Application of traditional Chinese medicine composition in intervention of IGF2BP1 gene in inhibition of liver cancer

Abstract

The invention discloses application of a traditional Chinese medicine composition in preparation of a medicine for treating cancers with abnormal IGF2BP1 gene expression. The medicine is designed by taking an IGF2BP1 gene as a target spot. The invention further discloses application of the traditional Chinese medicine composition in production of medicines for treating liver cancer. The traditional Chinese medicine composition comprises 30 g of astragalus membranaceus, 15 g of turtle shells, 15 g of rhizoma sparganii, 15 g of curcuma zedoary, 9 g of angelica sinensis, 18 g of safflower carthamus, 18 g of peach kernels, 6 g of raw liquorice, 3 g of coptis chinensis and 3 g of muskmelon pedicel. By adopting the traditional Chinese medicine composition with compatibility, the protein level and gene expression quantity of IGF2BP1 in Huh7 and MHCC-97H cell lines can be regulated and controlled. Various liver function indexes of rats are improved, and the tumor volume is reduced, so that the liver cancer progress is inhibited, and a foundation is laid for improving the survival rate and the survival quality of liver cancer patients.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and relates to the application of a traditional Chinese medicine composition in intervening in IGF2BP1 gene inhibition of liver cancer. Background Art

[0002] Liver cancer is a very harmful tumor, and its incidence rate is increasing year by year. The main causes of the disease include long-term drinking, chronic hepatitis, cirrhosis, etc., among which chronic hepatitis is the most important cause. The main clinical manifestations of liver cancer include pain in the liver area, weight loss, jaundice, etc. At present, chemotherapy is the conventional treatment for liver cancer, but the effect is not very ideal, mainly because liver cancer cells are resistant to chemotherapy drugs. Chemotherapy resistance is usually manifested as a weakened response of tumor cells to chemotherapy drugs or complete failure. The mechanism of chemotherapy resistance in liver cancer is complex and may be related to factors such as genetic mutations of liver cancer cells, changes in drug metabolism and excretion, etc.

[0003] With the development of molecular biology technology and the continuous improvement of human gene spectrum in recent years, people have a new understanding of the causes and pathogenesis of liver cancer, that is, the activation of liver cancer-related oncogenes and the inactivation and mutation of tumor suppressor genes are the root causes of liver cell carcinogenesis. Oncogenes and tumor suppressor genes in cells play a positive and negative regulatory role in cell proliferation and differentiation. Therefore, the changes in liver cancer-related oncogenes and tumor suppressor genes at the molecular level are the root causes of liver cancer. At the same time, people have proposed a new concept: gene therapy, which has brought new hope for the treatment of cancer. The so-called gene therapy for cancer refers to the introduction of cloned normal gene sequences into patients with gene defects based on genetic technology, so that the introduced genes can play a role and correct the gene defects, or design antisense sequences of mutated oncogenes to be introduced into patients to prevent the mutated oncogenes from exerting their effects. The ultimate goal of gene therapy for cancer is to kill tumor cells through different pathways, inhibit tumor growth and cause tumor regression. Therefore, gene therapy for cancer is to reverse the malignant transformation process of tumor cells and has no effect on normal cells.

[0004] Tumor metastasis is a multi-step, complex process. The research on the recurrence and metastasis of TCM against tumors mainly focuses on promoting blood circulation and removing blood stasis, strengthening the body and strengthening the foundation. Fuzheng Xiaozheng Recipe is composed of Astragalus, Angelica, Curcuma, Hedyotis diffusa, Coix seeds and Pinellia. The combination of these medicines can treat both the symptoms and the root causes, and regulate both the deficiency and the excess, which can replenish qi and blood, eliminate phlegm and eliminate symptoms, and clear away heat and detoxify. In view of the great potential of Fuzheng Xiaozheng Recipe in the treatment of liver cancer, it is imperative to explore the potential mechanism of Fuzheng Xiaozheng Recipe. However, due to the complex ingredients of the compound, it often acts on multiple parts and multiple targets in the body. Exploration from a single aspect or a certain pathway cannot fully explain the compound treatment mechanism. Transcriptome epigenetic modification and TCM treatment principles coincide to a certain extent, both of which regulate the body level from the overall level, thus providing a new idea for studying the mechanism of compound treatment of liver cancer. Therefore, research at the transcriptome gene expression level may be able to relatively fully explain the mechanism of action of Fuzheng Xiaozheng Recipe in the treatment of liver cancer. During this process, the inventors found that by optimizing and adjusting the composition of the Fuzheng Xiaozheng prescription for the important target gene IGF2BP1 in the epigenetic modification process, the intervention effect of liver cancer can be further enhanced. Summary of the invention

[0005] Based on this, the present invention first clarifies the role of Fuzheng Xiaozheng prescription in intervening in the inhibition of liver cancer by IGF2BP1 gene, and secondly, provides an expression inhibitor of IGF2BP1 gene - that is, adjusting the formula of Fuzheng Xiaozheng prescription, so as to prepare a drug that inhibits the metastasis and invasion of liver cancer cells, so as to improve the treatment effect of liver cancer.

[0006] The present invention is specifically achieved through the following technical means:

[0007] The present invention firstly discloses the application of a traditional Chinese medicine composition in preparing a medicine for treating cancer with abnormal IGF2BP1 gene expression.

[0008] Furthermore, the cancer is liver cancer.

[0009] Furthermore, the drug is designed with IGF2BP1 gene as the target.

[0010] The present invention also discloses an application of a Chinese medicine composition in the preparation of a drug for treating liver cancer, wherein:

[0011] The Chinese medicine composition comprises: 30g of Astragalus, 15g of Carapace of Turtle, 15g of Trigonosciadium japonicum, 15g of Curcuma zedoaria, 9g of Angelica sinensis, 18g of Carthamus tinctorius, 18g of Semen persica, 6g of Raw Licorice, 3g of Coptis chinensis, and 3g of Cucurbita chinensis.

[0012] Furthermore, in the application, the Chinese medicine composition can regulate the protein level and gene expression of IGF2BP1 in Huh7 and MHCC-97H cell lines.

[0013] The beneficial effects of the present invention are:

[0014] The present invention combines the previous studies - the mechanism of coptis chinensis inhibiting the proliferation and metastasis of hepatocellular carcinoma by promoting the ubiquitination of IGF2BP1, and the Allosteric Regulation of IGF2BP1 as a Novel Strategy for the Activation of Tumor Immune Microenvironment. It can be seen that coptis chinensis and citrus tangerine have an inhibitory effect on the proliferation of HuH7, 7404 and HepG2 cells, and HuH7 cells are more sensitive to the effect of coptis chinensis, while coptis chinensis is insensitive to the effect of normal human liver cells LO2. At the same time, IGF2BP1 is a potential key target of coptis chinensis and citrus tangerine on HCC. Coptis chinensis and citrus tangerine themselves are found to show significant inhibitory effects on subcutaneous tumors in nude mice when coptis chinensis or citrus tangerine is used alone; on this basis, the present invention adds coptis chinensis and citrus tangerine to the traditional Fuzheng Xiaozheng prescription to form a new Chinese medicine composition. In the intervention of rat primary liver cancer, it is found that its intervention effect on rat primary liver cancer is more obvious than that of the Fuzheng Xiaozheng prescription. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The effect of traditional Fuzheng Xiaozhi prescription on IGF2BP1 and invasion-related genes in Huh7 and MHCC-97H liver cancer cells, including: (A). IGF2BP1 protein expression level before and after the intervention of traditional Fuzheng Xiaozhi prescription; (B). IGF2BP1 gene expression level before and after the intervention of traditional Fuzheng Xiaozhi prescription; (C). The protein expression level of invasion-related genes before and after the intervention of traditional Fuzheng Xiaozhi prescription; (D). The gene expression level of invasion-related genes before and after the intervention of traditional Fuzheng Xiaozhi prescription. *, p < 0.05, **, p < 0.01, ***, p < 0.001 vs. 0 mg / mL; ns, no statistically significant difference vs. 0 mg / mL.

[0016] Figure 2 Identification of IGF2BP1 siRNA concentration and effect. (A). GAPDH siRNA transfection concentration gradient; (B). PCR quantitative identification of the transfection effect of three pairs of IGF2BP1 siRNA at a concentration of 30nM; (C). WB identification of the transfection effect of siIGF2BP1-2 at a concentration of 30nM; (D). WB quantification of the transfection effect of siIGF2BP1-2. Control, blank control group; NC, negative control group; *, p<0.05, **, p<0.01, ***, p<0.001 vs. NC; ns, no statistically significant difference vs. Control.

[0017] Figure 3The effect of IGF2BP1 knockout on the migration and invasion ability of Huh7 cells. (A). Scratch assay; (B). Scratch width statistics; (C). Transwell chamber assay; (D). Invading cell number statistics; (E). Changes in protein levels of invasion-related genes after IGF2BP1 knockout; (F). WB quantitative statistics of invasion-related genes after IGF2BP1 knockout; (G). qPCR quantitative analysis of invasion-related genes after IGF2BP1 knockout. NC, negative control group; *, p<0.05, **, p<0.01, ***, p<0.001 vs. NC; ns, no statistically significant difference vs. NC.

[0018] Figure 4 To identify IGF2BP1 overexpression and the intervention effect of traditional Fuzheng Xiaozheng prescription. (A). WB identification of IGF2BP1 overexpression; (B). WB quantification of IGF2BP1 overexpression, ***, p < 0.001 vs. NC; ns, no statistically significant difference vs. Control; (C). PCR quantification of IGF2BP1 overexpression, ***, p < 0.001 vs. NC; ns, no statistically significant difference vs. Control; (D). WB results of traditional Fuzheng Xiaozheng prescription intervention on IGF2BP1 overexpression; (E). WB quantification of traditional Fuzheng Xiaozheng prescription intervention on IGF2BP1 overexpression, #, p < 0.05 vs. NC; *, p < 0.05 vs. oeIGF2BP1; (F). PCR quantification of traditional Fuzheng Xiaozheng prescription intervention on IGF2BP1 overexpression, ###, p < 0.001 vs. NC; **, p < 0.01 vs. oeIGF2BP1. Control, blank control group; NC, negative control group; oe / oeIGF2BP1, IGF2BP1 overexpression group; oe+F / oeIGF2BP1+FZXZP, IGF2BP1 overexpression group intervened by traditional Fuzheng Xiaozheng prescription.

[0019] Figure 5The effects of IGF2BP1 overexpression and traditional Fuzheng Xiaozhi prescription intervention on MHCC-97H migration and invasion. (A). Scratch test; (B). Scratch width statistics; (C). Transwell chamber experiment; (D). Invading cell number statistics, ****, p < 0.0001; (E). PCR quantification of invasion-related genes after IGF2BP1 overexpression and traditional Fuzheng Xiaozhi prescription intervention; (F). Changes in protein levels of invasion-related genes after IGF2BP1 overexpression and traditional Fuzheng Xiaozhi prescription intervention; (G). WB quantification of invasion-related genes after IGF2BP1 overexpression and traditional Fuzheng Xiaozhi prescription intervention. NC, negative control group; oe / oeIGF2BP1, IGF2BP1 overexpression group; oe+F / oeIGF2BP1+FZXZP, IGF2BP1 overexpression group after traditional Fuzheng Xiaozhi prescription intervention. #, p<0.05, ##, p<0.01, ###, p<0.001 vs. NC; *, p<0.05, **, p<0.01, ***, p<0.001 vs. oeIGF2BP1; ns, no statistically significant difference vs. NC / oeIGF2BP1. DETAILED DESCRIPTION

[0020] In order to facilitate the understanding of the present invention, the present invention will be described more fully below, and the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0021] Unless otherwise defined, all technical and scientific terms used herein 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 herein are only for the purpose of describing specific embodiments and are not intended to limit the present invention.

[0022] Example 1

[0023] A Chinese medicine composition comprising:

[0024] Astragalus 30g, turtle shell 15g, Trillium 15g, Curcuma 15g, Angelica 9g, Carthamus tinctorius 18g, Peach kernel 18g, Raw licorice 6g, Coptis chinensis 3g, Cucurbita citrifolia 3g.

[0025] Comparative Example 1

[0026] Traditional prescription for strengthening the body and eliminating symptoms:

[0027] Astragalus 30g, turtle shell 15g, Trillium 15g, Curcuma 15g, Angelica 9g, Carthamus tinctorius 18g, Peach kernel 18g, and raw licorice 6g.

[0028] Test Example 1

[0029] Effects of traditional Fuzheng Xiaozheng prescription on IGF2BP1 and invasion-related genes

[0030] We screened out traditional Fuzheng Xiaozheng prescriptions to regulate the key target gene IGF2BP1 of liver cancer and verified it in liver cancer cell lines. The results showed that traditional Fuzheng Xiaozheng prescriptions can significantly reduce the protein level of IGF2BP1 in Huh7 and MHCC-97H cell lines ( Figure 1 A) and gene expression ( Figure 1 B), indicating that the traditional Fuzheng Xiaozheng prescription can reduce the expression of IGF2BP1 in both cell lines with high and low expression of IGF2BP1, thereby inhibiting the development of liver cancer. In addition, in order to clarify the effect of the traditional Fuzheng Xiaozheng prescription on invasion-related genes, the expression trend of the traditional Fuzheng Xiaozheng prescription in Huh7 and MHCC-97H cells was detected after intervention. Figure 1 C and 1D show that the traditional Fuzheng Xiaozheng recipe can significantly inhibit the gene and protein expression levels of matrix metalloproteinases such as MMP2, MMP3, and MMP9 and endothelial-mesenchymal transition-related indicators such as E-cadherin and N-cadherin in liver cancer cells, indicating that the traditional Fuzheng Xiaozheng recipe may inhibit the invasive ability of liver cancer cells by regulating the expression of invasion genes.

[0031] 1. Establishment of IGF2BP1 siRNA concentration and identification of knockout effect

[0032] Based on the above experimental basis, Huh7 cells have the highest expression of IGF2BP1, so Huh7 cells were selected as the IGF2BP1 knockout cell line. In IGF2BP1 siRNA transfection, in order to determine the best transfection efficiency, we selected GAPDH as a positive control and used GAPDH to explore the optimal siRNA transfection concentration, such as Figure 2 As shown in A, 24 hours after transfection, GAPDH expression inhibition was best when the siRNA concentration was 30 nM, so we continued to transfect IGF2BP1 siRNA in Huh7 cells at a concentration of 30 nM. Among the three pairs of independent siRNAs, siIGF2BP1-2 had the most obvious knockdown effect ( Figure 2 B), and the negative control had no significant effect on the expression of IGF2BP1, indicating that this siRNA is stable and usable. Therefore, 30nM siIGF2BP1-2 was selected as the transfection condition for IGF2BP1 knockout in Huh7 cells. WB results showed that the expression of IGF2BP1 in Huh7 cells was significantly decreased under this transfection condition ( Figure 2 CD), indicating that this transfection condition can achieve the best IGF2BP1 knockout effect.

[0033] 2. IGF2BP1 knockout significantly reduces the migration and invasion ability of liver cancer cells

[0034] Using the above transfection conditions, after IGF2BP1 siRNA transfection in Huh7 cells, the scratch width at 24h and 48h was significantly higher than that in the negative control group, indicating that knocking out IGF2BP1 reduced the migration ability of Huh7 cells ( Figure 3 AB). Similarly, the invasion ability of Huh7 cells was significantly reduced after IGF2BP1 silencing ( Figure 3 CD). WB results showed that after IGF2BP1 knockout, the expression levels of MMP2, MMP3, MMP9, E-cadherin, and N-cadherin were significantly reduced ( Figure 3 EF), and the PCR results also showed the same expression trend in the two groups ( Figure 3 G), all of which indicate that IGF2BP1 knockout can significantly reduce the migration and invasion ability of liver cancer cells.

[0035] 3. Traditional prescriptions for strengthening the body and eliminating symptoms can reverse the overexpression trend of IGF2BP1

[0036] In the above study, MHCC-97H had the lowest IGF2BP1 expression level, so MHCC-97H was selected as the IGF2BP1 overexpression cell to obtain a better overexpression effect. First, the total amount of IGF2BP1 plasmid transfection should be determined. Since the expression level of IGF2BP1 in MHCC-97H cells is very low, it is possible to achieve overexpression by transfecting a small amount of plasmid. The results showed that after transfecting 5ng of IGF2BP1 plasmid in a six-well plate, it was found that the overexpression effect could be achieved ( Figure 4 AC), and there was no significant difference in expression levels between the negative control and the blank control, indicating that this plasmid had no special effect on the expression of IGF2BP1 and the transfection system was stable. After successfully establishing the IGF2BP1 overexpression model, intervention with traditional Fuzheng Xiaozheng prescriptions was performed, and it was found that the expression trend of IGF2BP1 was reversed, that is, the protein and mRNA levels of IGF2BP1 were significantly reduced ( Figure 4 DF), which once again proved that IGF2BP1 is the key target of traditional prescriptions for strengthening the body and eliminating symptoms.

[0037] 4. Traditional Fuzheng Xiaozheng prescription inhibits the migration and invasion ability of IGF2BP1 overexpressing cell lines

[0038] After overexpression of IGF2BP1 in MHCC-97H, the scratch width of MHCC-97H at 24h and 48h decreased compared with the negative control, indicating that its migration ability was significantly enhanced. After intervention with the traditional Fuzheng Xiaozheng prescription, the scratch width at 24h and 48h increased compared with the overexpression group, indicating that its migration ability was reduced by the traditional Fuzheng Xiaozheng prescription ( Figure 5AB). Similarly, the invasion ability of MHCC-97H showed a trend of strengthening and weakening after IGF2BP1 overexpression and traditional Fuzheng Xiaozheng prescription intervention, indicating that the invasion ability of MHCC-97H was significantly increased after IGF2BP1 overexpression, and the traditional Fuzheng Xiaozheng prescription could still reduce the invasion ability of MHCC-97H ( Figure 5 CD). PCR( Figure 5 E) and WB( Figure 5 FG) Quantitative results showed that in the IGF2BP1 overexpression model, the expression levels of MMP2, MMP3, MMP9, E-cadherin, and N-cadherin were all increased, while their expression levels decreased after the intervention of the traditional Fuzheng Xiaozheng prescription, which indicated that the traditional Fuzheng Xiaozheng prescription could effectively inhibit the overexpression of IGF2BP1, thereby inhibiting the migration and invasion ability of liver cancer cells.

[0039] Test Example 2

[0040] Comparison of therapeutic effects of different drugs on rat liver cancer

[0041] 1. Establishment, grouping and drug administration of rat hepatocellular carcinoma model

[0042] 85 SD male rats were acclimated and fed at 23±2℃ and 50-60% humidity for 1 week, and then randomly divided into blank control group (10 rats), model group (30 rats), high-dose group (15 rats), low-dose group (15 rats), and positive control group (15 rats). Except for the blank group, the remaining groups were gavaged with 70mg / kg DEN aqueous solution once a week for 14 weeks to induce HCC, and the blank group was gavaged with the corresponding volume of normal saline once a week for 14 weeks. From the 12th week, rats in the high-dose and low-dose groups were gavaged with 25.2g / kg and 6.3g / kg of traditional Fuzheng Xiaozheng prescriptions, respectively, twice a day for 6 weeks. The doses of positive drugs used in this study were based on literature reports and slightly modified. Rats in the positive control group were intraperitoneally injected with sodium demethylcantharinate (1.5mg / kg) once a day for 6 consecutive weeks. Rats in the model group did not receive any additional treatment. Subsequently, starting from the 14th week of model induction, 3 rats were anesthetized with sodium pentobarbital (150 mg / kg) every two weeks in the model group to observe liver changes and related index detection, and evaluate the model progress. Finally, the model was successfully constructed at the 18th week, and the remaining number of rats in the blank group, model group, low-dose group, high-dose group, and positive control group were 10, 13, 10, 8, and 6, respectively. Then, all rats were anesthetized with intraperitoneal injection of sodium pentobarbital (150 mg / kg), and blood samples and liver tissues were collected and stored at -80°C for subsequent detection and analysis.

[0043] 2. Biochemical index detection

[0044] The collected blood samples were centrifuged at 3500rpm / min and 4℃ for 15min, and serum was extracted. Subsequently, the obtained serum was subjected to biochemical analysis using a fully automatic biochemical analyzer (BS-240VET) to detect alanine aminotransferase (ALT), total cholesterol (TC), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and serum bile acid determination (TBA) in rats of each group. At the same time, Ki67 and tumor size of rats were detected.

[0045] The traditional Fuzheng Xiaozheng prescription prepared in Comparative Example 1 was used to treat primary liver cancer in rats. The treatment results are shown in Table 1:

[0046] Table 1

[0047]

[0048]

[0049] The results of using Coptis chinensis to treat subcutaneous tumors in nude mice are shown in Table 2:

[0050] Table 2

[0051] Liver related indicators Blank group Model Group Low dose group 1g / kg / d High dose group 3g / kg / d Tumor size (diameter) 0cm 2.2cm 1.4cm 0.8cm Tumor weight 0 2.1g 1.2g 0.7g

[0052] The results of treatment with melon pedicles (3 g) (nude mouse subcutaneous tumor) are shown in Table 3:

[0053] Table 3

[0054] Liver related indicators Blank group Model Group Low dose group 1g / kg / d High dose group 3g / kg / d Tumor size (diameter) 0cm 0.9cm 0.75cm 0.5cm Tumor weight 0 0.7g 0.45g 0.36g

[0055] The Chinese medicine composition prepared in Example 1 was used to treat primary liver cancer in rats. The treatment results are shown in Table 4:

[0056] Table 4

[0057]

[0058] According to the results in Tables 2 and 3, it can be seen that the use of either Rhizoma Coptidis or Melon Seedlings alone showed significant inhibitory effects on subcutaneous tumors in nude mice, and the most significant reduction in tumor volume and weight was achieved with an intervention dose of 3g / kg / d. On this basis, the present invention adds Rhizoma Coptidis and Melon Seedlings to the traditional Fuzheng Xiaozheng prescription to form a new Chinese medicine composition. According to the results in Table 4, it can be seen that in the intervention of primary liver cancer in rats, the intervention effect of the present invention on primary liver cancer in rats is more obvious than that of the Fuzheng Xiaozheng prescription.

[0059] The above is only a specific implementation of the present application, but the protection scope of the present application is not limited thereto. Any technician familiar with the technical field can easily think of various equivalent modifications or replacements within the technical scope disclosed in the present application, and these modifications or replacements should be included in the protection scope of the present application. Therefore, the protection scope of the present application shall be based on the protection scope of the claims.

Claims

1. Use of a Chinese medicine composition in the preparation of a drug for treating cancer with abnormal IGF2BP1 gene expression.

2. The use according to claim 1, wherein: The cancer is liver cancer.

3. The use according to claim 1, wherein: The drug is designed with the IGF2BP1 gene as a target.

4. Use of a Chinese medicine composition in the preparation of a drug for treating liver cancer, wherein: The Chinese medicine composition comprises: 30g of Astragalus, 15g of Carapace of Turtle, 15g of Trigonosciadium japonicum, 15g of Curcuma zedoaria, 9g of Angelica sinensis, 18g of Carthamus tinctorius, 18g of Semen persica, 6g of Raw Licorice, 3g of Coptis chinensis, and 3g of Cucurbita chinensis.

5. The use according to any one of claims 1 to 4, wherein: The traditional Chinese medicine composition can regulate the protein level and gene expression amount of IGF2BP1 in Huh7 and MHCC-97H cell lines.

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