Application of SIRT3 and SIRT4 double-gene remodeling mitochondrial network to induction of cancer cell differentiation to inhibit malignant phenotypes

Abstract

Metabolic reprogramming mainly based on aerobic glycolysis occurs after cancerization of cancer cells. According to the invention, liver cancer cell differentiation is induced by the SIRT3 and SIRT4 double-gene remodeling mitochondrial network, the association between cancer cell mitochondrial network remodeling and induced differentiation is disclosed for the first time, and a new gene therapy strategy is provided for the differentiation therapy of malignant tumors. According to the method disclosed by the invention, the double-gene plasmid pSIRT3 / 4 is successfully constructed by utilizing connection of IRES, and co-expression of exogenous multiple genes in cells is realized. The liver cancer cell is proved to have a mitochondrial network with damaged mitochondrial function and broken mitochondrial function, and the quality and function of the mitochondrial are remarkably improved through transfer of double genes. And finally, carrying out malignant phenotype and differentiation index detection on the liver cancer cells, and proving that the SIRT3 and SIRT4 double genes achieve the purposes of reversing metabolism reprogramming and inducing liver cancer cell differentiation by improving mitochondrial dysfunction, so as to inhibit occurrence and development of the liver cancer cells, and provide a new theoretical basis for differentiation therapy of hepatocellular carcinoma and malignant tumors.

Description

Technical Field

[0001] The dual-gene plasmid constructed by the present invention remodels the mitochondrial network of liver cancer cells, affecting their metabolic reprogramming, and for the first time confirms the association between mitochondrial network remodeling and induced differentiation, thereby inhibiting the occurrence and development of liver cancer cells. Furthermore, the induced differentiation caused by acting on mitochondria in this study may be a new method for treating malignant tumors, providing a new theoretical basis and treatment strategy for tumor differentiation therapy. Background Art

[0002] The "Warburg" effect of liver cancer cells is a common feature of cell carcinogenesis, that is, the metabolic reprogramming of tumor cells, and the energy metabolism is transformed from mitochondrial-based ATP production to glycolysis. Therefore, reversing metabolic reprogramming is a highly promising tumor treatment method.

[0003] Mitochondria have independent genes, called mitochondrial DNA, which encode 13 proteins, including NADH-CoQ reductase, cytochrome C oxidase, subunits of ATP synthase, mitochondrial transcription factor A (TFAM), etc., which are mainly responsible for participating in oxidative phosphorylation and the formation of mtDNA. As the main site of oxidative phosphorylation in cells, most of ATP is generated in mitochondria. Mitochondrial damage is also a direct factor inducing cellular metabolic disorders. "Mitochondrial network" is a synonym for the sum of mitochondrial morphology, structure and quality. Mitochondrial network fragmentation, DNA mutation and changes in mitochondrial function are all considered to be one of the early events of tumorigenesis. In tumor tissues of patients with liver cancer, mtDNA copy number has also been shown to be lower than that of normal tissues, and mitochondrial dynamics, ROS, ATP content, etc. have changed to varying degrees. This abnormal mitochondrial change promotes the progression of HCC through metabolic disorders and signal transformation. It can be said that metabolic reprogramming closely related to mitochondria is a key factor in the occurrence and development of HCC.

[0004] Sirtuins are a class of NAD+-dependent deacetylases that promote mitochondrial function and remodel the mitochondrial network. The Sirtuins family consists of 7 members (Sirt1-7), among which SIRT3, SIRT4 and SIRT5 proteins are all localized in mitochondria after expression and affect the occurrence and development of tumors. At present, the research on the sirtuins family in hepatocellular carcinoma focuses on the impact on signaling pathways. For example, HCC patients with low expression of SIRT3 have a higher degree of tumor malignancy, and they are used as potential targets for the treatment of HCC by enhancing MnSOD activity or drug sensitivity such as sorafenib; SIRT4 participates in the inhibitory pathway of hepatocellular carcinoma and arrests the cycle of HCC; on the one hand, SIRT5 studies have shown that its desuccinylation ability affects the activity of ACOX enzymes to inhibit HCC, while on the other hand, SIRT5 is upregulated in some liver cancer cell lines and induces metabolic reprogramming of glycolysis. However, there is a lack of clear research reports in the field of directly remodeling the mitochondrial network and improving mitochondrial energy metabolism.

[0005] The effectiveness of gene therapy has been widely recognized, and a large number of studies have been conducted on the construction of corresponding vectors based on different genes. In order to significantly increase the therapeutic effect of exogenous genes on diseases, multiple studies have been conducted on multi-gene co-expression vectors, including internal promoters, self-cleaving 2A peptide elements, internal ribosome entry sites IRES and other construction strategies. Among them, the internal ribosome entry site IRES plays an important role in the translation process of most cell mRNAs. Both its upstream and downstream target genes can be translated and overcome the expression inhibition phenomenon between multiple promoters. On this theoretical basis, using it to construct multi-gene vectors will be a more effective strategy to achieve simultaneous co-expression.

[0006] The sirtuins family, SIRT3, SIRT4 and SIRT5 genes localized in mitochondria, are transfected into liver cancer cells, thereby reshaping the mitochondrial network, improving mitochondrial quality and function, and reversing the metabolic reprogramming of the "Warburg" effect. The main purpose of this invention is to observe whether this reversal can affect the differentiation and malignant phenotype of tumor cells and provide a new theoretical basis and treatment strategy for gene therapy of tumors. Summary of the invention

[0007] The present invention constructs SIRT3 and SIRT4 dual-gene plasmids by respectively detecting the effects of SIRT3, SIRT4 and SIRT5 genes on energy metabolism. The constructed dual-gene plasmid reshapes the mitochondrial network of liver cancer cells, thereby affecting their metabolic reprogramming, and for the first time confirms the association between mitochondrial network remodeling and induced differentiation, thereby inhibiting the occurrence and development of liver cancer cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1PCR amplification of SIRT3, SIRT4, and SIRT5 gene fragments in Example 1, M: DNA Marker, lane 1: SIRT3, lane 2: SIRT4, lane 3: SIRT5

[0009] Figure 2 This is the colony PCR identification of pSIRT3 and pSIRT4 plasmids in Example 1.

[0010] Figure 3 This is the colony PCR identification of the pSIRT5 plasmid in Example 1.

[0011] Figure 4 This is a Western blot detection of the expression of SIRT3, SIRT4, and SIRT5 proteins in L-O2 and HCCLM3 cells in Example 1.

[0012] Figure 5 The following are the test results of ATP content in each group of cells in Example 1.

[0013] Figure 6 The relative expression levels of IDH and α-KGDH mRNA in each group of cells in Example 1.

[0014] Figure 7 The relative expression levels of PK mRNA in each group of cells in Example 1.

[0015] Figure 8 This is the Western blot detection of mitochondrial function-related protein factors after transfection of pSIRT3 and pSIRT4 plasmids in Example 1.

[0016] Fig. 9 Example 2pIRES-SIRT3 / 4 plasmid map

[0017] Fig.10 This is the double enzyme digestion identification of the pIRES-SIRT3 / 4 plasmid in Example 2.

[0018] Fig.11 This is a diagram showing the sequencing results of the pIRES-SIRT3 / 4 plasmid in Example 2.

[0019] Fig.12 This is the Western blot detection of the target protein after plasmid transfection in Example 2.

[0020] Fig.13 The relative content of mtDNA in each group of cells in Example 2 (n=3, *p<0.05, **p<0.01, ***p<0.001 vs. Control; ns: not significant vs. Control.)

[0021] Fig.14 This is the fluorescence microscopy imaging and red-green ratio fluorescence intensity quantification of mitochondrial membrane potential observed using the JC-1 fluorescent probe in Example 2.

[0022] Fig.15 For Example 2, Western blot was used to detect mitochondrial biogenesis and autophagy factor expression.

[0023] Fig.16 This is Example 2: Laser confocal observation of mitochondrial network structure and fluorescence quantification in cells.

[0024] Fig.17 This is Example 2H2DCFDA probe method for observing ROS fluorescence microscopy imaging and fluorescence intensity quantification.

[0025] Fig.18 , 19 20 are the activity detection of the intracellular antioxidant enzymes SOD1, SOD2 and CAT in different groups of Example 2. (n=3, *p<0.05, **p<0.01 vs. Control; ns: not significant vs. Control.).

[0026] Fig.21 This is a bar graph showing the migration and invasion abilities and cell number of each group of cells detected by Transwell assay in Example 3 (n=3, *p<0.05, **p<0.01 vs. Control; ns: not significant vs. Control.).

[0027] Fig. 22 The relative viability of each group of cells was detected by CCK8 method as in Example 3 (Cell viability % = (OD of dosed cells / OD of control cells) x 100%).

[0028] Fig.23 The mRNA expression levels of the cell differentiation markers HNF3γ and HNF4α in each group of Example 3.

[0029] Fig.24 This is the ELISA test of the liver cancer marker protein AFP content in Example 3. DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with the embodiments.

[0031] Example 1: Effects of SIRT3, SIRT4 and SIRT5 genes on energy metabolism in normal and tumor liver cells

[0032] Materials and Methods

[0033] Strains and plasmids: Escherichia coli DH5α was purchased from Guangzhou Qingke Biotechnology Co., Ltd., human PBMC, L-O2 and HCCLM3 were cryopreserved from the Institute of Genomic Medicine, Jinan University; pIRES2-EGFP was purchased from Guangzhou Dingguo Biotechnology Co., Ltd.

[0034] Experimental reagents: agarose was purchased from Tiangen Biochemical Technology Co., Ltd.; mammalian genomic DNA extraction kit was purchased from Beijing Zhuangmeng International Biogene Technology Co., Ltd.; WB colorimetric solution was purchased from ADVANSTA; PVDF membrane was purchased from Millipre, USA; Nhe I, BamH I, BstX I, Not I, and AflII enzymes were all purchased from TaKaRa.

[0035] DNA extraction and amplification DNA from human PBMCs was extracted using a mammalian DNA extraction kit, and the gene sequences of SIRT3, SIRT4, and SIRT5 were amplified by PCR. The sequences were accessed from NCBI, and BstX I and Not I restriction sites were introduced at both ends of the SIRT3 gene, and Nhe I and BamH I restriction sites were introduced at both ends of the SIRT4 and SIRT5 genes. Primers were designed using PrimerBlast and synthesized by Shanghai Sangon Biotechnology Co., Ltd. The PCR reaction conditions were: 98°C for 10 s, 60°C for 5 s, and 72°C for 10 s, for 32 cycles. Total cell DNA was extracted using a mammalian genomic DNA extraction kit. Primers were designed according to Primer Blast and synthesized by Bioengineering. SYBR green Master Mix and the corresponding primer configuration system were added to each group for PCR, with β-actin as the internal reference gene. The PCR amplification primers are shown in the following table:

[0036] Agarose gel electrophoresis Electrophoresis: Pour 1% agarose gel solution into the gel plate, insert the gel comb, and let it stand until it solidifies. Add 1×TAE electrophoresis buffer to the electrophoresis tank, place the gel plate in the electrophoresis tank with the gel well facing the negative pole, mix the sirt3 and sirt4 gene amplification samples and markers with 10×Loading Buffer (5 μL each), and add them to the gel wells. Perform electrophoresis at 160V for 30 minutes. Take pictures with a gel imager. Fragment recovery: Cut the target gene under UV light and place it in a centrifuge tube, weigh it (the weight of the centrifuge tube and the gene), calculate the gel volume, add an equal volume of Binding Buffer, heat and melt it into a DNA-agarose solution. Load the DNA-agarose solution into the adsorption column and place it in the collection tube, centrifuge it at 12000rpm for 1min, and discard the liquid. Repeat once. Add SPW Wash buffer diluted with anhydrous ethanol, centrifuge it at 12000rpm for 1min, and discard the liquid. Repeat once to remove the residual ethanol. Put the adsorption column in the centrifuge tube, add 50μL EB to elute, centrifuge it at 12000rpm for 1min, collect the DNA, and store it at -20℃. DNA concentration = A260×50×dilution factor, unit ng / μL.

[0037] Construction of single gene plasmid The plasmid pIRES2-EGFP was double-digested with BstX I / Not I and Nhe I / BamH I, respectively, and then ligated with SIRT3, SIRT4, and SIRT5 fragments to construct pIRES-SIRT3-EGFP, pIRES-SIRT4-EGFP, and pIRES-SIRT5-EGFP plasmids, referred to as pSIRT3, pSIRT4, and pSIRT5.

[0038] Total protein extraction The cells were washed with pre-cooled PBS, centrifuged at 12000 rpm for 5 min at room temperature, the cell pellet was collected, and 1 mL of lysis buffer (RIPA: PMSF = 1 mL: 10 μL, 100 mM) was added, and the cells were lysed on ice for 30 min. The lysed cells were scraped off with a blade, and the cell suspension was transferred to a 1.5 mL centrifuge tube using a pipette, and then lysed on ice for 10 min, and centrifuged at 4°C, 12000 rpm for 5 min, the total protein supernatant was collected, and stored at -20°C.

[0039] BCA quantification Follow the instructions of the BCA protein quantification kit: Use PBS to prepare BSA into 0.05, 0.125, 0.25, 1, 2, 4 mg / mL standards, take 20 μL of the above concentration solutions and the protein to be tested to a 96-well plate, set up 2 replicates. Prepare the working solution with solution A and solution B at a volume ratio of 1:50, add 200 μL to each well, and incubate in a 37°C incubator for 30 minutes. Detect OD562nm and draw a standard curve. Dilute the total protein extracts of the three groups of cells 10 times, perform the absorbance detection at OD562nm as above, and calculate the total protein concentration of each group of cells according to the standard curve.

[0040] Western blot After boiling the total protein extract at 100℃ for 5min, mix it with the protein buffer. Add 10μL of internal reference and 15μL of sample to the prepared electrophoresis device. Electrophoresis was performed at 80V for 40min, and then continued at 120V for 45min. The electrophoresis was stopped when the electrophoresis buffer reached the top. A 0.22μm PVDF membrane was prepared, soaked in methanol for 1min, and the clamps were assembled in the order of "sponge-cardboard-glue-PVDF membrane-cardboard-sponge" to drive away bubbles. Insert the transfer tank and pour the transfer solution. Ice bath, 200mA current transfer for 60min. After the transfer, wash the membrane with TBST for 5min, block with 5% skim milk powder in PBS solution at room temperature for 1h, and wash the membrane 3 times with TBST, 5min each time. Add the primary antibody (dilution ratio 1:1000) and incubate overnight at 4℃. Wash the membrane three times according to the above operation, add the secondary antibody (dilution ratio 1:5000), incubate at room temperature for 2 hours, wash the membrane three times. Add ECL luminescent solution to the PVDF membrane, incubate for 3 minutes and then expose.

[0041] The cells were lysed and the ATP content was detected according to the ATP assay kit.

[0042] Real-time fluorescence quantitative analysis Total cell RNA was isolated using Trizol, and cDNA was synthesized using RNA reverse transcriptase. Primers were designed according to Primer Blast and synthesized by Bio-Industry. SYBR green Master Mix and the corresponding primer configuration system were added to each group for PCR, with β-actin as the internal reference gene, and three independent biological replicates were performed. The PCR reaction conditions were: 95℃15s, 60℃30s, 72℃30s, and 40 cycles. The relative expression content of the gene was calculated based on 2-ΔΔCt. The corresponding primer sequences are as follows

[0043] Experimental Results

[0044] Construction and identification of single plasmid Design and purchase the pIRES-EGFP gene vector carrying an internal ribosome entry site (IRES). First, amplify the human SIRT3, SIRT4, and SIRT5 genes ( Figure 1 ). The plasmids were identified by colony PCR, and pIRES-SIRT3-EGFP, pIRES-SIRT4-EGFP and pIRES-SIRT5-EGFP plasmids with target gene fragments were constructed ( Figure 2-3 ).

[0045] Identification of SIRT3 and SIRT4 double genes In the sirtuins family, SIRT3, SIRT4, and SIRT5 proteins are all located in mitochondria. We constructed three single genes and successfully transfected them into normal L-O2 human liver cell line and HCCLM3 liver cancer cell line ( Figure 4 ), and the ATP levels in each group of cells were checked after 24 hours of culture. The results showed that the three genes had a certain improvement effect on the ATP of liver cancer cells, but had no significant statistical significance for L-O2 cells except SIRT4 ( Figure 5 Next, we used RT-PCR to detect the expression levels of isocitrate dehydrogenase (IDH) and α-ketoglutarate dehydrogenase (α-KGDH), key enzymes of mitochondrial oxidative phosphorylation. Figure 6 As shown in Figure 2, SIRT3 and SIRT4 have a significantly greater impact on the key rate-limiting enzymes in mitochondria than SIRT5. By detecting the level of pyruvate kinase (PK), a key enzyme in glycolysis, it was found that SIRT5 enhanced the expression of PK ( Figure 7 ), suggesting that SIRT5 promotes glycolysis. Based on the research results of SIRT3 and SIRT4 on key mitochondrial enzymes, further detection of key proteins affecting mitochondrial function and autophagy found that SIRT3 acts on the key factor of mitochondrial biogenesis PGC-1α, and SIRT4 acts on the mitochondrial autophagy proteins Parkin and PINK1 ( Figure 8 ).

[0046] Example 2: pIRES-SIRT3 / 4 plasmid improves mitochondrial dysfunction in HCCLM3

[0047] Materials and Methods

[0048] Strains and plasmids: Human PBMC, L-O2 and HCCLM3 were cryopreserved from the Institute of Genomic Medicine, Jinan University; pIRES2-EGFP was purchased from Guangzhou Dingguo Biotechnology Co., Ltd.

[0049] Experimental reagents: DMEM cell culture medium was purchased from Gibco; trypsin was purchased from Sigma; ELISA kits for JC-1, antioxidant enzymes SOD1, SOD2 and CAT were purchased from RayBiotech; mitochondrial network detection kit was purchased from Beyotime

[0050] Construction of pIRES-SIRT3 / 4 plasmid The pIRES-SIRT3 / 4 plasmid was constructed by directly cloning SIRT3 into pIRES-SIRT4-EGFP after double restriction digestion with BstX I / Not I. All constructed plasmids were transfected into Escherichia coli DH5α competent cells for extraction and restriction digestion verification. The pIRES-SIRT3 / 4 plasmid was sent to Shanghai Sangon Biotechnology Co., Ltd. for double restriction digestion identification and sequencing.

[0051] qPCR detection of mtDNA The ND1 gene encoding the ND1 subunit of NADH dehydrogenase was used to quantify mtDNA, and β-actin was used to quantify nDNA. NCBI was used to query and design qPCR primers. The qPCR experiment was performed using the Light Cycler 480 System. The templates were extracted mtDNA and nDNA, respectively. The templates were amplified according to the manufacturer's instructions of the real-time fluorescence quantitative PCR kit, denaturation at 95°C for 5s, annealing at 60°C for 30s, extension at 72°C for 15s, thermal cycling for 42 times, and final extension at 72°C for 15s. The relative expression was calculated according to 2-ΔΔCt, and the relative content of mtDNA was calculated by the ratio of mtDNA / nDNA. Three replicate wells were set up in each group, and the average value was calculated. The primer sequences are as follows:

[0052] 1.2.6 Determination of mitochondrial membrane potential (JC-1 method) The mitochondrial membrane potential of each group was measured according to the instructions of the JC-1 detection kit. In brief, the prepared JC-1 staining working solution was transferred to the cells and incubated in an incubator for 20 min, then washed twice with ice-bathed JC-1 staining buffer (1×), added with complete culture medium, placed under an inverted fluorescence microscope for observation and photography, and the red fluorescence intensity was quantitatively analyzed using NIH Image J software.

[0053] Mitochondrial morphology observation (Mito-Tracker analysis) Use anhydrous DMSO to prepare Mito-Tracker Green solution into 1mM storage solution and store it in the dark at -20℃ for later use; transfer each group of cells to a laser confocal culture dish at a density of 1×105, and culture each group of cells routinely. After the culture is completed, remove the culture medium and add MitoTracker Green staining working solution preheated to 37℃ and incubate for 2h; after the incubation, replace the staining solution with fresh culture medium and observe the mitochondrial network structure under a laser confocal microscope.

[0054] Statistical analysis: All data were presented as arithmetic mean ± standard deviation (SD) and analyzed using GraphPadPrism version 9.3. One-way analysis of variance was used to compare multiple groups of samples. P < 0.05 was considered statistically significant.

[0055] Experimental Results

[0056] Construction and identification of pIRES-SIRT3 / 4 plasmid After confirming the SIRT3 and SIRT4 genes, the SIRT3 fragment was cloned into pIRES-SIRT4-EGFP to construct pIRES-SIRT3 / 4( Fig. 9 ). Shanghai Biotech performed double restriction enzyme digestion and sequencing of pIRES-SIRT3 / 4 plasmid. Double restriction enzyme digestion showed bands around 1170 bp and 952 bp respectively ( Fig.10 ), which are similar in size to SIRT3 and SIRT4 genes, indicating that SIRT3 and SIRT4 were successfully cloned into the plasmid pIRES-SIRT3 / 4. The sequencing results showed that the fragment cloned into the pIRES-SIRT3 / 4 plasmid was completely consistent with the sequence in the database ( Fig.11 ), indicating that the plasmid was successfully constructed.

[0057] pIRES-SIRT3 / 4 plasmid improves mitochondrial membrane potential and mtDNA levels SIRT3 gene affects mitochondrial biogenesis, SIRT4 gene affects mitochondrial autophagy. The remodeling of mitochondrial network depends on the combined effects of biogenesis and autophagy. To verify its effect on mitochondrial network, we transfected single gene plasmid and pIRES-SIRT3 / 4 double gene plasmid into HCCLM3 cells ( Fig.12 The results showed that the level of mtDNA in cells transfected with pIRES-SIRT3 / 4 plasmid was significantly higher than that in cells transfected with single gene plasmid ( Fig.13 ). Mitochondrial membrane potential is an important indicator of cell metabolism and mitochondrial quality. The JC-1 fluorescent probe method showed that the red:green fluorescence intensity in HCCLM3 cells was significantly enhanced after transfection with pSIRT3 / 4 ( Fig.14 ), indicating that dual-gene therapy has better efficacy.

[0058] pIRES-SIRT3 / 4 plasmid reshapes the mitochondrial network of HCCLM3 Western blot revealed that the expression level of key factors of mitochondrial function was high in normal cells, while the mitochondrial factors of liver cancer cells were significantly reduced. The results showed that cells transfected with pIRES-SIRT3 / 4 jointly increased the expression levels of mitochondrial biogenesis and autophagy factors ( Fig.15). Finally, we detected the mitochondrial network structure in L-O2 cells and HCCLM3 cells, respectively. The laser confocal system showed that the green fluorescence intensity of L-O2 cell mitochondria was high and the morphology was good, while the mitochondrial network did not change significantly after plasmid transfection. The mitochondrial fluorescence of HCCLM3 cells was weak and fragmented, indicating that the mitochondria were damaged. After transfection of SIRT3 and SIRT4 single genes, the mitochondrial fluorescence intensity was partially enhanced, but the morphology was imperfect and the number was small. The fluorescence in the pSIRT3 / 4 group was significantly enhanced, and its mitochondrial edges were clear and connected to each other to form a network with a certain shape ( Fig.16 ), indicating that the dual-gene plasmids can work together to more significantly reshape the mitochondrial network and improve mitochondrial quality.

[0059] pIRES-SIRT3 / 4 plasmid reduces oxidative stress in HCCLM3 cells Impaired mitochondrial respiratory chain function in liver cancer cells produces excessive ROS, leading to oxidative stress and promoting the occurrence and development of HCC. Our results confirmed that compared with L-O2, the fluorescence intensity in HCCLM3 cells was higher, indicating a higher level of ROS. The fluorescence of HCCLM3 cells was weakened to a certain extent after transfection with pSIRT3 and pSIRT4 plasmids, while the ROS level in the pSIRT3 / 4 group cells was significantly reduced ( Fig.17 ), showing the co-action of the two genes. The increase in ROS levels will inevitably cause changes in the activity of antioxidant enzymes in cells. To this end, we tested the activities of the antioxidant enzymes SOD and CAT in each group of cells. The results showed that the activities of SOD and CAT in the cells increased to varying degrees ( Fig.18 ). These results indicate that SIRT3 and SIRT4 proteins work together to reduce the oxidative stress level in HCCLM3 cells.

[0060] Example 3: pIRES-SIRT3 / 4 plasmid inhibits the malignant phenotype of HCCLM3 by inducing differentiation

[0061] Materials and Methods

[0062] Experimental reagents: CCK8 reagent was purchased from Biosharp, AFP ELISA kit was purchased from R&D-Systems, and SYBRgreen Master Mix was purchased from Beijing Kangrun Chengye Biotechnology Co., Ltd.

[0063] Transwell experiment The cell culture medium of each group was replaced with serum-free medium and cultured stably for 12 hours. The cells of each group were inoculated in the upper chamber of the Transwell with serum-free medium at a density of 10×105 per well. DMEM complete medium containing 10% FBS was added to the lower chamber. After 48 hours of culture, the medium in the well was discarded, and the cells were fixed with 4% paraformaldehyde for 30 minutes after washing with PBS and stained with 10% crystal violet for 30 minutes. The excess dye was washed with PBS, and the cells that did not pass through the surface of the polycarbonate permeable membrane were gently wiped off with a cotton swab, and the cells were observed under a microscope and photographed. In the invasion experiment, Matrigel was used to coat the upper chamber before the experiment, and the other steps were the same.

[0064] CCK-8 assay for cell proliferation Each group of cells was inoculated into a 96-well plate (3000 cells per well) and adhered to the wall. Complete medium was used as a blank control. Five replicate wells were set for each group and cultured in a 37°C, 5% CO2 incubator. According to the instructions, CCK-8 reagent (biosharp, Guangzhou, China) (10 μL per well) was used to measure and evaluate cell proliferation. The optical density (OD450) value of each well at 0h, 12h, 24h, 36h, 48h, and 72h was detected by an ELISA instrument at an absorption wavelength of 450nm.

[0065] RT-qPCR detection of liver cancer cell differentiation markers and hepatocyte differentiation factors Total RNA of cells was isolated by Trizol, and cDNA was synthesized by RNA reverse transcriptase. Primers for HNF4α and HNF3γ were designed according to Primer Blast, and the primers were synthesized by Bio-Industry. SYBR green Master Mix and the corresponding primer configuration system were added for PCR, with β-actin as the internal reference gene. The corresponding primer sequences are shown in Table 1, and three independent biological replicates were performed. The PCR reaction conditions were: 95℃15s, 60℃30s, 72℃30s, and 40 cycles. The relative expression content of the gene was calculated according to 2-ΔΔCt. PCR amplification primers are as follows:

[0066] ELISA protein concentration determination of liver cancer marker protein AFP ELISA kits were used to detect the protein expression levels of antioxidant enzymes and liver cancer cell differentiation indicators. The supernatants of cells from each group that had been stably cultured for 3 days were collected, and the protein concentration was determined using the AFP ELISA detection kit, and the absorbance was detected at a wavelength of 450 nm using an enzyme marker.

[0067] Experimental Results

[0068] pIRES-SIRT3 / 4 plasmid inhibits the malignant phenotype of hepatocellular carcinoma cells To verify the correlation between mitochondrial function and the occurrence and development of HCC, we further detected the proliferation and migration ability of HCC after transfection with pSIRT3, pSIRT4 or pSIRT3 / 4 plasmids. Transwell showed that the migration and invasion abilities of HCCLM3 cells decreased after transfection with pIRES-SIRT3 / 4 plasmids ( Fig.21 ), and the CCK8 transfection kit showed that the cell viability was significantly reduced after plasmid transfection ( Fig. 22 ).

[0069] pIRES-SIRT3 / 4 plasmid induces differentiation of hepatocellular carcinoma cells RT-PCR detection of HNF4α, a liver cancer cell differentiation marker, and HNF3γ, a liver cell differentiation factor, revealed that their expression was significantly increased ( Fig.23 ). The secretion of liver cancer marker protein AFP decreased ( Fig.24 ), the above results all show that the dual gene has a more significant tumor suppressive effect than the single gene. This indicates that the SIRT3 and SIRT4 dual gene plasmid can effectively induce liver cancer cell differentiation by remodeling the damaged mitochondrial network, thereby inhibiting the malignant phenotype of liver cancer cells.

Claims

1. The present invention constructs a dual-gene plasmid pIRES-SIRT3 / 4 through detection and screening of SIRT3, SIRT4 and SIRT5 genes, improves mitochondrial dysfunction of liver cancer cells, reverses metabolic reprogramming, induces differentiation of liver cancer cells, and achieves the effect of inhibiting malignant phenotypes.

2. The detection and screening of SIRT3, SIRT4 and SIRT5 genes according to claim 1, characterized in that: SIRT3 and SIRT4 improve key enzymes of oxidative phosphorylation in liver cancer cells and enhance mitochondrial respiratory function, while SIRT5 promotes PK, a key enzyme in glycolysis, thereby further promoting the "Warburg" effect.

3. The double gene plasmid pIRES-SIRT3 / 4 according to claim 1, characterized in that: The parent plasmid pIRES2-EGFP carrying the internal ribosome binding site IRES was selected, and the human SIRT3 and SIRT4 genes were cloned upstream and downstream of IRES. The stably expressed dual-gene expression plasmid pSIRT3 / 4 was successfully constructed to achieve simultaneous co-expression of SIRT3 and SIRT4 genes.

4. The SIRT3 and SIRT4 genes according to claim 3, characterized in that SIRT3 promotes the mitochondrial biogenesis factor PCG-1α in liver cancer cells, and SIRT4 regulates the mitophagy proteins PINK1 and Parkin. The two interact with each other and significantly reshape the damaged mitochondrial network in liver cancer cells.

5. The liver cancer cell differentiation according to claim 1, characterized in that: The mitochondrial network was significantly improved, mitochondrial respiratory function was restored, and liver cancer cells switched from aerobic glycolysis to mitochondrial oxidative phosphorylation. The differentiation markers HNF4α and HNF3γ of liver cancer cells increased, and the malignant phenotype of liver cancer cells was significantly inhibited.

6. The dual-gene plasmid pIRES-SIRT3 / 4 as described in claim 1, which induces differentiation by acting on mitochondria and may be a new method and strategy for treating malignant tumors, and can be used in the preparation of drugs for treating malignant tumors.

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