Application of miR-11085-5p, an inducer of ferroptosis in hepatocellular carcinoma cells, in the preparation of drugs for the treatment of hepatocellular carcinoma

Abstract

The present invention discloses the application of liver cancer cell ferroptosis inducer miR‑11085‑5p in the preparation of liver cancer treatment drugs, and innovative applications in the field of liver cancer treatment, specifically to the application of liver cancer cell ferroptosis inducer miR‑11085‑5p in the preparation of liver cancer treatment drugs. The present invention adopts lentiviral transfection technology to synthesize miR‑11085‑5p in vitro, and introduce it into liver cancer cells, and effectively induce liver cancer cells to undergo ferroptosis by precisely regulating the key signaling pathway of intracellular AMPKα / Nrf2, significantly inhibiting its proliferation activity. Therefore, for the disadvantages of low efficiency, large side effects and easy recurrence and metastasis of existing treatment methods for liver cancer, the miR‑11085‑5p provided by the present invention may become a biological preparation for the treatment of liver cancer, with potential drug development value, and provide a new solution for the treatment of liver cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to the application of miR-11085-5p, an inducer of ferroptosis of liver cancer cells, in the preparation of drugs for treating liver cancer, and specifically relates to the application of miR-11085-5p as an inducer of ferroptosis of liver cancer cells to induce ferroptosis of liver cancer cells through the AMPKα / Nrf2 signaling pathway in the preparation of drugs for treating liver cancer. Background Art

[0002] The incidence of liver cancer ranks fourth among malignant tumors and the mortality ranks second. Liver cancer accounts for about 13% of all cancer deaths. At present, surgery, interventional therapy, targeted therapy, radiotherapy, chemotherapy and immunotherapy are the main treatments for liver cancer. However, due to low efficiency, serious side effects and easy recurrence and metastasis, the clinical treatment of liver cancer still faces huge challenges.

[0003] MicroRNA (miRNA) is a type of non-coding small molecule RNA that can inhibit the expression of target genes or promote their degradation by binding to the 3' untranslated region of target genes, thereby widely participating in biological processes such as cell proliferation, differentiation, apoptosis and migration. In tumor research, miRNA has become one of the emerging directions of tumor treatment due to its important role in the occurrence, development and metastasis of cancer. By regulating key genes related to tumors, miRNA can effectively inhibit the growth and metastasis of tumor cells, thereby exerting an anti-tumor effect.

[0004] Therefore, discovering miRNAs with potential medical effects is of great significance for the research and development of nucleic acid drugs. Summary of the invention

[0005] In response to the clinical problems of poor efficacy and low overall survival rate of current liver cancer treatment options, the present invention innovatively proposes a new drug application, and provides the use of miR-11085-5p, an inducer of ferroptosis of liver cancer cells, in the preparation of drugs for treating liver cancer. Specifically, miR-11085-5p is used as an inducer of ferroptosis of liver cancer cells to induce ferroptosis of liver cancer cells via the AMPKα / Nrf2 signaling pathway, and the use of miR-11085-5p in the preparation of drugs for treating liver cancer.

[0006] The present invention provides a preparation method of miR-11085-5p and application of the miR-11085-5p in the preparation of anti-liver cancer drugs.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A miRNA is named as miR-11085-5p according to the miRNA naming principle, wherein miR represents miRNA, the nucleotide sequence of the miR-11085-5p is 5'-UCACCGGUGACGGAGCAGGGA-3', and the miR-11085-5p is used as an inducer of ferroptosis of liver cancer cells.

[0009] Furthermore, the miR-11085-5p can inhibit the proliferation of liver cancer cells HepG2 by inducing ferroptosis;

[0010] The miR-11085-5p can regulate the AMPKα / Nrf2 signaling pathway of liver cancer. Specifically, miR-11085-5p acts as an inducer of liver cancer cell ferroptosis and induces liver cancer cell ferroptosis through the AMPKα / Nrf2 signaling pathway.

[0011] The present invention also relates to the use of miR-11085-5p as an inducer of liver cancer cell ferroptosis in the preparation of a drug for treating liver cancer.

[0012] The preparation method of the miR-11085-5p comprises the following steps:

[0013] 1) Prepare ultraviolet light irradiation clean bench, related equipment and necessary reagents;

[0014] 2) Take cells in logarithmic growth phase. When the cells cover 80-90% of the bottom of the culture flask, discard the original culture medium, wash twice with PBS, discard the liquid, and add an appropriate amount of trypsin; observe the cells under a microscope. When the cell morphology becomes round and bright, aspirate the trypsin, add 3 times the volume of trypsin to complete culture medium, and gently blow the bottom of the culture flask to make the cells completely detach from the wall and become suspended; wipe the cell counting plate and cover glass with a 75% alcohol cotton ball;

[0015] 3) Mix the cell suspension evenly, use a pipette to absorb 10ul of the diluted cell suspension, and slowly drip it onto the cover glass along the edge to allow the suspension to freely penetrate and cover the counting plate;

[0016] Count the number of cells in the four grids of the counting plate under a microscope and calculate the cell concentration:

[0017] Cell concentration (cells / ml) = (sum of the number of cells in the four grids / 4) × 10 4 Pieces / ml;

[0018] 4) Dilute the cells to 4-5×10 according to the cell counting results. 4 / ml, gently blow and mix evenly, inoculate in a 6-well plate, add 2ml of suspension to each well, and place in an incubator for culture; culture in the incubator for 24 hours, observe under a microscope, and make the cells cover 40% of the bottom of the bottle;

[0019] 5) Take out the lentivirus from the -80°C refrigerator, place it on ice and slowly thaw it, add the virus to complete culture medium with MOI=20 as the standard, and make up to 2 ml;

[0020] Discard the culture medium in the 6-well plate, wash twice with PBS, add the prepared lentiviral infection reagent, mix well, and add to the incubator for continued culture;

[0021] 6) After 14 hours, discard the culture medium containing the lentiviral infection reagent, wash twice with PBS, add 2 ml of fresh culture medium, and continue culturing in an incubator;

[0022] After culturing for 72 hours, the fluorescence abundance of transfected cells was observed under a fluorescence microscope;

[0023] After 7 days of continuous culture with a medium containing 1ug / ml of puromycin, stably transfected cells were obtained, and the puromycin concentration was adjusted to 0.5ug / ml and the cells were continued to be cultured;

[0024] 7) RNAiso Plus was used to isolate total RNA from cells by performing real-time quantitative polymerase chain reaction (qRT-PCR), and then RNA concentration and purity were measured;

[0025] 8) Subsequently, the transcribed RNA was synthesized into cDNA as a template for qRT-PCR analysis; 2- ΔΔ Ct Methods The relative expression level of mRNA was determined.

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] The present invention discovered a new small molecule RNA that inhibits liver cancer ferroptosis, and prepared miR-11085-5p by lentiviral transfection. The results of the study showed that miR-11085-5p can promote ferroptosis and inhibit the proliferation of liver cancer cells by regulating the AMPKα-related signaling pathway of liver cancer. Therefore, in view of the shortcomings of existing liver cancer treatment methods such as low efficacy, large side effects, and easy recurrence and metastasis, the miR-11085-5p provided by the present invention may become a biological agent for the treatment of liver cancer, has potential drug development value, and provides a new solution for the treatment of liver cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used to explain the present invention together with the embodiments of the present invention and do not constitute a limitation of the present invention.

[0029] Figure 1is a diagram of the anti-liver cancer proliferation effect of miR-11085-5p verified by cell cloning experiments in the embodiments of the present invention;

[0030] Figure 2 is a diagram of the embodiment of the present invention verifying that miR-11085-5p promotes lipid peroxidation through ROS level measurement;

[0031] Figure 3 is another figure for verifying that miR-11085-5p promotes lipid peroxidation by measuring ROS levels in an embodiment of the present invention;

[0032] Figure 4 is a diagram showing changes in mitochondrial morphology under different treatments observed by transmission electron microscopy in an embodiment of the present invention;

[0033] Figure 5 This is a diagram of the embodiment of the present invention, in which the anti-liver cancer effect of miR-11085-5p is verified to be related to ferroptosis by measuring malondialdehyde (MDA) and glutathione (GSH) levels;

[0034] Figure 6 is a graph showing Western blot detection and relative protein expression of AMPKα in an embodiment of the present invention;

[0035] Figure 7 It is a graph of the relative protein expression of p-AMPKα and Keap-1 detected by Western blot in an embodiment of the present invention;

[0036] Figure 8 This is a graph showing the relative protein expression of Nrf2 and GPX4 detected by Western blot in an embodiment of the present invention. DETAILED DESCRIPTION

[0037] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Embodiment 1:

[0039] The preparation method of miR-11085-5p comprises the following steps:

[0040] Ultraviolet light irradiates the clean bench, related equipment and necessary reagents;

[0041] Take cells in logarithmic growth period, when the cells cover 80-90% of the bottom of the culture bottle, discard the original culture medium, wash twice with PBS, discard the liquid, and add an appropriate amount of trypsin; observe the cells under a microscope, when the cell morphology becomes round and bright, suck out the trypsin, add 3 times the volume of trypsin complete culture medium, gently blow the bottom of the culture bottle to make the cells completely detach from the wall and become suspended; wipe the cell counting plate and cover glass with 75% alcohol cotton balls; mix the cell suspension evenly, use a pipette to absorb 10ul of the diluted cell suspension, and slowly drip it onto the cover glass along the edge to allow the suspension to freely penetrate and cover the counting plate; count the number of cells in the four grids of the counting plate under a microscope, and calculate the cell concentration: cell concentration (cells / ml) = (the sum of the number of cells in the four grids / 4) × 10 4 Cells were diluted to 4-5×10 cells / ml according to the cell counting results. 4 / ml, gently blow and mix evenly, inoculate in a 6-well plate, add 2ml of suspension to each well, and place in an incubator for culture; culture in the incubator for 24 hours, observe under a microscope, and make the cells cover about 40% of the bottom of the bottle; take the lentivirus out of the -80℃ refrigerator, place it on ice to slowly melt, add the virus to the complete culture medium with MOI=20 as the standard, and make the volume to 2ml; discard the culture medium in the 6-well plate, wash it twice with PBS, add the prepared lentivirus infection reagent, mix it evenly, and add it to the incubator for continued culture; after 14 hours, discard the culture medium containing the lentivirus infection reagent, wash it twice with PBS and add 2ml of fresh culture medium, and place it in the incubator for continued culture; after continuing to culture for 72 hours, observe the fluorescence abundance of the transfected cells under a fluorescence microscope; after continuous culture for 7 days with a culture medium containing 1ug / ml puromycin, obtain stably transfected cells, adjust the puromycin concentration to 0.5ug / ml and continue to culture the cells; by performing real-time quantitative polymerase chain reaction (qRT-PCR), RNAiso Plus is used to isolate total RNA from cells and then measure RNA concentration and purity; subsequently, the transcribed RNA is synthesized into cDNA as a template for qRT-PCR analysis; using 2- ΔΔ Ct Methods The relative expression level of mRNA was determined.

[0042] Table 1 Primer sequences for constructing miR-11085-5p

[0043]

[0044] The results showed that the sequence containing miR-11085-5p was successfully integrated into HepG2 cells by lentiviral transfection technology, and then the miRNA was amplified by qRT-PCR technology, and the nucleotide sequence of miR-11085-5p was 5'-UCACCGGUGACGGAGCAGGGA-3'.

[0045] The single-chain miR11085-5p with 3′-OH terminal methylation was prepared into a 25 μM solution with DMEM culture medium. 2 ml of the prepared solution was added to a 6-well plate of adherent liver cancer cells, followed by 2500 ng of Lipofectamine 2000 reagent. After mixing, the plate was allowed to stand at room temperature for 15 minutes, and the 6-well plate was placed in an incubator. DMEM culture medium was replaced after 5 hours, and subsequent experiments were conducted after continued culture for 24 hours.

[0046] Embodiment 2:

[0047] miR-11085-5 inhibits the proliferation of HepG2 liver cancer cells

[0048] In the clean bench, use a sterile pipette to remove the original culture medium in the cell culture bottle, add 1ml of sterilized PBS solution, gently blow and wash the cells repeatedly to remove senescent cells, and aspirate the PBS solution. Repeat this step once. Add 1ml of trypsin cell digestion solution, observe under the microscope after 1min, when the cell morphology becomes round and can flow with the digestion solution, immediately remove the cell digestion solution, add 2ml of culture solution to terminate the digestion, blow the cells at the bottom of the bottle until they are suspended and count them, and then dilute them appropriately. Under the sterile operating table, add an appropriate amount of complete culture medium to each well of the 6-well culture plate, and then add the diluted cell suspension. Use a pipette to mix gently to ensure that the cells are evenly distributed, and then place the culture plate in a 5% CO2 incubator for group intervention culture. Within 12 days, observe the formation of cell clones under a microscope. When most cell colonies are >50 cells, fix them with 4% paraformaldehyde and stain them with 1% crystal violet. Use a camera to take pictures and record the data.

[0049] The results are as follows Figure 1 As shown, miR-11085-5 can significantly inhibit the proliferation of liver cancer cells HepG2.

[0050] Embodiment 3:

[0051] Detection of ROS, MDA, and GSH levels and observation of mitochondrial morphology under transmission electron microscopy verified that miR-11085-5 promoted ferroptosis in liver cancer cells HepG2

[0052] ROA level determination: In the ultra-clean operating table, use a sterile pipette to remove the original culture medium in the cell culture bottle, add 1ml of sterilized PBS solution, gently blow and wash the cells repeatedly to remove senescent cells, and aspirate the PBS solution. Repeat this step once. Add 1ml of trypsin cell digestion solution, observe under the microscope after 1min, when the cell morphology becomes round and can flow with the digestion solution, immediately remove the cell digestion solution, add 2ml of culture solution to terminate the digestion, blow the cells at the bottom of the bottle to a suspended state and count them, and then dilute them appropriately. Under the sterile operating table, add an appropriate amount of complete culture medium to each well of the 6-well plate, and then add the diluted cell suspension. Use a pipette to mix gently to ensure that the cells are evenly distributed, and then place the culture plate in a 5% CO2 incubator for culture. After the cells adhere to the wall, they are grouped and treated with drugs for intervention culture. Take out the DCFH-DA freeze-dried powder stored at low temperature, return to room temperature, and centrifuge at low speed to make the powder fall to the bottom of the bottle. Then add an appropriate amount of anhydrous DMSO solvent to prepare a 1-10mM storage solution. Dilute the storage solution with PBS to a working concentration of 5μM. Aspirate the culture medium and wash the cells twice with preheated PBS. Add 1 ml of DCFH-DA working solution to each well of the 6-well plate, and then place the cells in a 37°C cell culture incubator in the dark for 30 minutes. After incubation, wash the cells 3 times with serum-free culture medium to fully remove the DCFH-DA that has not entered the cells. Add an appropriate amount of miR-11085-5 diluted with PBS and incubate in a 37°C cell culture incubator in the dark. After induction, use a fluorescence microscope to take pictures. Resuspend the treated cells with an appropriate amount of cold PBS, transfer them to a flow tube, and detect them using a flow cytometer. Set the instrument excitation wavelength to 488 nm and the emission wavelength to 525 nm. Place the flow tube in a flow cytometer for detection and record the fluorescence signal intensity. Use the flow cytometry data analysis software FlowJ to analyze the test results, and analyze the level of intracellular reactive oxygen species (ROS) and its changing trend based on the changes in fluorescence intensity.

[0053] TEM observation of mitochondrial morphology: HepG2 cells in the control group and miR-11085-5 treatment group were digested with trypsin and collected by centrifugation, then washed with pre-cooled PBS buffer, and then added with pre-cooled 2.5% glutaraldehyde fixative, fixed at 4°C for more than 12 hours; then the fixed samples were placed in 30%, 50%, 70%, 80%, 90%, and 100% ethanol gradients in turn, and soaked for 10-15 minutes at each concentration. The dehydrated samples were placed in embedding agents such as epoxy resin and mixed thoroughly, placed in a mold, and the embedding agent was polymerized and solidified at a specific temperature. After that, the embedded samples were cut into 50-100nm slices using an ultrathin slicer to ensure that the slice thickness was uniform and complete. After the slices were completed, they were stained, first in uranyl acetate stain for 15-30 minutes, rinsed with distilled water, and then in lead citrate stain for 10-20 minutes, and finally rinsed with distilled water and dried. Finally, the stained thin slice is placed on the sample stage of a transmission electron microscope. Under a vacuum environment, the electron beam penetrates the thin slice to produce scattering to form an image, which is then magnified and focused and displayed on the imaging device. By adjusting the electron microscope parameters such as acceleration voltage, spot size, focal length, etc., the morphology, size, number, distribution and internal structure of the mitochondria can be clearly presented.

[0054] ELISA method for MDA level determination: After collecting cells from different groups, centrifuge at 1,000g for 20 minutes, take the supernatant and take out the required strips from the aluminum foil bag after equilibration at room temperature for 20 minutes, seal the remaining strips with a ziplock bag and return to 4°C, set up standard wells and sample wells, add 50μL of different concentrations of standard wells. Add 50μL of the sample to be tested to the sample well, and do not add to the blank well. Except for the blank well, add 100μL of horseradish peroxidase (HRP) labeled detection antibody to each well of the standard well and sample well. Seal the reaction wells with a sealing film and incubate in a 37°C water bath or incubator for 60 minutes. Discard the liquid, pat dry on absorbent paper, fill each well with washing solution (350μL), let stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat the washing 5 times. Add 50μL of substrate A and B to each well and incubate at 37°C in the dark for 15 minutes. Add 50 μL of stop solution to each well and measure the OD value of each well at a wavelength of 450 nm within 15 minutes. Use the OD value of the measured standard as the horizontal axis and the concentration value of the standard as the vertical axis to draw a standard curve on coordinate paper or using relevant software, and obtain a linear regression equation. Substitute the OD value of the sample into the equation to calculate the MDA level.

[0055] ELISA method for GSH level determination: GSH standard with known concentration is diluted into a series of concentrations with phosphate buffer or other buffers. At the same time, prepare the diluted enzyme-labeled antibody, the prepared substrate solution, the stop solution and the washing solution, and preheat the microplate reader, calibrate the pipette, and prepare the microplate. After the adherent cells are digested with trypsin, they are collected by centrifugation, and then GSH is extracted with the extraction reagent. Then add the sample, add 100μL of the standard solution to the microplate standard wells from low to high concentrations and set up 3 sets of duplicate wells, add 100μL of the diluted sample solution to the sample wells and set up 3 sets of duplicate wells, and add 100μL of PBS to the blank control wells. After adding the sample, seal the plate and incubate at 37℃ for 1-2 hours, then pour out the liquid and wash with PBST 3-5 times. Add 100μL of the diluted enzyme-labeled secondary antibody and incubate at 37℃ for 30-60 minutes and wash again. Then add 100μL of substrate solution and color at 37℃ in the dark for 10-30 minutes, and add 50μL of stop solution to terminate the reaction when appropriate. Finally, the microplate was placed on a microplate reader to detect the absorbance at 450 nm. A standard curve was made based on the concentration and absorbance of the standard, and the GSH level in the sample was calculated using a linear regression equation.

[0056] Statistical analysis: All data are expressed as mean ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test using GraphPad Prism 8 software (GraphPad software, USA). Statistical significance was established at p < 0.05.

[0057] The results show that:

[0058] pass Figure 2-Figure 5 It can be seen that the intracellular ROS level can be increased by miR-11085-5 treatment. Transmission electron microscopy analysis further showed that HepG2 cells treated with miR-11085-5p showed obvious mitochondrial damage, characterized by mitochondrial cristae changes and mitochondrial swelling. In addition, ELISA method quantified intracellular GSH and MDA levels, and it was found that GSH levels were reduced and MDA levels were increased.

[0059] Taken together, these results indicate that miR-11085-5p can promote oxidative stress-induced ferroptosis in HepG2 cells.

[0060] Embodiment 4:

[0061] miR-11085-5p can induce ferroptosis of liver cancer cells by regulating AMPKα / Nrf2 signaling pathway

[0062] Molecular biology experiments: ① Cell cloning experiment: the steps are the same as above; ② ROS detection: the steps are the same as above;

[0063] ③ Transmission electron microscopy observation of mitochondria: the steps are the same as above; ④ GSH and MDA detection: the steps are the same as above.

[0064] Western blot protein expression determination: The cells or tissues were then dissolved in chilled RIPA buffer (Beyotime, P0013B) containing 2% protease and phosphatase inhibitor cocktail (Beyotime, P1048). The protein concentration in the lysate was quantified using the BCA assay. The homogenate containing 20 μg of total protein was separated by SDS-PAGE on a 10-15% gradient gel and transferred to a polyvinylidene fluoride membrane. Blocked with 5% skim milk for 1 hour at room temperature and then washed three times with TBS containing 0.1% Tween. The membrane was incubated with specific primary antibodies overnight at 4°C with agitation and then incubated with enzyme-labeled secondary antibodies for 1 hour at room temperature. Protein bands were visualized using enhanced chemiluminescence. Band intensities were semi-quantified using ImageJ software version 1.8.0.

[0065] Statistical analysis: All data are expressed as mean ± SD and analyzed by one-way analysis of variance (ANOVA) followed by Tukey's post hoc multiple comparison test using GraphPad Prism 8 software (GraphPad software, USA). Statistical significance was established at p < 0.05.

[0066] The results show:

[0067] pass Figure 6-Figure 8It can be seen that compared with the control group, the number of cell clones in the AKPKα overexpression group (OE-AMPKα group) increased, the mitochondrial morphology was healthier, the mitochondria may be slightly elongated or increased, the function was relatively complete, the ROS level decreased, the GSH decreased, the MDA increased, and the expression of proteins AMPK, p-AMPK, GPX4, Nrf2, and keap-1 increased; the number of cell clones in the miR-11085-5p group decreased, the ROS level increased, the GSH increased, the MDA decreased, and the expression of proteins AMPKα, p-AMPKα, GPX4, Nrf2, and keap-1 decreased; the number of cell clones in the miR-11085-5p+OE-AMPKα group increased, the ROS level increased, the GSH decreased, the MDA, and the expression of proteins AMPKα, p-AMPKα, GPX4, Nrf2, and keap-1 increased. In this experiment, we set up a control group, an OE-AMPKα group, a miR-11085-5p group, and a miR-11085-5p+A OE-AMPKα group to explore the mechanism of miR-11085-5p inducing ferroptosis through the AMPKα / Nrf2 signaling pathway. The experimental results showed that compared with the control group, the OE-AMPKα group increased the number of cell clones, decreased the level of ROS, decreased GSH, increased MDA, and increased the expression of antioxidant-related proteins such as AMPKα, p-AMPKα, GPX4, Nrf2, and Keap-1, indicating that overexpression of AMPKα contributes to cell proliferation and survival, enhances the antioxidant capacity of cells, and slows down the process of ferroptosis. AMPKα (5'AMP-activated protein kinase), as a cellular energy sensor, plays an important role in responding to oxidative stress and maintaining cell homeostasis by regulating cellular energy metabolism. Its activated form, p-AMPKα, can inhibit excessive metabolic activity of cells, reduce the generation of ROS, and reduce oxidative damage by activating antioxidant enzymes such as GPX4, thereby inhibiting the occurrence of ferroptosis.

[0068] In the miR-11085-5p group, the number of cell clones decreased, mitochondria showed swelling, fragmentation, irregular morphology, black particles, increased ROS level, increased GSH, decreased MDA, and significantly decreased AMPKα, p-AMPKα, GPX4, Nrf2, and Keap-1 expression, indicating that miR-11085-5p aggravated oxidative stress and promoted ferroptosis by downregulating the AMPKα pathway and inhibiting the activity of antioxidant factors such as Nrf2. The effect of miR-11085-5p may inhibit the expression and activity of GPX4 by inhibiting the activation of AMPK and reducing the level of p-AMPKα. GPX4 is a key inhibitor of ferroptosis that can clear lipid peroxides in cells, and the inhibitory effect of miR-11085-5p may destroy this protective mechanism, making cells more susceptible to ferroptosis. At the same time, miR-11085-5p may also disable the antioxidant system by reducing the activity of Nrf2 (Nuclear factor erythroid 2-related factor 2). Nrf2 is a transcription factor that normally binds to Keap-1 to form a stable complex, but under oxidative stress, Nrf2 is released from Keap-1 and translocated to the nucleus to activate the expression of antioxidant genes and enhance the antioxidant capacity of cells. miR-11085-5p may inhibit the dissociation of Nrf2 from Keap-1 and reduce the transcriptional activity of Nrf2, thereby weakening the antioxidant response of cells, further increasing ROS accumulation and lipid peroxidation, and promoting ferroptosis.

[0069] In the miR-11085-5p+OE-AMPKα group, the number of cell clones increased compared with the miR-11085-5p group, and the mitochondrial morphology was improved compared with the miR-11085-5p group, showing mild swelling or elongation, less fragmentation, and overall integrity; AMPK overexpression significantly restored the expression of AMPKα, p-AMPKα, GPX4, Nrf2, Keap-1 and other proteins, and partially alleviated the oxidative damage induced by miR-11085-5p. Although the ROS level was still elevated, it was relatively low compared with the miR-11085-5p group, the GSH level was lower than the control group and miR-11085-5p group but higher than the OE-AMPKα group, and the MDA level was higher than the control group and miR-11085-5p group but lower than the OE-AMPKα group. OE-AMPKα activated the antioxidant pathway by enhancing the activity of p-AMPKα, increased the expression of GPX4 and Nrf2, thereby enhancing the antioxidant response of cells and partially inhibiting the oxidative stress and ferroptosis caused by miR-11085-5p. This indicates that OE-AMPKα can reduce the degree of ferroptosis by restoring the AMPKα / Nrf2 signaling pathway, inhibiting the accumulation of ROS, and slowing down lipid peroxidation.

[0070] In summary:

[0071] miR-11085-5p induces ferroptosis by downregulating the AMPKα / Nrf2 signaling pathway and inhibiting the expression of antioxidant factors such as GPX4 and Nrf2, leading to ROS accumulation and lipid peroxidation. The AMPKα / Nrf2 signaling pathway plays a key regulatory role in the ferroptosis induced by miR-11085-5p, and restoring the AMPK pathway can partially alleviate the occurrence of ferroptosis. AMPKα activation has broad application prospects by enhancing antioxidant responses and regulating lipid metabolism. It is not only expected to become a new anti-tumor treatment strategy. With the progress of delivery technology and clinical research, miR-11085-5p may bring new breakthroughs in the treatment of various diseases in the future.

[0072] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. Application of miR-11085-5p, an inducer of ferroptosis in liver cancer cells, in the preparation of a drug for treating liver cancer, characterized in that: The nucleotide sequence of miR-11085-5p is: 5'-UCACCGGUGACGGAGCAGGGA-3'; The miR-11085-5p inhibits the proliferation of liver cancer cells HepG2 by inducing ferroptosis; The miR-11085-5p can regulate the AMPKα / Nrf2 signaling pathway in liver cancer.

2. The use of the liver cancer cell ferroptosis inducer miR-11085-5p according to claim 1 in the preparation of a drug for treating liver cancer, characterized in that: The miR-11085-5p can regulate the AMPKα / Nrf2 signaling pathway of liver cancer, and miR-11085-5p induces ferroptosis of liver cancer cells through the AMPKα / Nrf2 signaling pathway.

3. The method for preparing the liver cancer cell ferroptosis inducer miR-11085-5p according to claim 1 or 2, characterized in that: The following steps are involved: 1) Prepare ultraviolet light irradiation clean bench, related equipment and necessary reagents; 2) Take cells in logarithmic growth phase. When the cells cover 80-90% of the bottom of the culture flask, discard the original culture medium, wash twice with PBS, discard the liquid, and add an appropriate amount of trypsin; observe the cells under a microscope. When the cell morphology becomes round and bright, aspirate the trypsin, add 3 times the volume of trypsin to complete culture medium, and gently blow the bottom of the culture flask to make the cells completely detach from the wall and become suspended; wipe the cell counting plate and cover glass with a 75% alcohol cotton ball; 3) Mix the cell suspension evenly, use a pipette to absorb 10ul of the diluted cell suspension, and slowly drip it onto the cover glass along the edge to allow the suspension to freely penetrate and cover the counting plate; Count the number of cells in the four grids of the counting plate under a microscope and calculate the cell concentration: Cell concentration (cells / ml) = (sum of the number of cells in the four grids / 4) × 10 4 Pieces / ml; 4) Dilute the cells to 4-5×10 according to the cell counting results. 4 / ml, gently blow and mix evenly, inoculate in a 6-well plate, add 2ml of suspension to each well, and place in an incubator for culture; culture in the incubator for 24 hours, observe under a microscope, and make the cells cover 40% of the bottom of the bottle; 5) Take out the lentivirus from the -80°C refrigerator, place it on ice and slowly thaw it, add the virus to complete culture medium with MOI=20 as the standard, and make up to 2 ml; Discard the culture medium in the 6-well plate, wash twice with PBS, add the prepared lentiviral infection reagent, mix well, and add to the incubator for continued culture; 6) After 14 hours, discard the culture medium containing the lentiviral infection reagent, wash twice with PBS, add 2 ml of fresh culture medium, and continue culturing in an incubator; After culturing for 72 hours, the fluorescence abundance of transfected cells was observed under a fluorescence microscope; After 7 days of continuous culture with a medium containing 1ug / ml of puromycin, stably transfected cells were obtained, and the puromycin concentration was adjusted to 0.5ug / ml and the cells were continued to be cultured; 7) RNAiso Plus was used to isolate total RNA from cells by performing real-time quantitative polymerase chain reaction (qRT-PCR), and then RNA concentration and purity were measured; 8) Subsequently, the transcribed RNA was synthesized into cDNA as a template for qRT-PCR analysis; 2- ΔΔ Ct Methods The relative expression level of mRNA was determined.

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