Application of genistein in preparation of anti-colorectal cancer medicine for inhibiting LINC00355 expression

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of genistein to preparation of an anti-colorectal cancer medicine for inhibiting LINC00355 expression. According to the present invention, the genistein is adopted as the LINC00355 expression inhibitor so as to effectively inhibit the expression of the LINC00355, such that the adsorption effect of the LINC00355 on the miR-150 is relieved, the expression of the miR-150 is up-regulated, the expression of the SGK1 is further inhibited by the miR-150, the activation of the downstream EGFR / PI3K / Akt and MAPK signal channels is finally inhibited, and the activity of the downstream EGFR / PI3K / Akt and MAPK signal channels is inhibited; and inhibition of colorectal cancer cell proliferation, inhibition of colorectal cancer cell cycle G0 / G1 phase, induction of colorectal cancer cell apoptosis and inhibition of colorectal cancer cell migration and invasion ability are realized, so that a better anti-colorectal cancer treatment effect is achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to the use of genistein in the preparation of an anti-colorectal cancer drug for inhibiting the expression of LINC00355. BACKGROUND

[0002] Colorectal cancer is the third most common cancer and the second leading cause of death worldwide, posing a serious threat to human public health. Due to the insidious early clinical symptoms, most patients are diagnosed at an advanced stage with distant metastasis, losing the opportunity for radical surgery. At present, radiotherapy and chemotherapy and targeted therapy are the main treatment methods for advanced colorectal cancer, but these treatment methods are often accompanied by obvious side effects, and the targeted drugs are expensive and only suitable for a small part of the population, which is greatly limited in clinical application.

[0003] Natural products have always been an important source of anticancer drugs, and more than 60% of clinically used anticancer drugs are derived from natural products. Flavonoids widely exist in plants and are an important class of natural anticancer compounds. Genistein, a major isoflavone isolated from soybeans, has been reported to have broad-spectrum antitumor activity, including inhibition of tumor cell proliferation, induction of apoptosis, inhibition of angiogenesis and metastasis, and reversal of multidrug resistance.

[0004] Long non-coding RNA LINC00355 is a newly discovered oncogenic RNA that has been shown to promote the progression of head and neck squamous cell carcinoma and colorectal cancer. It is involved in the regulation of competitive endogenous RNA networks in colorectal cancer and its expression is closely related to tumor stage and metastasis. MiR-150 is a recognized anticancer microRNA that regulates the occurrence and development of various cancers. SGK1 is a key downstream molecule of the PI3K pathway and is highly expressed in various tumors and promotes tumor progression. Therefore, there is an urgent need to provide a drug that can effectively inhibit the expression of LINC00355. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes the use of genistein in the preparation of a LINC00355 expression inhibitor.

[0006] The present application also proposes the use of genistein in the preparation of a product for treating colorectal cancer.

[0007] To achieve the above-mentioned purpose, the present application adopts the following technical solutions: In one aspect, the present application provides the use of genistein in the preparation of a LINC00355 expression inhibitor.

[0008] According to some embodiments of the present application, the LINC00355 expression inhibitor has at least one of the following functions: (1) promoting the expression of the glucocorticoid-induced kinase 1; (2) inhibiting the expression of miR-150; (3) promoting the expression of SGK1; (4) regulating the miR-150 / SGK1 axis; (4) regulating the extracellular signal-regulated kinase / mitogen-activated protein kinase pathway; (5) regulating the phosphatidylinositol 3-kinase / protein kinase B pathway; (6) resisting colorectal cancer.

[0009] In some embodiments of the present application, the regulation of the extracellular signal-regulated kinase / mitogen-activated protein kinase pathway is the activation of the extracellular signal-regulated kinase / mitogen-activated protein kinase pathway.

[0010] In some embodiments of the present application, the regulation of the phosphatidylinositol 3-kinase / protein kinase B pathway is the activation of the phosphatidylinositol 3-kinase / protein kinase B pathway.

[0011] In some embodiments of the present application, the resistance to colorectal cancer includes inhibiting the proliferation of colorectal cancer cells, arresting the G0 / G1 phase of the cell cycle of colorectal cancer cells, inducing the apoptosis of colorectal cancer cells, and / or inhibiting the migration and invasion ability of colorectal cancer cells.

[0012] According to a second aspect of the present application, the use of genistein in the preparation of a product for treating colorectal cancer is provided.

[0013] In some embodiments of the present application, the treatment of colorectal cancer includes inhibiting the proliferation of colorectal cancer cells, arresting the G0 / G1 phase of the cell cycle of colorectal cancer cells, inducing the apoptosis of colorectal cancer cells, and / or inhibiting the migration and invasion ability of colorectal cancer cells.

[0014] In some embodiments of the present application, the product is at least one of a drug, a health product, and a functional food.

[0015] In some embodiments of the present application, the product is a drug, and the drug further comprises a pharmaceutically acceptable excipient.

[0016] In some embodiments of the present application, the pharmaceutically acceptable excipient comprises at least one of a diluent, an excipient, a filler, a binder, a disintegrant, an absorption enhancer, a surfactant, an adsorption carrier, a lubricant, a sweetener, and a flavoring agent.

[0017] In some embodiments of the present application, the excipient comprises water.

[0018] In some embodiments of the present application, the filler comprises at least one of starch and sucrose.

[0019] In some embodiments of the present application, the binder comprises at least one of cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone.

[0020] In some embodiments of the present application, the wetting agent comprises glycerin.

[0021] In some embodiments of the present application, the disintegrant comprises at least one of agar, calcium carbonate and sodium bicarbonate.

[0022] In some embodiments of the present application, the absorption promoter comprises a quaternary ammonium compound.

[0023] In some embodiments of the present application, the surfactant comprises cetyl alcohol.

[0024] In some embodiments of the present application, the adsorptive carrier comprises at least one of kaolin and bentonite.

[0025] In some embodiments of the present application, the lubricant comprises at least one of talc, calcium stearate, magnesium stearate and polyethylene glycol.

[0026] In some embodiments of the present application, the dosage form of the pharmaceutical product is selected from tablets, capsules, granules, powders, liquid preparations, ointments, injection solutions or spray preparations.

[0027] In some embodiments of the present application, the pharmaceutical product can be administered to a patient in need of such treatment by oral or parenteral administration or the like. For example, when administered parenterally, it can be formulated into an injection. When formulated into an injection, it can be produced by using conventional methods in the field of pharmacy, and an aqueous solvent or a non-aqueous solvent can be used. The most commonly used aqueous solvent is water for injection, and 0.9% sodium chloride solution or other suitable aqueous solutions can also be used; the commonly used non-aqueous solvent is vegetable oil, mainly soybean oil for injection, and other aqueous solutions of ethanol, propylene glycol, polyethylene glycol, etc. When preparing an injection, no additional agent can be added, or a suitable additional agent can be added according to the nature of the drug, such as an osmotic pressure adjusting agent, a pH adjusting agent, a solubilizing agent, a filler, an antioxidant, a bacteriostatic agent, an emulsifying agent, a suspending agent, etc. When used orally, it can be formulated into conventional solid preparations, such as tablets, capsules, powders, pills, granules, etc.; or it can be formulated into oral liquid preparations, such as oral solution, oral suspension, syrup, etc. When formulated into oral preparations, suitable fillers, binders, disintegrants, lubricants, etc. can be added.

[0028] The specific frequency of drug administration in this invention can be determined by those skilled in the art through known techniques and by observing results obtained under similar conditions. Preferably, the frequency of drug administration is once to three times daily, or once every 2 to 30 days; for example, it can be once daily, twice daily, three times daily, once every two days, once every three days, once every four days, once every five days, once every six days, once every seven days, once every eight days, once every nine days, once every ten days, once every fifteen days, once every twenty days, once every twenty-five days, or once every thirty days.

[0029] Based on mouse body weight, the effective dose of the genistein for mice is 20 mg / kg / d to 100 mg / kg / d, specifically 50 mg / kg / d.

[0030] In this invention, the drug treatment targets mammals, including humans and mice. Those skilled in the art can estimate the human dose per unit body weight based on the equivalent dose conversion between humans and experimental animals. According to the dose-per-unit-body-weight conversion, the equivalent dose for mice is approximately nine times that for humans.

[0031] In some embodiments of the present invention, the mass fraction of the genistein in the product is 0.01% to 100%.

[0032] In some embodiments of the present invention, the mass fraction of the genistein in the product is 0.05% to 95%.

[0033] In some embodiments of the present invention, the mass fraction of the genistein in the product is 0.05% to 50%.

[0034] Compared with the prior art, some embodiments of the present invention have at least the following beneficial effects: This invention proposes the application of genistein in the preparation of LINC00355 expression inhibitors. By using genistein as a LINC00355 expression inhibitor, the expression of LINC00355 can be effectively inhibited, thereby relieving the "adsorption" effect of LINC00355 on miR-150, upregulating miR-150 expression, and further inhibiting SGK1 expression. Ultimately, this inhibits the activation of downstream EGFR / PI3K / Akt and MAPK signaling pathways, thereby achieving the inhibition of colorectal cancer cell proliferation, G0 / G1 phase arrest of colorectal cancer cell cycle, induction of apoptosis of colorectal cancer cells, and inhibition of the migration and invasion ability of colorectal cancer cells, resulting in better anti-colorectal cancer therapeutic effects.

[0035] Other features and advantages of the invention will be set forth in the description which follows, and will be apparent in part from the description, or may be learned by practicing the invention. Attached Figure Description

[0036] Figure 1 The figures shown are the results of detecting the inhibition of human colorectal cancer cell proliferation by genistein in the examples. A represents the chemical structure of genistein; B represents the MTT assay results of normal human colonic epithelial cells (NCM460) and human colorectal cancer cell lines (HCT-8, HCT-116, HT-29, LoVo, RKO, CACO2, SW620, SW480) treated with different concentrations of genistein (0, 10, 20, 40, 80, and 160 µM); C represents the results of evaluating the viability of HCT-116 cells under control conditions and after gene perturbation, assessing the effects of overexpression (OE) or knockdown (KD) of LINC00355, miR-150, or SGK1 in the presence of genistein; D and E represent the results of evaluating HCT-116 cells under control conditions and 14 days after gene perturbation. Image showing the results of cell clonogenic ability assay; data are expressed as mean ± standard deviation, ***p<0.001; Figure 2 The following is a graph showing the detection results of genistein regulating the cell cycle of human colorectal cancer cells via the LINC00355 / miR-150 / SGK1 axis in the examples. In each graph, the horizontal axis corresponds to DNA content (relative fluorescence intensity), with the unit being the number of channels (position indicates cell cycle phase: the first peak is G0 / G1 phase, the second peak is G2 / M phase, and the plateau region in the middle is S phase); the vertical axis represents the number of cells, with the unit being cell count. Figure 3 This is a graph showing the detection results of HCT-116 cell apoptosis detected by Annexin V-FITC / PI double staining flow cytometry in the example. Figure 4Figure for detection results of the detection of the regulation of genistein on cell cycle, apoptosis and apoptosis-related molecules of human colorectal cancer cells by LINC00355 / miR-150 / SGK1 axis in the examples, wherein A is the cycle distribution of HCT-116 cells after control and gene disturbance is analyzed by flow cytometry, the influence of overexpression or knockdown of LINC00355, miR-150 or SGK1 under the background of genistein treatment is evaluated, and the percentage of cell population in G0 / G1 phase, S phase and G2 phase is quantified, and the label description is as follows: a: p<0.05 compared with the Ctrl group, b: p<0.05 compared with the Gen group, c: p<0.05 compared with the Gen+LINC-OE group, d: p<0.05 compared with the Gen+miR-150-OE group, e: p<0.05 compared with the Gen+SGK1-OE group, f: p<0.05 compared with the Gen+LINC-KD group, g: p<0.05 compared with the Gen+miR-150-KD group, h: p<0.05 compared with the Gen+SGK1-KD group; B is the detection result figure of the apoptosis of HCT-116 cells under the same experimental conditions is detected by Annexin V-FITC / PI double staining flow cytometry, and the apoptosis rate is statistically analyzed; C, D, E are the detection result figures of the expression level of apoptosis-related proteins in HCT-116 cells are detected by Western Blot, GAPDH is used as an internal reference, and the relative protein expression amount is statistically analyzed; F, G are the detection result figures of the relative mRNA expression level of apoptosis-related genes in HCT-116 cells are detected by qRT-PCR under the control condition and gene disturbance combined with genistein treatment; the data is in the form of mean ± standard deviation; *p<0.05, **p<0.01, ***p<0.001; Figure 5Figure for the detection results of the inhibition of genistein on the migration and invasion of human colorectal cancer cells by LINC00355 / miR-150 / SGK1 axis in the examples, wherein A and C are the migration ability of HCT-116 cells under control conditions and genistein treatment combined with gene disturbance evaluated by scratch test, the left side shows representative scratch closure images at 0 hours, 24 hours, 48 hours and 72 hours, and the right side is the quantitative analysis of scratch closure rate, the annotations indicate that a: p<0.05 compared with the Ctrl group, b: p<0.05 compared with the Gen group, c: p<0.05 compared with the Gen+LINC-OE group, d: p<0.05 compared with the Gen+miR-150-OE group, e: p<0.05 compared with the Gen+SGK1-OE group, f: p<0.05 compared with the Gen+LINC-KD group, g: p<0.05 compared with the Gen+miR-150-KD group, h: p<0.05 compared with the Gen+SGK1-KD group; B and D are the detection results of the invasion ability of HCT-116 cells under the same experimental conditions by Transwell invasion test, B shows representative images of invasion cells stained with crystal violet, and D is the quantitative analysis of the number of invasion cells; the data is represented as mean ± standard deviation, **p<0.01, ***p<0.001; Figure 6 Figure for the detection results of the regulation of genistein on EGFR / PI3K / AKT and MAPK signaling pathways in human colorectal cancer cells by LINC00355 / miR-150 / SGK1 axis in the examples, wherein A-G are the protein expression levels of EGFR, PI3K, AKT, SGK1, MAPK and ERK1 / 2 in HCT-116 cells under control conditions, genistein treatment or genistein combined with gene disturbance conditions detected by immunoblotting, with GAPDH as the internal control for loading, the right side and the lower chart show the quantitative analysis results of relative protein expression, H-J are the relative mRNA expression levels of LINC00355, miR-150 and SGK1 in HCT-116 cells under the above experimental conditions detected by qRT-PCR; the data is represented as mean ± standard deviation, *p<0.05, **p<0.01, ***p<0.001; Figure 7The image shows the detection results of genistein inhibiting tumor growth in a human colorectal cancer xenograft model via the LINC00355 / miR-150 / SGK1 axis in the examples. In this image, A represents representative images of xenografts in nude mice from each group, and B and C represent quantitative analyses of tumor volume and weight in different groups: control group (Ctrl), genistein treatment group (Gen), genistein combined with LINC00355 overexpression group (LINC-OE), and genistein combined with miR-150 overexpression group (miR-150). The groups included R-150-OE, genistein combined with SGK1 overexpression (SGK1-OE), genistein combined with LINC00355 knockdown (LINC-KD), genistein combined with miR-150 knockdown (miR-150-KD), and genistein combined with SGK1 knockdown (SGK1-KD); D represents the change in body weight of nude mice in each group during the experiment; EJ represents the Western Blot analysis of the expression levels of EGFR, PI3K, AKT, SGK1, and MAPK proteins in tumor tissues and the quantitative analysis of their relative protein expression. GAPDH was used as an internal control; KM represents the real-time quantitative PCR analysis of the relative mRNA expression levels of LINC00355, miR-150, and SGK1 in tumor tissues; data are expressed as mean ± standard deviation; *p<0.05, **p<0.01, ***p<0.001; Figure 8 The images show the immunohistochemical staining results of PI3K, AKT, SGK1, and MAPK in a human colorectal cancer xenograft model in this embodiment, along with quantitative analysis of their relative protein expression. In the images, A represents the immunohistochemical staining results, B represents the quantitative analysis results of PI3K relative protein expression, C represents the quantitative analysis results of AKT relative protein expression, D represents the quantitative analysis results of SGK1 relative protein expression, and E represents the quantitative analysis results of MAPK relative protein expression. *p<0.05, **p<0.01, ***p<0.001. Figure 9 This is a schematic diagram illustrating the molecular mechanism by which genistein acts on colorectal cancer cells. Detailed Implementation

[0037] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0038] Unless otherwise specified, in this invention, "about" means that the allowable error is within ±10%, and further, within ±5%.

[0039] When a numerical range is disclosed herein, the range is intended to include the minimum and maximum values, and every stated value between the minimum and maximum values, as well as each integer within that range. Further, when a range is provided, it is intended to include every subrange between and including the minimum value of the range and the maximum value of the range, as well as every integer within that range. Additionally, when multiple ranges are provided to describe a feature or characteristic, the ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein are to be understood to be inclusive of any and all subranges subsumed therein.

[0040] Unless otherwise indicated, conventional methods or those modifications known to those of ordinary skill in the art were used. Unless otherwise indicated, all reagents or instruments were conventional products available commercially.

[0041] Example 1: Use of genistein as an LINC00355 expression inhibitor and its application in the preparation of a drug for treating colorectal cancer This example provides the use of genistein as an LINC00355 expression inhibitor and its application in the preparation of a drug for treating colorectal cancer, and the specific verification steps are as follows: 1. In vitro cell experiment (1) Material preparation Cell lines: Human normal colon epithelial cell line NCM460 and colorectal cancer cell lines HCT-8, HCT-116, HT-29, LoVo, RKO, CACO2, SW620, and SW480 were purchased from the Chinese Academy of Sciences Cell Bank.

[0042] Reagents: Genistein (purity ≥98%) was purchased from Sigma-Aldrich (structure shown in FIG. A of Figure 1 ); RPMI1640 medium, fetal bovine serum (FBS), and trypsin were purchased from Gibco; MTT reagent, Annexin V-FITC / PI apoptosis detection kit, and cell cycle detection kit were purchased from Biyun Tian Biotechnology Co., Ltd.; Anti-BCL2, BAX, EGFR, PI3K, AKT, SGK1, MAPK, ERK1 / 2, and GAPDH antibodies were purchased from Proteintech Group; qRT-PCR kit was purchased from Baobioengineering (Dalian) Co., Ltd.; LINC00355, miR-150, and SGK1 overexpression and knockdown lentiviral vectors were constructed by Guangzhou Mai Zhi Biotechnology Co., Ltd.

[0043] (2) Experimental method Cell culture: Human normal colon epithelial cell line NCM460 and different colorectal cancer cell lines were cultured in RPMI1640 medium containing 10% FBS and 1% penicillin-streptomycin, and placed in a constant temperature incubator at 37℃, 5% CO2.

[0044] Drug treatment: Cells were seeded in 96-well plates, 6-well plates or Transwell chambers, and then treated with different concentrations (0, 10, 20, 40, 80, 160 μmol / L) of genistein for a specified time.

[0045] 1) Effect of genistein on cell viability MTT assay for cell viability: Cells (human normal colon epithelial cell line NCM460 and different colorectal cancer cell lines) were seeded in 96-well plates at a density of 5×10 3 μL MTT solution (5 mg / mL) was added to each well, incubated for 4 h, the supernatant was discarded, 150 μL dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and the absorbance value at 490 nm was determined using a microplate reader to calculate the cell viability.

[0046] The results are shown in Figure B of Figure 1 , from which it can be seen that genistein inhibited the proliferation of all tested colorectal cancer cell lines in a dose-dependent manner, and had little effect on the normal colon epithelial cell line NCM460, with the strongest inhibitory effect on HCT-116 cells, with an IC 50 of about 50 μmol / L.

[0047] 2) Gene interference experiment Gene interference experiment: HCT-116 cells were infected with lentiviral vectors (LINC00355, miR-150, SGK1 overexpression and knockdown lentiviral vectors, respectively) for 48 h, and then selected with puromycin (2 μg / mL) for 7 days to establish stable cell lines. The stable cell lines were verified by sequencing to ensure correct construction. HCT-116 stable cell lines were seeded in 6-well plates at a density of 500 cells / well, and then treated with genistein at a final concentration of 50 μmol / L for 2 h for subsequent experiments.

[0048] The grouping of each group in the gene interference experiment is shown as follows: a blank control group (Ctrl), a genistein group (Gen), a genistein + LINC00355 overexpression group (Gen+ LINC-OE), a genistein + miR-150 overexpression group (Gen+ miR-150-OE), a genistein + SGK1 overexpression group (Gen+ SGK1-OE), a genistein + LINC00355 knockdown group (Gen+ LINC-KD), a genistein + miR-150 knockdown group (Gen+ miR-150-KD), and a genistein + SGK1 knockdown group (Gen+ SGK1-KD).

[0049] The results are shown in C of FIG. 1, and it can be seen from the figure that genistein significantly reduces the viability of HCT-116 cells, overexpression of LINC00355, SGK1 or knockdown of miR-150 can weaken the inhibitory effect, and knockdown of LINC00355, SGK1 or overexpression of miR-150 can enhance the inhibitory effect. Figure 1

[0050] 3) Cloning experiment After HCT-116 cells were infected with lentiviral vectors (LINC00355, miR-150, SGK1 overexpression and knockdown lentiviral vectors, respectively) for 48 h, they were screened with puromycin (2 μg / mL) for 7 days to establish stable cell lines. After the stable cell line construction was verified by sequencing, the HCT-116 stable cell line was seeded in a 6-well plate at a density of 500 cells / well, then treated with genistein at a final concentration of 50 μmol / L for 2 h, and the medium was changed for 14 days. After cloning, the cells were fixed with 4% paraformaldehyde for 30 min and stained with 0.1% crystal violet for 15 min. The number of clones with cell numbers ≥50 was counted under a microscope.

[0051] The grouping is shown as follows: a blank control group (Ctrl), a genistein group (Gen), a genistein + LINC00355 overexpression group (Gen+ LINC-OE), a genistein + miR-150 overexpression group (Gen+ miR-150-OE), a genistein + SGK1 overexpression group (Gen+ SGK1-OE), a genistein + LINC00355 knockdown group (Gen+ LINC-KD), a genistein + miR-150 knockdown group (Gen+ miR-150-KD), and a genistein + SGK1 knockdown group (Gen+ SGK1-KD).

[0052] The results are shown in C of FIG. 1, and it can be seen from the figure that genistein significantly reduces the viability of HCT-116 cells, overexpression of LINC00355, SGK1 or knockdown of miR-150 can weaken the inhibitory effect, and knockdown of LINC00355, SGK1 or overexpression of miR-150 can enhance the inhibitory effect. Figure 1 ​As shown in the D-E diagram in FIG. 5, it can be seen from the diagram that genistein significantly reduced the number of HCT-116 cell clones, overexpression of LINC00355, SGK1 or knockdown of miR-150 can weaken the inhibitory effect, and knockdown of LINC00355, SGK1 or overexpression of miR-150 can enhance the inhibitory effect.

[0053] 4) Cell cycle and apoptosis detection experiment Flow cytometry was used to detect cell cycle and apoptosis. In the cell cycle detection, the HCT-116 cells (the treatment method of each group of cells was consistent with the above step 2) were collected, washed with PBS, fixed with 70% ethanol at -20°C overnight, then stained with propidium iodide (PI) solution for 30 min, and analyzed by flow cytometry. In the apoptosis detection, the HCT-116 cells (the treatment method of each group of cells was consistent with the above step 2) were collected, washed with PBS, stained with Annexin V-FITC and PI staining solution, and incubated in the dark for 15 min, and then detected by flow cytometry.

[0054] The results are shown in FIG. 6. Figures 2-4 As shown in the diagram, genistein can induce G0 / G1 phase arrest in HCT-116 cells, significantly increase the apoptosis rate, down-regulate the expression of anti-apoptotic gene BCL2, and up-regulate the expression of pro-apoptotic gene BAX. Overexpression of LINC00355 or SGK1 can reverse the above effects, overexpression of miR-150 can enhance the above effects, knockdown of LINC00355 or SGK1 can produce a synergistic effect with genistein, and knockdown of miR-150 can reverse the effect.

[0055] 5) Scratch test and cell invasion detection experiment The cell migration ability was detected by scratch test, and the steps were as follows: HCT-116 stable cell lines (the treatment method of each group of cells was consistent with the above step 2) were inoculated in a 6-well plate at a density of 1×10 6 μmol / L, and then replaced with serum-free medium for continuous culture. The scratch images were taken under a microscope at 0, 24, 48 and 72 h, and the scratch healing rate was calculated.

[0056] The groups are as follows: blank control group (Ctrl), genistein group (Gen), genistein + LINC00355 overexpression group (Gen + LINC-OE), genistein + miR-150 overexpression group (Gen + miR-150-OE), genistein + SGK1 overexpression group (Gen + SGK1-OE), genistein + LINC00355 knockdown group (Gen + LINC-KD), genistein + miR-150 knockdown group (Gen + miR-150-KD), and genistein + SGK1 knockdown group (Gen + SGK1-KD).

[0057] Transwell experiment to detect cell invasion ability: Transwell chamber (pore size 8 μm) was coated with Matrigel matrix glue in advance, 1×10 5 HCT-116 stable cell lines (the treatment method of each group of cells is consistent with the above step 2) gene interference experiment) were treated with genistein at a final concentration of 50 μmol / L for 2 h, suspended in serum-free medium and added to the upper chamber, and the lower chamber was added with medium containing 10% FBS. After incubation for 24 h, the cells on the surface of the upper chamber membrane were wiped off with a cotton swab, and the invasive cells on the surface of the lower chamber membrane were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Under a microscope, 5 fields of view were randomly selected to count the number of invasive cells.

[0058] The experimental results are shown in Figure 5 From the figure, it can be seen that genistein significantly reduces the scratch healing rate and the number of invasive cells of HCT-116 cells, overexpression of LINC00355 or SGK1 can reverse its inhibitory effect on cell migration and invasion, overexpression of miR-150 can enhance the inhibitory effect, and knockdown of LINC00355 or SGK1 can produce a synergistic effect with genistein, and knockdown of miR-150 can reverse the effect.

[0059] 6) qRT-PCR to detect the relative mRNA expression level of apoptosis-related genes in HCT-116 cells, and Western Blot to detect the expression level of apoptosis-related proteins in HCT-116 cells qRT-PCR to detect gene expression: total RNA was extracted from the collected HCT-116 cells (the treatment method of each group of cells is consistent with the above step 2) gene interference experiment) using TRIzol reagent, cDNA was synthesized using a reverse transcription kit, and qRT-PCR was performed using SYBR Green PCR Master Mix, with GAPDH as an internal reference gene. The relative expression of LINC00355, miR-150, and SGK1 genes was calculated using the 2 -ΔΔCt method.

[0060] Primer sequences used for qRT-PCR: Primer sequences for detecting LINC00355: Upstream primer: 5'-TCCAGTGTGTTTCCAGAGCC-3' (SEQ ID NO: 1); Downstream primer: 5'-GGCTGACCTCATCCCTGTAA-3' (SEQ ID NO: 2); Primer sequences for detecting miR-150: Upstream primer: 5'-GCGCGCTTCTCCCAACC-3' (SEQ ID NO: 3); Downstream primer: 5'-GTGCAGGGTCCGAGGT-3' (SEQ ID NO: 4); Primer sequences for detecting SGK1: Upstream primer: 5'-AGATCGCAAGGATGGCACTA-3' (SEQ ID NO: 5); Downstream primer: 5'-CAGGAACGATGGCTTGGTAG-3' (SEQ ID NO: 6); Primer sequences for detecting GAPDH: Upstream primer: 5'-GGAGCGAGATCCCTCCAAAAT-3' (SEQ ID NO: 7); Downstream primer: 5'-GGCTGTTGTCATACTTCTCATGG-3' (SEQ ID NO: 8).

[0061] Western blot detection of protein expression: Total protein was extracted from HCT-116 cells (consistent with the above step 2) gene interference experiment) using RIPA lysis buffer containing protease and phosphatase inhibitors, and the protein concentration was determined by BCA method. Equal amounts of protein were separated by SDS-PAGE electrophoresis and transferred to PVDF membranes. The membranes were blocked with 5% skim milk for 1 h, and primary antibodies were added for incubation at 4°C overnight. Then secondary antibodies were added for incubation at room temperature for 1 h. ECL chemiluminescence kit was used for development, and ImageJ software was used for quantitative analysis of protein band gray value. GAPDH was used as the internal reference protein to calculate the relative expression of protein.

[0062] Results are as follows Figure 6As shown in the A-G graphs, Western blot analysis showed that genistein could down-regulate the protein expression levels of EGFR, PI3K, AKT, SGK1, MAPK and ERK1 / 2 in HCT-116 cells. Gene regulation experiments showed that overexpression of LINC00355 or SGK1 could reverse the down-regulation of these proteins by genistein, while overexpression of miR-150 could enhance the down-regulation effect; knockdown of LINC00355 or SGK1 could produce a synergistic effect with genistein, further down-regulating the expression of these proteins, while knockdown of miR-150 could reverse the effect.

[0063] As shown in the H-J graphs, qRT-PCR results showed that genistein could down-regulate the mRNA expression levels of LINC00355 and SGK1, and up-regulate the mRNA expression level of miR-150; overexpression of LINC00355, miR-150 or SGK1 could reverse the regulation of these molecules by genistein, while knockdown of these molecules could enhance the regulation effect.

[0064] 7) Genistein regulates EGFR / PI3K / AKT and MAPK signaling pathways in human colorectal cancer cells through the LINC00355 / miR-150 / SGK1 axis The protein expression levels of EGFR, PI3K, AKT, SGK1, MAPK and ERK1 / 2 in HCT-116 cells (the treatment method of each group of cells is the same as step 2 above) under control conditions, genistein treatment or genistein combined with gene disturbance conditions were detected by immunoblotting, with GAPDH as the loading control, and the quantitative analysis results of relative protein expression were detected; the relative mRNA expression levels of LINC00355, miR-150 and SGK1 in HCT-116 cells under the above experimental conditions were detected by qRT-PCR.

[0065] The results are shown in Figure 6 As shown in the graphs, genistein can regulate EGFR / PI3K / AKT and MAPK signaling pathways in human colorectal cancer cells through the LINC00355 / miR-150 / SGK1 axis.

[0066] 2. In vivo animal experiments (1) Material preparation Experimental animals: 4-6 week old BALB / C nude mice (body weight 20-22 g), purchased from the Experimental Animal Center of Guangxi Medical University, all animal experiments were approved by the Animal Ethics Committee of the institution.

[0067] Cells and reagents: HCT-116 cells, genistein and other related reagents are the same as described above in 1, in vitro cell experiment.

[0068] Establishment of HCT-116 cell lines stably overexpressing and knocking down LINC00355, miR-150 and SGK1 using lentiviral vectors: Lentiviral vectors for overexpression of LINC00355, miR-150, SGK1 and knocking down their expression by RNA interference (RNAi) were purchased. Lentiviruses were packaged in 293T cells and then transduced into HCT-116 cells (MOI = 10 (LINC00355 overexpression), MOI = 8 (miR-150 overexpression), MOI = 15 (SGK1 overexpression), MOI = 15 (LINC00355 knockdown), MOI = 20 (miR-150 knockdown), MOI = 18 (SGK1 knockdown), respectively). After transduction, appropriate antibiotics (such as puromycin) were used for screening to establish stable polyclonal HCT-116 cell populations. The efficiency of overexpression and knockdown was verified by real-time quantitative PCR (qPCR) and / or Western blot analysis.

[0069] (2) Experimental methods Construction of animal models: HCT-116 cells stably expressing the target genes (5 x 10 6 animals (8 groups, 6 animals per group) were injected subcutaneously into the right dorsal of nude mice, and 7 days after inoculation, the nude mice in each group were injected with genistein.

[0070] The groups were as follows: control group (Ctrl injected with the same amount of normal saline solution), genistein group (Gen, 50 mg / kg, 200 μL, intraperitoneal injection once a day), genistein + LINC00355 overexpression group (Gen + LINC-OE, 50 mg / kg, 200 μL, intraperitoneal injection once a day), genistein + miR-150 overexpression group (Gen + miR-150-OE, 50 mg / kg, 200 μL, intraperitoneal injection once a day), genistein + SGK1 overexpression group (Gen + SGK1-OE, 50 mg / kg, 200 μL, intraperitoneal injection once a day), genistein + LINC00355 knockdown group (Gen + LINC-KD, 50 mg / kg, 200 μL, intraperitoneal injection once a day), genistein + miR-150 knockdown group (Gen + miR-150-KD, 50 mg / kg, 200 μL, intraperitoneal injection once a day), genistein + SGK1 knockdown group (Gen + SGK1-KD, 50 mg / kg, 200 μL, intraperitoneal injection once a day).

[0071] Experimental monitoring: every 2-3 days, the tumor volume was measured with vernier caliper, and the tumor volume was calculated according to the formula V = length x width2 / 2; the body weight of nude mice was recorded every 2 days.

[0072] Sample processing: 30 days after inoculation, the nude mice were anesthetized with 1% sodium pentobarbital and sacrificed, and the tumor tissue was stripped and weighed. Part of the tumor tissue was fixed with 10% formalin for IHC detection; the rest of the tissue was stored at -80°C for qRT-PCR and Western blot detection.

[0073] IHC (immunohistochemistry) detection: the tumor tissue fixed with 10% formalin was paraffin-embedded, cut into 4 μm thick sections, deparaffinated, hydrated, and then antigen-repaired with citrate buffer. The sections were incubated with 3% H2O2 for 10 min to block endogenous peroxidase activity, blocked with 5% BSA for 30 min, and then incubated with anti-PI3K, AKT, SGK1, and MAPK primary antibodies at 4°C overnight. After incubation with secondary antibodies, DAB color development was performed, and hematoxylin counterstaining was performed. The images were taken under a microscope at 100x and 200x magnification, respectively.

[0074] qRT-PCR and Western blot detection: the methods were consistent with the above 1, in vitro cell experiment.

[0075] (3) Experimental results The experimental results are shown in Figures 7-8 From Figure 7 , it can be seen that compared with the control group, the tumor volume and weight of the genistein group of nude mice were significantly reduced (as shown in Figure 7 A-C); there was no significant difference in the body weight of nude mice in each group, indicating that the dose of genistein used had no obvious toxic side effects (as shown in Figure 7 D); overexpression of LINC00355, SGK1 or knockdown of miR-150 can weaken the inhibitory effect of genistein on tumor growth, and knockdown of LINC00355, SGK1 or overexpression of miR-150 can enhance its inhibitory effect; genistein can down-regulate the mRNA and protein expression levels of LINC00355 and SGK1 in tumor tissue, up-regulate the mRNA expression level of miR-150, and down-regulate the protein expression levels and positive expression levels of EGFR, PI3K, AKT, SGK1 and MAPK; gene regulation can change the regulatory effect of genistein on the expression of these molecules (as shown in Figure 7 E-M).

[0076] The results of immunohistochemical staining of PI3K, AKT, SGK1 and MAPK in human colorectal cancer xenograft tumor models are shown in Figure 8As shown in the figure, it can be seen from the figure that genistein regulates key signal molecules in tumor tissues through the LINC00355 / miR-150 / SGK1 axis.

[0077] The schematic diagram of the molecular mechanism of the effect of genistein on colorectal cancer cells is shown in FIG. 3. Figure 9 As shown in the figure, it can be seen from the figure that genistein inhibits LINC00355, LINC00355 promotes the expression of serum and glucocorticoid-induced kinase 1 and inhibits miR-150, and miR-150 can inhibit SGK1. At the same time, the epidermal growth factor receptor is activated, thereby activating the extracellular signal-regulated kinase / mitogen-activated protein kinase pathway and the phosphatidylinositol 3-kinase / protein kinase B pathway. Ultimately, these signal cascades cause regulatory changes in the nucleus, thereby inhibiting the proliferation of colorectal cancer cells.

[0078] The above describes the embodiments of the present application in detail, but the present application is not limited to the above-described embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. Application of genistein in the preparation of LINC00355 expression inhibitors.

2. The application according to claim 1, characterized in that, The LINC00355 expression inhibitor has at least one of the following functions: (1) Promotes the expression of glucocorticoid-induced kinase 1; (2) Inhibit the expression of miR-150; (3) Promotes SGK1 expression; (4) Adjust the miR-150 / SGK1 axis; (4) Regulation of extracellular signal-regulated kinase / mitogen-activated protein kinase pathway; (5) Regulates the phosphatidylinositol 3-kinase / protein kinase B pathway; (6) Anti-colorectal cancer.

3. The application according to claim 2, characterized in that, The regulation of the extracellular signal-regulated kinase / mitogen-activated protein kinase pathway is the activation of the extracellular signal-regulated kinase / mitogen-activated protein kinase pathway.

4. The application according to claim 2, characterized in that, The regulation of the phosphatidylinositol 3-kinase / protein kinase B pathway is the activation of the phosphatidylinositol 3-kinase / protein kinase B pathway.

5. The application according to claim 2, characterized in that, The anti-colorectal cancer treatment includes inhibiting the proliferation of colorectal cancer cells, arresting the G0 / G1 phase of the colorectal cancer cell cycle, inducing apoptosis of colorectal cancer cells, and / or inhibiting the migration and invasion abilities of colorectal cancer cells.

6. Application of genistein in the preparation of products for treating colorectal cancer.

7. The application according to claim 6, characterized in that, The treatment of colorectal cancer includes inhibiting the proliferation of colorectal cancer cells, arresting the G0 / G1 phase of the colorectal cancer cell cycle, inducing apoptosis of colorectal cancer cells, and / or inhibiting the migration and invasion abilities of colorectal cancer cells.

8. The application according to claim 6, characterized in that, The product is at least one of pharmaceuticals, health products, and functional foods; Preferably, the product is a pharmaceutical product, and the pharmaceutical product further includes pharmaceutically acceptable excipients.

9. The application according to claim 8, characterized in that, The dosage form of the drug is selected from tablets, capsules, granules, powders, liquid preparations, ointments, injections, or sprays.

10. The application according to claim 6, characterized in that, The mass fraction of the genistein in the product is 0.01% to 100%.

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