 stably knocked down cell lines in MHCC97H (left) and HLF (right) cell lines. Figure 2D: CCK8 experiment shows the proliferation promotion effect comparison of overexpression of FHL3. Figure 2 E: CCK8 experiment shows the proliferation inhibition effect comparison of shRNA of FHL3. Figure 2 F: EdU experiment shows the proliferation promotion effect comparison of overexpression of FHL3. Figure 2 G: EdU experiment shows the proliferation inhibition effect comparison of shRNA of FHL3. Figure 2 H: Transwell experiment shows the invasion promotion effect comparison of overexpression of FHL3. Figure 2 I: Transwell experiment shows the invasion inhibition effect comparison of shRNA of FHL3. Figure 2 J: Scratch experiment shows the invasion promotion effect comparison of overexpression of FHL3. Figure 3 K: Scratch experiment shows the invasion inhibition effect comparison of shRNA of FHL3.

[0022] Figure 3 To implement the in vivo efficacy of FHL3 overexpression or shFHL3 in regulating liver cancer progression in Example 3. Figure 3 A: Hep3B cell line stably overexpressing FHL3 subcutaneous tumor model, and tumor weight and volume statistics. Figure 3 B: MHCC97H cell line stably knocking down FHL3 subcutaneous tumor model (top), and tumor weight and volume statistics. Figure 3 C: Representative images of H&E staining and immunohistochemical staining of Hep3B cell line subcutaneous tumor tissue (left), and Ki67 positive cell rate statistics (right). Figure 3 D: Representative images of H&E staining and immunohistochemical staining of MHCC97H cell line subcutaneous tumor tissue (left), and Ki67 positive cell rate statistics (right). Figure 3 E: Representative images of Hep3B cell line orthotopic tumor model mouse imaging (left), and fluorescence intensity statistics (right). Figure 3 F: Representative images of Hep3B cell line orthotopic tumor model in liver, H&E staining panorama, magnified image and Ki67 immunohistochemical staining, and tumor volume and Ki67 positive proportion statistics (right). Figure 3 G: Representative images of MHCC97H cell line orthotopic tumor model mouse imaging (left), and fluorescence intensity statistics (right). Figure 3 H: Representative images of MHCC97H cell line orthotopic tumor model in liver, H&E staining panorama, magnified image and Ki67 immunohistochemical staining, and tumor volume and Ki67 positive proportion statistics (right). Figure 3 I: Representative images of Hep3B cell line orthotopic tumor model lung tissue H&E staining and lung metastasis statistics. Figure 4J: Representative images of H&E staining of lung tissue of orthotopic transplantation tumor model of HCC97H cell line and lung metastasis statistics.

[0023] Figure 4 Verification of FHL3 and YAP clinical prognosis synergy in Example 4. Figure 4 A: Immunohistochemical staining shows the correlation between YAP and FHL3 protein expression in the same tissue. Figure 4 B: YAP and FHL3 immunohistochemical staining scores show that the two protein abundances are correlated. Figure 4 C: Kaplan-Meier curve of overall survival rate of YAP differentially expressed group in Tongji cohort. Figure 4 D: Kaplan-Meier curve of overall survival rate of YAP and FHL3 differentially expressed group in Tongji cohort. Figure 4 E: CCK8 experiment of knocking down FHL3 based on YAP overexpression in Hep3B cell line. Figure 4 F: EdU incorporation experiment of knocking down FHL3 based on YAP overexpression in Hep3B cell line. Figure 4 G: Transwell experiment of knocking down FHL3 based on YAP overexpression in Hep3B cell line. Figure 4 H: Scratch experiment of knocking down FHL3 based on YAP overexpression in Hep3B cell line. Figure 4 I: Subcutaneous transplantation tumor weight statistics chart. Figure 4 J: Subcutaneous transplantation tumor volume change measured every three days. Figure 4 K: Ki67 positive cell rate statistics. Figure 4 L: Total fluorescence intensity statistics of liver orthotopic transplantation model. Figure 4 M: YAP-driven spontaneous tumor model established after knocking down FHL3 using rAAV8 adenovirus method. Figure 4 N: Representative liver images and H&E staining images after knocking down FHL3 in YAP-driven mouse spontaneous tumor model. Figure 1 O: Western blotting detects YAP, FHL3, and KRAS protein expression in liver tissue of animal model. Figure 1 P: Effect of FHL3 knockdown on YAP-induced HCC mouse prognosis. DETAILED DESCRIPTION

[0024] The advantages and various effects of the present application will be more clearly presented from the following specific embodiments and examples. Those skilled in the art should understand that these specific embodiments and examples are used to illustrate the present application, not to limit the present application.

[0025] Throughout the specification, unless otherwise specifically indicated, the terms used are intended to be understood as commonly used in the field of the present application. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. If there is a conflict between the present specification and the definitions, the present specification takes precedence.

[0026] Unless otherwise specifically indicated, various materials, reagents, instruments and equipment and the like used in the present application can be purchased on the market or can be obtained by existing methods.

[0027] The application of FHL3 gene in the preparation of a medicament for preventing and / or treating hepatocellular carcinoma will be described in detail below in combination with examples and experimental data.

[0028] Example 1, FHL3 can be used as a diagnostic marker for liver cancer 1. Experimental method (1) Sample source: TCGA database hepatocellular carcinoma data set (n=374); Shanghai Tongji Hospital hepatocellular carcinoma tissue chip (n=110) and paired cancer / paracancer samples (n=50 pairs).

[0029] (2) Detection technology: qPCR adopts TaqMan probe method: Primer sequence: qPCR-FHL3-F: 5'-GAGTCCCTGTATGGACGCAA-3' (SEQ ID NO. 3); qPCR-FHL3-R: 5'-GCATGACAGTCTCCCCACAA-3' (SEQ ID NO. 4); Immunohistochemistry (IHC): anti-FHL3 antibody (1:200) staining, scored according to H-score (0-12 points).

[0030] Western Blot: YAP / c-MYC / p-AKT / FHL3 antibody was used.

[0031] (3) Statistical analysis: Survival analysis: comparison of survival rates between high expression group (H-score≥6) and low expression group (H-score<6) (Kaplan-Meier, Log-rank test).

[0032] Diagnostic efficiency: ROC curve analysis was performed.

[0033] 2. Experimental results ​​(1) TCGA data: HCC tissue FHL3 mRNA expression median 2.8 vs adjacent 0.9 (*P<0.001, Figure 1 F), indicating that HCC tissue FHL3 expression was significantly higher than adjacent (*P<0.001).

[0034] (2) Clinical samples: Protein level: 6.5 ± 1.1 vs 1.2 ± 0.4 (*P<0.001, Figure 1 H); mRNA level: 6.8 ± 1.2 vs 1.5 ± 0.3 (*P<0.001, Figure 1 I); (3) Prognostic value: TCGA cohort: FHL3 high expression group 5-year survival rate 32% vs low expression group 72% (HR=2.1, P<0.001, Figure 2 G) The 5-year overall survival rate of the high expression group was significantly lower than that of the FHL3 low expression group, and the expression level of FHL3 was an independent risk factor for patient prognosis (HR=2.46, 95% CI: 1.10-5.49, *P<0.001, Figure 2 L, M).

[0035] (4) Diagnostic efficiency: Single marker: AUC=0.84 (95% CI: 0.78-0.90) FHL3+YAP combination: ROC area under the curve (AUC) increased to 0.91 (sensitivity 92.5%, specificity 88.3%), and the critical value was H-score≥6 (FHL3) and YAP nuclear positive rate≥30%.

[0036] Example 2: shFHL3 inhibits liver cancer in vivo and in vitro I. In vitro experiment: 1. Method shFHL3-1, shFHL3-2 (see Table 1) were transfected into liver cancer cells MHCC97H, HLF.

[0037] Table 1

[0038] Meanwhile, FHL3 overexpression stable strain was constructed as a control group, which was transfected into liver cancer cells Hep3B, Huh7.

[0039] CCK-8 method proliferation detection and metastasis detection (Transwell, scratch healing experiment) were performed on the two groups of cell lines (shFHL3 group and overexpression group).

[0040] 2、Results In multiple hepatoma cell lines, Figure 2 A), by constructing FHL3 overexpression stable strains (Hep3B / Huh7, protein expression increased 5.3-6.1 times, Figure 2 B) and shFHL3 knockdown stable strains (MHCC97H / HLF, knockdown efficiency >80%, Figure 2 C), it was found that FHL3 significantly promoted the malignant phenotype of hepatoma: (1) Proliferation effect: Overexpression of FHL3 increased the cell proliferation rate by 58% (CCK-8 detection, Figure 2 D), EdU positive cells increased by 2.3 times ( Figure 2 F); On the contrary, the shFHL3 group inhibited the proliferation rate by 65-68% (CCK-8, *P<0.001, Figure 2 E), EdU positive cells decreased by 71% ( Figure 2 G).

[0041] (2) Metastasis effect: In the Transwell experiment, the number of membrane-penetrating cells in the overexpression group increased by 2.1 times ( Figure 2 H), while the shFHL3 group reduced the membrane-penetrating cells by 74% (125±18 to 32±6 / field, *P<0.001, Figure 3 I); Wound healing experiment showed that the healing rate of the overexpression group increased to 85% ( Figure 3 J), while the shFHL3 group decreased to 28% ( Figure 3 K).

[0042] In summary, targeted inhibition of FHL3 expression can significantly reverse the malignant phenotype.

[0043] II. In vivo experiment: 1. Method: (1) Establishment of animal model Mouse subcutaneous tumor model: BALB / c nude mice (female, 6 weeks old, body weight 18-20g) were used, and 5×10 6 liver cancer cells (overexpression group: Hep3B-FHL3; knockdown group: MHCC97H-shFHL3; control: empty vector cells) were injected subcutaneously, and treatment was started at 7 days after inoculation (n=8 / group).

[0044] Orthotopic transplantation tumor model of hepatoma: After intraperitoneal injection of pentobarbital anesthesia in mice, the liver was exposed by laparotomy, and 2×10 6 Hep3B-luc or MHCC97H-luc cells (expressing luciferase) were injected into the left lobe of the liver, and the mice were fed after suture.

[0045] (2) Treatment plan Treatment group: AAV8-shFHL3 (containing SEQ ID NO.1 / 2 sequence, dose 1×10 11 vg / kg, once a week × 4 weeks); Control group: injected with the same amount of AAV8-scramble virus.

[0046] (3) Evaluation indicator monitoring Tumor growth: The subcutaneous tumor volume was measured every 3 days using a vernier caliper (formula: V = 0.5 × length × width). 2 ); In vivo imaging: Orthotopic tumor models were intraperitoneally injected with D-luciferin (150 mg / kg) weekly, and the fluorescence intensity was quantified using an IVIS Spectrum small animal imaging system. Metastasis assessment: At the end of the experiment, lung tissue was collected, fixed with 4% paraformaldehyde, and embedded in paraffin. Metastatic nodules were counted after H&E staining (≥0.1 mm was defined as metastatic lesions); Histological analysis: Tumor tissue sections were subjected to H&E and Ki67 immunohistochemical staining (anti-Ki67 antibody 1:200), and the positive rate was counted by two pathologists in a double-blind manner.

[0047] 2. Results: (1) In an in vivo model, FHL3 significantly regulates the progression of liver cancer: ① In the subcutaneous tumor model, overexpression of FHL3 increased tumor weight by 2.3 times ( Figure 3 A), while the tumor volume of the shFHL3 group decreased by 76% (1250±210 mm³→300±75 mm³, *P<0.001, Figure 3 B), the Ki67 positive cell rate decreased from 45%±6% to 12%±3% ( Figure 3 D); ② The orthotopic transplant tumor model showed that the liver fluorescence intensity of the overexpression group increased by 3.2 times ( Figure 3 E), the fluorescence intensity of the shFHL3 group decreased by 82% ( Figure 4 G, and H&E staining confirmed that the tumor boundaries in the treatment group were clear and necrosis was reduced ( Figure 4 F,H); ③ In terms of lung metastasis inhibition, the number of metastatic nodules in the overexpression group reached 18.3±3.2 per mouse, while that in the shFHL3 group was reduced to 2.1±0.9 (a decrease of 88%, *P<0.001, Figure 4 J).

[0048] In summary, targeting FHL3 can simultaneously inhibit primary tumor growth and distant metastasis, and significantly improve survival.

[0049] (2) FHL3 synergizes with YAP to drive malignant progression of HCC: Clinical relevance: Nuclear localization of YAP and expression of FHL3 were strongly correlated in HCC tissues (r = 0.78, *P < 0.001, Fig. 1A, B), and patients with double high expression had a 5-year survival rate of only 15% vs 85% in the double low group (HR = 5.2, *P < 0.001, Fig. 1C, D); Figure 4 Figure 4 Molecular mechanism: shFHL3 still inhibited proliferation (CCK-8 inhibition rate 61%, Fig. 2A, B) and reduced transmembrane cells by 70% (Transwell, Fig. 2C, D) under YAP overexpression; Figure 4 Synergistic treatment in vivo: shFHL3 reduced the volume of YAP-driven tumors by 68% (Fig. 3A, B), the Ki67 positive rate from 52% to 18% (Fig. 3C, D), and significantly prolonged the survival period in spontaneous models (Fig. 3E, F); Figure 4 AAV8-shFHL3 down-regulated the expression of FHL3 / YAP / KRAS proteins (Fig. 4A, B). Figure 4 Figure 4 Figure 4 ​

[0050] Tumor volume in the treatment group was reduced by 76% compared with the control group (*P < 0.001, Fig. 5A, B), and the survival rate of mice was significantly improved (*P < 0.001, Fig. 5C, D). ​ ​

[0051] In summary: FHL3 is a key effector factor downstream of the YAP pathway, and targeted inhibition can break through YAP-mediated drug resistance, providing a new strategy for precise treatment of HCC.

[0052] Example 3: Preparation of a pharmaceutical preparation 1. shFHL3 sustained-release preparation: AAV8-shFHL3 (SEQ ID NO. 1-SEQ ID NO. 2) was mixed with liposomes (DOPE:CHEMS = 7:3) to form a complex.

[0053] After adding a lyoprotectant (trehalose / PEG 4000), it was lyophilized, and reconstituted with physiological saline before use.

[0054] 2. Quality controlDynamic light scattering was used to detect the particle size (110 ± 15 nm), and transmission electron microscopy was used to confirm that the encapsulation efficiency was >95%.

[0055] ​​​​​​​​​Finally, it should be noted that the terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but can also include other elements not expressly listed or inherent to such process, method, article, or apparatus.

[0056] While the preferred embodiments of the application have been described, additional variations and modifications can be made to the preferred embodiments by those of skill in the art once they have the benefit of the present disclosure without departing from the spirit and scope of the application. Accordingly, the appended claims are intended to encompass within their scope all such variations and modifications as are within the scope of the application.

[0057] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. Use of the FHL3 gene as a target gene in screening drugs for preventing, alleviating and / or treating hepatocellular carcinoma, characterized in that: The screening method includes screening for substances capable of inhibiting the expression of the FHL3 gene.

2. Use of a substance that inhibits FHL3 gene expression in the preparation of a drug for preventing, alleviating and / or treating hepatocellular carcinoma.

3. The use according to claim 2, characterized in that The substance that inhibits FHL3 gene expression includes shRNA for knockout.

4. The use according to claim 3, characterized in that The sequence of the shRNA is shown in SEQ ID NO. 1-2.

5. The use according to any one of claims 1 to 4, characterized in that: The drug prevents, alleviates and / or treats hepatocellular carcinoma by at least one of the following actions: inhibiting the proliferation or metastasis of liver cancer.

6. A drug for preventing, alleviating and / or treating hepatocellular carcinoma, characterized in that: The drug contains a substance that inhibits the expression of the FHL3 gene.

7. The drug according to claim 6, characterized in that The drug also includes pharmaceutically acceptable excipients.

8. Application of FHL3 as a molecular marker in the preparation of products for the diagnosis of hepatocellular carcinoma.

9. Use of FHL3 detection reagents in the preparation of products for diagnosing hepatocellular carcinoma.

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