Construction method of in-vitro liver cancer liver invasion and metastasis experimental model

Abstract

The invention discloses a construction method of an in-vitro liver cancer liver invasion and metastasis experimental model, and relates to the technical field of model construction, and the construction method comprises the following steps: culturing liver cancer cells and hepatic stellate cells, carrying out fluorescence labeling, generating a space coordinate set by using the fluorescence labeled liver cancer cells and hepatic stellate cells, and calculating the in-vitro liver cancer liver invasion and metastasis experimental model. The method comprises the following steps of: printing liver cancer cells and hepatic stellate cells according to a space coordinate set to generate a three-dimensional cell-matrix structure, pouring photoresponse hydrogel into the three-dimensional cell-matrix structure, and inducing HOTAIR long non-coding RNA concentration by using a transforming growth factor to obtain the three-dimensional cell-matrix structure integrated with the photoresponse hydrogel. The space coordinate set is generated through the Markov model, accurate simulation of radial distribution of the highly invasive liver cancer cell core area and the hepatic stellate cells is achieved, and an accurate position template is provided for printing of a three-dimensional cell-matrix structure.

Description

Technical Field

[0001] This invention relates to the field of model construction technology, and in particular to a method for constructing an in vitro experimental model of liver cancer invasion and metastasis. Background Technology

[0002] In vitro experimental models of hepatocellular carcinoma (HCC) invasion and metastasis are important tools for studying the tumor microenvironment, cell behavior, and molecular mechanisms. Their development has evolved from two-dimensional cell culture to three-dimensional tissue engineering. Early studies relied on two-dimensional monolayer cell culture technology, culturing highly aggressive HCC cells and hepatic stellate cells, and using methods such as Western blot and enzyme-linked immunosorbent assay (ELISA) to detect the expression of matrix metalloproteinases, vimentin, and cytokines, thus initially revealing the molecular mechanisms of HCC invasion and metastasis. In recent years, three-dimensional cell culture technologies (such as mixtures of basement membrane matrix and collagen matrix) combined with the introduction of microfluidic chips have significantly improved the spatial simulation and dynamic control capabilities of the models, partially simulating the cell distribution characteristics of the tumor microenvironment and mimicking the mechanical changes in the progression of cirrhosis.

[0003] Despite significant advancements in existing methods, limitations remain that restrict the accurate simulation of clinical hepatocellular carcinoma (HCC) invasion and metastasis mechanisms using in vitro models. Firstly, current three-dimensional cell culture models largely rely on random or empirical cell arrangement, lacking precise spatial coordinate optimization based on pathological slide data. This results in low correlation between cell spatial arrangement and the actual characteristics of the core region of highly invasive HCC cells and the radial distribution of hepatic stellate cells within the clinical HCC microenvironment, impacting the model's biological reliability. Furthermore, while existing microfluidic technologies can regulate stiffness through matrix perfusion, they lack dynamic regulatory mechanisms linked to real-time gene expression (e.g., HOTAIR long non-coding RNA), making it difficult to simulate the dynamic changes in gene-mechanical interactions during the progression of cirrhosis. Summary of the Invention

[0004] In view of the aforementioned existing problems, the present invention is proposed.

[0005] Therefore, this invention provides a method for constructing an in vitro liver cancer invasion and metastasis experimental model to solve the problem that existing three-dimensional cell culture models mostly rely on random or empirical cell arrangement and lack real-time linkage with gene expression.

[0006] To solve the above-mentioned technical problems, the present invention provides the following technical solution: In a first aspect, the present invention provides a method for constructing an in vitro experimental model of liver cancer invasion and metastasis, comprising, Hepatocellular carcinoma cells and hepatic stellate cells were cultured and fluorescently labeled. A spatial coordinate set was generated using the fluorescently labeled hepatocellular carcinoma cells and hepatic stellate cells. The hepatocellular carcinoma cells and hepatic stellate cells were printed according to the spatial coordinate set to generate a three-dimensional cell-matrix structure. A three-dimensional cell-matrix structure was perfused with a photoresponsive hydrogel, and the concentration of HOTAIR long non-coding RNA was induced by transforming growth factor to obtain a three-dimensional cell-matrix structure integrating a photoresponsive hydrogel. In a three-dimensional cell-matrix structure integrating a photoresponsive hydrogel, the intensity of ultraviolet light was adjusted based on the concentration of HOTAIR long non-coding RNA to enhance the stiffness of the photoresponsive hydrogel. The migration speed of fluorescently labeled liver cancer cells was monitored to verify the correlation between gene expression and stiffness, thus generating a dynamic microenvironment. By integrating the dynamic microenvironment into the dynamic perfusion unit, adjusting the stiffness of the basement membrane matrix and collagen mixed matrix based on the Markov decision process model, detecting invasion and metastasis indicators, and generating an in vitro experimental model of liver cancer invasion and metastasis.

[0007] As a preferred embodiment of the method for constructing the in vitro liver cancer invasion and metastasis experimental model described in this invention, the culture of liver cancer cells and hepatic stellate cells refers to culturing highly invasive liver cancer cells and hepatic stellate cells in a culture flask using fetal bovine serum and penicillin-streptomycin DMEM medium, respectively, to obtain suspensions of highly invasive liver cancer cells and hepatic stellate cells.

[0008] As a preferred embodiment of the method for constructing the in vitro liver cancer invasion and metastasis experimental model described in this invention, the fluorescent labeling refers to transfecting highly invasive liver cancer cells with green fluorescent protein and hepatic stellate cells with red fluorescent protein. After transfection, green fluorescent protein-positive and red fluorescent protein-positive cells are sorted by flow cytometry, and the labeling stability is confirmed by fluorescence microscopy to obtain a suspension of fluorescently labeled highly invasive liver cancer cells and hepatic stellate cells.

[0009] As a preferred embodiment of the method for constructing the in vitro liver cancer invasion and metastasis experimental model described in this invention, the generation of the spatial coordinate set specifically includes the following steps. Spatial distribution data of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells were extracted from publicly available hepatocellular carcinoma pathology slide databases. A target spacing was set, a Markov random field model was constructed, and a preliminary spatial coordinate set was generated after defining the energy function. The preliminary spatial coordinate set was validated. After the correlation between the spatial distribution of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells and the spatial arrangement of cells in the publicly available hepatocellular carcinoma pathology slide database met the requirements, Gibbs sampling was performed to generate the spatial coordinate set. The spatial coordinate set includes the radiation zones of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells.

[0010] As a preferred embodiment of the method for constructing the in vitro liver cancer invasion and metastasis experimental model described in this invention, the step of printing liver cancer cells and hepatic stellate cells according to a spatial coordinate set to generate a three-dimensional cell-matrix structure refers to loading a suspension of fluorescently labeled highly invasive liver cancer cells and hepatic stellate cells into a microdroplet printing unit, using a mixture of basement membrane matrix and collagen as the matrix, printing the core area of ​​highly invasive liver cancer cells and the radiation area of ​​hepatic stellate cells according to a spatial coordinate set, and forming a three-dimensional cell-matrix structure after solidification.

[0011] As a preferred embodiment of the method for constructing the in vitro liver cancer invasion and metastasis experimental model described in this invention, the step of perfusing a photoresponsive hydrogel into a three-dimensional cell-matrix structure and inducing HOTAIR concentration with transforming growth factor specifically includes the following steps. A photoresponsive hydrogel, prepared by a photoresponsive hydrogel precursor solution of polyethylene glycol diacrylate and HOTAIR aptamer, was infused into a three-dimensional cell-matrix structure in the cell culture area of ​​a microfluidic chip, forming a three-dimensional cell-matrix structure integrating the photoresponsive hydrogel. Transforming growth factor was added to a microfluidic chip to induce the expression of HOTAIR long non-coding RNA in highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein, and the concentration of HOTAIR long non-coding RNA was detected by real-time quantitative PCR.

[0012] As a preferred embodiment of the method for constructing the in vitro liver cancer invasion and metastasis experimental model described in this invention, the following steps are taken: In a three-dimensional cell-matrix structure integrating a photoresponsive hydrogel, HOTAIR long non-coding RNA is bound to HOTAIR aptamers to enhance fluorescence resonance energy transfer efficiency; the intensity of fluorescence resonance energy transfer signal is recorded using a fluorescence microscope; the intensity of ultraviolet light is adjusted by a light-controlled device to increase the hardness of the photoresponsive hydrogel; the range of hardness variation of the photoresponsive hydrogel is verified; and the hardness data of the photoresponsive hydrogel are obtained. The correlation between the concentration of HOTAIR long non-coding RNA and the hardness data of photoresponsive hydrogel was calculated to obtain the correlation results between the two. We recorded the migration velocity data of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein, verified the correlation between the migration velocity data of highly invasive hepatocellular carcinoma cells and the concentration of HOTAIR long non-coding RNA and the hardness data of photoresponsive hydrogels, and generated a dynamic microenvironment regulated by gene-physical property feedback.

[0013] As a preferred embodiment of the method for constructing the in vitro hepatocellular carcinoma invasion and metastasis experimental model described in this invention, the step of adjusting the stiffness of the basement membrane matrix and collagen mixed matrix based on a Markov decision process model and detecting invasion and metastasis indicators specifically includes the following steps. A microfluidic chip in a dynamic microenvironment with gene-physical property feedback regulation is connected to a dynamic perfusion unit to perfuse a matrix solution, wherein the matrix solution has initial hardness. A Markov decision process model was constructed using the recorded migration speed of highly invasive hepatocellular carcinoma cells and the expression ratio of epithelial cadherin to neurocadherin. The matrix solution ratio was adjusted to increase the hardness of the photoresponsive hydrogel, which induced the invasion of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein. The migration trajectory was recorded, the number of invading cells was counted, and dynamic groups, static groups, and evaluation indicators were set. The changes in evaluation indicators between the dynamic group and the static matrix control group were compared by t-test. Combined with the correlation results of cell spatial arrangement in the three-dimensional cell-matrix structure and the correlation results of HOTAIR long non-coding RNA concentration and photoresponsive hydrogel hardness data, a complete in vitro liver cancer invasion and metastasis experimental model was generated.

[0014] In a second aspect, the present invention provides a computer device, including a memory and a processor, wherein the memory stores a computer program, wherein: when the computer program is executed by the processor, it implements any step of the method for constructing an in vitro liver cancer invasion and metastasis experimental model as described in the first aspect of the present invention.

[0015] Thirdly, the present invention provides a computer-readable storage medium having a computer program stored thereon, wherein: when the computer program is executed by a processor, it implements any step of the method for constructing an in vitro liver cancer invasion and metastasis experimental model as described in the first aspect of the present invention.

[0016] The beneficial effects of this invention are as follows: By constructing a Markov random field model and generating a highly realistic spatial coordinate set, the precise simulation of the core region of highly invasive hepatocellular carcinoma cells and the radial distribution of hepatic stellate cells is achieved, providing a precise positional template for printing three-dimensional cell-matrix structures. This simulates the real cell arrangement characteristics of the clinical hepatocellular carcinoma microenvironment, significantly improving the biological reliability of the in vitro model and ensuring a high degree of consistency between the three-dimensional cell-matrix structure and pathological sections. This provides a realistic microenvironmental basis for studying the interaction between highly invasive hepatocellular carcinoma cells and hepatic stellate cells. In addition, by preparing a photoresponsive hydrogel containing HOTAIR aptamers and perfusing it into the three-dimensional cell-matrix structure, the concentration of HOTAIR long non-coding RNA is used to adjust the ultraviolet light intensity through fluorescence resonance energy transfer signals, realizing a dynamic microenvironment of gene-mechanical interaction. This simulates the real-time linkage between gene expression and matrix stiffness in the process of cirrhosis, achieving the effect of quantifying the regulatory effect of HOTAIR long non-coding RNA on stiffness and invasive behavior, and improving the ability to simulate the dynamics of the hepatocellular carcinoma microenvironment. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a flowchart illustrating the method for constructing an in vitro experimental model of liver cancer invasion and metastasis.

[0019] Figure 2 Generate a flowchart for spatial coordinates.

[0020] Figure 3 This is a flowchart of the light response regulation process.

[0021] Figure 4 This is a flowchart for intrusion verification. Detailed Implementation

[0022] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0023] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0024] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0025] Reference Figures 1-4 This is one embodiment of the present invention, which provides a method for constructing an in vitro experimental model of liver cancer invasion and metastasis, comprising the following steps: S1: Culture liver cancer cells and hepatic stellate cells and label them with fluorescence. Use the fluorescently labeled liver cancer cells and hepatic stellate cells to generate a spatial coordinate set. Print the liver cancer cells and hepatic stellate cells according to the spatial coordinate set to generate a three-dimensional cell-matrix structure. Specifically, the steps include the following: S1.1. Highly invasive hepatocellular carcinoma cells (MHCC97-H) and hepatic stellate cells (LX-2) were cultured in T75 culture flasks using DMEM medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The flasks were placed at 37 degrees Celsius and 5% carbon dioxide. Cell growth was observed regularly until the density of highly invasive hepatocellular carcinoma cells and hepatic stellate cells reached 80%.

[0026] Highly aggressive hepatocellular carcinoma cells and hepatic stellate cells with a density of 80% were digested with 0.25% trypsin. After digestion, the cells were centrifuged at 1,000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in DMEM medium to obtain a suspension of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells.

[0027] Fluorescent labeling of suspensions of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells was performed using lentiviral vectors. The lentiviral vectors consisted of pLenti-green fluorescent protein (GFP) transfected into highly aggressive hepatocellular carcinoma cells and pLenti-red fluorescent protein (RBF) transfected into RBF cells. Forty-eight hours after transfection, GFP-positive and RBF-positive cells were sorted by flow cytometry. The sorted highly aggressive hepatocellular carcinoma cells and RBF cells were passaged three times consecutively. Fluorescence microscopy confirmed that the GFP and RBF signals did not attenuate, verifying the labeling stability and yielding fluorescently labeled suspensions of highly aggressive hepatocellular carcinoma cells and RBF cells.

[0028] S1.2 Extract spatial distribution data of highly invasive hepatocellular carcinoma cells and hepatic stellate cells from publicly available liver cancer pathology slide databases. Determine the relative positions of highly invasive hepatocellular carcinoma cells and hepatic stellate cells in the tumor microenvironment through pathology slide images. Record the distribution characteristics of the core area formed by highly invasive hepatocellular carcinoma cells and the radial distribution pattern of hepatic stellate cells around the core area. Measure the intercellular distance and set the target distance as fifty micrometers.

[0029] Based on the spatial distribution data of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells, a Markov random field model was constructed. The energy function was defined as the sum of squares of the deviations between the distance between hepatic stellate cells and highly aggressive hepatocellular carcinoma cells and the target distance of 50 micrometers. Specifically, the difference between the actual distance between each cell pair and 50 micrometers was calculated, squared, and summed. The expression is as follows: ; in, Represents the energy function. This indicates highly aggressive liver cancer cells. Indicates hepatic stellate cells, This represents the actual spatial distance between highly aggressive liver cancer cells and hepatic stellate cells.

[0030] Minimizing the energy function is used as the optimization objective to ensure that the Markov random field model reflects the true spatial relationship between the core area of ​​highly aggressive hepatocellular carcinoma cells and the radial distribution of hepatic stellate cells in pathological sections.

[0031] Gibbs sampling was performed on the Markov random field model using Python's PyMC3 library. The number of sampling iterations was set, and the random positions of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells were initialized. The position of each cell was updated iteratively, and the probability distribution was calculated based on an energy function, using a Boltzmann distribution. The expression is as follows: ; in, Indicates the corresponding position The probability, Indicates the corresponding position The energy function, This represents the temperature parameter.

[0032] Using Python's PyMC3 library, Gibbs sampling was employed to iteratively update the location of each cell, extracting high-probability locations and gradually optimizing the spatial layout of the cells. This resulted in highly aggressive liver cancer cells forming a core region with a diameter of 200 micrometers, and hepatic stellate cells being distributed radially. After sampling, the set of cell locations that satisfied the minimization of the energy function was extracted, yielding a preliminary set of spatial coordinates. The initial spatial coordinate set was validated by comparing the spatial distribution of highly invasive hepatocellular carcinoma cells and hepatic stellate cells with the cell spatial arrangement in publicly available hepatocellular carcinoma pathology slide databases. For example, based on the requirements of clinical pathology simulation, the correlation should reach 0.92. The correlation of cell spatial arrangement reflects that the spatial distribution of highly invasive hepatocellular carcinoma cells and hepatic stellate cells in the in vitro three-dimensional cell-matrix structure should highly reproduce the cell arrangement characteristics in clinical hepatocellular carcinoma pathology slides. A correlation of 0.92 indicates that the deviation between the cell spatial distribution of the in vitro model and the clinical data is minimal. If the correlation of cell spatial arrangement is less than 0.92, the Gibbs sampling parameters are adjusted, such as increasing the number of samplings or optimizing the initial position, until the optimal spatial coordinate set is generated. The optimal spatial coordinate set contains a core area with a diameter of 200 micrometers formed by highly invasive hepatocellular carcinoma cells, with hepatic stellate cells arranged radially around the core area of ​​highly invasive hepatocellular carcinoma cells, and the intercellular spacing gradient is 30 to 50 micrometers.

[0033] Fluorescently labeled suspensions of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells were loaded into a microdroplet printing unit. A 1:1 mixture of basement membrane matrix and collagen with a hardness of 5 kPa was prepared as the printing matrix. The core area of ​​highly aggressive hepatocellular carcinoma cells and the radiation area of ​​hepatic stellate cells were printed according to the optimal spatial coordinate set. After printing, the cells were cured at 37 degrees Celsius for 30 minutes to form a three-dimensional cell-matrix structure.

[0034] Furthermore, the precise spatial coordinate set generated through Gibbs sampling and a Markov random field model ensures that the radial distribution of the core region of highly invasive hepatocellular carcinoma cells and hepatic stellate cells closely matches the characteristics of pathological sections, accurately simulating the spatial dynamics of cells within the tumor microenvironment. This high-precision three-dimensional cell-matrix structure better reflects the interaction between highly invasive hepatocellular carcinoma cells and hepatic stellate cells during invasion and metastasis, providing a reliable in vitro platform for studying the molecular mechanisms, cellular behavior, and microenvironmental influences of hepatocellular carcinoma invasion and metastasis.

[0035] S2: Photoresponsive hydrogel was perfused into a three-dimensional cell-matrix structure, and the HOTAIR concentration was detected to obtain a three-dimensional cell-matrix structure integrating photoresponsive hydrogel; Specifically, the steps include the following: S2.1: Prepare a solution containing 10% polyethylene glycol diacrylate, dissolve it in phosphate buffer, add 0.5% azobenzene derivative crosslinking agent, 0.1% photoinitiator and 10 μmol HOTAIR aptamer, mix thoroughly to form a photoresponsive hydrogel precursor solution; perform photopolymerization under ultraviolet light to form a photoresponsive hydrogel with an initial hardness of 4 kPa.

[0036] In vitro, a photoresponsive hydrogel was incubated with HOTAIR long non-coding RNA at concentrations ranging from 0 to 100 nanomoles. HOTAIR long non-coding RNA is a non-coding RNA molecule involved in regulating gene expression and is associated with the invasion and metastasis of liver cancer. Fluorescence resonance energy transfer was used to detect conformational changes of the HOTAIR aptamer. By measuring the fluorescence intensity ratio of the donor to the acceptor, the specific binding of the HOTAIR aptamer to the HOTAIR long non-coding RNA was confirmed. The affinity constant was less than 10 nanomoles, and there was no cross-reactivity. A photoresponsive hydrogel of the HOTAIR aptamer with verified specific binding ability was obtained.

[0037] S2.2: The three-dimensional cell-matrix structure is placed in the cell culture area of ​​the microfluidic chip, which includes a cell culture area, a matrix perfusion area, and a light control area; the photoresponsive hydrogel with specific binding ability of HOTAIR aptamer is perfused into the cell culture area of ​​the microfluidic chip to ensure that the photoresponsive hydrogel completely encapsulates the three-dimensional cell-matrix structure, forming a three-dimensional cell-matrix structure with integrated photoresponsive hydrogel.

[0038] Transforming growth factor-β was added to the cell culture region of a microfluidic chip within a three-dimensional cell-matrix structure integrated with a photoresponsive hydrogel. This increased or activated the expression of HOTAIR long non-coding RNA in highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein in an in vitro environment. After 24 hours of culture, RNA was extracted from the three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel using an RNA extraction reagent, and the concentration of HOTAIR long non-coding RNA was detected by real-time quantitative PCR.

[0039] Further explanation reveals that the integrated three-dimensional cell-matrix structure of the photoresponsive hydrogel, combined with highly realistic cell spatial arrangement and the gene-responsive properties of the photoresponsive hydrogel, achieves an initial hardness of 4 kPa, mimicking the physical properties of normal liver tissue. The specific binding ability of the HOTAIR aptamer enables it to respond to changes in HOTAIR long non-coding RNA concentration, triggering dynamic regulation of hardness. This structure provides a functional carrier for subsequent steps, simulating microenvironmental changes from normal liver tissue to advanced cirrhosis. The generation of HOTAIR long non-coding RNA concentration data further provides a trigger signal for hardness regulation, ensuring the dynamic correlation between microenvironmental changes and gene expression, and providing a reliable platform for studying the microenvironment-dependent nature of liver cancer invasion and metastasis.

[0040] S3: In the three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel, the intensity of ultraviolet light was adjusted based on the concentration of HOTAIR long non-coding RNA to enhance the hardness of the photoresponsive hydrogel. The migration speed of fluorescently labeled liver cancer cells was monitored to verify the correlation between gene expression and hardness, thus generating a dynamic microenvironment. Specifically, the steps include the following: S3.1: In the three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel, the change in the concentration of HOTAIR long non-coding RNA specifically binds to the HOTAIR aptamer in the photoresponsive hydrogel. The HOTAIR long non-coding RNA molecule pairs with the nucleic acid sequence of the HOTAIR aptamer, causing the HOTAIR aptamer to change from the initial conformation to the bound conformation. This results in a shortening of the spatial distance between the fluorescent donor and acceptor in the photoresponsive hydrogel, thereby enhancing the fluorescence resonance energy transfer efficiency.

[0041] The three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel undergoing HOTAIR aptamer conformational changes was observed using fluorescence microscopy. Fluorescence resonance energy transfer (FRET) signal intensity was recorded every 4 hours, and the ratio of donor fluorescence intensity (green fluorescence) to acceptor fluorescence intensity (red fluorescence) was measured to calculate the FRET efficiency. FRET is a non-radiative energy transfer process. When the emission spectrum of the donor fluorophore overlaps with the absorption spectrum of the acceptor fluorophore (red fluorophore), and the distance between them is less than 10 nm, the donor transfers energy to the acceptor after excitation, resulting in a decrease in donor fluorescence intensity and an increase in acceptor fluorescence intensity. In this experiment, the binding of HOTAIR long non-coding RNA to the HOTAIR aptamer induced a conformational change in the aptamer, shortening the distance between the donor and acceptor fluorophores and enhancing the FRET efficiency. The FRET efficiency reflects the degree of energy transfer and is directly related to the concentration of HOTAIR long non-coding RNA. The expression for calculating the FRET efficiency is: ; in, Indicates fluorescence resonance energy transfer efficiency. This indicates the donor fluorescence intensity in the presence of an acceptor. This indicates the donor fluorescence intensity in the absence of an acceptor.

[0042] Record changes in fluorescence resonance energy transfer (FRET) signal intensity data to ensure that the FRET signal intensity data corresponds to changes in HOTAIR long noncoding RNA concentration. The FRET signal intensity data includes donor fluorescence intensity, acceptor fluorescence intensity, FRET energy ratio, and FRET efficiency.

[0043] The fluorescence resonance energy transfer signal intensity data is transmitted through a light-controlled device. The light-controlled device automatically adjusts the intensity of the ultraviolet light source according to the changes in the fluorescence resonance energy transfer signal intensity. The ultraviolet light irradiates the azobenzene cross-linking agent in the photoresponsive hydrogel, enhancing the cis-trans isomerization reaction of the azobenzene cross-linking agent and promoting the increase of cross-linking density. This causes the hardness of the photoresponsive hydrogel in the three-dimensional cell-matrix structure integrating the photoresponsive hydrogel to dynamically increase from 4 kPa to 9 kPa. The irradiation is continuous and the light intensity is adjusted every four hours to ensure that the hardness is dynamically adjusted according to the changes in the concentration of HOTAIR long non-coding RNA.

[0044] The hardness of the photoresponsive hydrogel in the three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel was measured using atomic force microscopy. Five uniformly distributed measurement points were selected in the cell culture area of ​​the microfluidic chip every 4 hours. A constant force was applied to record the probe indentation depth, Young's modulus was calculated, and the hardness value of the photoresponsive hydrogel was determined. The hardness data of each measurement were recorded to confirm that the hardness variation range covered 4 kPa to 9 kPa.

[0045] By comparing the hardness measurement data of photoresponsive hydrogels with the concentration data of HOTAIR long non-coding RNA, the correlation between hardness changes and HOTAIR long non-coding RNA concentration was analyzed. Statistical methods were used to calculate the correlation coefficient, quantify the strength of the association, and generate correlation coefficient results. Based on the correlation coefficient results, it was confirmed that the correlation coefficient was positive and significant. Regression analysis was used to verify that the hardness of the photoresponsive hydrogel dynamically increases with the increase of HOTAIR long non-coding RNA concentration, generating a validation conclusion to ensure the regulatory effect and ensure that the hardness dynamically increases with the increase of HOTAIR long non-coding RNA concentration. This validates the hardness regulation to simulate the changes in physical characteristics from normal liver tissue (hardness of about 4 kPa) to late-stage cirrhosis (hardness of about 9 kPa). If the hardness range does not cover 4 kPa to 9 kPa, the ultraviolet light intensity is adjusted, and the hardness measurement is repeated until the dynamic range of hardness is confirmed to meet the requirements, generating validated hardness data.

[0046] S3.2: In a three-dimensional cell-matrix structure of an integrated photoresponsive hydrogel with a verified hardness range of 4 kPa to 9 kPa, highly aggressive liver cancer cells labeled with green fluorescent protein were observed in real time using a fluorescence microscope. Fluorescence images of the cell culture area of ​​the microfluidic chip were captured every hour. The position coordinates of at least 10 highly aggressive liver cancer cells in the three-dimensional cell-matrix structure were recorded, the displacement distance per hour was calculated, and data on the migration speed of highly aggressive liver cancer cells were generated.

[0047] Pearson correlation coefficients were calculated using statistical software between the migration velocity data of highly invasive hepatocellular carcinoma cells and the concentration data of HOTAIR long non-coding RNA. For example, based on biological experimental data, a correlation coefficient greater than 0.85 was used to confirm a positive correlation, quantifying the promoting effect of HOTAIR long non-coding RNA as a gene regulator on the migration of highly invasive hepatocellular carcinoma cells. Pearson correlation coefficients were also calculated between the migration velocity data of invasive hepatocellular carcinoma cells and the hardness data of photoresponsive hydrogels. For example, based on statistical standards, a correlation coefficient greater than 0.8 was used to confirm a positive correlation, quantifying the mechanical regulatory effect of photoresponsive hydrogel hardness on the migration of highly invasive hepatocellular carcinoma cells and verifying the biological significance of hardness ranging from 4 kPa to 9 kPa simulating the progression of liver cirrhosis. Linear regression analysis was used to further analyze the migration velocity data of HOTAIR long non-coding RNA. Using HOTAIR long non-coding RNA concentration data as the independent variable and the migration speed of highly invasive hepatocellular carcinoma cells as the dependent variable, we verified that increased HOTAIR long non-coding RNA concentration increases migration speed. Using photoresponsive hydrogel hardness data as the independent variable and the migration speed of highly invasive hepatocellular carcinoma cells as the dependent variable, we verified that increasing hardness from 4 kPa to 9 kPa promotes the migration of highly invasive hepatocellular carcinoma cells. Through multivariate linear regression, using HOTAIR long non-coding RNA concentration data and photoresponsive hydrogel hardness data as independent variables and highly invasive hepatocellular carcinoma cell migration speed data as the dependent variable, we confirmed the positive association between HOTAIR long non-coding RNA expression and the regulation of photoresponsive hydrogel hardness, forming a dynamic microenvironment of gene-physical property feedback regulation, in which hardness is regulated in real time with HOTAIR long non-coding RNA expression.

[0048] Further explanation reveals that the dynamic microenvironment regulated by gene-physical feedback is driven by the concentration of HOTAIR long non-coding RNA, resulting in changes in the stiffness of the photoresponsive hydrogel. Combined with the highly realistic cell arrangement of the three-dimensional cell-matrix structure integrated with the photoresponsive hydrogel, the complex interactions between gene expression, matrix stiffness, and cell invasion behavior in the hepatocellular carcinoma (HCC) microenvironment can be dynamically reproduced. The stiffness regulation range from 4 kPa to 9 kPa simulates the pathological changes in matrix stiffness during the progression of cirrhosis. The migration velocity data of highly invasive HCC cells labeled with green fluorescent protein provides a direct indicator of invasion behavior, enabling the microenvironment to accurately reflect the regulatory role of HOTAIR long non-coding RNA in HCC invasion and metastasis. This significantly enhances the ability of the in vitro model to simulate the clinical mechanisms of HCC invasion and metastasis, providing a reliable tool for in-depth research on molecular mechanisms and screening of anti-metastasis strategies.

[0049] S4: Integrate the dynamic microenvironment into the dynamic perfusion unit, adjust the stiffness of the basement membrane matrix and collagen mixed matrix based on the Markov decision process model, detect invasion and metastasis indicators, and generate an in vitro liver cancer invasion and metastasis experimental model.

[0050] Specifically, the steps include the following: S4.1: Connect the microfluidic chip in the dynamic microenvironment regulated by gene-physical characteristic feedback to the dynamic perfusion unit (IBIDI pump), prepare a matrix solution containing basement membrane matrix and collagen (1:1 ratio, hardness 5 kPa), and perfuse it into the matrix perfusion area of ​​the microfluidic chip to stabilize the distribution and hardness of the matrix solution.

[0051] In a microfluidic chip with an initial hardness of 5 kPa, the displacement of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein was recorded hourly using a fluorescence microscope. The migration velocity of these cells was calculated. The calculation process was as follows: For each highly invasive hepatocellular carcinoma cell, the coordinates were recorded every hour, and the displacement distance was calculated. The expression for this was: ; in, Highly aggressive liver cancer cells in the first Displacement distance within the measurement time period. This indicates that highly aggressive liver cancer cells were present in the first... The two-dimensional coordinates of the second measurement Axis value, This indicates that highly aggressive liver cancer cells were present in the first... The two-dimensional coordinates of the second measurement Axis value, This indicates that highly aggressive liver cancer cells were present in the first... The two-dimensional coordinates of the second measurement Axis value, This indicates that highly aggressive liver cancer cells were present in the first... The two-dimensional coordinates of the second measurement Axis value.

[0052] The expression for calculating the migration velocity of highly aggressive liver cancer cells is: ; in, This indicates that highly aggressive liver cancer cells were present in the first... Migration speed within the measurement time period This indicates a time interval, here it is one hour.

[0053] By connecting the displacement coordinates at consecutive time points, the migration trajectories of highly invasive liver cancer cells are formed; these trajectories represent the invasion pathways. Simultaneously, epithelial cadherin (representing epithelial cells) is labeled with green fluorescent protein (GFP) and neurocadherin (representing neurocadherin) with red fluorescent protein (GFP) using lentivirus. The ratio of green fluorescence intensity to red fluorescence intensity is measured to calculate the expression ratio of epithelial cadherin to neurocadherin. Epithelial cadherin, an adhesion molecule of epithelial cells, is labeled with GFP; neurocadherin, an adhesion molecule of mesenchymal cells, is labeled with red fluorescent protein (GFP). The relative changes in their expression levels reflect the epithelial-mesenchymal transition (EMT) status. In liver cancer invasion and metastasis, epithelial cadherin expression is often decreased, while neurocadherin expression is increased. The expression ratio is quantified by measuring the ratio of green fluorescence intensity (representing epithelial cadherin) to red fluorescence intensity (representing neurocadherin), with fluorescence intensity being directly proportional to protein expression level. The expression ratio of epithelial cadherin to neurocadherin is expressed by the formula: ; in, The ratio of epithelial cadherin to neural cadherin expression reflects the relative expression level of epithelial cadherin compared to neural cadherin. The intensity of the green fluorescence of epithelial cadherin is indicated. The intensity of the red fluorescence of neurotrophin is indicated.

[0054] Record the migration rate of highly aggressive hepatocellular carcinoma cells and the expression ratio of epithelial cadherin to neurocadherin every two hours.

[0055] Based on data on the migration velocity of highly invasive hepatocellular carcinoma cells and the expression ratio of epithelial cadherin to neurocadherin, a Markov decision process model was constructed. The state was defined as the migration velocity of highly invasive hepatocellular carcinoma cells and the expression ratio of epithelial cadherin to neurocadherin. The action was defined as adjusting the ratio of basement membrane matrix to collagen, changing it by 5% each time, for example, from 1:1 to 1:105, with a stiffness increment of 0.5 kPa. The reward function was the structural similarity index between the invasion path and clinical cirrhosis samples, for example, a target of ≥90%. The metastasis probability was based on a publicly available database of hepatocellular carcinoma invasion experiments. A value iteration algorithm was implemented using Python's PyMDPtoolbox library. The optimal strategy was calculated from the input state data, action, reward function, and metastasis probability, and the next basement membrane matrix to collagen ratio was output every two hours.

[0056] Based on the ratio of basement membrane matrix to collagen every two hours, a basement membrane matrix and collagen matrix solution with the corresponding ratio is prepared and perfused into the matrix perfusion area of ​​the microfluidic chip through a dynamic perfusion unit. The matrix solution ratio is updated every two hours, so that the hardness of the photoresponsive hydrogel gradually increases from 5 kPa to 8 kPa. Perfusion is continued and the hardness is measured every four hours using an atomic force microscope to confirm that the hardness change is in line with expectations, thus simulating the process of liver cirrhosis.

[0057] S4.2: DMEM medium containing 10% fetal bovine serum was added to the lower layer of the culture zone of the microfluidic chip with dynamically regulated hardness to induce invasion of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein. The cells were cultured for 48 hours at 37 degrees Celsius and 5% carbon dioxide to ensure the activity of the highly invasive hepatocellular carcinoma cells.

[0058] In a microfluidic chip with dynamically regulated stiffness to induce invasion, the migration trajectories of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein were recorded hourly using a fluorescence microscope, and the displacement paths of at least ten highly invasive hepatocellular carcinoma cells were calculated. Simultaneously, the fluorescence intensity of epithelial cadherin labeled with green fluorescent protein and neurocadherin labeled with red fluorescent protein were measured, the expression ratio of epithelial cadherin to neurocadherin was calculated, and the migration trajectory data and expression ratio data were recorded hourly.

[0059] In a microfluidic chip with dynamically regulated stiffness to induce invasion, after 48 hours of culture, highly invasive liver cancer cells on the lower membrane of the microfluidic chip were stained with 0.5% crystal violet solution. The number of invasive cells in each field of view was observed and counted using an optical microscope. The average number of invasive cells in at least five fields of view was calculated and the invasive cell count data were recorded. A static matrix control group was set up (the static matrix control group had a fixed stiffness of 5 kPa and a basement membrane matrix to collagen ratio of 1:1). The same staining and counting operations were repeated to obtain the invasive cell count data of the control group.

[0060] Proteins from highly invasive hepatocellular carcinoma cells were extracted from a microfluidic chip with dynamically regulated stiffness to induce invasion. The expression levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and vimentin were detected using Western blot. Specifically, after electrophoretic separation, the proteins were transferred to a polyvinylidene fluoride membrane, incubated with specific antibodies, and then subjected to chemiluminescence imaging. The intensity of the protein bands was analyzed by grayscale, and the protein expression level data were recorded. The same protein extraction and Western blot detection were repeated for highly invasive hepatocellular carcinoma cells in a static matrix control group to obtain the control group's protein expression level data.

[0061] Culture supernatant from a microfluidic chip with dynamically regulated stiffness to induce invasion was collected. The secretion levels of interleukin-6 and transforming growth factor-β were detected using an enzyme-linked immunosorbent assay (ELISA). Specifically, the supernatant was added to a pre-coated antibody-based ELISA plate, followed by the addition of the detection antibody and substrate. The absorbance at 450 nm was measured, and the secretion level was calculated. The procedure was as follows: culture supernatant was collected, a pre-coated antibody-based ELISA plate was added, incubated, and then the detection antibody and substrate were added. The absorbance was measured using an ELISA reader. Simultaneously, a standard curve was established using known concentrations of interleukin-6 and transforming growth factor-β standards. The fitting equation was: ; in, Indicates absorbance value. This represents the slope of the standard curve. Indicates the concentration of secretion. This represents the intercept of the standard curve.

[0062] The sample absorbance values ​​were substituted into the standard curve equation to calculate the concentration. The experiment was repeated three times, and the average value was taken to record the secretion data of interleukin-6 and transforming growth factor-β. The same enzyme-linked immunosorbent assay was repeated on the culture supernatant of the static matrix control group to obtain the secretion data of the control group. The expression levels of matrix metalloproteinase-2, matrix metalloproteinase-9, and vimentin reflect the invasive ability and epithelial-mesenchymal transition status of highly aggressive hepatocellular carcinoma cells. The secretion levels of interleukin-6 and transforming growth factor-β reflect the inflammation and signal transduction activity in the microenvironment and are related to the invasion and metastasis of hepatocellular carcinoma. Matrix metalloproteinase-2 and matrix metalloproteinase-9 degrade the matrix to promote cell migration, vimentin marks the epithelial-mesenchymal transition, and interleukin-6 and transforming growth factor-β regulate microenvironment signals. These indicators are directly related to the molecular mechanisms of hepatocellular carcinoma invasion and metastasis, i.e., they are the core indicators.

[0063] The t-test was used to compare the number of invasive cells, matrix metalloproteinase-2, matrix metalloproteinase-9, and vimentin expression levels, and interleukin-6 and transforming growth factor-β secretion levels in the dynamic group and the static matrix control group. The p-value was calculated, and the significance level was confirmed to be less than 0.05 to verify the increase in invasion depth in the dynamic group compared with that in the static matrix control group. The basis for confirming the significance level of less than 0.05 is the conventional threshold of the t-test in statistics. It was used to compare the number of invasive cells, matrix metalloproteinase-2, matrix metalloproteinase-9, vimentin expression levels, interleukin-6, and transforming growth factor-β secretion levels in the dynamic group (hardness 5 kPa to 8 kPa) and the static matrix control group (hardness 5 kPa). A significance level less than 0.05 indicates that the difference between the two groups is significant at a 95% confidence level, verifying the biological effect of dynamic stiffness promoting invasive behavior. A significance level less than 0.05 is a standard for verifying differences between experimental and control groups in biomedical research, ensuring the statistical reliability of the increased invasion depth in the dynamic group and reflecting the promoting effect of stiffness changes in the liver cirrhosis microenvironment on invasion. The structural similarity index between the migration trajectory data of highly invasive liver cancer cells and clinical liver cirrhosis samples was calculated, confirming that the structural similarity is greater than or equal to 90%. Since the in vitro model needs to highly simulate clinical pathological features, the structural similarity greater than 90% is quantified by dynamic time regularization to ensure that the in vitro migration path (displacement distance, directionality) is highly consistent with the cancer cell migration pattern of clinical samples. Combining the correlation of cell spatial arrangement and the correlation of HOTAIR regulation of stiffness, a complete in vitro liver cancer invasion and metastasis experimental model was generated.

[0064] This embodiment also provides a computer device applicable to the construction method of an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model, comprising: a memory and a processor; the memory is used to store computer-executable instructions, and the processor is used to execute the computer-executable instructions to realize the construction method of the in vitro hepatocellular carcinoma liver invasion and metastasis experimental model proposed in the above embodiment.

[0065] The computer device can be a terminal, comprising a processor, memory, communication interface, display screen, and input devices connected via a system bus. The processor provides computing and control capabilities. The memory includes non-volatile storage media and internal memory. The non-volatile storage media stores the operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs stored in the non-volatile storage media. The communication interface is used for wired or wireless communication with external terminals; wireless communication can be achieved through Wi-Fi, carrier networks, NFC (Near Field Communication), or other technologies. The display screen can be an LCD screen or an e-ink screen. The input devices can be a touch layer covering the display screen, buttons, a trackball, or a touchpad on the computer device's casing, or an external keyboard, touchpad, or mouse.

[0066] This embodiment also provides a storage medium storing a computer program, which, when executed by a processor, implements the method for constructing an in vitro liver cancer invasion and metastasis experimental model as proposed in the above embodiments. The storage medium can be implemented by any type of volatile or non-volatile storage device or a combination thereof, such as Static Random Access Memory (SRAM), Electrically Erasable Programmable Read-Only Memory (EEPROM), Erasable Programmable Read Only Memory (EPROM), Programmable Red-Only Memory (PROM), Read-Only Memory (ROM), magnetic storage, flash memory, magnetic disk, or optical disk.

[0067] In summary, this invention: By constructing a Markov random field model and generating a highly realistic spatial coordinate set, it achieves accurate simulation of the core region of highly invasive hepatocellular carcinoma cells and the radial distribution of hepatic stellate cells, providing a precise location template for printing three-dimensional cell-matrix structures. It simulates the real cell arrangement characteristics of the clinical hepatocellular carcinoma microenvironment, significantly improving the biological reliability of the in vitro model and ensuring a high degree of consistency between the three-dimensional cell-matrix structure and pathological sections. This provides a realistic microenvironmental basis for studying the interaction between highly invasive hepatocellular carcinoma cells and hepatic stellate cells. Furthermore, by preparing a photoresponsive hydrogel containing HOTAIR aptamers and perfusing it into the three-dimensional cell-matrix structure, and adjusting the ultraviolet light intensity based on the concentration of HOTAIR long non-coding RNA through fluorescence resonance energy transfer signals, it achieves a dynamic microenvironment of gene-mechanical interaction, simulating the real-time linkage between gene expression and matrix stiffness during the process of cirrhosis. This quantifies the regulatory effect of HOTAIR long non-coding RNA on stiffness and invasive behavior, enhancing the ability to simulate the dynamics of the hepatocellular carcinoma microenvironment.

[0068] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A method for constructing an in vitro experimental model of liver cancer invasion and metastasis, characterized in that: include, Hepatocellular carcinoma cells and hepatic stellate cells were cultured and fluorescently labeled. A spatial coordinate set was generated using the fluorescently labeled hepatocellular carcinoma cells and hepatic stellate cells. The hepatocellular carcinoma cells and hepatic stellate cells were printed according to the spatial coordinate set to generate a three-dimensional cell-matrix structure. Photoresponsive hydrogels were perfused into a three-dimensional cell-matrix structure, and the concentration of HOTAIR long non-coding RNA was induced by transforming growth factor to obtain a three-dimensional cell-matrix structure integrating photoresponsive hydrogels. In the three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel, the intensity of ultraviolet light was adjusted based on the concentration of HOTAIR long non-coding RNA to enhance the stiffness of the photoresponsive hydrogel. The migration speed of fluorescently labeled liver cancer cells was monitored to verify the correlation between gene expression and stiffness, thus generating a dynamic microenvironment. By integrating the dynamic microenvironment into the dynamic perfusion unit, adjusting the stiffness of the basement membrane matrix and collagen mixed matrix based on the Markov decision process model, detecting invasion and metastasis indicators, and generating an in vitro experimental model of liver cancer invasion and metastasis.

2. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 1, characterized in that: The culture of hepatocellular carcinoma cells and hepatic stellate cells refers to the culture of highly invasive hepatocellular carcinoma cells and hepatic stellate cells in DMEM medium containing fetal bovine serum and penicillin-streptomycin in culture flasks, respectively, to obtain suspensions of highly invasive hepatocellular carcinoma cells and hepatic stellate cells.

3. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 2, characterized in that: The fluorescent labeling process involves transfecting highly aggressive hepatocellular carcinoma cells with green fluorescent protein (GFP) and hepatic stellate cells with red fluorescent protein (RFP). After transfection, GFP-positive and RFP-positive cells are sorted by flow cytometry, and the labeling stability is confirmed by fluorescence microscopy to obtain a suspension of fluorescently labeled highly aggressive hepatocellular carcinoma cells and hepatic stellate cells.

4. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 3, characterized in that: The generation of the spatial coordinate set specifically includes the following steps. Spatial distribution data of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells were extracted from publicly available hepatocellular carcinoma pathology slide databases. A target spacing was set, a Markov random field model was constructed, and a preliminary spatial coordinate set was generated after defining the energy function. The preliminary spatial coordinate set was validated. After the correlation between the spatial distribution of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells and the spatial arrangement of cells in the publicly available hepatocellular carcinoma pathology slide database met the requirements, Gibbs sampling was performed to generate the spatial coordinate set. The spatial coordinate set includes the radiation zones of highly aggressive hepatocellular carcinoma cells and hepatic stellate cells.

5. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 4, characterized in that: The process of printing liver cancer cells and hepatic stellate cells according to a spatial coordinate set to generate a three-dimensional cell-matrix structure refers to loading a suspension of fluorescently labeled highly invasive liver cancer cells and hepatic stellate cells into a microdroplet printing unit, using a mixture of basement membrane matrix and collagen as the matrix, and printing the core area of ​​highly invasive liver cancer cells and the radiation area of ​​hepatic stellate cells according to a spatial coordinate set. After solidification, a three-dimensional cell-matrix structure is formed.

6. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 5, characterized in that: The process of perfusing a photoresponsive hydrogel into a three-dimensional cell-matrix structure and inducing HOTAIR concentration with transforming growth factor specifically includes the following steps. A photoresponsive hydrogel, prepared by a photoresponsive hydrogel precursor solution of polyethylene glycol diacrylate and HOTAIR aptamer, is infused into a three-dimensional cell-matrix structure in the cell culture area of ​​a microfluidic chip to form a three-dimensional cell-matrix structure integrating the photoresponsive hydrogel. Transforming growth factor was added to a microfluidic chip to induce the expression of HOTAIR long non-coding RNA in highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein, and the concentration of HOTAIR long non-coding RNA was detected by real-time quantitative PCR.

7. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 6, characterized in that: By adjusting the concentration of HOTAIR long non-coding RNA to regulate ultraviolet light intensity, enhancing the hardness of photoresponsive hydrogels, and monitoring the migration speed of fluorescently labeled liver cancer cells, the correlation between gene expression and hardness was verified, thus generating a dynamic microenvironment. The specific steps include the following: In the three-dimensional cell-matrix structure of the integrated photoresponsive hydrogel, HOTAIR long non-coding RNA was bound to HOTAIR aptamers to enhance fluorescence resonance energy transfer efficiency. The intensity of fluorescence resonance energy transfer signal was recorded using a fluorescence microscope. The intensity of ultraviolet light was adjusted by a light control device to increase the hardness of the photoresponsive hydrogel. The range of hardness variation of the photoresponsive hydrogel was verified, and the hardness data of the photoresponsive hydrogel were obtained. The correlation between the concentration of HOTAIR long noncoding RNA and the hardness data of photoresponsive hydrogel was calculated to obtain the correlation results between the two. We recorded the migration velocity data of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein, verified the correlation between the migration velocity data of highly invasive hepatocellular carcinoma cells and the concentration of HOTAIR long non-coding RNA and the hardness data of photoresponsive hydrogels, and generated a dynamic microenvironment regulated by gene-physical property feedback.

8. The method for constructing an in vitro hepatocellular carcinoma liver invasion and metastasis experimental model as described in claim 7, characterized in that: The method of adjusting the stiffness of the basement membrane matrix and collagen mixed matrix based on a Markov decision process model and detecting invasion and metastasis indicators specifically includes the following steps. A microfluidic chip in a dynamic microenvironment with gene-physical property feedback regulation is connected to a dynamic perfusion unit to perfuse a matrix solution; the matrix solution has initial hardness. A Markov decision process model was constructed using the recorded migration speed of highly invasive hepatocellular carcinoma cells and the expression ratio of epithelial cadherin to neurocadherin. The matrix solution ratio was adjusted to increase the hardness of the photoresponsive hydrogel, which induced the invasion of highly invasive hepatocellular carcinoma cells labeled with green fluorescent protein. The migration trajectory was recorded, the number of invading cells was counted, and dynamic groups, static groups, and evaluation indicators were set. The changes in evaluation indicators between the dynamic group and the static matrix control group were compared by t-test. Combined with the correlation results of cell spatial arrangement in the three-dimensional cell-matrix structure and the correlation results of HOTAIR long non-coding RNA concentration and photoresponsive hydrogel hardness data, a complete in vitro liver cancer invasion and metastasis experimental model was generated.

9. A computer device comprising a memory and a processor, wherein the memory stores a computer program, characterized in that: When the processor executes the computer program, it implements the steps of the method for constructing the in vitro liver cancer invasion and metastasis experimental model as described in any one of claims 1 to 8.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by the processor, it implements the steps of the method for constructing the in vitro liver cancer invasion and metastasis experimental model as described in any one of claims 1 to 8.

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