Application of ACSL3 gene in the treatment of liver cancer and a nano drug

Abstract

The present invention provides the use of an agent for inhibiting the expression of the ACSL3 gene in the preparation of a drug for treating liver cancer patients and a drug for treating liver cancer patients and / or patients with lung metastasis of liver cancer. The present invention has found for the first time through research that the expression of the ACSL3 gene in the lung metastasis pathological sections of liver cancer metastases is significantly higher than that in the primary liver cancer pathological sections, and the level of ACSL3 expression is significantly correlated with the overall survival (OS) of liver cancer patients. Further studies have found that knocking down the expression of the ACSL3 gene can effectively inhibit the growth, migration and invasion of liver cancer cells, promote their apoptosis, and inhibit the lung metastasis of liver cancer cells. Therefore, the ACSL3 gene can be used for the prognosis assessment of liver cancer patients, and can be used as a therapeutic target for liver cancer patients, especially patients with lung metastasis of liver cancer.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to application of ACSL3 gene in treating liver cancer and a nano drug. Background Art

[0002] Primary liver cancer is the fourth most common malignant tumor in my country. Because the liver has adequate blood supply, tumor cells can enter the pulmonary circulation through blood reflux. However, low lung pressure and hyperactivity of the fibrinolytic system can easily cause tumor cell retention and colonization. The lung is the most common organ for extrahepatic metastasis after liver cancer surgery, accounting for about 50% of all extrahepatic metastases. There is currently no unified standard for how to treat lung metastasis after liver cancer surgery. Most liver cancer lung metastases are multiple and scattered, and cannot be radically removed. Interventional and chemotherapy are the only options, but these methods require the patient to be in good general condition and have a greater risk of invasive treatment. Therefore, no effective therapeutic targets have been found clinically to improve the situation of lung metastasis in liver cancer patients.

[0003] There are many cancer-driving genes in tumor cells. The expression of cancer-driving genes in tumors is higher than that in normal cells, and even higher in metastatic lesions than in primary lesions. They are related to the occurrence, development, metastasis, and invasion of tumors. At present, there is a lack of sensitive indicators to identify the occurrence of early liver cancer lung metastasis in clinical practice.

[0004] RNA interference RNA interference (RNAi) technology has developed rapidly in recent years and has great potential in the treatment of various diseases. Compared with small molecule targeted drugs, siRNA has better selectivity for targets, can specifically bind to target genes and downregulate target gene expression, and will not affect the expression of other normal genes in cells. However, the biggest problem in the clinical transformation of RNAi technology is the lack of low-toxic and efficient vectors to deliver siRNA to lesions and cells. For cancer treatment based on RNAi technology, siRNA needs to break through a series of physiological barriers during the delivery process, such as how to target tumor cells, penetrate tumor tissues and cell membranes, efficiently escape from endosomes, and effectively release siRNA in the cytoplasm. Although the use of traditional viral vectors such as adenovirus and retrovirus to deliver siRNA can overcome the above-mentioned physiological barriers, it has the disadvantages of difficult preparation, small siRNA capacity, poor targeting specificity, and strong immunogenicity. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide the application of ACSL3 gene in the treatment of liver cancer. ACSL3 gene is highly expressed in lung metastases of liver cancer and can be used as a target for inhibiting lung metastasis of liver cancer, and can be used to predict the overall survival time and disease-free time of liver cancer patients.

[0006] To achieve the above object, the present invention adopts the following technical solution.

[0007] Use of an agent for inhibiting ACSL3 gene expression in the preparation of a drug for treating liver cancer patients.

[0008] In some embodiments, the agent for inhibiting ACSL3 gene expression is selected from at least one of an ACSL3 gene inhibitor and siRNA; preferably, the siRNA is selected from siRNA1 or siRNA2; wherein siRNA1 comprises a sense chain as shown in SEQ ID NO.1 and an antisense chain as shown in SEQ ID NO.2, and SEQ ID NO.1 and SEQ ID NO.2 have 2 dT modifications at their 3' ends; siRNA2 comprises a sense chain as shown in SEQ ID NO.3 and an antisense chain as shown in SEQ ID NO.4, and SEQ ID NO.3 and SEQ ID NO.4 have 2 dT modifications at their 3' ends.

[0009] The present invention also provides the use of the ACSL3 protein neutralizer in preparing a medicine for treating liver cancer patients.

[0010] In some embodiments, the drug has at least one of the following effects: inhibiting the growth of liver cancer cells, inhibiting the migration of liver cancer cells, inhibiting the invasive ability of liver cancer cells, promoting the apoptosis of liver cancer cells, and inhibiting the lung metastasis of liver cancer cells.

[0011] In some embodiments, the liver cancer patient is a liver cancer lung metastasis patient.

[0012] The present invention also provides a medicine for treating patients with liver cancer and / or patients with lung metastasis of liver cancer, wherein the medicine comprises an agent for inhibiting ACSL3 gene expression and / or an ACSL3 protein neutralizer, and pharmaceutically acceptable excipients.

[0013] In some embodiments, the agent that inhibits ACSL3 gene expression is selected from at least one of an ACSL3 gene inhibitor and siRNA.

[0014] In some embodiments, the drug is a nanoparticle loaded with siRNA; the nanoparticle is prepared by the following method: (1) dissolving the polymer Meo-PEG-b-PDPA in DMF to form a solution 1 with a concentration of 10 mg / mL; (2) dissolving siRNA in water to form a solution 2 with a concentration of 0.1 nmol / μL; dissolving G0-C14 in DMF to form a solution 3 with a concentration of 5 mg / mL; (3) mixing the solution 1, solution 2 and solution 3 at a volume ratio of 20:1:5 to obtain a mixture, and then adding the mixture dropwise into water under stirring to form a suspension; (4) transferring the suspension to an ultrafiltration device, centrifuging to remove the organic solvent and free compounds, and then washing with a PBS solution to obtain the nanoparticle.

[0015] In some embodiments, the stirring speed is 1000-1800 rpm.

[0016] In some embodiments, the siRNA is selected from siRNA1 or siRNA2; wherein siRNA1 comprises a sense strand as shown in SEQ ID NO.1 and an antisense strand as shown in SEQ ID NO.2, and SEQ ID NO.1 and SEQ ID NO.2 have 2 dT modifications at their 3' ends; siRNA2 comprises a sense strand as shown in SEQ ID NO.3 and an antisense strand as shown in SEQ ID NO.4, and SEQ ID NO.3 and SEQ ID NO.4 have 2 dT modifications at their 3' ends.

[0017] The present invention also provides the use of a reagent for detecting the expression level of ACSL3 gene and / or the content of ACSL3 protein in preparing a kit for evaluating the prognosis of patients with liver cancer and / or patients with lung metastasis of liver cancer.

[0018] The present invention has found for the first time that the expression of the ACSL3 gene in the pathological sections of liver cancer metastasis and lung metastasis is significantly higher than that in the pathological sections of primary liver cancer, and the level of ACSL3 expression is significantly correlated with the overall survival (OS) of liver cancer patients. Further studies have found that knocking down the expression of the ACSL3 gene can effectively inhibit the growth, migration and invasion of liver cancer cells, promote their apoptosis, and inhibit the lung metastasis of liver cancer cells. Therefore, the ACSL3 gene can be used for the prognosis assessment of liver cancer patients, and can be used as a therapeutic target for liver cancer patients, especially patients with liver cancer lung metastasis.

[0019] The present invention also provides a nano drug loaded with siRNA targeting the ACSL3 gene, which has long circulation ability in the blood environment and high accumulation in the tumor site. The nano carrier can be quickly released under the acidic conditions of the lysosome, efficiently silences the target gene, and effectively inhibits the occurrence of liver cancer lung metastasis. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The size distribution diagram of nanoparticles loaded with siRNA1.

[0021] Figure 2 The potential diagram of nanoparticles loaded with siRNA1.

[0022] Figure 3 This is an electron micrograph of nanoparticles loaded with siRNA1.

[0023] Figure 4 The release curve of nanoparticles loaded with siRNA1 in PBS solutions with different pH concentrations.

[0024] Figure 5 This is a heat map analysis of the patient's primary liver cancer lesions and lung metastases.

[0025] Figure 6 The results of univariate and multivariate COX analysis of ACSL3 expression and OS in TCGA.

[0026] Figure 7 This is a graph showing the relationship between ACSL3 expression and clinical prognosis (OS, DFS) in TCGA liver cancer.

[0027] Figure 8 The graph shows the expression results of ACSL3 in liver cancer cell lines.

[0028] Fig. 9 This is a graph showing the growth inhibition results of MHCC97H cells by knocking down ACSL3.

[0029] Fig.10 This figure shows the effect of knocking down ACSL3 on the migration, invasion and apoptosis of MHCC97H cells.

[0030] Fig.11 The results show that silencing ACSL3 inhibits tumor lung metastasis in vivo.

[0031] Fig.12 This is a graph showing the knockdown efficiency of ACSL3 by nanoparticles at different siRNA concentrations.

[0032] Fig.13 The figure shows the result of ACSL3 silencing inhibiting the metastasis of MHCC97H cells.

[0033] Fig.14 Figure 3 is the growth curve of each treatment group in orthotopic tumors and PDX tumors. DETAILED DESCRIPTION

[0034] The experimental methods in the following examples of the present invention, where no specific conditions are specified, are usually carried out under conventional conditions or under conditions recommended by the manufacturers. The various commonly used chemical reagents used in the examples are all commercially available products.

[0035] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0036] The terms "including" and "having" and any variations thereof of the present invention are intended to cover non-exclusive inclusions. For example, a process, method, device, product or equipment comprising a series of steps is not limited to the listed steps or modules, but may optionally include steps not listed, or may optionally include other steps inherent to these processes, methods, products or equipment.

[0037] In the present invention, the term "multiple" refers to two or more than two. "And / or" describes the association relationship of associated objects, indicating that three relationships may exist. For example, A and / or B can represent the following three situations: A exists alone, A and B exist at the same time, and B exists alone. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0038] The following describes the invention in conjunction with specific embodiments.

[0039] Example 1

[0040] The present embodiment provides a nano drug loaded with siRNA for ACSL3 gene, which is prepared by nano precipitation method, specifically comprising the following steps: nanoparticles encapsulating siRNA are prepared by nano precipitation method, polymer Meo-PEG-b-PDPA (preparation method can refer to: Ultra pH-Responsive and Tumor-Penetrating Nanoplatform for Targeted siRNA Delivery with Robust Anti-Cancer Efficacy.) is dissolved in DMF to form a uniform solution with a concentration of 10 mg / mL. Subsequently, a mixture of 10 μL siRNA (0.1 nmol / μL aqueous solution) and 50 μL G0-C14 (5 mg / mL in DMF) is prepared, and then mixed with 200 μL Meo-PEG-b-PDPA solution. Under vigorous stirring (1000 rpm), the mixture is added dropwise to 5 mL deionized water. The formed NP suspension is transferred to an ultrafiltration device (EMDMillipore, MWCO100K) and centrifuged to remove organic solvents and free compounds. After washing three times with phosphate buffered saline (PBS) solution (3×5 mL), the obtained siRNA-loaded NPs (denoted as NPs(siACSL3)) were suspended in 1 mL of PBS solution.

[0041] The siRNA can be siRNA1 (denoted as siACSL3-1) or siRNA2 (denoted as siACSL3-2). siACSL3-1 comprises a sense strand and an antisense strand; siACSL3-2 comprises a sense strand and an antisense strand. The siRNA is coated with a Cy5 label.

[0042] siACSL3-1 positive strand: 5′-GGUUAUUCUUGGACAGUAU (SEQ ID NO. 1) dTdT-3′;

[0043] siACSL3-1 antisense strand: 5′-AUACUGUCCAAGAAUAACC (SEQ ID NO. 2) dTdT-3′;

[0044] siACSL3-2 positive strand: 5′-GAUGUGAUACUUUAGAUAA (SEQ ID NO. 3) dTdT-3′;

[0045] siACSL3-2 antisense strand: 5′-UUAUCUAAAGUAUCACAUC (SEQ ID NO. 4) dTdT-3′.

[0046] The size and zeta potential of the encapsulated siACSL3-1 nanoparticles were determined by dynamic light scattering (DLS, Malvern, USA). Figure 1 and 2 The NP morphology was visualized under a transmission electron microscope (TEM) (TecnaiG2SpiritBioTWIN), as shown. Figure 3 shown.

[0047] After multiple washings, the siACSL3-1 nanoparticles were dispersed in 1 ml PBS, the nanoparticle solution was transferred to a dialysis bag, and the dialysis bag was placed in a beaker containing PBS solutions of different pH concentrations (pH 7.4 and pH 6.0). At the specified time point, 5 μL of the nanoparticle solution was taken out and added to 100 μL DMSO, and the fluorescence intensity was detected using a microplate reader. The cumulative release rate of siRNA was calculated according to the following formula: Cumulative release amount (%) = (M t / M ∞ )×100; where M t is the siRNA released from the nanoparticles at a given time point, M ∞ is the total amount of siRNA loaded in the nanoparticles. The release curves of siRNA-loaded nanoparticles in PBS solutions with different pH concentrations are shown in Figure 2. Figure 4 shown.

[0048] Example 2

[0049] Three pairs of matched patients' primary liver cancer pathological sections and metastatic lung cancer pathological sections were collected and analyzed by proteomic sequencing. The results showed that ACSL3 was highly expressed in metastatic lesions (e.g. Figure 5 ), and in univariate and multivariate COX analysis, the expression of ACSL3 was significantly correlated with overall survival (OS) (as shown in Figure 6 In addition, in the TCGA database, survival analysis indicated that high expression of ACSL3 led to shorter overall survival (OS) and disease-free survival (DFS) in patients with liver cancer (as shown in Figure 7 These results indicate that ACSL3 is significantly upregulated in metastatic lesions and is associated with poor prognosis in patients with HCC.

[0050] Example 3

[0051] Knockdown of ACSL3 significantly inhibited the biological functions of liver cancer cells.

[0052] ACSL3 is highly expressed in the hepatoma cell line MHCC97H (e.g. Figure 8 Therefore, we used siRNA to silence its expression in this cell line to study its effect on liver cancer function.

[0053] Lipofectamine3000 was used to transfect siACSL3-1 and siACSL3-2 into MHCC97H cells. The cell proliferation was detected by alarmblue method on the 1st, 3rd, 5th and 7th days after transfection. The results showed that knocking down ACSL3 significantly inhibited the growth ability of liver cancer cells (such as Fig. 9 After transfection, the migration and invasion abilities of MHCC97H cells were detected by crystal violet staining 2 days after transfection. 2 days after transfection, the apoptosis of MHCC97H cells was detected by flow cytometry. The results showed that knockdown of ACSL3 inhibited the migration and invasion abilities of MHCC97H cells and increased the apoptosis of MHCC97H cells (as shown in Figure 2). Fig.10 shown).

[0054] In addition, we also constructed a cell line with stable knockdown of ACSL3 using the lentiviral Lucifer tag, and injected shNC and shACSL3-1 cell lines into the tail vein of mice to evaluate the in vivo inhibitory effect of this gene on lung metastasis, with an injection dose of 2 million cells (200 μl).

[0055] The results are as follows Fig.11 As shown, silencing ACSL3 inhibited the ability of tumors to metastasize to the lungs in vivo. Therefore, knocking down ACSL3 inhibited the biological progression of tumors and the process of lung metastasis.

[0056] Example 4

[0057] In vitro and in vivo inhibitory results of siACSL3-1-loaded nanoparticles.

[0058] The liver cancer MHCC97H cells were inoculated in a 6-well plate (100,000 cells per well), and 2 mL of a culture medium containing 10% fetal bovine serum was added and incubated for 24 hours to allow them to fully adhere to the wall. Subsequently, nanoparticles loaded with siACSL3-1 at different concentrations (siRNA concentrations were: 10 nM, 30 nM, 50 nM, respectively) were added, and the nanoparticle preparation method was the same as in Example 1. The experimental groups were as follows: (1) PBS group; (2) NakedsiACSL3 group: 50nM siACSL3-1 was directly added; (3) NPs(siNC) group: nanoparticles loaded with 50nM scrambled siRNA; (4) NPs(siACSL3)-10nM group: nanoparticles loaded with 10nM siACSL3-1; (5) NPs(siACSL3)-30nM group: nanoparticles loaded with 30nM siACSL3-1; (6) NPs(siACSL3)-50nM group: nanoparticles loaded with 50nM siACSL3-1. After incubation of cells in serum-free medium for 24 hours, the cells were replaced with medium containing fetal bovine serum and incubated for another 48 hours. After RNA and protein extraction, the expression of ACSL3 was detected by PCR and Western blot.

[0059] like Fig.12 As shown in the figure, the nanoparticles loaded with siACSL3-1 can knock down the expression of the target gene ACSL3 at different siRNA concentrations. At the same time, the effect of ACSL3 in inhibiting tumor metastasis was detected. Taking the NPs (siACSL3)-50nM group as an example, Fig.13 As shown, ACSL3 silencing inhibited the invasion and metastasis ability of MHCC97H cells.

[0060] Example 5

[0061] Orthotopic tumor and PDX treatment models: Nanoparticles loaded with siACSL3-1 can inhibit liver cancer growth.

[0062] We verified the effect of nanoparticles loaded with siACSL3-1 (prepared in the same way as in Example 1) in vivo: 20 nude mice were divided into 5 groups, and MHCC97H cells were injected in situ. After the tumor grew to a size where fluorescent signals could be observed by in vivo imaging, we gave treatment to each group. In addition, a PDX model was constructed using human tumor tissue. After the third generation of transplantation, the tumor was grown to 100 mm. 3Afterwards, treatment was carried out. The treatment groups were as follows: 1) Injection of PBS 200uL, Control group; 2) Injection of naked siACSL3-1 200uL (siACSL3-1 is 1nmoL), NakedsiACSL3 group; 3) Injection of nanomaterials loaded with scrambled siRNA (siNC) 200uL (siRNA is 1nmoL), NPs (siNC) group; 4) Injection of nanomaterials loaded with siACSL3-1 200uL (siACSL3-1 is 1nmoL), NPs (ACSL3) group. Inject 200uL (siACSL3 is 1nmoL) through the tail vein every other day. After 3 injections, observe for 2 weeks, 4 weeks and record the growth curve of the nude mice. Fig.14 As shown, we found that the NPs (ACSL3) group had the best therapeutic effect, indicating that nanoparticles loaded with siACSL3-1 can significantly inhibit the growth of liver cancer in vivo.

[0063] In summary, the ACSL3 gene can be used for the prognosis assessment of patients with liver cancer and can be used as a therapeutic target for patients with liver cancer, especially those with lung metastasis. Silencing the ACSL3 gene can effectively inhibit the growth, invasion and metastasis of liver cancer cells, promote their apoptosis, and inhibit their lung metastasis.

[0064] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0065] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. Use of an agent for inhibiting ACSL3 gene expression in the preparation of a drug for treating liver cancer patients, characterized in that: The reagent for inhibiting ACSL3 gene expression includes siRNA; the siRNA is selected from siRNA1 or siRNA2; wherein siRNA1 comprises a sense chain as shown in SEQ ID NO.1 and an antisense chain as shown in SEQ ID NO.2, and SEQ ID NO.1 and SEQ ID NO.2 have 2 dT modifications at their 3' ends; siRNA2 comprises a sense chain as shown in SEQ ID NO.3 and an antisense chain as shown in SEQ ID NO.4, and SEQ ID NO.3 and SEQ ID NO.4 have 2 dT modifications at their 3' ends.

2. The use according to claim 1, characterized in that The drug has at least one of the following effects: inhibiting the growth of liver cancer cells, inhibiting the migration of liver cancer cells, inhibiting the invasive ability of liver cancer cells, promoting the apoptosis of liver cancer cells, and inhibiting the lung metastasis of liver cancer cells.

3. The use according to claim 1, characterized in that The liver cancer patient is a patient with liver cancer lung metastasis.

4. A drug for treating patients with liver cancer and / or patients with lung metastasis of liver cancer, characterized in that: The drug comprises an agent for inhibiting the expression of ACSL3 gene and a pharmaceutically acceptable excipient; the agent for inhibiting the expression of ACSL3 gene is siRNA; the siRNA is selected from siRNA1 or siRNA2; wherein siRNA1 comprises a sense chain as shown in SEQ ID NO.1 and an antisense chain as shown in SEQ ID NO.2, and SEQ ID NO.1 and SEQ ID NO.2 have 2 dT modifications at their 3' ends; siRNA2 comprises a sense chain as shown in SEQ ID NO.3 and an antisense chain as shown in SEQ ID NO.4, and SEQ ID NO.3 and SEQID NO.4 have 2 dT modifications at their 3' ends.

5. The drug according to claim 4, characterized in that The drug is a nanoparticle loaded with siRNA; the nanoparticle is prepared by the following method: (1) dissolving a polymer Meo-PEG-b-PDPA in DMF to form a solution 1 with a concentration of 10 mg / mL; (2) dissolving siRNA in water to form a solution 2 with a concentration of 0.1 nmol / μL; dissolving G0-C14 in DMF to form a solution 3 with a concentration of 5 mg / mL; (3) mixing the solution 1, solution 2 and solution 3 at a volume ratio of 20:1:5 to obtain a mixture, and then adding the mixture dropwise into water under stirring conditions to form a suspension; (4) transferring the suspension to an ultrafiltration device, centrifuging to remove organic solvents and free compounds, and then washing with a PBS solution to obtain the nanoparticle.

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