CN105521482B

CN105521482B - Combined application of HNF1 α, HNF4 α and FOXA3 for inducing differentiation and treating hepatocellular carcinoma

Info

Publication number: CN105521482B
Application number: CN201510974601.7A
Authority: CN
Inventors: 丁劲; 王红阳; 程卓; 李恒宇; 宁北芳
Original assignee: Second Military Medical University SMMU
Current assignee: Second Military Medical University SMMU
Priority date: 2015-12-22
Filing date: 2015-12-22
Publication date: 2020-07-14
Anticipated expiration: 2035-12-22
Also published as: CN105521482A

Abstract

The invention relates to a method for inducing differentiation and treating hepatocellular carcinoma by combined application of HNF1 α, HNF4 α and FOXA3, in particular to a method and application for inducing differentiation of human malignant hepatocellular carcinoma by using hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 so as to be applied to treatment of malignant solid tumors.

Description

Combined application of HNF1 α, HNF4 α and FOXA3 for inducing differentiation and treating hepatocellular carcinoma

Technical Field

The invention relates to the fields of molecular biology, cell biology and medicine, in particular to a method and application for inducing differentiation of human hepatocellular carcinoma by using hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 so as to treat the hepatocellular carcinoma.

Background

Liver cancer is one of the most common malignant tumors in the world, and about half of patients are distributed in China. Despite the continuous progress of the diagnosis and treatment technology of liver cancer in recent years, the current situations of difficult early diagnosis, high recurrence and metastasis rate and insensitivity to radiotherapy and chemotherapy are not fundamentally improved. The only approved target treatment drug for the liver cancer is sorafenib, and the treatment effect is not ideal. Therefore, the research on treating liver cancer needs to adjust the thought to find more effective treatment means.

Differentiation therapy is a therapy method which has been developed in recent years, and the purpose of treating tumors is achieved by inducing poorly differentiated tumor cells to differentiate toward a mature phenotype, inhibiting proliferation and metastasis thereof, and promoting apoptosis thereof. Until now, the best application of differentiation therapy is to treat acute promyelocytic leukemia by using all-trans retinoic acid, and a large number of patients obtain good treatment effect. The success of the differentiation therapy of the blood tumor also drives the differentiation therapy research of the solid tumor. The research of applying all-trans retinoic acid, dimethyl sulfoxide and arsenic agents to treat breast cancer, lung cancer and colon cancer is presented successively, but the research of differentiation treatment of liver cancer is still few at present.

Liver Cell nuclear factor (HNF) is a transcription factor which is relatively specifically and highly expressed in liver cells and plays an important role in liver development and liver Cell function maintenance, family members comprise HNF1, HNF3, HNF4, HNF6, CCAAT/enhancer binding protein (C/EBP) and the like, and the transcription factors are a series of transcription factors which are preferentially expressed in liver (Schrem H, L driver-induced transcription factors in liver functions and degeneration), part I: the liver Cell nuclear factor gene vector and liver tissue expression, Pharmacological research markers 2002, 54 (63129-58) and the inventor reports that the liver Cell nuclear factor 4 α and liver cancer cells have a very good liver cancer function in liver cancer research group, liver cancer cells are transformed into liver cells, liver cancer cells have a very good liver cancer function recovery effect (HNliver cancer cells of liver cancer cells, liver cancer cells are transformed into liver cells), liver cancer cells have a very good liver cancer Cell Differentiation effect after liver cancer cells are transformed into liver cells, liver cancer cells are transformed into liver cells, liver cancer cells are transformed into liver cancer cells, liver cancer.

Therefore, the aim in the field is to develop and jointly apply a plurality of hepatocyte nuclear factors to induce the differentiation of the hepatoma cells, and provide a new method for treating the hepatoma.

Disclosure of Invention

The invention also aims to provide novel medical application of three hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 genes and product proteins thereof.

In a first aspect of the invention, there is provided the use of three hepatocyte nuclear factors HNF1 α, HNF4 α, FOXA3 genes and/or proteins, to induce hepatocellular carcinoma differentiation.

The invention provides application of three hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 genes and/or proteins in preparation of a reagent or a composition for inducing (malignant) hepatocellular carcinoma differentiation.

The application refers to the combined action (combination) of three hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA 3.

The liver cancer differentiation treatment medicine provided by the invention is used for inhibiting proliferation and transfer of liver cancer cells and promoting apoptosis of the liver cancer cells by inducing the low-differentiation liver cancer cells to differentiate towards mature phenotypes, so that the aim of treating liver cancer is fulfilled.

The reagent of the present invention is a reagent for increasing the expression levels of HNF1 α, HNF4 α and FOXA 3.

The composition is a pharmaceutical composition.

The pharmaceutical composition contains proteins of three hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3, a coding sequence or an expression vector containing the coding sequence and a pharmaceutically acceptable carrier or excipient.

The dosage form of the pharmaceutical composition of the invention is preferably injection.

The hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 are human hepatocyte nuclear factors.

GENBANK ID of HNF1 α NM-001306179.1;

GENBANK ID of HNF4 α NM-000457.4;

GENBANK ID of FOXA 3: NM _ 004497.2.

In a second aspect of the present invention, there is provided a pharmaceutical combination, or "combination", comprising as active ingredients any one of:

(a) HNF1 α protein, HNF4 α protein, and FOXA3 protein;

(b) a coding sequence of HNF1 α protein, a coding sequence of HNF4 α protein and a coding sequence of FOXA3 protein;

(c) an expression vector containing a coding sequence of HNF1 α protein, a coding sequence of HNF4 α protein and a coding sequence of FOXA3 protein.

The expression vector can simultaneously or respectively express the coding sequences of the three factors.

The expression vector includes but is not limited to viral vectors such as adenovirus, lentivirus, retrovirus and adeno-associated virus, and non-viral vectors.

The combination drug also comprises a pharmaceutically acceptable carrier or excipient.

The invention also provides the application of the combined medicament in preparing a liver cancer treatment medicament, in particular to a liver cancer differentiation treatment medicament.

The invention provides a combination of HNF1 α, HNF4 α and FOXA3, which can be used for inhibiting the formation of hepatocyte tumor in vivo.

The combination drug of the invention can also comprise a chemotherapeutic agent.

The combined drug comprises three protein, coding sequences or expression vectors containing the coding sequences of hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3, which are simultaneously or sequentially applied with effective dose.

The pharmaceutically acceptable carrier or excipient of the present invention refers to additives commonly used in the pharmaceutical field, other than active ingredients, such as diluents (starches, sugars, celluloses, and inorganic salts), excipients, and the like, fillers such as starch, sucrose, binders such as water, ethanol, cellulose derivatives, gelatin, and polyvinylpyrrolidone, disintegrants such as dry starch and sodium carboxymethyl starch, solubilizers such as polysorbates and polyoxyethylene fatty acid esters, and the like, absorption enhancers, surfactants such as tween, span, adsorption carriers, lubricants such as magnesium stearate, aerosil, and the like. In addition, other adjuvants such as flavoring agent, sweetener, etc. can also be added into the composition.

The combination according to the invention may be administered in the form of a pharmaceutical composition by oral, nasal inhalation, rectal, parenteral or transdermal administration to a patient in need of such treatment. For oral administration, it can be made into conventional solid preparations such as tablet, powder, granule, capsule, pill, sustained release pellet, solid dispersion, clathrate, etc., and liquid preparations such as suspension, emulsion, melt agent, syrup, mixture, solution, etc., for parenteral administration, it can be made into solution for injection, aqueous or oily suspension, emulsion, liposome, microcapsule, microsphere, nanoparticle, etc., or it can be made into various sustained release and controlled release preparations. The preferred form is an injection, particularly one with site-specific targeted release.

The pharmaceutical composition and the combined medicament are also used for inhibiting the formation of solid tumors in vivo.

In a third aspect of the present invention, there is provided a method of inducing or promoting differentiation of hepatocellular carcinoma in a mammal, i.e., a novel method of treating liver cancer.

The method comprises the steps of introducing combined application of hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 proteins, coding sequences thereof or expression vectors of the coding sequences into the hepatoma cells, inhibiting proliferation and metastasis of the hepatoma cells and promoting apoptosis of the hepatoma cells.

The liver cancer of the present invention is preferably human primary hepatocellular carcinoma.

The invention provides a new medical application of hepatocyte nuclear factor.

The invention also provides a novel method for inducing the differentiation of the liver cancer cells and then treating the liver cancer by cooperatively introducing a plurality of hepatocyte nuclear factors into the liver cancer cells to play a role.

Drawings

FIG. 1 shows Western blot detection of protein expression of three factors after 2 days of infection of three adenovirus-infected hepatoma cell lines L M3, P L C and HepG2 cells by Ad HNF1 α, AdHNF4 α and AdFOXA 3.

FIG. 2 is a diagram of the quantitative analysis of the mRNA expression of the functional molecule related to the liver cell 2 days after the detection of the combined infection of the liver cancer cell with the three factors by Realtime-PCR, wherein A is L M3 cell line, B is P L C cell line, C is HepG2 cell line, and D is SMMC-7721 cell line.

FIG. 3 shows the change of cell morphology of the liver cancer cells L M315 after infection with the three-factor adenovirus, wherein A is L M3 plus GFP adenovirus cell morphology, and B is L M3 plus three-factor adenovirus cell morphology.

FIG. 4 shows glycogen synthesis in the presence of L M315 days after infection of hepatoma cells with three-factor adenovirus, where A is staining of L M3 plus GFP adenovirus PAS (periodic acid-Schiff staining), and B is staining of L M3 plus three-factor adenovirus PAS.

FIG. 5 shows fat synthesis at L M315 days after infection of hepatoma cells with three-factor adenovirus, where A is L M3 plus GFP adenovirus oil red staining and B is L M3 plus three-factor adenovirus oil red staining.

FIG. 6 shows urea secretion after 315 days of infection of hepatoma cells with a three-factor adenovirus L M.

FIG. 7 shows the inhibition of tumor cell growth after infection of hepatocarcinoma cell L M3 with three-factor adenovirus.

FIG. 8 shows the inhibition of tumor cell migration after infection of hepatocarcinoma cell L M3 with three-factor adenovirus, wherein A is L M3 plus GFP adenovirus transmembrane migration, B is L M3 plus three-factor adenovirus transmembrane migration, and C is the quantitative analysis of both transmembrane migration.

FIG. 9 shows the flow analysis of EpCAM positive rate after infection of hepatoma cells by the three-factor adenovirus, wherein A is L M3 plus GFP adenovirus EpCAM positive rate, and B is L M3 plus three-factor adenovirus EpCAM positive rate.

FIG. 10 is a subcutaneous tumor-bearing experiment from patients treated with intratumoral injection of three-factor adenovirus.

Detailed Description

The invention is further illustrated by the following figures and examples, which are intended to be illustrative only and not limiting, and the simple modification of the process of the invention, which is based on the idea underlying the invention, is within the scope of the invention as claimed.

The reagents and starting materials used in the present invention are commercially available or may be prepared according to literature procedures for experiments not specifying the particular conditions in the examples which follow, generally according to conventional conditions such as those described in Sambrook et al, molecular cloning, laboratory Manual (New York: Cold Spring Harbor L laboratory Press, 1989), or according to conventional conditions, or according to the conditions recommended by the manufacturer.

Example 1 three adenoviruses expressing HNF1 α, HNF4 α and FOXA3 were constructed, and the expression of three-factor adenoviruses infecting hepatoma cell lines L M3, P L C and HepG2 cells was examined by western blot.

For example, FOXA3 is packaged at 35mm²293A cells were cultured to 80% -90% confluence, and backbone plasmid pBHG11, vector plasmid pAdTrack-CMV-FOXA3() were prepared. The pBHG11 plasmid and the pAdTrack-CMV-FOXA3 plasmid were ligated in the order of 2: 1 into 100 mul PBS to prepare solution A; mixing 6 μ l PEI into 100 μ l PBS to prepare solution B; and (3) fully and uniformly mixing the solution A and the solution B, standing at room temperature for 20min, and transfecting 293A cells. After 12h, the DMEM medium containing 5% of the pseudobovine serum was replaced. 7-10 days later, after obvious plaque is formed, sucking plaque cell and adding it into 293A-inoculated 24-well plate, amplifying virus clone and harvesting cell, repeatedly freezing-thawing at-80 deg.C and 37 deg.C for 3 times to obtain adenovirus supernatantTaking 5ul for PCR amplification (PCR primer provided by kit), observing cells in a 24-well plate, if the cells float completely, sucking all the cells and culture medium to infect 293A cells with 80% confluence rate in a 10cm culture plate, after 36h, collecting the cells, repeatedly freezing and thawing for 3 times, harvesting virus lysate, purifying by using an adenovirus purification kit of BD company, collecting virus particles, and determining the titer of adenovirus by using an adenovirus titer determination kit of BD company, wherein the preparation methods of two adenoviruses HNF1 α and HNF4 α are the same as above.

Ad HNF1 α, AdHNF4 α and AdFOXA3 were mixed and infected with L M3, HepG2, P L C, after 4 hours, the cell lysate was collected into whole cell protein, after 24 hours, after the protein standard was quantified, 20ug each of the proteins was electrophoretically separated in 10% SDS-PADE, the lower filter, nitrocellulose (NC membrane), electrophoresis gel and upper filter were sequentially superposed and equilibrated in transfer Buffer (Transferring Buffer), and then placed in a semidry electric transfer apparatus, 15V, 70min, after 1 hour of blocking the membrane at room temperature with 5% skim milk 20ml, and after TBST washing for 3 6855 min, HNF1 α (from Santa Cruz, sc-10791), HNF4 α (from Abcam, 92ab), FOXA3 (from Santa Cruz, sc-74424) primary antibody (1:1000) was incubated with rabbit monoclonal antibody (1:1000), and after incubation with fluorescent light beam light, gamma, and fluorescent light, fluorescent, light.

The results show that the expression of HNF1 α, HNF4 α and FOXA3 can be obviously improved after the human liver cancer cell line is infected by the adenovirus of the three factors (figure 1).

Example 2: the three factors adenovirus infect liver cancer cell, real-time quantitative PCR detects the expression of liver cell relative function gene.

(1) L M3, P L C, HepG2 and SMMC-7721 are put in a 6-well plate and cultured until the density is 50-60%, then AdHNF1 α, AdHNF4 α and AdFOXA3 infect cells by MOI100, 10% fetal calf serum fresh culture medium is replaced in 4 hours, (2) TRIZO L1 ml is added after 2 days, the mixture is stood at room temperature for 3min, (3) 200 mul chloroform is added, the mixture is vigorously shaken for 30sec and stood at room temperature for 3min, (4)4 ℃, 12,000rpm and centrifuged for 15min, (5) an upper layer water phase containing RNA is sucked in a new 1.5ml Ep tube, 500 mul isopropanol is added to precipitate the RNA, the mixture is vigorously shaken for 30sec and stood on ice for 10min, (6)4 ℃, 12,000rpm, centrifuged for 15min, (7) a supernatant is discarded, 0.5ml ethanol is added to wash the RNA precipitate, 4 ℃, 12,000rpm, 5min is centrifuged, (8) the ethanol is removed, the RNA is stored in a blower, the RNA is dried under a drier, the condition that the RNA content of the RNA is dissolved in a first ultraviolet light, and the RNA is dissolved in a first sample (20 ℃) after the RNA is generated:

(11) the obtained cDNA is used for fluorescent quantitative PCR detection. The relative expression quantity of the target gene is analyzed after internal reference.

Real-Time PCR step

(12) Mixing, pre-denaturing at 95 deg.C for 10min, denaturing at 95 deg.C for 30sec, annealing at 60 deg.C for 30sec, extending at 72 deg.C for 40sec, and extending at 72 deg.C for 10min for 40 cycles. The results were analyzed using Applied Biosystems 7300/7900Fast Real-Time PCRSystem software.

TABLE 1 human primer sequences for liver function

Albumin (albumin, A L B), cytochrome P450 family 1 α 2 (cytochromes P4501 α 2, CYP1 α 2), phosphoenolpyruvate carboxykinase (PEPCK), glucose-6-phosphatase (glucose-6-phosphatase, G-6-P), Glutamine Synthetase (GS), α -antitrypsin (α 1-antitrypsin, AAT), multidrug resistance-associated protein 2(multidrug resistance-associated proteins 2, MRP 2).

The results show that the combined effect of the three factors in improving liver functions of liver cancer is obviously better than the effect of single factors and pairwise combination (1+4, 1+ F and 4+ F) of HNF1 α, HNF4 α and FOXA3 (figure 2).

Example 3 cytomorphological Observation of hepatocarcinoma cells L M315 days after infection of three-factor adenovirus

L M3/GFP and L M3/3F cells infected with three factors and GFP adenovirus for 15 days are respectively planted in a 6-well plate according to 2000 wells, after 7 days, the morphology of living cells is observed in an inverted phase contrast microscope (× 200 times), L M3/GFP cells can be observed through a light microscope to be small in morphology and round and closely arranged (shown in figure 3A), and L M3/3F cells are larger and flat and wide in morphology (shown in figure 3B).

Example 4 Effect of three factors adenovirus on glycogen synthesis at day L M315 after infection of hepatoma cells.

Respectively planting L M3/GFP and L M3/3F cells in a 12-hole plate, removing a culture medium after adhering to the wall for one day, washing for 2 times by PBS, adding 10% neutral formaldehyde for fixing for 10min, washing for 1min by water flow, washing for 5min by 1% periodic acid at room temperature, washing for 5min by distilled water, adding a Schiff's liquid chamber for 15min, washing for 5min by distilled water, and observing the dyeing condition under an optical microscope.

The results showed that L M3/3F cells were more red and glycogen synthesized more than L M3/GFP cells (FIG. 4).

Example 5 Effect of three factors adenovirus on fat synthesis in L M315 days after infection of hepatoma cells.

Respectively planting L M3/GFP and L M3/3F cells in a 12-hole plate, removing a culture medium after adhering to the wall for one day, washing for 2 times by PBS, adding 10% neutral formaldehyde for fixing for 30min, washing for 1min by 70% ethanol, dyeing for 15min by oil red dye prepared by 70% ethanol, washing for 1min by 70% ethanol, dyeing for 90sec by hematoxylin, performing water flow for 15sec, and observing the dyeing condition under an optical microscope.

The results showed that L M3/3F cells contained more lipid droplets than L M3/GFP cells (FIG. 5).

Example 6 Effect of three factors adenovirus on Urea secretion in L M315 days after infection of hepatoma cells.

L M3/GFP and L M3/3F cells are cultured according to 10000 cells per well and 96 wells, each cell is provided with 3 multiple wells, after the cells are attached to the wall, each well is replaced by a serum-free culture medium of 100 mu L, the cells are placed in a 37 ℃ and 5% CO2 incubator for 24h and then supernatant is sucked, Urea is measured according to the instructions of Quantit ChromTM Urea Assay Kit (purchased from Bioassay Systems company), 100 mu L CCK8 working solution is added to each remaining cell, after the cells are cultured for 1h in the incubator, an enzyme reader is placed for detecting at the wavelength of 450nm, the value of each well is read and the average value is calculated, the OD L M3/GFP cell proliferation condition is taken as the standard, the relative proliferation condition of L M3/3F is normalized, and the measured Urea content is divided by the relative CCK8 value to obtain the relative Urea secretion value of the cells.

The results showed that L M3/3F cells were able to secrete more urea than L M3/GFP cells (FIG. 6).

Example 7 inhibition of tumor cell growth after infection of hepatocarcinoma cell L M3 with adenovirus containing three factors.

L M3 cells in logarithmic growth phase are planted in 2 6-hole plates and are respectively infected with three factors and GFP adenovirus, liquid is changed after 4h, the cells are inoculated into a 96-hole plate according to the density of 5000 cells in each hole after 24h, 3 multiple holes are arranged, 200 mu L culture medium is added into each hole, the 96-hole plate is placed in a 5% CO2 incubator at 37 ℃ for 0d,1d,2d,3d,4d and 5d, the culture medium is sucked and removed at each time point, 100 mu L CCK8 working solution is added into each hole, the culture medium is placed in the incubator for 1h, an ELISA reader is used for detecting at the wavelength of 450nm, the OD value of each hole is read, the average value is calculated, and the time is used as the horizontal coordinate, and the OD value average value is used as the vertical coordinate, and a growth curve is drawn.

After continuous measurement of the growth curves of the two cells for multiple days, the expression of the three factors has obvious inhibition effect on the proliferation of the liver cancer cells, the proliferation of the cells of the control group is obvious along with the time, and the proliferation of the cells of the three factors is smooth (figure 7).

Example 8 inhibition of tumor cell migration after infection of hepatocarcinoma cell L M3 with adenovirus containing three factors.

L M3 cells in logarithmic growth phase are planted in 2 6-hole plates and are respectively infected with three factors and GFP adenovirus, the solution is changed after 4 hours, the cells are inoculated into an upper chamber of a transwell according to the density of 2 × 105 cells per hole after 24 hours, a culture medium with 200 mu L per hole is kept in each hole and is placed in a 24-hole plate, a culture medium containing 20% of calf serum is injected into a lower 24-hole plate and is placed in a 37 ℃ and 5% CO2 incubator for culture, the transwell is taken out after 20 hours, the culture medium of the upper chamber is sucked out, PBS is washed for 2 times, the cells are fixed for 10 minutes by 10% neutral formaldehyde, PBS is washed for 2 times, 0.1% crystal violet is dyed for 15 minutes, PBS is washed for 2 times, the cells dyed in the upper chamber are carefully erased by a cotton stick, and the cells are observed by an inverted phase contrast microscope (× 100 times).

The results show that the three-factor cell can obviously inhibit the migration ability of the liver cancer cell (figure 8).

Example 9: EpCAM expression after the liver cancer cells are infected by the three factors.

L M3 cells in a logarithmic growth phase are planted in 2 well plates, infected with three factors and GFP adenovirus respectively, the solution is changed after 4h, the cells are collected on the 2 nd day, washed by PBS for 2 times, EpCAM (purchased from Bio L egend company) is used as primary antibody to be incubated at 4 ℃ for 30min, the cells are flicked once every 5min to be fully contacted, and the proportion of the EpCAM + cells is detected by a flow cytometer.

The results showed a significant decrease in the cell proportion of EpCAM + in hepatoma cells after three-factor infection (fig. 9). EpCAM + is a specific marker of the tumor stem cells, and the proportion of EpCAM + cells is reduced, which indicates that the three factors can promote the differentiation of the tumor stem cells.

Example 10 three-factor adenovirus treatment of patients derived from subcutaneous tumor-bearing tumors.

The patient is prepared from liver cancer tissue at a concentration of about 0.5cm³Inoculating to the underarm skin of nude mouse, and allowing the tumor to grow to 500cm after 1 month³Post intratumoral injection of a total pfu (plaque forming unit) of 1 × 10¹⁰Three-factor adenovirus, the control group was injected with the same pfu (plaque forming unit) amount of GFP adenovirus, and the change in size of tumor body was measured by injection every other day.

The results showed that two weeks after treatment, the experimental nude mice had significantly reduced tumor volume compared to the control group (fig. 10).

While the preferred embodiments of the present invention have been described in detail, it will be understood by those skilled in the art that the invention is not limited thereto, and that various changes and modifications may be made without departing from the spirit of the invention, and the scope of the appended claims is to be accorded the full scope of the invention.

Claims

1. The application of the combination of three hepatocyte nuclear factor HNF1 α, HNF4 α and FOXA3 genes and/or proteins in preparing a reagent or a composition for inducing liver cancer differentiation is to induce the differentiation of poorly differentiated liver cancer cells to mature phenotype, inhibit the proliferation and transfer of the liver cancer cells and promote the apoptosis of the liver cancer cells.

2. The use of the combination of the three hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 genes and/or proteins according to claim 1 in the preparation of an agent or a composition for inducing liver cancer differentiation, wherein the agent is an agent for increasing the expression level of HNF1 α, HNF4 α and FOXA 3.

3. The use of the combination of the three hepatocyte nuclear factors HNF1 α, HNF4 α, FOXA3 genes and/or proteins according to claim 1 in the preparation of an agent or a composition for inducing differentiation of liver cancer, wherein the composition is a pharmaceutical composition, and comprises the proteins, coding sequences or expression vectors containing the coding sequences of the three hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3, and pharmaceutically acceptable carriers or excipients.

4. The use of the combination of three hepatocyte nuclear factors HNF1 α, HNF4 α, FOXA3 genes and/or proteins according to claim 1, wherein the hepatocyte nuclear factors HNF1 α, HNF4 α and FOXA3 are human hepatocyte nuclear factors.

5. The application of the combined medicine in preparing the liver cancer differentiation treatment medicine is to induce the poorly differentiated liver cancer cells to differentiate towards mature phenotype, inhibit the proliferation and transfer of the liver cancer cells and promote the apoptosis of the liver cancer cells; the active ingredients of the combined medicament comprise any one of the following components:

(a) HNF1 α protein, HNF4 α protein, and FOXA3 protein;

6. The use of the combination as claimed in claim 5 in the preparation of a medicament for the differentiation therapy of liver cancer, wherein the expression vector comprises adenovirus, lentivirus, retrovirus and adeno-associated virus and non-viral vectors.

7. The use of the combination according to claim 5 for the preparation of a medicament for the differentiation therapy of liver cancer, wherein the combination further comprises a pharmaceutically acceptable carrier or excipient.

8. The use of the combination according to claim 5 in the preparation of a medicament for the differentiation treatment of liver cancer, wherein the combination further comprises a chemotherapeutic agent.