Application of interference HSP90 alpha in treatment of fatty liver related liver cancer

Abstract

The invention relates to the field of biomedicine, in particular to application of interference HSP90 alpha in treatment of fatty liver related HCC. Through fatty acid treated liver cancer cell experiments and fatty acid-rich diet induced fatty liver related HCC animal model researches, HSP90 alpha expression is significantly up-regulated, and the protein is a novel protein for promoting fatty liver related HCC proliferation. The expression of the HSP90 alpha is interfered, so that the HCC proliferation induced by fatty acid can be effectively weakened. The result shows that the HSP90 alpha is possibly a key factor for promoting the progress of the fatty liver-related hepatocellular carcinoma, and inhibition of the HSP90 alpha provides a new treatment strategy for clinical treatment of the fatty liver-related HCC.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to the application of interfering with HSP90α in treating fatty liver-related liver cancer. Background Art

[0002] Liver cancer is characterized by high morbidity, strong invasiveness, difficulty in cure, poor prognosis and high mortality, and is an important public health issue that affects people's health. Liver cancer develops in the context of chronic liver disease. With the improvement of residents' living standards, fatty liver has become an important factor inducing liver cancer. The prevalence of metabolic risk factors for liver cancer, including obesity and non-alcoholic fatty liver disease (NAFLD), is becoming an important cause of liver cancer, and may become the main cause of liver cancer worldwide in the near future. Therefore, it is of great clinical significance to explore new biomolecular markers to predict the progression of fatty liver-related liver cancer and guide precision treatment to improve patients' quality of life and prolong survival.

[0003] Heat shock protein 90α (HSP90α) is a molecular chaperone protein that exists in various organs of the human body. Its N-terminal ATP binding site is involved in regulating the synthesis of polypeptides, protein folding and degradation, and the regulation of cell heat response. Many studies have shown that the expression of HSP90α in tumors is significantly higher than that in normal adjacent tissues. It is a chaperone protein that plays an important role in tumor occurrence and development, and helps cancer cells survive under harsh physiological and stress conditions. The application of interfering with HSP90α in fatty liver-related liver cancer has not been reported. Exploring its expression and potential value is of great significance for the clinical diagnosis and treatment of fatty liver-related liver cancer and drug development. Summary of the invention

[0004] The purpose of the present invention is to analyze the expression and role of HSP90α in fatty liver-related liver cancer. HSP90α is highly expressed in fatty liver-related liver cancer. Interference with it can inhibit tumor proliferation and provide a new therapeutic target for the clinical treatment of fatty liver-related liver cancer.

[0005] In order to achieve the above object, the present invention adopts the following technical means:

[0006] In a first aspect, the present invention provides a method for interfering with HSP90α in fatty liver-related liver cancer.

[0007] Among them, preferably, the liver cancer cells are human liver cancer cell lines PLC5, HepG2, and Huh7 treated with fatty acids.

[0008] Preferably, the fatty liver-related HCC animal model is an in situ liver cancer model in nude mice that is fed with a fatty acid-rich diet and constructed using MHCC-97H cells.

[0009] In a second aspect, the present invention provides an application for inhibiting the progression of fatty liver-related liver cancer.

[0010] (1) Used to inhibit the proliferation of fatty liver-related liver cancer;

[0011] (2) Prepare drugs for improving the efficacy of fatty liver-related liver cancer.

[0012] Among them, preferably, the HSP90α is expressed more in liver cancer cells treated with fatty acids than in the control group, and the progression of fatty liver-related liver cancer can be predicted based on it.

[0013] Among them, preferably, the interference of HSP90α can inhibit the progression of fatty liver-related liver cancer, as shown in that in in vitro experiments, a long-term dynamic cell imaging system detected a decrease in the relative growth percentage of cells, indicating that cell proliferation was inhibited. In in vivo experiments, under fatty acid feeding conditions, mice with HSP90α knockdown had reduced liver weight, reduced tumor burden, and reduced PCNA expression compared with the control group.

[0014] The beneficial effects of the present invention are:

[0015] The present invention studies fatty liver-related liver cancer through in vivo and in vitro experiments, and finds that fatty acid treatment promotes the proliferation of liver cancer, and the HSP90α expression in the fatty acid treatment group is significantly increased compared with the control group. By interfering with HSP90α expression, fatty acid-induced liver cancer proliferation can be eliminated, and the progression of fatty liver-related liver cancer can be inhibited, highlighting the significance of interfering with HSP90α in the prediction and treatment of fatty liver-related liver cancer. In short, HSP90α is a protein related to fatty liver-related liver cancer, which can provide a predictive marker and therapeutic target for the treatment of clinical fatty liver-related liver cancer. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 High-fat diet promotes the proliferation of orthotopic liver cancer in mice. A is the liver tumor image and liver coefficient of orthotopic liver cancer mice. B is the H&E staining of liver tissue sections and tumor area ratio of mice in the NCD and HFD groups. C is the PCNA staining results of tissues of mice in the NCD and HFD groups and the average number of positive cells in the field of view. (* is p<0.05; ** is p<0.01; *** is p<0.001; **** is p<0.0001.)

[0017] Figure 2The proliferation of human liver cancer cells Huh7 and PLC5 is promoted by fatty acid treatment. A is the relative growth rate of cells after 48 hours of fatty acid treatment detected by the dynamic cell imaging system. B is the effect of fatty acids on the proliferation of Huh7 cells and PLC5 cells measured by CCK8. (* is p<0.05; ** is p<0.01; *** is p<0.001; **** is p<0.0001.)

[0018] Figure 3 The expression of HSP90α in human liver cancer cells PLC5 and HepG2 was upregulated after fatty acid treatment. A is the transcription level of HSP90α mRNA in PLC5 and HepG2 cells measured by q-PCR. B is the expression level of HSP90α in the control and fatty acid treated cells detected by immunofluorescence staining and quantitative analysis of fluorescence area. (* is p<0.05; ** is p<0.01; *** is p<0.001; **** is p<0.0001.)

[0019] Figure 4 Interference with HSP90α weakened the proliferation of human liver cancer cells PLC5 and HepG2 promoted by fatty acids. A and B are the cell proliferation of the control group and fatty acid group measured by dynamic imaging system and CCK8 after treatment with AAG and NS, respectively. C is the knockdown efficiency of HSP90α in two liver cancer cell lines verified by Western blotting. D is the growth of PLC5 and HepG2 cells detected by dynamic cell imaging system and CCK8 after interference with HSP90α. (* is p<0.05; ** is p<0.01; *** is p<0.001; **** is p<0.0001.)

[0020] Figure 5 The effect of HSP90α knockdown on fatty acid-induced liver cancer development was verified in the mouse orthotopic liver cancer model. A is the liver tumor image and liver coefficient analysis of the control group and HSP90α knockdown mice fed with a high-fat diet. B is the PCNA staining results of the liver tissue of the control group and HSP90α knockdown mice fed with a high-fat diet and the quantitative analysis of the average number of positive cells in the field of view. (* is p<0.05; ** is p<0.01; *** is p<0.001; **** is p<0.0001.) Specific implementation plan

[0021] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments.

[0022] The experimental methods involved in the following examples are conventional methods unless otherwise specified, and the experiments are performed according to the technical methods described in the literature in the field or according to the product instructions. The reagents and consumables used in the following examples can be obtained from commercial channels unless otherwise specified.

[0023] 1. Materials and Methods

[0024] 1. Cell lines and cell culture

[0025] Human hepatoma cell lines PLC5, Huh7, and HepG2 were obtained from the Chinese Academy of Sciences (Shanghai, China) and cultured in a medium containing 10% FBS and 1% penicillin-streptomycin in a humidified incubator at 37°C and 5% CO2.

[0026] 2. Experimental animals and high-fat diet animal model construction

[0027] The 4-week-old male BALB / c-nu nude mice used in the experiment were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0028] (1) The mice were numbered and randomly divided into two groups, with 9 mice in each group. After adapting to the animal center feed for one week, they were fed with a normal diet or a high-fat diet rich in oleic acid (Whitbit, China), respectively.

[0029] (2) MHCC-97H cells were cultured in a 37°C, 5% CO2 humidified incubator.

[0030] (3) The mice were starved for 8 h one day before surgery.

[0031] (4) On the day of surgery, MHCC-97H cells were digested and mixed with Matrigel (356234, Corning, USA) at a ratio of 1:1 and mixed with a pipette. Mice were anesthetized with 4 μl / g chloral hydrate (Shengang, China), and the abdomen was opened. Then, 25 μl of the mixture of cells and Matrigel was sucked out with an insulin needle and slowly injected into the left lobe of the mouse liver, and then sutured.

[0032] (5) After being fed a normal diet or a high-fat diet for one month, liver tissues of mice were obtained for experiments.

[0033] 3. PCNA staining

[0034] (1) Dewaxing and hydration. Place tissue sections in a 60°C incubator to melt wax for 20 minutes. Soak in xylene I and II for 20 minutes each, and then soak in anhydrous ethanol, 85% ethanol, 70% ethanol, and distilled water for 5 minutes each.

[0035] (2) Antigen retrieval: The tissue sections were immersed in 0.01 M citrate buffer (Dingguo Changsheng, China), heated in a microwave oven for 15 min, and then cooled for 30 min, and repeated twice.

[0036] (3) Block endogenous peroxidase: Add 3% H2O2 to the tissue sections at room temperature for 30 minutes, then wash with PBS for 3 minutes, and repeat 3 times.

[0037] (4) PCNA primary antibody (10205-2-AP, proteintech, China, 1:200) was added dropwise and incubated in a humidified box at 4°C overnight.

[0038] (5) Place the slices in a 37°C incubator to rewarm for 10 min. Add the reaction enhancement solution to the slices and incubate them in a 37°C incubator for 1 h. After that, wash them with PBS three times, 3 min each time.

[0039] (6) Goat anti-rabbit secondary antibody (Zhongshan Jinqiao, China) was added dropwise and incubated at 37°C for 1 h. Afterwards, the cells were washed three times with PBS, each time for three minutes.

[0040] (7) Add hematoxylin (Biyuntian, China) for staining, rinse with water after 1 min to terminate the staining, and rinse with running water for 10 min.

[0041] (8) DAB staining was used and staining was terminated when PCNA was positive in the cell nucleus.

[0042] (9) Soak in distilled water, 70% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 min.

[0043] (9) Seal the slides with neutral gum.

[0044] 4. H&E staining

[0045] (1) The initial step is the same as the PCNA staining step (1).

[0046] (2) Add hematoxylin stain (Biyuntian, China) on the sections, let stand for 2 min, rinse with water to stop staining, and rinse with running water for 10 min.

[0047] (3) Add eosin stain solution, let it stand for two minutes, and then rinse with running water.

[0048] (4) Soak in distilled water, 70% ethanol, 85% ethanol, 95% ethanol, and anhydrous ethanol for 5 min in sequence.

[0049] (5) Allow to dry in a fume hood and seal with neutral gum.

[0050] 5. Preparation of fatty acid storage solution

[0051] Weigh 2.0g of BSA and add it to 20.4ml of PBS to make a 10% BSA solution, and place it in a 37℃ water bath for later use. Add 0.015222g of sodium oleate (Sigma, USA) to 1ml of distilled water and place it in a 60℃ water bath until it is completely dissolved. Then add the oleic acid solution to the BSA solution and mix well. At this time, the oleic acid concentration is 3mM. Filter it with a 0.2μm filter and place it in a -20℃ refrigerator for later use.

[0052] 6. Long-term dynamic imaging system to monitor cell proliferation

[0053] (1) Dilute the cell density to 3×10 4 / ml, four concentration gradients of oleic acid were set, and a total of 5 groups were added with the control group. 100 μl of cell suspension was added to each well and seeded in a 96-well plate.

[0054] (2) When cells are in good condition, starve the cells for 12 h.

[0055] (3) 50 μM, 100 μM, 200 μM, and 400 μM oleic acid were added to the wells, respectively, and treated for 48 h. The cell proliferation was photographed and recorded using a long-term cell dynamic monitoring system.

[0056] 7. Cell viability assay (CCK-8)

[0057] The seeding process was the same as the steps for monitoring cell proliferation using a long-term dynamic imaging system. After a certain period of oleic acid treatment, 10% CCK-8 solution (Biyuntian, China) was added to the wells and incubated for 90 min in an incubator at 37°C and 5% CO2, and the absorbance at 450 nm was measured using a microplate reader.

[0058] 8. q-PCR

[0059] Total cellular RNA was extracted using Trizol (TaKaRa, Japan), and then reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Japan). Real-time quantitative PCR was then performed using SYBR Green Premix (Biomarker, China) to calculate the relative expression of the target gene in the cDNA (with β-actin as the internal reference gene).

[0060] 9. Cell Immunofluorescence Staining

[0061] (1) Cells were seeded onto slides, n = 3, and allowed to adhere for 24 h. The cells were starved for 12 h, treated with 200 μM oleic acid for 24 h, and cultured in a 37°C, 5% CO2 incubator.

[0062] (2) Wash the plate with PBS three times for 3 min each time, and add 1 ml of 4% paraformaldehyde to each well for fixation for 15 min.

[0063] (3) Wash with PBS three times and block with 5% BSA for 30 min at room temperature.

[0064] (4) Prepare the primary antibody (ab282108, Abcam, 1:100), add 50 μl of antibody to each well, and incubate at 4°C overnight.

[0065] (5) Wash three times with PBS, prepare fluorescent secondary antibody, add 50 μl to each well, and incubate at room temperature for 60 min.

[0066] (6) Wash three times with PBS, cover the slides with DAPI containing fluorescence quencher, and take pictures under a microscope.

[0067] 10. Knockdown of liver cancer cell lines

[0068] To construct a HSP90α knockdown liver cancer cell line, siRNA oligo and RNAiMAX transfection reagent (Invitrogen, USA) were co-transfected into the target cells. siRNA: HSP90α-1 (5′-UAUGGCAUGACAACUACUUUATT-3′); HSP90α-2

[0069] (5-UACUUGGAGGAACGAAGAAUATT-3); HSP90α-3(5-

[0070] GUUAUCCUACACCUGAAAGAATT-3).

[0071] 11. Western Blot

[0072] Cell samples were lysed with RIPA lysis buffer (containing 1% phosphatase inhibitor and 1% protease inhibitor), and protein samples were separated by electrophoresis in SDS-PAGE gel, followed by conventional blocking, transfer, incubation with primary antibody, incubation with secondary antibody, and finally chemiluminescence for color development, and the gray value of the target protein was analyzed using ImageJ software. The antibodies used are as follows: antiHSP90αantibody (GTX109753, Gene Tex, 1:2000), anti-Actin antibody (bs0061R, Bioss, 1:5000).

[0073] 12. Data Analysis

[0074] All data were analyzed using GraphPad Prism9 software. The data were expressed as mean ± SEM. T-test was used to compare the two groups. The difference was considered statistically significant when p < 0.05.

[0075] 2. Results

[0076] 1. A high-fat diet promotes the progression of liver cancer in mice.

[0077] In the constructed in situ liver cancer model, the liver coefficient of mice given a diet rich in oleic acid was significantly increased compared with mice in the control group with a normal diet. H&E staining results showed that a high-fat diet promoted tumor growth in mice and increased the tumor area. In addition, a high-fat diet promoted the expression of PCNA-positive cells in mouse liver tissue. The above results all indicate that a high-fat diet can promote the development of liver cancer.

[0078] 2. Fatty acids promote the proliferation of liver cancer cells.

[0079] PLC5 and Huh7 cells were treated with oleic acid at concentrations of 50μM, 100μM, 200μM, and 400μM, respectively, and it was found that oleic acid promoted the proliferation of liver cancer cells in a dose-dependent manner. In addition, CCK-8 proliferation experiments also confirmed that compared with the control group, oleic acid treatment was significantly beneficial to the growth of liver cancer cells.

[0080] 3. HSP90α expression is increased in fatty acid-treated cells

[0081] The levels of HSP90α in PLC5 and Hep-G2 cells were detected by q-PCR and immunofluorescence. The results showed that the mRNA and protein levels of HSP90α in the liver cancer cells in the oleic acid treatment group were significantly higher than those in the control group, indicating that fatty acids can increase the level of HSP90α in liver cancer cells.

[0082] 4. Inhibition of HSP90α expression can reverse fatty acid-enhanced liver cancer cell growth

[0083] After adding HSP90α inhibitors AAG and NS, the ability of fatty acids to promote the proliferation of liver cancer cells PLC5 and HepG2 was weakened. The same conclusion was reached by knocking down the expression of HSP90α. It can be seen that interfering with HSO90α can reverse the progression of liver cancer promoted by fatty acids.

[0084] 5. HSP90α knockdown inhibits fatty acid-induced liver cancer proliferation

[0085] Knocking down HSP90α under high-fat feeding conditions can inhibit tumor growth in mice, while the number of PCNA-positive cells in liver cancer tissue decreases, inhibiting the proliferation of fatty liver-related liver cancer.

Claims

1. Application of interfering HSP90α in the treatment of fatty liver-related liver cancer.

2. The use according to claim 1, characterized in that: The liver cancer is fatty liver-related liver cancer.

3. The use according to claim 2, characterized in that: The liver cancer cells are human-derived liver cancer cells PLC / PRF / 5, Huh7 and HepG2 cells treated with oleic acid.

4. The application according to claim 1, characterized in that: The HSP90α is upregulated in fatty liver-related liver cancer.

5. The application of interfering with HSP90α in fatty liver-related liver cancer is a means to alleviate the progression of fatty liver-related liver cancer and provide a new strategy for the clinical treatment of patients with fatty liver-related liver cancer.

6. The use according to claim 5, characterized in that The expression of HSP90α in liver cancer increased after oleic acid treatment, promoting liver cancer proliferation, while inhibiting the expression of HSP90α eliminated the oleic acid-induced liver cancer cell proliferation. HSP90α has the potential to become a new biomarker and effective therapeutic target for fatty liver-related liver cancer.

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