Research method for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REG gamma signal channel

Abstract

The invention belongs to the technical field of biomedicine, and particularly relates to a research method for inhibiting liver cancer by regulating glycolysis through an SIK1 / PKM2 / REG gamma signal pathway, which comprises the following steps: S1, in-vitro cytology experiment; s2, in-vivo animal experiments are carried out, lentiviral vector plasmids interfering or stably expressing SIK1, REG gamma and PKM2 are constructed, after corresponding stable low-expression and high-expression cell strains are obtained, subcutaneous tumor models in the logarithmic phase are taken, subcutaneous injection is carried out on the subcutaneous tumor models, the subcutaneous tumor models are inoculated to the two sides of the back of 4-5-week-old BALB / c nude mice according to 2 * 10 < 6 > / mouse, and 10 mice are inoculated into each group; the formation condition of the subcutaneous tumor is observed every week, the diameter of the tumor is measured, the volume of the tumor is calculated, and the volume of the tumor is equal to long diameter * short diameter square / 2; and S3, clinical verification. According to the application disclosed by the invention, the correlation of the expression levels of SIK1, PKM2, phosphorylated PKM2 and REG gamma in tissues of clinical hepatocellular carcinoma patients is clarified, and the relationship between the expression levels and whether the hepatocellular carcinoma is metastatic or not is clarified in combination with follow-up visit data, so that a scientific basis is provided for targeted therapy of clinical hepatocellular carcinoma.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to a research method for inhibiting liver cancer by regulating glycolysis through a SIK1 / PKM2 / REGγ signaling pathway. Background Art

[0002] Salt-inducible kinase (SIK) includes three members: SIK1, SIK2 and SIK3. SIK is a serine / threonine kinase, which is classified into the same family because its amino acid sequence is similar to the subunit of the heterotrimer of adenylate-activated protein kinase (AMP-activated protein kinase, AMPK). Some articles have confirmed that SIK1 expression is reduced in hepatocellular carcinoma cell lines, and overexpression of SIK1 can inhibit the occurrence and metastasis of hepatocellular carcinoma. In addition, according to literature reports, SIK1 plays an important role in the growth of glomerular mesangial cells induced by high glucose, which highly suggests that SIK1 is closely related to glucose metabolism. At the same time, Johanna et al. found that SIK2 has a great relationship with the homeostasis of glucose metabolism, and is mainly involved in the regulation of blood glucose concentration by insulin and glucagon.

[0003] Pyruvate kinase muscle isozyme M2 (PKM2) is essential for tumor cell growth. PKM2 is an enzyme that catalyzes glycolysis and is a key enzyme in promoting tumor metabolism and growth. There are four PK subtypes in mammalian cells: PKM1, PKM2, PKL, and PKR. PKM2 plays a role in the occurrence and development of tumors. Its main functions are: first, as a low-activity enzyme in the cytoplasm, it maintains the glycolytic phenotype and promotes the production of intermediate metabolites in the synthesis of biomacromolecules; second, it translocates into the nucleus and acts as a nuclear protein that phosphorylates specific protein kinases. Studies have found that insulin can also induce PKM gene expression by activating the PI3K / AKT / mTOR / HIF-1α signaling pathway; and silencing tumor suppressor genes during the occurrence of hepatocellular carcinoma can also promote PKM2 expression by activating AKT to promote mitogen-activated protein kinase (MAPK) activity. In addition, it has been confirmed in bladder cancer, breast cancer, colon cancer and lung cancer that the methylation status of PKM2 exons is closely related to PKM2 expression, proving that DNA methylation plays an important role in the regulation of PKM2 expression.

[0004] REGγ is a member of the 11S family and a proteasome activator of the 20S core proteasome. It can promote the degradation of Ki antigens. It is a multi-protein, including p53 and MDM2, and exists independently in the form of ubiquitinase. In recent years, there have been many reports on the anti-cancer effect of REGγ. Studies have shown that the REGγ gene sequence has different degrees of mutations in breast cancer, endometrial cancer, oral cancer, and skin cancer, and its mutation may be closely related to the occurrence of tumors. In the latest domestic and foreign studies, Chai F et al. found that AP-1, a downstream effector of MAPK / p38, can directly bind to the REGγ promoter and activate REGγ transcription when stimulated by TPA. Its anti-cancer mechanism is mainly as follows: REGγ can activate the Wnt / β-catenin signaling pathway by promoting the degradation of GSK-3β, and increase the expression of β-catenin downstream genes c-myc and CyclinD1, inhibit MAPK / p38 activation of REGγ, and inhibit TPA-induced increase in CyclinD1 and c-myc expression. This study shows that REGγ can activate the Wnt / β-catenin signaling pathway by regulating MAPK / p38 and plays an important role in skin tumorigenesis.

[0005] However, there are currently no reports addressing the relationship between SIK1, REGγ and PKM2, and how SIK1 regulates glycolysis to control the occurrence and development of hepatocellular carcinoma. Summary of the invention

[0006] 1. Technical problems to be solved

[0007] The purpose of the present invention is to solve the problem that there is no report in the prior art on the relationship between SIK1, REGγ and PKM2, and to propose a research method for inhibiting liver cancer by regulating glycolysis through the SIK1 / PKM2 / REGγ signaling pathway.

[0008] 2. Technical solution

[0009] In order to achieve the above object, the present invention adopts the following technical solutions:

[0010] The research method of inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway includes the following steps:

[0011] S1: In vitro cytological experiment, including the following steps:

[0012] S1.1: Cell culture;

[0013] S1.2: Mass spectrometry LC-MS analysis of target proteins that may bind to SIK1;

[0014] S1.3: Co-immunoprecipitation CO-IP verifies the binding of SIK1 to REGγ and PKM2;

[0015] S1.4: GST-pull down to verify the binding of SIK1 to REGγ and PKM2;

[0016] S1.5: CO-IP and GST-pull down to verify the specific domains of PKM2, REGγ and SIK1 binding;

[0017] S1.6: Identification of SIK1 phosphorylation sites of PKM2 by isotope and point mutation experiments;

[0018] S2: In vivo animal experiment, construct lentiviral vector plasmids that interfere with or stably express SIK1, REGγ, and PKM2, obtain corresponding stable low-expression and high-expression cell lines, and take the logarithmic growth period. Subcutaneous tumor model: subcutaneous injection, 2×106 / mouse inoculated into both sides of the back of 4-5 week-old BALB / c nude mice, 10 / group; detect subcutaneous tumor: observe subcutaneous tumor formation every week, measure tumor diameter, and calculate tumor volume, tumor volume = long diameter × short diameter square / 2;

[0019] S3: Clinical verification: detect the expression of SIK1, REGγ, PKM2 and phospho-PKM2 in normal tissues, adjacent tissues and hepatocellular carcinoma tissues, and analyze the correlation of their expression levels; combine clinical follow-up data to analyze whether their expression levels are related to the prognosis of hepatocellular carcinoma patients.

[0020] Preferably, the cell culture in S1.1 is as follows: cells of hepatocellular carcinoma cell lines MHCC97H, HepG2, SMMC-7721, Hep3B, HCCLM3, etc. are cultured in DMEM culture medium containing 10% fetal bovine serum and penicillin / streptomycin, and stable high-expression / low-expression cell lines are constructed by amplifying the target gene from the cDNA library or synthesizing the DNA oligo of the target gene by PCR, and the target gene is directionally exchanged with the linear vector cut by enzyme, and the product is transformed into bacterial competent cells, and the grown clones are first identified by colony PCR, and then the clones identified positive by PCR are sequenced and compared. The clones with correct comparison are the successfully constructed plasmids, and finally the constructed fusion protein expression vector is ultrapurified to remove endotoxin and extract to improve the purity of the plasmid, according to Invitrogen's Lipofectamine 293T cells were co-transfected with 2000 according to the instructions. The culture medium was replaced with complete medium 8 hours after transfection. After culturing for 48 hours, the cell supernatant rich in lentiviral particles was collected and concentrated to obtain a high-titer lentiviral concentrate. The virus titer MOI was measured and calibrated in 293T cells. The target cells were infected according to the most suitable MOI. The transfection efficiency was observed under a fluorescence microscope 96 hours later, and the total cell RNA and protein were extracted. The above experimental methods were used to apply real-time quantitative RT-PCR technology and Western blot technology to detect the changes in the mRNA level and protein level of the target gene after transfection to further verify the cell transfection efficiency.

[0021] Preferably, the mass spectrometry LC-MS analysis of the target protein that may bind to SIK1 in S1.2 is specifically as follows: Flag-SIK1 is transfected into the hepatocellular carcinoma cell line HCCLM3, the protein is extracted after 48 hours, the Flag-SIK1 complex in the cells is precipitated using Flag-Beads, the cells are shaken overnight at 4°C, and the cells are centrifuged at 14000g for 5s the next day, the precipitate is collected, and washed three times with pre-cooled washing buffer or pre-cooled PBS, the supernatant is collected, a portion of the protein sample is electrophoresed on SDS-PAGE and silver stained to verify that the precipitated protein is a Flag-SIK1 complex, the target protein band is sent to mass spectrometry analysis, and the protein bound to SIK1 is identified.

[0022] Preferably, the immunoprecipitation CO-IP in S1.3 is specifically as follows: HA-SIK1 and GFP-REGγ or Flag-PKM2 are co-transfected into the hepatocellular carcinoma cell line MHCC97H HepG2; after 48 hours, the cells are washed twice with pre-cooled PBS, pre-cooled RIPA lysis buffer is added, 1 ml is added for 107 cells, the cells are scraped off the culture medium with a pre-cooled cell scraper, and transferred to a clean 1.5 ml EP tube; placed on a low-speed shaker, slowly shaken at 4°C for 15 min, and centrifuged at 4°C, 14000g for 15 min; the supernatant is immediately transferred to a new centrifuge tube; the Protein A / G-agarose microspheres are washed twice with PBS; a 50% protein A / G-agarose working solution is prepared with PBS, and 50% Protein A / G is added to every 1 ml at a ratio of 100 μl in the product. agarose working solution, shake on a horizontal shaker for 10 minutes, centrifuge at 14000g for 15 minutes at 4°C; transfer the supernatant to a new centrifuge tube, remove the protein A / G-agarose microspheres, use the BCA method or other methods to determine the concentration of total protein, dilute the total protein to 1μg / μl with PBS to reduce the concentration of detergent in the lysate, add a certain volume of primary antibody to a total volume of about 500μl, shake the antigen-antibody mixture slowly on a shaker, incubate at 4°C overnight, centrifuge at 14000g for 5s, collect the precipitate; and wash 3 times with pre-cooled washing buffer (or pre-cooled PBS), adding 800μl each time, collect the supernatant, and detect the expression of HA and Flag by WB.

[0023] Preferably, the GST-pull down verification of the binding of SIK1 to REGγ and PKM2 in S1.4 is specifically as follows: using a protein purification system to obtain purified GST-SIK1, HA-REGγ and His-PKM2 proteins, fully mixing the GST-SIK1 protein with the His-PKM2 and HA-REGγ proteins in a reaction solution, adding GST magnetic beads, shaking at 4°C for 2h, then centrifuging at 13000rpm for 10s at 4°C, discarding the supernatant, retaining the magnetic beads; and washing 3 times with pre-cooled washing buffer or pre-cooled PBS, adding SDS-PAGE, boiling and detecting GST, His and HA with WB.

[0024] Preferably, the specific domains of PKM2, REGγ and SIK1 bound by CO-IP and GST-pull down in S1.5 are specifically as follows: the domain of the target protein is obtained according to ExPASy, different domains of the target gene are amplified by PCR, and after connecting with the vector, expression plasmids of different domains are constructed, and the specific domains of PKM2, REGγ and SIK1 bound are verified again by the above method.

[0025] Preferably, the isotope and point mutation experiments in S1.6 to identify the SIK1 phosphorylation site of PKM2 are specifically as follows: using NetPhos 2.0Server and phosphonet to predict the SIK1 phosphorylation site of PKM2, adding ATP, SIK1 kinase and PKM2 purified protein labeled with isotope 32P to the isotope reaction buffer, performing isotope autoradiography after half an hour, and using the QuikChange II Site-Directed Mutagenesis kit Stratagene to construct a possible PKM2 phosphorylation site point mutation expression plasmid, and repeating the above isotope experiment.

[0026] Preferably, S2 also includes:

[0027] S2.1: To clarify the effect of SIK1 on hepatocellular carcinoma metastasis in vivo, construct a lentiviral vector plasmid sh-Luc-SIK1 with fluorescence interference of SIK1 expression, and infect the highly malignant human hepatocellular carcinoma cell line MHCC97H; and construct a lentiviral vector plasmid LV-Luc-SIK1 with fluorescence stable expression of SIK1, and infect the low-grade malignant human hepatocellular carcinoma cell line HepG2. Use the BALB / C nude mouse hepatocellular carcinoma orthotopic carcinoma metastasis model and hepatocellular carcinoma lung metastasis model to compare the metastatic tumor volume, weight, survival of tumor-bearing mice, and immunohistochemical detection in the control group, SIK1 high expression group, and SIK1 low expression group;

[0028] S2.2: To clarify the effect of phosphorylated PKM2 on hepatocellular carcinoma metastasis in vivo, a lentiviral vector plasmid LV-Luc-SA-PKM2 with fluorescent stable expression of continuously phosphorylated PKM2 was constructed and infected with the low-grade human hepatocellular carcinoma cell line HepG2. The BALB / C nude mouse hepatocellular carcinoma orthotopic carcinoma metastasis model and hepatocellular carcinoma lung metastasis model were used to compare the metastatic tumor volume, weight, survival of tumor-bearing mice and immunohistochemistry detection in the control group, SIK1 high expression group and SIK1 low expression group. In the nude mouse orthotopic hepatocellular carcinoma metastasis model, 2×106 / 50ul cells in the logarithmic growth phase were taken and injected into the liver lobe of 4-5 week-old BALB / c nude mice. After 6 weeks, the in vitro imaging system was used to detect the metastasis of tumor cells to the abdominal organs. In the nude mouse hepatocellular carcinoma lung metastasis model, 1×106 / 100ul cells in the logarithmic growth phase were taken and injected into the tail vein of 4-5 week-old BALB / c nude mice. After 6 weeks, the in vitro imaging system was used to detect the metastasis of tumor cells to the lungs.

[0029] 10. Preferably, S3 also includes:

[0030] S3.1: Real-time quantitative RT-PCR was used to detect the expression of SIK1, REGγ and PKM2 mRNA in fresh cancer tissues and adjacent tissues. Total RNA was extracted from fresh cancer tissues and adjacent tissues using the Trizol one-step method. RNA was reverse transcribed into cDNA using the Tapman PerfectReal Time kit. Primers and taqman probe sequences for SIK1, REGγ, PKM2 and internal reference GAPDH were synthesized according to the standard real-time quantitative RT-PCR primer design principles. The final results were expressed as the ratio of SIK1, REGγ, PKM2 to internal reference GAPDH.

[0031] S3.2: Western Blot was used to determine the protein expression of SIK1, REGγ and PKM2 in fresh cancer tissues and paracancerous tissues. About 100 mg of fresh cancer tissues and paracancerous tissues were collected, lysate was added, homogenized in an ice bath, centrifuged for 10 min, and the supernatant was subjected to protein determination by the modified Lowry method. An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples, and the samples were heated at 100°C or in a boiling water bath for 3-5 min to fully denature the proteins. 50 μg of protein was taken for sodium dodecyl sulfate-polyacrylamide gel SDS-PAGE electrophoresis, transferred to the membrane, and rinsed with PBST after blocking. Rabbit anti- or mouse anti-SIK1, REGγ, PKM2 and GAPDH monoclonal antibodies from Abcam were added respectively, and incubated at 4°C overnight. After rinsing with PBST, hybridization was performed with goat anti-rabbit or anti-mouse IgG secondary antibodies labeled with horseradish peroxidase, and the samples were shaken at 37°C for 2 h. The filter membrane was washed 3 times with PBST by shaking, and the color was developed using an enhanced chemiluminescence color development system, and developed on film in a dark room.

[0032] S3.3: Immunohistochemistry was used to detect paraffin cancer tissue specimens and adjacent tissue specimens. Paraffin-embedded cancer tissues and adjacent tissue specimens were selected for 5 μm continuous sectioning, dewaxed, hydrated, and incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase; then placed in EDTA and reacted in a microwave oven at 90°C for 10 min for antigen repair, cooled to room temperature and rinsed with PBS three times; rabbit anti- or mouse anti-SIK1, REGγ, PKM2 monoclonal antibodies were added dropwise, PBS was used as a negative control, and incubated at 4°C overnight; after rinsing with PBS, horseradish peroxidase-labeled goat anti-rabbit secondary antibodies were added dropwise, incubated at 37°C for 20 min, and diaminobenzidine was used for color development;

[0033] S3.4: Statistical analysis: SPSS13.0 statistical software was used for analysis. Mann-Whitney U rank sum test was used to analyze the differences in the expression of SIK1, REGγ, and PKM2 proteins in cancer tissues and adjacent tissues. The relationship between the different levels of SIK1, REGγ, and PKM2 protein expression and the clinical pathological parameters of patients was also analyzed. The Kaplan-Meier survival analysis method was used to analyze the effects of high and low expression of SIK1, REGγ, and PKM2 on the disease-free survival and overall survival of patients. The Log-Rank test was used for survival curve analysis.

[0034] 3. Beneficial effects

[0035] Compared with the prior art, the advantages of the present invention are:

[0036] (1) The present invention first reveals that SIK1 negatively regulates glycolysis in hepatocellular carcinoma, thereby affecting the occurrence and development of hepatocellular carcinoma. It first reveals that SIK1 can phosphorylate PKM2, and phosphorylated PKM2 binds to REGγ and then ubiquitinates and degrades SIK1, thereby forming a negative feedback loop.

[0037] (2) In this invention, the SIK1 phosphorylation site on PKM2 was identified for the first time, and the effect of this site on glycolysis in hepatocellular carcinoma was revealed. At the same time, the corresponding phosphorylation antibody was prepared based on this site, which has long-term translational value;

[0038] (3) In the present invention, the correlation between the expression levels of SIK1, PKM2, phosphorylated PKM2 and REGγ in the tissues of clinical hepatocellular carcinoma patients is further clarified, and the relationship between their expression levels and whether hepatocellular carcinoma metastasizes is clarified in combination with follow-up data, which will provide a scientific basis for the targeted treatment of clinical hepatocellular carcinoma. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 A schematic diagram of the technical route of the research method proposed in the present invention for inhibiting liver cancer by regulating glycolysis through the SIK1 / PKM2 / REGγ signaling pathway;

[0040] Figure 2 This is a schematic diagram of the results of mass spectrometry identification of SIK1 binding proteins proposed in the present invention;

[0041] Figure 3 This is a result diagram showing the inhibition of sugar consumption, lactic acid production and glycolysis efficiency of hepatocellular carcinoma cells by SIK1 proposed in the present invention;

[0042] Figure 4 The SIK1 phosphorylation motif diagram on the PKM2 protein sequence proposed in the present invention;

[0043] Figure 5 This is the binding diagram of SIK1, PKM2 and REGγ proposed in the present invention;

[0044] Figure 6 This is a graph showing the expression results of SIK1 and PKM2 in tumor tissues proposed in the present invention. DETAILED DESCRIPTION

[0045] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments.

[0046] Embodiment 1:

[0047] Reference Figure 1 The research method of inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway comprises the following steps:

[0048] S1: In vitro cytological experiment, including the following steps:

[0049] S1.1: Cell culture;

[0050] S1.2: Mass spectrometry LC-MS analysis of target proteins that may bind to SIK1;

[0051] S1.3: Co-immunoprecipitation CO-IP verifies the binding of SIK1 to REGγ and PKM2;

[0052] S1.4: GST-pull down to verify the binding of SIK1 to REGγ and PKM2;

[0053] S1.5: CO-IP and GST-pull down to verify the specific domains of PKM2, REGγ and SIK1 binding;

[0054] S1.6: Identification of SIK1 phosphorylation sites of PKM2 by isotope and point mutation experiments;

[0055] S2: In vivo animal experiment, construct lentiviral vector plasmids that interfere with or stably express SIK1, REGγ, and PKM2, obtain corresponding stable low-expression and high-expression cell lines, and take the logarithmic growth period. Subcutaneous tumor model: subcutaneous injection, 2×106 / mouse inoculated into both sides of the back of 4-5 week-old BALB / c nude mice, 10 / group; detect subcutaneous tumor: observe subcutaneous tumor formation every week, measure tumor diameter, and calculate tumor volume, tumor volume = long diameter × short diameter square / 2;

[0056] S3: Clinical verification: detect the expression of SIK1, REGγ, PKM2 and phospho-PKM2 in normal tissues, adjacent tissues and hepatocellular carcinoma tissues, and analyze the correlation of their expression levels; combine clinical follow-up data to analyze whether their expression levels are related to the prognosis of hepatocellular carcinoma patients.

[0057] In this example, it is revealed for the first time that SIK1 negatively regulates glycolysis in hepatocellular carcinoma, thereby affecting the occurrence and development of hepatocellular carcinoma. It is revealed for the first time that SIK1 can phosphorylate PKM2, and phosphorylated PKM2 binds to REGγ and then ubiquitinates and degrades SIK1, thereby forming a negative feedback loop.

[0058] In this example, the SIK1 phosphorylation site on PKM2 was identified for the first time, and the effect of this site on glycolysis in hepatocellular carcinoma was revealed. At the same time, the corresponding phosphorylation antibody was prepared based on this site, which has long-term translational value;

[0059] In this example, the correlation between the expression levels of SIK1, PKM2, phosphorylated PKM2 and REGγ in clinical hepatocellular carcinoma patient tissues was further clarified, and the relationship between their expression levels and whether hepatocellular carcinoma metastasized was explained in combination with follow-up data, which will provide a scientific basis for the targeted treatment of clinical hepatocellular carcinoma.

[0060] Embodiment 2:

[0061] In this embodiment, the cell culture is specifically as follows: the hepatocellular carcinoma cell lines MHCC97H, HepG2, SMMC-7721, Hep3B, HCCLM3 and other cells are cultured in DMEM culture medium, the culture medium contains 10% fetal bovine serum and penicillin / streptomycin, and the stable high expression / low expression cell line is constructed: the target gene is amplified from the cDNA library by PCR method or the DNA oligo of the synthesized target gene is directed to exchange the target gene with the restriction linear vector, and the product is transformed into bacterial competent cells, and the grown clones are first identified by colony PCR, and then the clones identified positive by PCR are sequenced and compared. The clones with correct comparison are the successfully constructed plasmids, and finally the constructed fusion protein expression vector is ultrapurified to remove endotoxin and extract to improve the purity of the plasmid, according to Invitrogen's Lipofectamine 293T cells were co-transfected with 2000 according to the instructions. The culture medium was replaced with complete medium 8 hours after transfection. After culturing for 48 hours, the cell supernatant rich in lentiviral particles was collected and concentrated to obtain a high-titer lentiviral concentrate. The virus titer MOI was measured and calibrated in 293T cells. The target cells were infected according to the most suitable MOI. The transfection efficiency was observed under a fluorescence microscope 96 hours later, and the total cell RNA and protein were extracted. The above experimental methods were used to apply real-time quantitative RT-PCR technology and Western blot technology to detect the changes in the mRNA level and protein level of the target gene after transfection to further verify the cell transfection efficiency.

[0062] Embodiment 3:

[0063] In this embodiment, mass spectrometry LC-MS analysis of target proteins that may bind to SIK1 is specifically as follows: Flag-SIK1 is transfected into the hepatocellular carcinoma cell line HCCLM3, protein is extracted after 48 hours, Flag-SIK1 complex in the cells is precipitated using Flag-Beads, shaken overnight at 4°C, centrifuged at 14000g for 5s the next day, the precipitate is collected, and washed three times with pre-cooled washing buffer or pre-cooled PBS, the supernatant is collected, a portion of the protein sample is electrophoresed on SDS-PAGE and silver stained to verify that the precipitated protein is a Flag-SIK1 complex, the target protein band is sent to mass spectrometry analysis, and the protein bound to SIK1 is identified.

[0064] In this embodiment, the immunoprecipitation CO-IP in S1.3 verifies the binding of SIK1 with REGγ and PKM2 as follows: HA-SIK1 and GFP-REGγ or Flag-PKM2 are co-transfected into the hepatocellular carcinoma cell line MHCC97H HepG2; 48 hours later, the cells are washed twice with pre-cooled PBS, pre-cooled RIPA lysis buffer is added, 1 ml is added for 107 cells, the cells are scraped off the culture medium with a pre-cooled cell scraper, and transferred to a clean 1.5 ml EP tube; the tube is placed on a low-speed shaker, slowly shaken at 4°C for 15 min, and centrifuged at 14000g for 15 min at 4°C; the supernatant is immediately transferred to a new centrifuge tube; the Protein A / G-agarose microspheres are washed twice with PBS; a 50% protein A / G-agarose working solution is prepared with PBS, and 50% Protein A / G is added to each 1 ml at a ratio of 100 μl in the product. agarose working solution, shake on a horizontal shaker for 10 minutes, centrifuge at 14000g for 15 minutes at 4°C; transfer the supernatant to a new centrifuge tube, remove the protein A / G-agarose microspheres, use the BCA method or other methods to determine the concentration of total protein, dilute the total protein to 1μg / μl with PBS to reduce the concentration of detergent in the lysate, add a certain volume of primary antibody to a total volume of about 500μl, shake the antigen-antibody mixture slowly on a shaker, incubate at 4°C overnight, centrifuge at 14000g for 5s, collect the precipitate; and wash 3 times with pre-cooled washing buffer (or pre-cooled PBS), adding 800μl each time, collect the supernatant, and detect the expression of HA and Flag by WB.

[0065] In this embodiment, the GST-pull down in S1.4 verifies the binding of SIK1 to REGγ and PKM2 as follows: using a protein purification system, purified GST-SIK1, HA-REGγ and His-PKM2 proteins are obtained, the GST-SIK1 protein is fully mixed with the His-PKM2 and HA-REGγ proteins in a reaction solution, GST magnetic beads are added, and the mixture is shaken at 4°C for 2 h, followed by centrifugation at 13,000 rpm for 10 s at 4°C, the supernatant is discarded, and the magnetic beads are retained; and the mixture is washed three times with pre-cooled washing buffer or pre-cooled PBS, added to SDS-PAGE, boiled, and then GST, His and HA are detected using WB.

[0066] In this embodiment, the specific domains of PKM2, REGγ and SIK1 binding verified by CO-IP and GST-pull down in S1.5 are as follows: the domain of the target protein is obtained according to ExPASy, the different domains of the target gene are amplified by PCR, and after connecting with the vector, expression plasmids of different domains are constructed, and the specific domains of PKM2, REGγ and SIK1 binding are verified again by the above method.

[0067] In this embodiment, refer to Figure 2 and 3 It can be concluded that after Flag-SIK1 was transfected in the MHCC97H cell line, the protein bound to SIK1 was extracted by immunoprecipitation and identified by LC-MS. SIK1 may be related to phosphorylated PKM2. SIK1 was overexpressed in the MHCC97H cell line, and the glucose consumption (B) and lactate consumption (C) were detected. The glycolysis rate was detected. The results showed that overexpression of SIK1 inhibited the consumption of glucose and lactate and the relative glycolysis rate.

[0068] Embodiment 4:

[0069] In this embodiment, refer to Figure 4 , the isotope and point mutation experiments in S1.6 identified the SIK1 phosphorylation site of PKM2 as follows: NetPhos 2.0Server and phosphonet were used to predict the SIK1 phosphorylation site of PKM2, ATP, SIK1 kinase and PKM2 purified protein labeled with isotope 32P were added to the isotope reaction buffer, and isotope autoradiography was performed after half an hour. At the same time, the QuikChange II Site-Directed Mutagenesis Kit Stratagene was used to construct a possible PKM2 phosphorylation site point mutation expression plasmid, and the above isotope experiment was repeated.

[0070] Embodiment 5:

[0071] In this embodiment, immunoprecipitation was used to verify that SIK1 can bind to REGγ, and S2 also includes: S2.1: clarifying the effect of SIK1 on hepatocellular carcinoma metastasis in vivo, constructing a lentiviral vector plasmid sh-Luc-SIK1 with fluorescence interference SIK1 expression, infecting the highly malignant human hepatocellular carcinoma cell line MHCC97H; and constructing a lentiviral vector plasmid LV-Luc-SIK1 with fluorescence stable expression SIK1, infecting the low-grade malignant human hepatocellular carcinoma cell line HepG2, using a BALB / C nude mouse hepatocellular carcinoma orthotopic carcinoma metastasis model and a hepatocellular carcinoma lung metastasis model, comparing the metastatic tumor volume, weight, survival of tumor-bearing mice, and immunohistochemical detection in the control group, SIK1 high expression group, and low expression group;

[0072] S2.2: To clarify the effect of phosphorylated PKM2 on hepatocellular carcinoma metastasis in vivo, a lentiviral vector plasmid LV-Luc-SA-PKM2 with fluorescent stable expression of continuously phosphorylated PKM2 was constructed and infected with the low-grade human hepatocellular carcinoma cell line HepG2. The BALB / C nude mouse hepatocellular carcinoma orthotopic carcinoma metastasis model and hepatocellular carcinoma lung metastasis model were used to compare the metastatic tumor volume, weight, survival of tumor-bearing mice and immunohistochemistry detection in the control group, SIK1 high expression group and SIK1 low expression group. In the nude mouse orthotopic hepatocellular carcinoma metastasis model, 2×106 / 50ul cells in the logarithmic growth phase were taken and injected into the liver lobe of 4-5 week-old BALB / c nude mice. After 6 weeks, the in vitro imaging system was used to detect the metastasis of tumor cells to the abdominal organs. In the nude mouse hepatocellular carcinoma lung metastasis model, 1×106 / 100ul cells in the logarithmic growth phase were taken and injected into the tail vein of 4-5 week-old BALB / c nude mice. After 6 weeks, the in vitro imaging system was used to detect the metastasis of tumor cells to the lungs.

[0073] In this embodiment, refer to Figure 5 As can be seen, (A) Flag-SIK1 and HA-PKM2 were co-transfected in MHCC97H, and the binding of SIK1 and PKM2 was verified by co-immunoprecipitation (B) Flag-SIK1 and HA-REGγ were co-transfected in HepG2 cells.

[0074] Embodiment 6:

[0075] In this embodiment, S3 also includes: S3.1: Real-time quantitative RT-PCR detection of SIK1, REGγ and PKM2 mRNA expression in fresh cancer tissues and adjacent tissues, Trizol one-step method to extract total RNA from fresh cancer tissues and adjacent tissue cells, using Tapman Perfect Real Time kit to reverse transcribe RNA into cDNA, according to standard real-time quantitative RT-PCR primer design principles, synthesize primers and taqman probe sequences of SIK1, REGγ, PKM2 and internal reference GAPDH, and the final results are expressed as the content ratio of SIK1, REGγ, PKM2 and internal reference GAPDH.

[0076] S3.2: Western Blot was used to determine the protein expression of SIK1, REGγ and PKM2 in fresh cancer tissues and paracancerous tissues. About 100 mg of fresh cancer tissues and paracancerous tissues were collected, lysate was added, homogenized in an ice bath, centrifuged for 10 min, and the supernatant was subjected to protein determination by the modified Lowry method. An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples, and the samples were heated at 100°C or in a boiling water bath for 3-5 min to fully denature the proteins. 50 μg of protein was taken for sodium dodecyl sulfate-polyacrylamide gel SDS-PAGE electrophoresis, transferred to the membrane, and rinsed with PBST after blocking. Rabbit anti- or mouse anti-SIK1, REGγ, PKM2 and GAPDH monoclonal antibodies from Abcam were added respectively, and incubated at 4°C overnight. After rinsing with PBST, hybridization was performed with goat anti-rabbit or anti-mouse IgG secondary antibodies labeled with horseradish peroxidase, and the samples were shaken at 37°C for 2 h. The filter membrane was washed 3 times with PBST by shaking, and the color was developed using an enhanced chemiluminescence color development system, and developed on film in a dark room.

[0077] S3.3: Immunohistochemistry was used to detect paraffin cancer tissue specimens and adjacent tissue specimens. Paraffin-embedded cancer tissues and adjacent tissue specimens were selected for 5 μm continuous sectioning, dewaxed, hydrated, and incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase; then placed in EDTA and reacted in a microwave oven at 90°C for 10 min for antigen repair, cooled to room temperature and rinsed with PBS three times; rabbit anti- or mouse anti-SIK1, REGγ, PKM2 monoclonal antibodies were added dropwise, PBS was used as a negative control, and incubated at 4°C overnight; after rinsing with PBS, horseradish peroxidase-labeled goat anti-rabbit secondary antibodies were added dropwise, incubated at 37°C for 20 min, and diaminobenzidine was used for color development;

[0078] S3.4: Statistical analysis: SPSS13.0 statistical software was used for analysis. Mann-Whitney U rank sum test was used to analyze the differences in the expression of SIK1, REGγ, and PKM2 proteins in cancer tissues and adjacent tissues. The relationship between the different levels of SIK1, REGγ, and PKM2 protein expression and the clinical pathological parameters of patients was also analyzed. The Kaplan-Meier survival analysis method was used to analyze the effects of high and low expression of SIK1, REGγ, and PKM2 on the disease-free survival and overall survival of patients. The Log-Rank test was used for survival curve analysis.

[0079] In this embodiment, refer to Figure 6 , it can be seen that (A) in clinical hepatocellular carcinoma patient specimens, SIK1 expression is high, PKM2 expression is low, and vice versa. (B) The data show that the expression of SIK1 and PKM2 is negatively correlated.

[0080] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. A research method for inhibiting liver cancer by regulating glycolysis via SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The following steps are involved: S1: In vitro cytological experiment, including the following steps: S1.1: Cell culture; S1.2: Mass spectrometry LC-MS analysis of target proteins that may bind to SIK1; S1.3: Co-immunoprecipitation CO-IP verifies the binding of SIK1 to REGγ and PKM2; S1.4: GST-pull down to verify the binding of SIK1 to REGγ and PKM2; S1.5: CO-IP and GST-pull down to verify the specific domains of PKM2, REGγ and SIK1 binding; S1.6: Identification of SIK1 phosphorylation sites of PKM2 by isotope and point mutation experiments; S2: In vivo animal experiment, construct lentiviral vector plasmids that interfere with or stably express SIK1, REGγ, and PKM2, obtain corresponding stable low-expression and high-expression cell lines, and take the logarithmic growth period. Subcutaneous tumor model: subcutaneous injection, 2×106 / mouse inoculated into both sides of the back of 4-5 week-old BALB / c nude mice, 10 / group; detect subcutaneous tumor: observe subcutaneous tumor formation every week, measure tumor diameter, and calculate tumor volume, tumor volume = long diameter × short diameter square / 2; S3: Clinical verification: detect the expression of SIK1, REGγ, PKM2 and phospho-PKM2 in normal tissues, adjacent tissues and hepatocellular carcinoma tissues, and analyze the correlation of their expression levels; combine clinical follow-up data to analyze whether their expression levels are related to the prognosis of hepatocellular carcinoma patients.

2. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The cell culture in S1.1 is as follows: the hepatocellular carcinoma cell lines MHCC97H, HepG2, SMMC-7721, Hep3B, HCCLM3 and other cells are cultured in DMEM culture medium containing 10% fetal bovine serum and penicillin / streptomycin, and stable high-expression / low-expression cell lines are constructed: the target gene is amplified from the cDNA library by PCR or the DNA oligo of the synthesized target gene is directed to exchange the target gene with the enzyme-cut linear vector, and the product is transformed into bacterial competent cells, and the grown clones are first identified by colony PCR, and then the clones identified positive by PCR are sequenced and compared. The clones with correct comparison are the successfully constructed plasmids, and finally the constructed fusion protein expression vector is ultrapurified to remove endotoxin and extract to improve the purity of the plasmid, according to Invitrogen's Lipofectamine 293T cells were co-transfected with 2000 according to the instructions. The culture medium was replaced with complete medium 8 hours after transfection. After culturing for 48 hours, the cell supernatant rich in lentiviral particles was collected and concentrated to obtain a high-titer lentiviral concentrate. The virus titer MOI was measured and calibrated in 293T cells. The target cells were infected according to the most suitable MOI. The transfection efficiency was observed under a fluorescence microscope 96 hours later, and the total cell RNA and protein were extracted. The above experimental methods were used to apply real-time quantitative RT-PCR technology and Western blot technology to detect the changes in the mRNA level and protein level of the target gene after transfection to further verify the cell transfection efficiency.

3. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The mass spectrometry LC-MS analysis of the target protein that may bind to SIK1 in S1.2 is specifically as follows: Flag-SIK1 is transfected into the hepatocellular carcinoma cell line HCCLM3, the protein is extracted after 48 hours, the Flag-SIK1 complex in the cells is precipitated using Flag-Beads, the cells are shaken overnight at 4°C, and the precipitate is centrifuged at 14000g for 5s the next day, the precipitate is collected, and washed three times with pre-cooled washing buffer or pre-cooled PBS, the supernatant is collected, a portion of the protein sample is taken for electrophoresis on SDS-PAGE and silver staining is used to verify that the precipitated protein is a Flag-SIK1 complex, the target protein band is sent for mass spectrometry analysis, and the protein bound to SIK1 is identified.

4. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The immunoprecipitation CO-IP in S1.3 is specifically as follows: HA-SIK1 and GFP-REGγ or Flag-PKM2 are co-transfected into the hepatocellular carcinoma cell line MHCC97H HepG2; 48 hours later, the cells are washed twice with pre-cooled PBS, pre-cooled RIPA lysis buffer is added, 1 ml is added for 107 cells, the cells are scraped off the culture medium with a pre-cooled cell scraper, and transferred to a clean 1.5 ml EP tube; placed in a low-speed shaker, slowly shaken at 4°C for 15 minutes, and centrifuged at 4°C, 14000g for 15 minutes; the supernatant is immediately transferred to a new centrifuge tube; the Protein A / G-agarose microspheres are washed twice with PBS; Prepare 50% protein A / G-agarose working solution with PBS, add 50% protein A / G agarose working solution at a ratio of 100 μl per 1 ml, shake on a horizontal shaker for 10 min, and centrifuge at 14000g for 15 min at 4°C; transfer the supernatant to a new centrifuge tube, remove the protein A / G-agarose microspheres, determine the concentration of total protein using the BCA method or other methods, dilute the total protein to 1 μg / μl with PBS to reduce the concentration of detergent in the lysate, add a certain volume of primary antibody to a total volume of about 500 μl, shake the antigen-antibody mixture slowly on a shaker, incubate at 4°C overnight, centrifuge at 14000g for 5s, collect the precipitate; and wash 3 times with pre-cooled washing buffer (or pre-cooled PBS), adding 800 μl each time, collect the supernatant, and detect the expression of HA and Flag by WB.

5. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The GST-pull down verification of the binding of SIK1 to REGγ and PKM2 in S1.4 is specifically as follows: using a protein purification system to obtain purified GST-SIK1, HA-REGγ and His-PKM2 proteins, the GST-SIK1 protein is fully mixed with His-PKM2 and HA-REGγ proteins in a reaction solution, GST magnetic beads are added, and the mixture is shaken at 4°C for 2 hours, followed by centrifugation at 13000 rpm for 10 seconds at 4°C, the supernatant is discarded, and the magnetic beads are retained; and the mixture is washed three times with pre-cooled washing buffer or pre-cooled PBS, added to SDS-PAGE, boiled, and then GST, His and HA are detected using WB.

6. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The specific domains of PKM2, REGγ and SIK1 binding verified by CO-IP and GST-pull down in S1.5 are as follows: the domain of the target protein is obtained according to ExPASy, different domains of the target gene are amplified by PCR, and after connecting with the vector, expression plasmids of different domains are constructed, and the specific domains of PKM2, REGγ and SIK1 binding are verified again by the above method.

7. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The isotope and point mutation experiments in S1.6 to identify the SIK1 phosphorylation site of PKM2 are specifically as follows: NetPhos 2.0 Server and phosphonet are used to predict the SIK1 phosphorylation site of PKM2, ATP, SIK1 kinase and PKM2 purified protein labeled with isotope 32P are added to the isotope reaction buffer, and isotope autoradiography is performed after half an hour. At the same time, the QuikChange II Site-Directed Mutagenesis Kit Stratagene is used to construct a possible PKM2 phosphorylation site point mutation expression plasmid, and the above isotope experiment is repeated.

8. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The S2 also includes: S2.1: To clarify the effect of SIK1 on hepatocellular carcinoma metastasis in vivo, construct a lentiviral vector plasmid sh-Luc-SIK1 with fluorescence interference of SIK1 expression, and infect the highly malignant human hepatocellular carcinoma cell line MHCC97H; and construct a lentiviral vector plasmid LV-Luc-SIK1 with fluorescence stable expression of SIK1, and infect the low-grade malignant human hepatocellular carcinoma cell line HepG2. Use the BALB / C nude mouse hepatocellular carcinoma orthotopic carcinoma metastasis model and hepatocellular carcinoma lung metastasis model to compare the metastatic tumor volume, weight, survival of tumor-bearing mice, and immunohistochemical detection in the control group, SIK1 high expression group, and SIK1 low expression group; S2.2: To clarify the effect of phosphorylated PKM2 on hepatocellular carcinoma metastasis in vivo, a lentiviral vector plasmid LV-Luc-SA-PKM2 with fluorescent stable expression of continuously phosphorylated PKM2 was constructed and infected with the low-grade human hepatocellular carcinoma cell line HepG2. The BALB / C nude mouse hepatocellular carcinoma orthotopic carcinoma metastasis model and hepatocellular carcinoma lung metastasis model were used to compare the metastatic tumor volume, weight, survival of tumor-bearing mice and immunohistochemistry detection in the control group, SIK1 high expression group and SIK1 low expression group. In the nude mouse orthotopic hepatocellular carcinoma metastasis model, 2×106 / 50ul cells in the logarithmic growth phase were taken and injected into the liver lobe of 4-5 week-old BALB / c nude mice. After 6 weeks, the in vitro imaging system was used to detect the metastasis of tumor cells to the abdominal organs. In the nude mouse hepatocellular carcinoma lung metastasis model, 1×106 / 100ul cells in the logarithmic growth phase were taken and injected into the tail vein of 4-5 week-old BALB / c nude mice. After 6 weeks, the in vitro imaging system was used to detect the metastasis of tumor cells to the lungs.

9. The research method of claim 1 for inhibiting liver cancer by regulating glycolysis through SIK1 / PKM2 / REGγ signaling pathway, characterized in that: The S3 also includes: S3.1: Real-time quantitative RT-PCR was used to detect the expression of SIK1, REGγ and PKM2 mRNA in fresh cancer tissues and adjacent tissues. Total RNA was extracted from fresh cancer tissues and adjacent tissues using the Trizol one-step method. RNA was reverse transcribed into cDNA using the Tapman Perfect RealTime kit. Primers and taqman probe sequences for SIK1, REGγ, PKM2 and internal reference GAPDH were synthesized according to the standard real-time quantitative RT-PCR primer design principles. The final results were expressed as the ratio of SIK1, REGγ, PKM2 to internal reference GAPDH. S3.2: Western Blot was used to determine the protein expression of SIK1, REGγ and PKM2 in fresh cancer tissues and paracancerous tissues. About 100 mg of fresh cancer tissues and paracancerous tissues were collected, lysate was added, homogenized in an ice bath, centrifuged for 10 min, and the supernatant was subjected to protein determination by the modified Lowry method. An appropriate amount of concentrated SDS-PAGE protein loading buffer was added to the collected protein samples, and the samples were heated at 100°C or in a boiling water bath for 3-5 min to fully denature the proteins. 50 μg of protein was taken for sodium dodecyl sulfate-polyacrylamide gel SDS-PAGE electrophoresis, transferred to the membrane, and rinsed with PBST after blocking. Rabbit anti- or mouse anti-SIK1, REGγ, PKM2 and GAPDH monoclonal antibodies from Abcam were added respectively, and incubated at 4°C overnight. After rinsing with PBST, hybridization was performed with goat anti-rabbit or anti-mouse IgG secondary antibodies labeled with horseradish peroxidase, and the samples were shaken at 37°C for 2 h. The filter membrane was washed 3 times with PBST by shaking, and the color was developed using an enhanced chemiluminescence color development system, and developed on film in a dark room. S3.3: Immunohistochemistry was used to detect paraffin cancer tissue specimens and adjacent tissue specimens. Paraffin-embedded cancer tissues and adjacent tissue specimens were selected for 5 μm continuous sectioning, dewaxed, hydrated, and incubated with 3% hydrogen peroxide for 5 min to block endogenous peroxidase; then placed in EDTA and reacted in a microwave oven at 90°C for 10 min for antigen repair, cooled to room temperature and rinsed with PBS three times; rabbit anti- or mouse anti-SIK1, REGγ, PKM2 monoclonal antibodies were added dropwise, PBS was used as a negative control, and incubated at 4°C overnight; after rinsing with PBS, horseradish peroxidase-labeled goat anti-rabbit secondary antibodies were added dropwise, incubated at 37°C for 20 min, and diaminobenzidine was used for color development; S3.4: Statistical analysis: SPSS13.0 statistical software was used for analysis. Mann-Whitney U rank sum test was used to analyze the differences in the expression of SIK1, REGγ, and PKM2 proteins in cancer tissues and adjacent tissues. The relationship between the different levels of SIK1, REGγ, and PKM2 protein expression and the clinical pathological parameters of patients was also analyzed. The Kaplan-Meier survival analysis method was used to analyze the effects of high and low expression of SIK1, REGγ, and PKM2 on the disease-free survival and overall survival of patients. The Log-Rank test was used for survival curve analysis.

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