AUH acetylation site mutant and application thereof in preparation of liver cancer drugs

Abstract

The invention relates to an AUH acetylation site mutant and application thereof in preparation of liver cancer drugs, the amino acid sequence of the AUH acetylation site mutant is as shown in SEQ ID NO: 1, and the amino acid at the 109th site is arginine R instead of lysine K. According to the AUH acetylation site mutant obtained through screening, the activation level of a leucine-mTOR signal channel in liver cancer is reduced by increasing the enzyme catalytic activity of AUH; the mRNA-LNP medicine obtained by packaging has the effect of inhibiting the growth and progress of liver cancer in both in-vivo and in-vitro experiments, and reveals the possibility of becoming a novel treatment medicine for treating the non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention belongs to the technical field of liver cancer treatment, and particularly relates to an AUH acetylation site mutant and application thereof in the preparation of liver cancer drugs. Background Art

[0002] Liver cancer occupies an important position in the global cancer incidence and is one of the sixth most common cancers. According to the International Agency for Research on Cancer (IARC), the incidence and mortality of liver cancer are both among the highest, with more than 800,000 new cases of liver cancer each year. The incidence of liver cancer in Asia and Africa is significantly higher than that in Europe and the United States, especially in China, Vietnam and South Korea. The 5-year survival rate of liver cancer is low, usually less than 20%. Early diagnosis and treatment can significantly improve the survival rate, but many patients have lost the best treatment opportunity when they are discovered, which brings a huge burden to society. The high incidence and serious disease burden of liver cancer pose a major challenge to global public health. Through effective preventive measures, early screening and timely treatment, the incidence and mortality of liver cancer can be reduced and the disease burden can be reduced.

[0003] The development and application of liver cancer drugs face multiple difficulties: First, liver cancer is highly heterogeneous, resulting in significant differences in drug efficacy; second, the liver is the main organ for drug metabolism, and many drugs are rapidly degraded in the liver, affecting the therapeutic effect; in addition, liver cancer is usually diagnosed in the late stage, further limiting the treatment window. The lack of effective targets and biomarkers also increases the complexity of drug development. Therefore, clarifying the important mechanisms of liver cancer metabolic regulation is of great significance for the development of liver cancer drugs. The mTOR (mammalian target of rapamycin) pathway plays an important role in cell growth, proliferation, and survival, and is a potential target for liver cancer treatment. However, the traditional application of liver cancer drugs targeting the mTOR pathway has the following limitations:

[0004] 1. Development of drug resistance: Many patients with liver cancer develop resistance to mTOR inhibitors, resulting in reduced therapeutic efficacy. 2. Tumor heterogeneity: The high heterogeneity of liver cancer makes it impossible for a single therapy targeting the mTOR pathway to fully inhibit all tumor cells. 3. Side effects: mTOR inhibitors may cause a variety of side effects, including immunosuppression, metabolic disorders, and oral ulcers, which limit their long-term use. 4. Limited monotherapy efficacy: mTOR inhibitors alone have limited efficacy and usually need to be used in combination with other treatments, but the optimization of combined therapy has not yet been fully determined. 5. Incomplete inhibition: The mTOR pathway is complex, with multiple feedback mechanisms and cross-pathways. Complete inhibition of mTOR signaling may require combined inhibition of other pathways. These limitations suggest that further research and development of new treatment strategies are needed to improve the efficacy and safety of mTOR-targeted drugs in the treatment of liver cancer.

[0005] Previous studies have shown that AUH gene expression is decreased in a variety of epithelial tumors, including liver cancer (including hepatocellular carcinoma, intrahepatic cholangiocarcinoma), pancreatic cancer, intestinal cancer, and renal cancer. Overexpression of AUH can increase the leucine catabolism in tumor cells, thereby reducing the level of leucine in tumors, and inhibit tumor cell proliferation and metastasis, suggesting that AUH is a potential target for tumor treatment. In order to achieve tumor treatment, it is necessary to ensure the long-term, stable and efficient expression of HNF4α in tumor cells; due to the rapid proliferation of tumors, tumor treatment requires repeated administration to ensure the therapeutic effect. However, due to the pre-existing immunity to adenovirus in the population, adenovirus vectors can also induce the human immune system to produce an antiviral response, thereby rejecting the expression of the target gene mediated by the viral vector in tumor cells, thus limiting the frequency of administration of adenovirus vectors to overexpress HNF4α for tumor treatment. On the other hand, previous studies have also shown that adenovirus vectors have defects in safety and tissue targeting, and their preparation costs are high, so the actual application potential of adenovirus vector-mediated HNF4α overexpression for tumor treatment is low.

[0006] mRNA technology is a revolutionary gene delivery technology developed in recent years. This technology synthesizes mRNA molecules with specific sequences by in vitro transcription, and uses lipid nanoparticles (LNP) to encapsulate mRNA and transport it into human cells. It relies on the cell's own translation system to translate mRNA into target proteins. Compared with the existing viral vector-based delivery systems, it has good efficiency and safety. If combined with the screening and modification of nanolipid particles, the tissue specificity of the delivery system can be increased. The potential application directions of the currently known mRNA technology are mainly concentrated in the development of vaccines for infectious diseases, therapeutic tumor vaccines, protein replacement therapy for protein deficiency caused by gene defects, and gene editing to treat genetic diseases or transform immune cells. However, since mRNA can only mediate transient high expression of target genes in cells, there are significant deficiencies in expression duration and expression efficiency, and it cannot meet the needs of relatively long-term and stable expression of target proteins in tumor cells with rapid growth ability. Summary of the invention

[0007] The technical problem to be solved by the present invention is to provide an AUH acetylation site mutant and its application in the preparation of liver cancer drugs. The screened AUH acetylation site mutant reduces the activation level of the leucine-mTOR signaling pathway in liver cancer by increasing the enzymatic catalytic activity of AUH.

[0008] The present invention provides an AUH acetylation site mutant, whose amino acid sequence is shown in SEQ ID NO: 1, and the amino acid at position 109 is R instead of K.

[0009] The present invention also provides a nucleic acid encoding the AUH acetylation site mutant, or a nucleic acid having the same length as the encoding nucleic acid and completely complementary to the encoding nucleic acid.

[0010] The present invention also provides a vector for expressing the AUH acetylation site mutant.

[0011] The present invention also provides an application of an AUH acetylation site mutant in the preparation of liver cancer drugs.

[0012] Furthermore, the drug is the AUH acetylation site mutant encapsulated by lipid nanoparticles.

[0013] Further, the drug includes but is not limited to an mRNA-LNP drug encapsulating the mutant, denoted as AUH K109R -LNP.

[0014] Furthermore, the drug also contains pharmaceutically acceptable carriers and / or excipients.

[0015] Furthermore, the pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a humectant, an adsorption carrier, a lubricant, a filler, and a disintegrant.

[0016] Beneficial Effects

[0017] The AUH acetylation site mutant screened by the present invention reduces the activation level of the leucine-mTOR signaling pathway in liver cancer by increasing the enzymatic catalytic activity of AUH; the packaged mRNA-LNP drug has the effect of inhibiting the growth and progression of liver cancer in both in vivo and in vitro experiments, revealing the possibility of becoming a new therapeutic drug for the treatment of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The CCK8 method was used to detect the inhibitory effect of AUH mutants on cell growth.

[0019] Figure 2 To verify AUH using LC-MS K109R Effect on AUH enzyme activity.

[0020] Figure 3 For empty LNP (eGFP-LNP) and mutant LNP (AUH K109R -LNP) PDI measurement results.

[0021] Figure 4 For empty LNP (eGFP-LNP) and mutant LNP (AUH K109R -LNP) particle size distribution.

[0022] Figure 5 For mutant LNP (AUH K109R -LNP) electron micrograph.

[0023] Figure 6 For empty LNP (eGFP-LNP) and mutant LNP (AUH K109R -LNP) is overexpressed in tumor cells.

[0024] Figure 7 For empty LNP (eGFP-LNP) and mutant LNP (AUH K109R -LNP) inhibited liver cancer cells.

[0025] Figure 8 For mutant LNP (AUH K109R -LNP) in vivo experimental results. DETAILED DESCRIPTION

[0026] The present invention will be further described below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application. All data in the text are analyzed and mapped using GraphPad Prism 8 software. All data error values ​​are displayed as mean ± standard error (± SEM). The comparison between the two groups of data adopts t test, and the statistical test is two-tailed, wherein p<0.05 is considered to be statistically significant, * represents p<0.05, ** represents p<0.01, and *** represents p<0.001.

[0027] Example 1

[0028] Five AUH site mutants were designed, and the CCK8 method was used to detect the inhibitory effect of AUH mutants on cell growth, and the mutant AUH with the best inhibitory effect on liver cancer cells was screened out. K109R , whose amino acid sequence is shown in SEQ ID NO:1 ( Figure 1 ). AUH was verified using LC-MS K109R The effect of AUH enzyme activity was determined by Figure 2 As you can see, AUH K109R Significantly increased its catalytic activity.

[0029] Example 2

[0030] LNP encapsulation and in vitro expression verification:

[0031] Preparation method:

[0032] Four lipids were dissolved in anhydrous ethanol at a specific molar ratio (50:10:38.5:1.5, SM102, DSPC, cholesterol, and PEG2000-DMG, respectively) to form a lipid mixture. K109R Dilute the AUH to a concentration of 111 ng / μl using DEPC water containing 50 mM sodium citrate. K109R The lipid mixture was mixed using NanoAssemblr Ignite+ (Precision NanoSystems, Canada) in a volume ratio of 3:1. The mixed product was immediately received with the same volume of DEPC water containing 50mM sodium citrate. Finally, the formulation was dialyzed in PBS (pH 7.4) for at least 15 hours at room temperature. The dialyzed product was filtered through a 0.22 micron filter and concentrated using an Amicon Ultra centrifugal filter (MilliporeSigma, USA) to obtain mutant LNPs (AUH K109R -LNP), and empty LNP (eGFP-LNP) was used as control. The size and PDI of LNP were measured by Zetasizer Pro (Zetasizer, Malvern Panalytical, USA). Figure 3 and Figure 4 As shown in the figure, we can see that AUH K109R The PDI increased and the particle size was within the normal range.

[0033] The images were acquired using a Talos F200C G2 transmission electron microscope (Thermo Fisher, USA) at 200 kV and a magnification of 36,000. The pixel size of the sample is captured. The transmission electron microscope shows the encapsulation of LNP and the morphology of nanoparticles. The transmission electron microscope imaging was performed by the Instrumental Analysis Center of Shanghai Jiao Tong University. The results are shown in Figure 5 As shown, it can be seen from the figure that the liposomes are uniform and stable.

[0034] Detection of empty LNP (eGFP-LNP) and mutant LNP (AUH K109R -LNP) overexpression in tumor cells:

[0035] Hepatoma cell Huh7 was seeded into 6-well plates at 1x 105 cells / well and cultured overnight. After washing with 1ml PBS, 1ml Opti-MEM diluted control eGFP-LNP, AUH K109R-LNP (final concentration was 2ng RNA / 200 cells). The cells were cultured in DMEM medium containing 10% FBS. After 2 days, the transfected cells were collected with RIPA protein lysis buffer and Western blot was used to detect the protein level of AUH in the cells. The results are shown in Figure 6 As shown in the figure, we can see that AUH K109R It has been successfully transfected into cells and overexpressed.

[0036] AUH K109R -LNP inhibits the growth of liver cancer cells:

[0037] Hepatoma cell Huh7 was used to verify the inhibition of malignant phenotypes such as tumor cell proliferation and clone formation by the gene delivery system. 3 Cells were seeded in 6-well plates at a density of 1.54 × 10 cells / well. After overnight culture, equal amounts of eGFP-LNP and AUH were added. K109R -LNP treatment. After 24 hours of treatment, the cells were inoculated into 6-well plates at a cell density of 1000 / well. The culture medium was changed every 3 days, and the cloning of tumor cells was observed under a microscope. After the clones were formed, they were washed twice with PBS, stained with crystal violet for 20 minutes, washed with PBS, dried and photographed, and the number of clones was counted by image J. The results are as follows Figure 7 Compared with control cells, AUH K109R -LNP significantly inhibited the cloning ability of liver cancer cells.

[0038] Example 3

[0039] LNP in vivo experimental validation:

[0040] AUH K109R -LNP inhibits the growth of subcutaneous tumors in mice:

[0041] Four-week-old male nude mice (BALB / c immunodeficient strain) were purchased from Shanghai Slake Company and were raised in a specific pathogen-free environment in an SPF animal room with a 12-hour on-off light cycle. 6 Huh-7 cells were injected subcutaneously into the right axilla of male nude mice, and the tumor size was measured in two dimensions using a vernier caliper. The volume was calculated using the following formula: volume = length × (width)2 × 1 / 2. When the average tumor volume reached approximately 100 mm 3At the same time, the mice were randomly divided into four groups (seven animals in each group). eGFP-LNP and AUHK109R-LNP or saline (solvent control) were injected into the mouse tumor at a final volume of 50μl at 0.25mg / kg. The tumor volume was measured once a day after injection, and the tumor growth curve was drawn. Five days after RNA injection, the mice were killed, the tumors were removed and weighed, and the tumor inhibition rate was calculated. Part of the tumor tissue was paraffin embedded, and the expression of AUH was detected by western blot and immunohistochemistry, and the proliferation state of tumor cells was evaluated by Ki-67 staining. Figure 8 As shown, the results showed that intratumoral injection of AUH K109R -LNP slowed down the tumor growth, and the tumor inhibition rate exceeded 50%. Western-blot and immunohistochemistry results showed that AUH K109R -LNP significantly increased the protein expression of AUH in tumor tissues, and the expression of Ki-67, a tumor cell proliferation indicator, was significantly decreased. These results indicate that AUH K109R -LNP can effectively induce the expression of HNF4α in cancer cells and inhibit the growth of liver cancer implants in mice.

Claims

1. An AUH acetylation site mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO: 1, and the amino acid at position 109 is arginine R instead of lysine K.

2. A nucleic acid encoding the AUH acetylation site mutant as claimed in claim 1, or a nucleic acid having the same length as the encoding nucleic acid and completely complementary to the encoding nucleic acid.

3. A vector expressing the AUH acetylation site mutant as claimed in claim 2.

4. Use of the AUH acetylation site mutant according to claim 1 in preparing liver cancer drugs.

5. The use according to claim 4, characterized in that: The drug is the AUH acetylation site mutant encapsulated by lipid nanoparticles.

6. The use according to claim 4, characterized in that: The drug includes but is not limited to the mRNA-LNP drug encapsulating the mutant, denoted as AUH K109R -LNP.

7. The use according to claim 4, characterized in that: The medicine further comprises a pharmaceutically acceptable carrier and / or excipient.

8. The use according to claim 7, characterized in that: The pharmaceutically acceptable carrier and / or excipient includes at least one of a diluent, a binder, a surfactant, a wetting agent, an adsorption carrier, a lubricant, a filler, and a disintegrant.

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