Application of HIF-3alpha-3 gene or protein coded by HIF-3alpha-3 gene in preparation of medicine for preventing and treating bladder cancer

Abstract

The invention provides an application of an HIF-3alpha-3 gene or a protein coded by the HIF-3alpha-3 gene in preparation of a medicine for preventing and treating bladder cancer, and belongs to the technical field of biological medicines. The invention reveals that HIF-3alpha-3 in bladder cancer up-regulates PHD3 gene expression from a transcriptional regulation level, so that the hydroxylation level of HIF-1alpha in bladder cancer cells is increased, and the protein stability of HIF-1alpha is reduced for the first time. Meanwhile, overexpression of the HIF-3alpha-3 gene or the coded protein can effectively inhibit proliferation and in-vivo tumorigenicity of bladder cancer cells. Therefore, the invention provides the application of the HIF-3alpha-3 gene or the protein coded by the HIF-3alpha-3 gene or the PHD3 gene or the protein coded by the PHD3 gene in preparation of medicines for preventing and treating bladder cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological medicines, and particularly relates to the application of HIF-3α-3 gene or the protein encoded by it in the preparation of medicines for preventing and treating bladder cancer. Background Art

[0002] Hypoxia inducible factor (HIF), a transcriptional regulator discovered in the 1990s, is a key player in the cellular response to hypoxia. It plays a crucial role in tumor cell glycolysis, angiogenesis, and apoptosis by mediating the transcriptional expression of up-clock target genes. HIF-1 was the first HIF discovered and plays a crucial role in the entire hypoxia response. The subsequent discovery of its homologous proteins, HIF-2 and HIF-3, has garnered significant attention for HIF research.

[0003] Hypoxia-inducible factor-3α (HIF-3α) exists in at least five different splice forms in humans, and its intracellular expression is regulated by oxygen partial pressure. Under normoxic conditions, HIF-3α is expressed in both the nucleus and cytoplasm, whereas its expression increases under hypoxia. Studies have shown that HIF-3α is highly expressed in human renal cell carcinoma cells and may be involved in regulating HIF-1α expression (Expression and Clinical Significance of HIF-1α and HIF-3α in Renal Cell Carcinoma, Li Zhi, Master's Thesis, Huazhong University of Science and Technology, April 2011). However, relatively few reports exist on the role of HIF-3α in bladder cancer. Summary of the Invention

[0004] In view of this, the purpose of the present invention is to provide a use of the HIF-3α-3 gene or the protein it encodes in the preparation of a drug for preventing and / or treating bladder cancer. The HIF-3α-3 gene or the protein it encodes can upregulate PHD3 gene expression, reduce the stability of HIF-1α protein, reduce the hypoxia adaptability of bladder cancer, and inhibit cancer cell proliferation, thereby providing a basis for the development of new anti-bladder cancer drugs.

[0005] The present invention provides the use of HIF-3α-3 gene or protein encoded by HIF-3α-3 gene in preparing medicine for preventing and / or treating bladder cancer.

[0006] Preferably, the HIF-3α-3 gene includes at least one of the following forms:

[0007] A. HIF-3α-3 gene with a nucleotide sequence as shown in SEQ ID NO: 1;

[0008] B. HIF-3α-3 gene encoding the protein with the amino acid sequence shown in SEQ ID NO: 2;

[0009] C. The amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, only the codons are optimized to form the HIF-3α-3 gene;

[0010] D. A genetically engineered product comprising at least one HIF-3α-3 gene selected from A, B, and C.

[0011] Preferably, the genetic engineering product includes at least one of the following: a recombinant expression vector, a recombinant strain and a recombinant virus.

[0012] Preferably, the protein encoded by the HIF-3α-3 gene includes at least one of the following:

[0013] 1. HIF-3α-3 protein with the amino acid sequence shown in SEQ ID NO: 2;

[0014] II. HIF-3α-3 protein that has been chemically modified based on item I but whose biological function remains unchanged.

[0015] The present invention provides an application of an agent for promoting the expression of HIF-3α-3 gene or protein encoded by the HIF-3α-3 gene in the preparation of a drug for preventing and / or treating bladder cancer.

[0016] Preferably, the agent comprises a molecule that enhances the transcriptional function of the HIF-3α-3 gene.

[0017] The present invention provides the use of PHD3 protein, a gene encoding PHD3 protein or an agent activating a PHD3 gene promoter in the preparation of a drug for preventing and / or treating bladder cancer.

[0018] Preferably, the PHD3 protein comprises at least one of the following:

[0019] S1. PHD3 protein with the amino acid sequence shown in SEQ ID NO: 3;

[0020] S2. PHD3 protein chemically modified based on item S1 without changing its biological function.

[0021] Preferably, the gene encoding the PHD3 protein includes at least one of the following forms:

[0022] A. PHD3 gene with the nucleotide sequence shown in SEQ ID NO: 4;

[0023] B. a PHD3 gene encoding a PHD3 protein having an amino acid sequence as shown in SEQ ID NO: 3;

[0024] C. The amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, only the PHD3 gene is codon-optimized;

[0025] D. A genetically engineered product comprising at least one PHD3 gene from A, B, and C.

[0026] Preferably, the agent for activating the PHD3 gene promoter includes a protein encoded by the HIF-3α-3 gene.

[0027] The present invention provides the use of the HIF-3α-3 gene or the protein encoded by the HIF-3α-3 gene in the preparation of a drug for the prevention and / or treatment of bladder cancer. Experiments have shown that HIF-3α-3 is expressed in human bladder cancer cell lines 5637, T24, RT112 and normal urothelial immortalized cells SV-HUC1. Using SV-HUC1 as a control cell line, it was found that the protein expression level of HIF-3α-3 in 5637, T24, and RT112 cells was significantly lower than that in SV-HUC1. The present invention detected the specific low expression of HIF-3α-3 in bladder cancer at both the tissue and cellular levels. At the same time, in vivo experiments, in vitro experiments, and organoid experiments verified that overexpression of HIF-3α-3 can significantly inhibit the proliferation or tumorigenicity of bladder cancer cells and reduce the viability of cancer cells under hypoxic conditions. Furthermore, the present invention further investigates the molecular mechanism of bladder cancer inhibition. HIF-3α-3 activates the PHD3 promoter to upregulate PHD3 gene expression, thereby facilitating HIF-1α protein degradation and downregulating HIF-1α protein stability, thereby inhibiting the development of bladder cancer. This invention provides new insights into drug development for bladder cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The results of specific expression of HIF-3α-3 protein and mRNA in bladder cancer, where A is the Western Blot assay for HIF-3α-3 protein expression in 8 pairs of cancer tissues (BT) and corresponding para-cancer tissues (BN); B is the RT-qPCR assay for HIF-3α-3 mRNA expression in 22 pairs of cancer tissues (BT) and corresponding para-cancer tissues (BN); C, D are the detection of HIF-3α-3 protein and mRNA expression levels in bladder cancer cell lines (5637, T24, RT112) and normal urothelial cells (SV-HUC1), respectively, **p<0.01;

[0029] Figure 2 The results of HIF-3α-3 overexpression level verification are shown in Figure 1. A is a Western Blot assay to detect HIF-3α-3 after lentiviral infection and screening with puromycin. OE The expression level of HIF-3α-3 protein in 5637 and T24 cells of group and vector group; B is the RT-qPCR assay to detect HIF-3α-3 OEResults of HIF-3α-3 mRNA expression in 5637 and T24 cells of the control group and the vector group, ***p<0.001;

[0030] Figure 3 The results of clone formation activity test of 5637 cells and T24 cells after HIF-3α-3 overexpression, where A and C are 2000 HIF-3α-3 cells seeded per well. OE Crystal violet staining results of 5637 and T24 cells in the A and B groups after culturing for 2 weeks; B and D show the colony counts of cells in each group, and the relative colony formation rates are used to represent the colony formation abilities of 5637 and T24 cells in different treatment groups, **p<0.01;

[0031] Figure 4 The results of the proliferation ability test of 5637 cells and T24 cells after HIF-3α-3 overexpression, where A and B are the results of the EDU imaging experiment to detect HIF-3α-3 OE The proliferation capacity and fluorescence-labeled positive cells of 5637 and T24 cells in the A and B groups under normal oxygen levels; C and D are the results of the number of EDU-positive cells in each field of view, and the proliferation capacity of cells in each group is expressed as the EDU-positive cell rate, *p<0.05, **p<0.01;

[0032] Figure 5 Figure 3 shows the growth trend of patient-derived bladder cancer organoids after HIF-3α-3 overexpression. A shows the growth status of three bladder cancer organoids infected with lentivirus and selected with puromycin, observed under white light field for 7 days after the first passage. B and C show the growth trend of HIF-3α-3 every 3 days after the first passage after the screening. OE Results of organoid number and diameter size in the WT group and Vector group (scale bar is 100 μm), *p<0.05, **p<0.01;

[0033] Figure 6 Figure 3: HIF-3α-3 inhibits bladder cancer growth in an in vivo subcutaneous tumor model. A shows tumor volume in the subcutaneous tumor model; B shows tumor length and width, **p<0.01; C shows tumor weight in each group, **p<0.01; D shows H&E staining to observe tumor cell morphology and immunohistochemical detection of Ki-67, PCNA and other molecular markers.

[0034] Figure 7 The results indicate that HIF-3α-3 is involved in regulating the various biological behaviors of BLCA;

[0035] Figure 8 The results showed that HIF-3α-3 protein levels showed a downward trend during chemically induced hypoxia;

[0036] Figure 9 The results show that HIF-3α-3 overexpression further inhibits the viability of BLCA cells under hypoxia, where A and B represent HIF-3α-3 OE Group and Vector group 5637 cell stably transfected lines; C, D are the OD values ​​of cells in different treatment groups detected by microplate reader, *p<0.05, **p<0.01;

[0037] Figure 10 The results show that HIF-3α-3 overexpression further inhibits the proliferation of BLCA cells under hypoxia; A and B show the results of HIF-3α-3 overexpression under hypoxia (addition of 300μM CoCl2) OE 5637 cells and T24 cells in the vector group and the vector group were stably transfected for 48 hours, EDU solution was added, and they were incubated at 37℃ for 2 hours, fixed, stained, and photographed under a microscope; C and D are the counting and statistics of HIF-3α-3 OE The EDU-positive cell rates of 5637 cells and T24 cells in the group and vector group, **p<0.01;

[0038] Figure 11 Figure 3 shows that HIF-3α-3 overexpression promotes HIF-1α protein degradation under hypoxia. A shows the analysis of HIF-1α gene expression levels and patient survival prognosis. B shows HIF-1α protein expression. C shows the inhibition of HIF-1α protein expression by the LW6 inhibitor. D shows the cell viability assay under hypoxia using CCK-8. ns indicates no significant statistical difference, **p<0.01.

[0039] Figure 12 This is the result that HIF-3α-3 does not participate in the regulation of HIF-1α protein degradation;

[0040] Figure 13 This is the result of HIF-3α-3 regulating the synthesis of HIF-1α protein;

[0041] Figure 14 Figure 3 is the result of HIF-3α-3 participating in the transcriptional regulation process under hypoxia. A and B are gene enrichment analysis (GSEA) results showing that HIF-3α-3 participates in the transcriptional regulation process of RNA polymerase II under hypoxia and the gene expression levels of each signaling molecule (top 10) included in this signaling pathway. The blue area indicates that the gene was downregulated in all three replicate samples, and the red area indicates that the gene was upregulated.

[0042] Figure 15The results show that HIF-3α-3 upregulates the protein and mRNA levels of PHD3. A and B show the mRNA expression of PHD3 detected by RT-qPCR, *p<0.05, **p<0.01; B shows the effect of upregulation of PHD3 protein level on OH-HIF-1α protein; C shows the inhibition of PHD3 protein expression by MK8617 inhibitor; D shows the change of cell viability of T24 stable cell lines detected by CCK-8 assay, ns means no statistically significant difference, **p<0.01;

[0043] Figure 16 The results show that HIF-3α-3 binds to the PHD3 promoter region and upregulates its gene expression level, where A is the dual luciferase reporter gene experiment to verify HIF-3α-3 OE Figure 3. Changes in transcriptional activation of the PHD3 promoter in 293T cells in the control and vector groups under normoxia and hypoxia. ns: no statistical difference. **p<0.01. B: correlation analysis results between HIF-3α and PHD3 gene expression. R value: 0.4≤0.51<0.6, indicating a moderate correlation. DETAILED DESCRIPTION

[0044] The present invention provides the use of HIF-3α-3 gene or protein encoded by HIF-3α-3 gene in preparing medicine for preventing and / or treating bladder cancer.

[0045] In the present invention, the HIF-3α-3 gene preferably includes at least one of the following forms:

[0046] A. HIF-3α-3 gene with a nucleotide sequence as shown in SEQ ID NO: 1;

[0047] The nucleotide sequence encoding the HIF-3α-3 gene is:

[0048]

[0049] B. HIF-3α-3 gene encoding the protein with the amino acid sequence shown in SEQ ID NO: 2;

[0050] The amino acid sequence encoding the HIF-3α-3 protein is:

[0051] (SEQ ID NO: 2);

[0052] C. The amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, only the codons are optimized to form the HIF-3α-3 gene;

[0053] D. A genetically engineered product comprising at least one HIF-3α-3 gene selected from A, B, and C.

[0054] In the present invention, the amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, and only the codons in the HIF-3α-3 gene are optimized, preferably according to the type of host to be expressed.

[0055] In the present invention, the genetically engineered product preferably includes at least one of the following: a recombinant expression vector, a recombinant bacterial strain, and a recombinant virus. The present invention does not impose any particular restrictions on the type of backbone vector used in the recombinant expression vector; any well-known backbone vector used in gene therapy can be used. The host bacteria for the recombinant bacterial strain preferably include a prokaryotic expression system or a eukaryotic expression system. The prokaryotic expression system preferably includes Escherichia coli or lactic acid bacteria; the eukaryotic expression system preferably includes yeast. The recombinant virus preferably includes a recombinant lentivirus or a recombinant adenovirus, more preferably a recombinant lentivirus.

[0056] In the present invention, the protein encoded by the HIF-3α-3 gene preferably includes at least one of the following:

[0057] 1. HIF-3α-3 protein with the amino acid sequence shown in SEQ ID NO: 2;

[0058] II. HIF-3α-3 protein that has been chemically modified based on item I but whose biological function remains unchanged.

[0059] The present invention does not impose any particular limitation on the method of chemical modification, and any method known in the art for replacing or substituting functional groups without changing the function of the protein may be used.

[0060] In the present invention, the dosage form of the drug is preferably an injection. The present invention has no particular limitation on the preparation method of the injection, and any preparation method of the injection well known in the art can be used.

[0061] In the present invention, the HIF-3α-3 gene, HIF-3α-3 mRNA, or protein encoded by the HIF-3α-3 gene is specifically underexpressed in bladder cancer, and overexpression of the HIF-3α-3 gene inhibits the clonogenicity and proliferation of bladder cancer cells in vitro, simultaneously inhibits the growth of bladder cancer organoids, and inhibits the increase in tumor volume in mice in vivo. This indicates that the HIF-3α-3 gene, HIF-3α-3 mRNA, or protein encoded by the HIF-3α-3 gene exerts an anti-bladder cancer effect through positive regulation. Therefore, the present invention preferably provides the use of the HIF-3α-3 gene, HIF-3α-3 mRNA, or protein encoded by the HIF-3α-3 gene in the treatment of bladder cancer.

[0062] The present invention provides an application of an agent for promoting the expression of HIF-3α-3 gene or protein encoded by the HIF-3α-3 gene in the preparation of a drug for preventing and / or treating bladder cancer.

[0063] In the present invention, the agent preferably includes a molecule that enhances the transcriptional function of the HIF-3α-3 gene. The molecule includes at least one of the following genetic elements or a recombinant expression vector containing such genetic elements: a promoter, an enhancer, a facilitator, and a transcription factor. Given that overexpression of the HIF-3α-3 gene can effectively inhibit bladder cancer, increasing the expression of HIF-3α-3 mRNA and the corresponding protein by enhancing the transcriptional function of the HIF-3α-3 gene can also achieve the desired anti-bladder cancer effect. The drug is a gene drug. The present invention does not specifically limit the preparation method of the gene drug; methods well known in the art can be used to prepare the gene drug.

[0064] To further explore the molecular mechanism by which the HIF-3α-3 gene or the encoded protein exerts its anti-bladder cancer effects, the present invention first investigated the role of the HIF-3α-3 gene in hypoxic regulation of bladder cancer. Experiments demonstrated that, when wild-type bladder cancer cells are exposed to hypoxic conditions, HIF-1α protein expression levels increase while HIF-3α-3 protein expression levels decrease. Furthermore, overexpression of the HIF-3α-3 gene or protein further reduces the viability and proliferation of bladder cancer cells under hypoxic conditions. Secondly, the present invention investigated the effect of overexpression of the HIF-3α-3 gene or protein on HIF-1α protein, a prognostic biomarker for bladder cancer. The results demonstrated that, under hypoxic conditions, overexpression of HIF-3α-3 significantly reduces HIF-1α protein expression in cells. HIF-1α inhibitors can counteract the inhibitory effects of HIF-3α-3 on cell proliferation. In summary, HIF-3α-3 inhibits bladder cancer cell proliferation by reducing HIF-1α expression. Thirdly, to verify whether there is a direct interaction between HIF-3α-3 and HIF-1α, bladder cancer cells were treated with the tripeptide aldehyde inhibitor MG132 under hypoxic conditions to verify whether HIF-3α-3 regulates the synthesis of HIF-1α protein. The results showed that the effect of HIF-3α-3-mediated low protein expression of HIF-1α was not through intervention in the protein translation process. The present invention also conducted an assessment of factors affecting HIF-1α protein stability. The half-life of HIF-1α protein in the HIF-3α-3 high expression group was shorter than that in the control group. HIF-3α-3 overexpression caused widespread accumulation of OH-HIF-1α protein. Based on this, it can be determined that HIF-3α-3 inhibits the stability of HIF-1α protein by increasing the hydroxylation level of HIF-1α. In addition, under hypoxic conditions, HIF-3α-3 overexpressing cells participate in transcriptional regulation, increasing the expression levels of multiple genes including PHD3. PHD3 is a key oxygen sensor and inhibitory factor that controls the stability of HIF-1α. The results of the dual-luciferase reporter gene experiment showed that HIF-3α-3 may directly bind to the PHD3 promoter region to upregulate the expression of this gene.

[0065] Given that the HIF-3α-3 gene exerts its anti-bladder cancer effect by directly acting on the PHD3 protein or the expression level of the gene encoding it, the present invention provides the use of the PHD3 protein, the gene encoding the PHD3 protein, or an agent that activates the PHD3 gene promoter in the preparation of a drug for preventing and / or treating bladder cancer.

[0066] In the present invention, the PHD3 protein preferably includes at least one of the following:

[0067] S1. PHD3 protein with the amino acid sequence shown in SEQ ID NO: 3;

[0068] S2. PHD3 protein chemically modified based on item S1 without changing its biological function.

[0069] The present invention does not impose any particular limitation on the method of chemical modification, and any method known in the art for replacing or substituting functional groups without changing the function of the protein may be used.

[0070] In the present invention, the gene encoding the PHD3 protein preferably includes at least one of the following forms: A. a PHD3 gene having a nucleotide sequence as shown in SEQ ID NO: 4;

[0071] The nucleotide sequence of the PHD3 gene is:

[0072]

[0073] B. a PHD3 gene encoding a PHD3 protein having an amino acid sequence as shown in SEQ ID NO: 3;

[0074] The amino acid sequence of the PHD protein is:

[0075] MPLGHIMRLDLEKIALEYIVPCLHEVGFCYLDNFLGEVVGDCVLERVKQLHCTGALRDGQLAGPRAGVSKRHLRGDQITWIGGNEEGCEAISFLLSLIDRLVLYCGSRLGKYYVKERSKAM VACYPGNGTGYVRHVDNPNGDGRCITCIYYLNKNWDAKLHGGILRIFPEGKSFIADVEPIFDRLLFFWSDRRNPHEVQPSYATRYAMTVWYFDAEERAEAKKKFRNLTRKTESALTED(SEQ ID NO:3);

[0076] C. The amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, only the codons are optimized in the PHD3 gene;

[0077] D. A genetically engineered product comprising at least one PHD3 gene from A, B, and C.

[0078] In the present invention, the agent for activating the PHD3 gene promoter preferably includes the protein encoded by the HIF-3α-3 gene.

[0079] In the present invention, the drug can effectively inhibit the proliferation of bladder cancer cells in vivo and effectively reduce the size of bladder cancer tumors in vitro, thereby improving the survival rate. Therefore, the present invention provides a new approach for the development of new gene therapy drugs for the targeted treatment of bladder cancer.

[0080] The following describes in detail the use of the HIF-3α-3 gene or the protein encoded by it in the preparation of drugs for preventing and treating bladder cancer, in conjunction with the examples provided by the present invention. However, these examples should not be construed as limiting the scope of protection of the present invention.

[0081] Example 1

[0082] 1. Construction of HIF-3α-3 gene overexpression vector and lentiviral packaging

[0083] GenScript Biotech was commissioned to chemically synthesize a DNA fragment of the nucleotide sequence of the HIF-3α-3 gene (SEQ ID NO: 1). The GFP tag of the stop codon fusion vector was removed and inserted into the pcDNA3.1(+)-C-eGFP vector cut with EcoRV enzyme. The resulting HIF-3α-3 overexpression plasmid and the empty vector were simultaneously packaged with lentiviral vectors. Specifically, 293T cells were seeded in 75 mm culture flasks and transfected the next day using Lipofectamine 2000 (ThermoFisher, 11668019) according to the instructions, with the target plasmid, packaging plasmid psPAX2, and pMD2.G to prepare a lipid transfer system. The cell culture medium was replaced 16 h after transfection. The viral supernatant was collected 48 h and 72 h after transfection, respectively. Cell debris was removed by filtration using a 40 μm cell mesh, and the virus was collected by ultracentrifugation. The aliquots were stored at -80°C until use.

[0084] 2. Construction of overexpression cell lines

[0085] According to 5×10 per well 5 5637 cells and T24 cells were seeded into 6-well plates and cultured overnight. When the cells were completely attached and the confluence rate reached 60% to 70%, the cells were divided into the HIF-3α-3 overexpression experimental group (HIF-3α-3 OE ) and empty vector (Vector) control groups. Add 20μg / mL Polybrene transfer agent to each well, add the corresponding volume of virus according to the specific titer of each group of lentivirus at an MOI of 1:50, place in a 5% CO2, 37°C cell culture incubator, incubate for 12 hours, and then change the medium and add normal RPMI-1640 complete medium containing 1% double antibody and 10% FBS. Continue to culture in a 5% CO2, 37°C cell culture incubator, and expand the culture normally. After the third day, change to RPMI-1640 complete medium containing 2μg / mL puromycin to screen for stable expression cell lines.

[0086] 3. Real-time fluorescence quantitative PCR detection

[0087] 3.1 RNA extraction

[0088] TRIzol reagent was used to extract total genomic RNA from bladder cancer cell lines. First, 1×10 6 HIF-3α-3 OE5637 and T24 cells from the ELISA and vector groups were placed in 1.5 mL EP tubes. 1 mL of TRIzol solution was then added, the tubes were resuspended and mixed, and the tubes were placed on ice for 5 minutes to allow for complete lysis. 200 μL of chloroform solution was added to each tube, and the tubes were mixed by rapid inversion 15 times. The tubes were then placed on ice for 10 minutes to allow the layers to separate. The tubes were then placed symmetrically in a pre-cooled 4°C centrifuge and centrifuged at 12,000 rcf for 15 minutes. The top layer of the clear solution was transferred to a new, labeled 1.5 mL EP tube. An equal volume of isopropanol solution was added, the tubes were mixed by inversion, and the tubes were placed symmetrically in a pre-cooled 4°C centrifuge at 12,000 rcf for 10 minutes. The supernatant was then discarded. 1 mL of 75% ethanol was added to each tube, and the RNA pellet was washed twice. The supernatant was discarded. 20 μL of DEPC water was added to each sample tube to further dissolve the RNA pellet and determine RNA concentration.

[0089] 3.2 Reverse transcription

[0090] This step uses Ⅲ1st StrandcDNA Synthesis Super Mix for qPCR (gDNAdigester plus) reagent, and reverse transcription experiments were performed according to the instructions of the product.

[0091] 3.2.1 Removal of residual genomic DNA

[0092] Prepare the mixture according to Table 1 and place it in an RNase-free centrifuge tube. Gently pipette to mix thoroughly. Centrifuge for 1 minute to concentrate the liquid at the bottom of the tube. Incubate at 42°C for 2 minutes.

[0093] Table 1 Reverse transcription reaction system

[0094] Components Usage <![CDATA[RNasefreeddH2O]]> Add to 15 μL 5×gDNAdigesterMix 3μL Total RNA 1 μg

[0095] 3.2.2 Reverse transcription reaction system configuration and reverse transcription program settings

[0096] Table 2 Reverse transcription reaction procedure

[0097] temperature time 25℃ 5min 55℃ 15min 85℃ 5min

[0098] Add directly to the reaction tube III SuperMixplus, gently pipette to mix, then centrifuge for 1 min in a small centrifuge to concentrate the liquid in the tube at the bottom of the tube, and finally set the reverse transcription reaction program steps according to Table 2.

[0099] 3.3RT-qPCR

[0100] The products obtained by reverse transcription were subjected to a three-step real-time quantitative polymerase chain reaction (RT-qPCR) test. The reaction system was as follows: 1 μg of cDNA template, 0.6 μg of upstream and downstream primers, 5 μL of 2×Taq Pro Universal SYBR qPCR Master Mix, and finally RNase-free ddH2O was added to make the total volume of the liquid in the tube 10 μL. The reaction procedure steps of this experiment are shown in Table 3, and the sequences of the primers used are shown in Table 4. The mRNA expression levels of each gene measured were expressed as relative expression. The internal reference gene used in this experiment was the GAPDH gene. △CT experimental = CT treatment - CT treatment internal reference; △CT control = CT control - CT control internal reference; △△CT = △CT experimental - △CT control; the relative expression calculation formula is 2 -△△CT .

[0101] Table 3 qPCR reaction procedure

[0102]

[0103] Table 4 Primer sequences

[0104] Gene name 5' end sequence 3' end sequence HIF-3α-3 CCTGTGACCAAGAGGAGCTT(SEQ ID NO:5) TCATGCGCAAGGAGAAGCA(SEQ ID NO:6) PHD3 TCCCTAGCAGTCTTTCTT(SEQ ID NO:7) TACGTGCTCAATACCAAG(SEQ ID NO:8) TRIM21 GTATGTGCCCAGTCTCGGAA(SEQ ID NO:9) AGCTTCTCCTGGTACTCCTG(SEQ ID NO:10) HIF-1α GCAGCAACGACACAGAAACT(SEQ ID NO:11) CGTTTCAGCGGTGGGTAATG(SEQ ID NO:12) BAX TCCACCAAGAAGCTGAGCGAG(SEQ ID NO:13) GTCCAGCCCATGATGGTTCT(SEQ ID NO:14) Bcl2 ACTTCGCCGAGATGTCCAGC(SEQ ID NO:15) TACAGTTCCAAAAGGCATCCC(SEQ ID NO:16) GAPDH GGAGCGAGATCCCTCCAAAAT(SEQ ID NO:17) GGCTGTTGTCATACTTCTCATGG(SEQ ID NO:18)

[0105] 4. Protein Concentration Determination and Immunoblotting

[0106] 4.1 Protein extraction

[0107] 1) Take HIF-3α-3 OE 1×10 cells in each of the group and vector group 6 Cells were placed in 1.5 mL EP tubes, and 100 μL of RIPA cell lysis mixture containing protease inhibitors and phosphatase inhibitors (protease inhibitors: phosphatase inhibitors: RIPA cell lysis buffer = 1:1:100) was added to each tube to resuspend the cells.

[0108] 2) Place the EP tube on ice for 20 minutes (during this process, occasionally shake the liquid in the EP tube to ensure more complete lysis). Then place the EP tube symmetrically in a pre-cooled 4°C centrifuge at 13,000 rcf for 20 minutes. After centrifugation, collect the upper layer of solution into a newly labeled 1.5 mL EP tube.

[0109] 4.2 Protein concentration determination (BCA method)

[0110] 1) Prepare a standard protein solution with a certain concentration gradient in advance (concentration gradient is 0, 125, 250, 500, 1000, 2000 ng / μL) and add it to a 96-well plate in the order of the concentration gradient. The loading volume of the standard protein solution is 5 μL, and 3 replicates are set for each concentration.

[0111] 2) Mark the 96-well plate in advance, take 5 μL of the cell protein sample to be tested and add it to the marked corresponding position of the 96-well plate, and set up three replicate wells.

[0112] 3) Prepare BCA reaction working solution using Solution A and Solution B in a 50:1 volume ratio. Add 200 μL of BCA reaction working solution to each well of a 96-well plate. Wrap the 96-well plate in foil and incubate in a 37°C incubator to protect from light for 30 minutes. Then, remove the lid of the 96-well plate and place it in a microplate reader to measure the absorbance of each sample well at 562 nm. Draw a protein concentration standard curve based on the absorbance corresponding to the standard protein solution measured in the same batch. Calculate the concentration of the protein sample according to the standard curve formula.

[0113] 4) Based on the specific protein concentration of each sample measured in the above steps, dilute each protein sample with cell protein lysis buffer to a final concentration of 1000 ng / μL. Then, transfer 10 μL of the adjusted protein solution to a new 1.5 mL EP tube. Add 25 μL of 5× Protein Loading Buffer to each tube and mix thoroughly. Then, heat the EP tube in a 100°C dry bath for 10 min. After protein denaturation, store in a -20°C refrigerator.

[0114] 4.3 Electrophoresis

[0115] 1) Place 100 mL of 10× Tris / glycine / SDS electrophoresis buffer in a 2 L beaker. Add double-distilled water to a total volume of 1 L. Place a magnetic stir bar in the beaker and mix thoroughly on a magnetic stirrer at room temperature for 5 min.

[0116] 2) Add the diluted and mixed electrophoresis buffer to the electrophoresis tank, evenly pull out the comb of the 10% precast electrophoresis gel, add 10 μL of denatured protein sample to the wells of the precast electrophoresis gel, and add 5 μL of prestained protein marker to the empty wells at both ends of all the sample wells.

[0117] 3) Set the voltage to 100 V and start electrophoresis for 1.5 hours.

[0118] 4.4 Transfer and Blocking

[0119] 1) Place 100 mL of 10× Transfer Buffer in a 2 L beaker. Add double-distilled water until the total volume is 1 L. Place a magnetic stir bar in the beaker and place it on a magnetic stirrer to mix thoroughly at room temperature. Then, pre-cool the thoroughly mixed 1× Transfer Buffer in a 4°C refrigerator.

[0120] 2) Cut a polyvinylidene fluoride transfer membrane (PVDF transfer membrane) according to experimental requirements, place the membrane in an appropriate amount of methanol, and shake it on a shaker at low speed for 1 minute to activate the membrane.

[0121] 3) Soak the activated PVDF transfer membrane, sponge, and filter paper in the transfer solution for 5 minutes to fully soak them.

[0122] 4) Arrange the "sandwich" model in the order of sponge, filter paper, electrophoresis gel, PVDF transfer membrane, filter paper, and sponge. Ensure there are no bubbles between the PVDF transfer membrane and the electrophoresis gel. Set a constant current of 150 mA and transfer for 1.5 hours. (During transfer, place the transfer tank in an ice bath to prevent heat from overheating the transfer buffer and affecting the transfer process.)

[0123] 5) After the transfer is completed, place the PVDF transfer membrane in a small box containing 25 mL of 5% BSA solution on a shaker and block the membrane at room temperature for 30 minutes.

[0124] 4.5 Antibody incubation and exposure

[0125] 1) Discard the blocking buffer and cut the membrane into small strips based on the molecular size of the target protein and the internal control protein. Place the membrane in an incubation box, add 5 mL of 3% BSA solution, and add the corresponding primary antibody solution according to the antibody manufacturer's instructions. In this experiment, the working concentrations of the primary antibodies used are as follows: β-actin antibody concentration of 1:5000, HIF-3A antibody concentration of 1:1000, HIF-1A antibody concentration of 1:1000, Hydroxy-HIF1A antibody concentration of 1:1000, PHD3 antibody concentration of 1:2000, Bcl2 antibody concentration of 1:1000, BAX antibody concentration of 1:1000.

[0126] 2) Place the antibody incubation box on a shaker in a 4°C refrigerator to incubate the primary antibody overnight.

[0127] 3) The next day, recover the primary antibody solution and add 5 mL of 1×TBST solution containing Tween 20 to the antibody incubation box. Place the box on a shaker and wash at room temperature for 10 minutes, three times.

[0128] 4) Aspirate the 1× TBST solution in the incubation box, add 5 mL of the corresponding secondary antibody solution (secondary antibody dilution concentration is 1:2000), place the antibody incubation box on a shaker, and incubate at room temperature for 1 hour.

[0129] 5) After the secondary antibody incubation, recover the secondary antibody solution and add 5 mL of 1×TBST solution containing Tween 20 solution to the antibody incubation box. Place it on a shaker and wash at room temperature for 10 minutes, a total of 3 times.

[0130] 6) Prepare the chemiluminescent reaction working solution with a volume ratio of 1:1 between solution A and solution B of the chemiluminescent detection solution. Slowly add the chemiluminescent reaction working solution dropwise to the front of the membrane at a uniform speed and expose the membrane for imaging using a chemiluminescent gel imaging system.

[0131] 7) Data processing: The internal reference protein used in this experiment is human β-actin protein. ImageJ software was used to analyze and count the grayscale of the bands corresponding to each loaded protein, and the ratio of the target protein band to the internal reference protein band was calculated.

[0132] 5. Determination of cell viability (CCK-8 method)

[0133] Take a certain amount of HIF-3α-3 in the logarithmic growth phase OE 5637 cells and T24 cells in the vector group were resuspended in 1 mL of RPMI-1640 complete medium and counted. 100 μL of cells were added to a 96-well plate. The number of cells was 4 × 10 3 The cell suspension was prepared and the 96-well plate was pre-cultured in a 5% CO2, 37°C cell culture incubator for a period of time. After the cells were fully attached, the solution in the wells was aspirated and the mixture of 300μM CoCl2 (hypoxia treatment), different concentrations of LW6 (HIF-1α specific inhibitor), and MK8617 (PHD3 inhibitor) was added to the 96-well plate. Five replicate wells were set for each drug concentration, and corresponding blank control wells were set. The plate was then returned to the 5% CO2, 37°C cell culture incubator for continued culture. After 48 hours of drug treatment, the liquid in the wells was discarded and CCK-8 reaction solution was prepared at a ratio of 1:9 with CCK-8 solution and RPMI-1640 complete medium. 100 μL was added to each well (be careful not to form bubbles in the wells, otherwise it will affect the absorbance reading of the microplate reader). After wrapping with tin foil, the 96-well plate was placed in a cell culture incubator with 5% CO2 and 37°C and incubated for 1-4 hours. The 96-well plate cover was removed and placed in a microplate reader to measure the absorbance (OD value) at a wavelength of 450 nm. This experiment was repeated three times in parallel, and the OD value measured at a wavelength of 450 nm was used to represent the relative cell viability.

[0134] 6. Clone Formation Assay

[0135] Take a certain amount of HIF-3α-3 in the logarithmic growth phase OEFor the 5637 and T24 cells in the vector group and the 5637 and T24 cells in the vector group, 1 mL of complete RPMI-1640 medium was added and gently pipetted to a single-cell suspension. 10 μL of the cell suspension was then collected for cell counting. 2000 cells per well of each group were seeded into 6-well plates. The plates were gently shaken to evenly distribute the cells within the wells. The 6-well plates were incubated in a cell culture incubator at 5% CO2, 37°C, and saturated humidity for 2 weeks. During this period, cell growth within the wells was frequently monitored, and the medium was changed every 3 days until visible colonies of cells appeared within the 6-well plates. The old medium was aspirated and carefully rinsed twice with 1 mL of PBS per well. The cells were then fixed with 1 mL of 4% PFA for 15 minutes at room temperature. The fixative was aspirated and 1.5 mL of 0.02% crystal violet stain was added to each well. The 6-well plates were placed on a horizontal shaker and stained for 30 minutes at room temperature. Rinse each well slowly with tap water to remove the staining solution, let it dry, and then take a picture and count the colonies. Calculate the colony formation rate according to Formula 1.

[0136] Clone formation rate = 100% × (number of clones / number of seeded cells) Formula I.

[0137] 7. EdU cell proliferation imaging detection

[0138] 7.1 Cell culture and treatment

[0139] Take HIF-3α-3 in the logarithmic growth phase OE After counting the 5637 cells and T24 cells in the Vector group, the number of cells per well was 4×10 3 The cells were seeded into a 96-well plate. After the cells were completely attached to the wall, the corresponding drugs were added for treatment. The 96-well plate was placed in a cell culture incubator at 5% CO2 and 37°C and incubated for 48 hours.

[0140] 7.2EDU Marking

[0141] Dilute the EDU solution (reagent A) with RPMI-1640 complete medium at a volume ratio of 2000:1, add 100 μL of the diluted EDU reaction solution to each well, place the 96-well plate at 37°C and incubate for 2 h, aspirate the liquid in the wells, add 100 μL of PBS solution and wash the adherent cells in the wells for 3 min, a total of 1-2 times.

[0142] 7.3 Cell Fixation

[0143] Add 50 μL of 4% PFA solution to each well, place the 96-well plate on a horizontal shaker, incubate at room temperature for 10 minutes, and discard the fixative in the wells; add 50 μL of 2 mg / mL glycine solution to each well, place the plate on a horizontal shaker and incubate at room temperature for 5 minutes, and discard the glycine solution; add 100 μL of PBS solution to each well, place the culture plate on a horizontal shaker and wash for 5 minutes, for a total of 2 times; add 100 μL of permeabilization solution (PBS solution containing 0.5% TritonX-100) to each well, place the culture plate on a horizontal shaker, incubate at room temperature for 10 minutes, and then wash with PBS for 5 minutes, for a total of 2 times.

[0144] 7.4 Appollo staining

[0145] Prepare 1×Appollo staining reaction solution according to the Appollo staining reaction solution reference table in the instruction manual, and add 100 μL of For the staining reaction solution, wrap the 96-well plate with tin foil and place it on a horizontal shaker. Incubate at room temperature in the dark for 30 minutes, then discard the staining reaction solution. Add 100 μL of permeabilization solution (PBS containing 0.5% Triton X-100) to each well and incubate on a shaker for 10 minutes. Discard the permeabilization solution.

[0146] 7.5 DNA staining

[0147] After Appollo staining, wash away the staining solution with 100 μL of PBS solution per well, then add 100 μL of 1× HOEchst 33258 staining solution. Incubate the wells on a horizontal shaker at room temperature for 30 minutes. After staining, discard the staining solution and wash each well with 100 μL of PBS solution for 5 minutes three times. Photograph and analyze the results using a fluorescence microscope.

[0148] 8. RNA Sequencing and Analysis

[0149] TRIzol reagent was used to separate and extract the whole genome RNA of T24 cells in each group after gene editing. OE Three RNA samples from normoxic and hypoxic T24 cells in each group (both the two groups) were sequenced using an Agilent 2100 Bioanalyzer at Novogene (China). Sequence reads were mapped to the reference genome using HISAT2 software, and transcript abundance (number of reading frames) was calculated using Subread software. DESeq2 in the R language was used to normalize genomic mRNA expression levels, analyze differential expression, and create heat maps.

[0150] 9. Dual luciferase reporter system experiment

[0151] 9.1 Cell Treatment

[0152] HIF-3α-3 OE Human embryonic kidney 293T cells in the vector group were cultured in a 5% CO2, 37°C cell culture incubator. Cells in the logarithmic growth phase were digested and resuspended in DMEM complete medium to form a cell suspension. The number of cells per well was 5×10 4 Each group of cells was seeded into a 24-well plate, and transfection was performed when the confluence of 293T cells in the well was about 70%. OE 293T cells in the two groups (pGL3.1-PHD3 promoter plasmid, pGL3.1-Basic plasmid, and Renilla luciferase reporter gene plasmid) were co-transfected for 48 hours. After transfection, the medium was changed. The old solution in the 24-well plate was removed, and 500 μL of PBS solution was added to each well. The wells were gently rinsed twice. 200 μL of cell lysis buffer was added to the 24-well plate. The plate was incubated on ice for 5 minutes. The entire volume of the wells was transferred to a 1.5 mL EP tube. The tubes were placed symmetrically in a pre-cooled 4°C centrifuge at 10,000 rpm for 1 minute. The supernatant was transferred to a newly labeled 1.5 mL EP tube and the lysate was stored at -20°C.

[0153] 9.2 Fluorescence Detection

[0154] 1) Take appropriate amounts of 50× firefly luciferase substrate and 50× Renilla luciferase substrate and dilute them with the corresponding reaction buffer to make 1× firefly luciferase reaction working solution and 1× Renilla luciferin reaction solution, respectively, and incubate them to room temperature for use.

[0155] 2) Perform fluorescence detection according to the instructions in the manual. Take 20 μL of lysate and add it to a black, opaque, flat-bottom 96-well plate (set up three replicate wells, leaving a blank spacer between each well to avoid interference between the fluorescence values ​​of adjacent wells).

[0156] 3) Add 100 μL of firefly luciferase reaction solution to each sample well containing the lysate. Gently shake the plate to mix thoroughly. Measure the corresponding firefly luciferase fluorescence value of each sample well using a microplate reader as soon as possible.

[0157] 4) Add 100 μL of Renilla luciferase working solution to each well, gently shake the plate to mix, and immediately measure the Renilla luciferase fluorescence value corresponding to each sample well using a microplate reader.

[0158] 5) Collect the measured fluorescence values ​​and analyze the data, among which HIF-3α-3 OE The ratio of the experimental group to the vector group was calculated according to formula II; the reporter gene expression fold was equal to the experimental group ratio / the control group ratio.

[0159] Ratio = (fluorescence value measured by firefly luciferase - firefly fluorescence background value) / (fluorescence value measured by Renilla luciferase - Renilla fluorescence background value) Formula II

[0160] 10. Mouse subcutaneous tumor formation experiment

[0161] HIF-3α-3 in the logarithmic growth phase OE BIU87 cells in the vector group were digested with 0.25% trypsin and counted, with a count of 3×10 cells per nude mouse. 6 The corresponding cells were removed and resuspended in a mixture of PBS and Matrigel (PBS to Matrigel ratio of 1:1). 100 μL of the cell suspension was injected subcutaneously near the right axilla of nude mice using a 0.2 μm insulin needle. The length (l) and width (w) of the subcutaneous tumor of the mice were measured every 3 days with an electronic vernier caliper. The tumor volume was calculated as V = lw 2 The mice were weighed and their weights were recorded. After 14 days, the mice were euthanized, photographed according to group, and the tumors were removed by autopsy. Tumors were photographed and weighed, and the subcutaneous tumor volumes of the mice in the different groups were calculated, and P values ​​were calculated. Graphpad 8.0 software was used to generate graphs of tumor volume and body weight changes in the two groups over 14 days for analysis.

[0162] 11. H&E staining and immunohistochemistry

[0163] 1) Tissue block pretreatment: Fresh tumor tissue was placed in a 4% PFA solution and fixed overnight in a 4°C refrigerator. The tissue was then dehydrated and transparentized by soaking in 30% ethanol, 50% ethanol, 70% ethanol, 80% ethanol, 90% ethanol, and 100% ethanol for 30 minutes each. The tissue was then soaked in xylene for 10 minutes twice.

[0164] 2) Paraffin Embedding of Tissue: First, melt the paraffin block in a 56°C thermostat. Then, pour a small amount of melted liquid paraffin into a paper mold. Wait for the paraffin in the paper mold to cool slightly, and then gently place the tissue block to be embedded in the paraffin in the paper mold. During this process, pay attention to arrange the tissue blocks neatly. Then, add an appropriate amount of melted liquid paraffin and place it at room temperature until the paraffin block in the paper mold is completely frozen and becomes solid.

[0165] 3) Tissue sectioning: Remove the cooled solid tissue wax block from the paper mold, place the wax block on the paraffin slicer, and adjust the direction of the paraffin block installed on the slicer to keep the tissue and cutting directions consistent. Then adjust the thickness of the slice to 5μm. If the slice is not cut well, adjust the thickness of the slice appropriately. Use a brush to gently pull the cut tissue slice outwards, and at the same time use tweezers to lay the slice with complete tissue slice flat on the surface of 40℃ warm water (note that before placing the tissue slice in 40℃ warm water, the bubbles in the water bath should be driven away first to prevent the bubbles in the water from floating up due to heat and sticking to the tissue slice and damaging the tissue slice).

[0166] 4) Retrieving tissue slices: When the tissue slices are heated and spread out on the surface of 40℃ warm water, use the lower half of the slide to retrieve the tissue slices. Usually, two slices of tissue are retrieved from each slide. This will reduce the error in the experiment. Then place the slides with the tissue slices on a slide rack and put them into a 37℃ constant temperature box to dry.

[0167] 11.1 H&E staining

[0168] 1) Dewaxing: Place the dried slides on a slide rack and soak them in a container of xylene for 15 minutes, repeat twice. Then, soak the slide rack in 100% ethanol I, 100% ethanol II, 95% ethanol, 80% ethanol, and 70% ethanol, respectively, for 5 minutes each.

[0169] 2) Hematoxylin staining: Wash the slides once in tap water for 5 minutes; stain them in hematoxylin solution for 3-5 minutes, then wash them in tap water. Then, soak the slides in blueing solution for 1-2 seconds, and then soak them in tap water for 5 minutes.

[0170] 3) Eosin staining: Dehydrate in 80% ethanol for 5 minutes, then stain in eosin alcohol solution for 10-15 minutes.

[0171] 4) Mounting: Soak the slides in 95% ethanol and then 100% ethanol twice for 1 minute each. Finally, soak them in xylene twice for 3 minutes each. Add a small amount of neutral resin to the front of the slide and slowly cover it with a coverslip (be careful not to allow air bubbles to form on the tissue during the mounting process). Place the mounted slides in a fume hood to dry.

[0172] 11.2 Immunohistochemistry

[0173] 1) Dewaxing: Place the slides prepared in advance for tissue sections in a slide rack. Place the slide rack in xylene I, xylene II, 100% ethanol I, 100% ethanol II, 95% ethanol, 90% ethanol, 80% ethanol, and 70% ethanol solutions in sequence, soaking in each solution for 5-10 minutes.

[0174] 2) Antigen retrieval: First, place the dewaxed slide in a large beaker filled with 1× citric acid buffer (pH 5.8). Wipe the surface and bottom of the beaker dry, then microwave on high for 3 minutes (until the solution in the beaker boils). Place the slide in the large beaker, cover the beaker with plastic wrap, and microwave on low for 15-20 minutes. Remove the beaker from the microwave and cool to room temperature (cooking is to expose the antigen sites on the tissue). Rinse slowly with tap water for 2-3 minutes, then soak in 3% H2O2 solution for 10 minutes (to remove endogenous catalase). Rinse again in tap water twice, 5 minutes each time.

[0175] 3) Serum Blocking: Immerse the slide in PBS twice, 5 minutes each time. Wipe away any liquid surrounding the tissue on the slide with absorbent paper. Use a water-blocking pen to draw a water-blocking circle around the tissue. Immediately add an appropriate amount of serum blocking solution diluted 10-fold with PBS to the tissue (to block nonspecific sites). Then, incubate the slide in a 37°C incubator for 30 minutes.

[0176] 4) Primary Antibody Incubation: Remove the slide from the 37°C incubator and wipe dry the liquid on the back of the slide and around the water-blocking ring on the front with absorbent paper. Add an appropriate amount of primary antibody solution to the water-blocking ring. Simultaneously, set up a blank control group. Add PBS solution to another tissue on the same slide. Then, place the slide in a humidified chamber and incubate with the primary antibody overnight at 4°C.

[0177] 5) Add Reaction Enhancement Solution: Remove the slide from the 4°C refrigerator, shake off the primary antibody solution, and place the slide on a slide rack. Place the slide in a beaker of PBS solution on a decolorizing shaker and rinse three times, 5 minutes each time. After washing, wipe the PBS solution around the water-blocking ring with absorbent paper. Then, add an appropriate amount of Reaction Enhancement Solution and incubate the slide in a humidified chamber at room temperature for 20 minutes.

[0178] 6) Incubation with Secondary Antibody: After incubation in the reaction enhancement solution, place the slides in a slide rack and place them in a beaker of PBS solution on a decolorizing shaker. Wash three times, 5 minutes each time. After washing, wipe the PBS solution around the water barrier with absorbent paper. Then, add an appropriate amount of enhanced enzyme-labeled goat anti-rabbit IgG polymer. Place the slides in a humidified chamber and incubate at room temperature for 1 hour.

[0179] 7) DBA Color Development: After secondary antibody incubation, place the slides in a slide rack, place them in a beaker of PBS solution, and place the beaker on a decolorizing shaker. Wash the slides three times, 5 minutes each time. After washing, wipe the PBS solution around the water-blocking ring with absorbent paper. Then, add an appropriate amount of freshly prepared DAB color development solution and incubate at room temperature for 5-10 minutes.

[0180] 8) Re-staining: After staining, rinse the slide with tap water for 3-5 minutes, then place the slide in hematoxylin solution for re-staining, differentiate, and rinse to return to blue.

[0181] 9) Dehydration and transparentization: Rinse the counterstained slides in tap water, then soak them in 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, 100% ethanol I, 100% ethanol II, xylene I, and xylene II solutions for 5 minutes (xylene soaking should be done in a fume hood).

[0182] 10) Sealing: Add a small amount of neutral resin to the tissue on the front of the slide, then slowly cover it with a coverslip (do not allow bubbles to form on the tissue during the sealing process). Place the sealed slide in a fume hood to dry.

[0183] 12. Statistical Analysis

[0184] GraphPad Prism 8.0 software was used for graphical analysis. Data conforming to a normal distribution are expressed as x ± standard error of the mean (SEM). Unpaired t-tests were used for comparisons between two groups, and one-way analysis of variance was used for comparisons between multiple groups. *p < 0.05, **p < 0.01, ***p < 0.001 indicate statistically significant differences between two or more groups.

[0185] 13. Results

[0186] 13.1 HIF-3α-3 is specifically underexpressed in bladder cancer

[0187] First, samples of cancerous tissue (BT) and their corresponding paracancerous tissue (BN) were obtained from 8 patients with bladder cancer after surgical resection. After pretreatment, protein was extracted from the tissue samples and Western Blot analysis was performed. The experimental results showed that the expression level of HIF-3α-3 protein was higher in the patients' paracancerous tissue samples, while the expression level of this protein was lower in the cancer tissue samples. The comparison showed that the expression level of HIF-3α-3 protein in cancer tissue was significantly lower than that in normal tissue ( Figure 1 Middle A).

[0188] Further, the expression difference of HIF-3α-3 was detected at the mRNA level. RNA was extracted from cancer tissues and corresponding adjacent tissues of 22 bladder cancer patients, and the relative expression of HIF-3α-3 mRNA was detected by RT-qPCR. It was found that the mRNA expression level of HIF-3α-3 in cancer tissues was significantly lower than that in adjacent tissues. This trend was consistent with the expression difference at the protein level ( Figure 1 Middle B).

[0189] At the same time, the experiment found that HIF-3α-3 was expressed in human bladder cancer cell lines 5637, T24, RT112 and normal urothelial immortalized cells SV-HUC1. Using SV-HUC1 as a control cell line, the protein expression levels of HIF-3α-3 in 5637, T24, and RT112 cells were significantly lower than those in SV-HUC1 ( Figure 1 C), and this trend was also verified at the mRNA level ( Figure 1 Through the above analysis, the experiment detected that HIF-3α-3 was specifically underexpressed in bladder cancer at both the tissue and cell levels.

[0190] 13.2 HIF-3α-3 overexpression inhibits bladder cancer cell proliferation

[0191] Stable cell lines constructed through lentiviral infection have high overexpression efficiency and stable cell states, making them more convenient for in vitro experimental operations and subsequent exploration of biological signaling molecules. Therefore, 5637 cells and T24 cells with stable overexpression of HIF-3α-3 were constructed. Based on the specific low expression of HIF-3α-3 in bladder cancer, clone formation experiments and EDU experiments were considered to verify the specific functional role of HIF-3α-3 in bladder cancer.

[0192] Under a fluorescence microscope, all cells in each group that had completed lentiviral infection showed red fluorescent signals, indicating that the fluorescently labeled HIF-3α-3 lentiviral vector was stably expressed in the cells. Western Blot and RT-qPCR experiments were further used to verify the effect of HIF-3α-3 gene overexpression in 5637 cells and T24 cells that were infected with lentivirus and screened with puromycin. The experimental results showed that the protein and mRNA levels of HIF-3α-3 in 5637 cells and T24 cells were stably and continuously expressed at high levels ( Figure 2 (A, B).

[0193] The clone formation rate can be used to relatively quantitatively analyze the proliferation potential and independent viability of cells. OE 5637 cells and T24 cells in the vector group were seeded into 6-well plates. After culturing for 2 weeks, the adherent cells in the wells were fixed and stained with crystal violet. The number of clones of 5637 cells and T24 cells in each group was statistically analyzed. It was found that the number of clones formed by the two cell lines was significantly higher than that of HIF-3α-3. OE The number of colonies in the group was significantly less than that in the Vector group ( Figure 3 A, C), the statistical results also showed that HIF-3α-3 OE The clone formation rates of 5637 cells and T24 cells in the group were lower than those in the Vector group ( Figure 3Therefore, it was judged that HIF-3α-3 overexpression significantly inhibited the cell clone formation ability and proliferation potential.

[0194] The thymidine nucleoside analog (5-Ethynyl-2'-deoxyuridine, EDU) can replace thymine and be incorporated into DNA replication chains during cell proliferation. By combining with corresponding fluorescent dyes, DNA replication activity can be rapidly detected, indirectly reflecting cell proliferation. EDU experiments were further conducted to verify whether HIF-3α-3 overexpression affects the proliferation of 5637 and T24 cells.

[0195] After cell counting, take the same amount of HIF-3α-3 OE The 5637 cells and T24 cells in the vector group were plated in 96-well plates and cultured in a cell culture incubator for 48 hours. The cell proliferation activity of each group of cells was detected by EDU assay. After EDU staining, HIF-3α-3 OE The number of EDU-stained cells in 5637 cells and T24 cells in the group was significantly lower than that in the Vector group ( Figure 4 A, B), and the results of counting analysis also showed that HIF-3α-3 OE The EDU-positive cell rates of 5637 cells and T24 cells in the group were even lower ( Figure 4 (C, D) further confirmed the specific functional role of HIF-3α-3 in in vitro cell experiments, namely, inhibiting the proliferation of bladder urothelial cells.

[0196] 13.3 HIF-3α-3 inhibits the growth of bladder cancer organoids

[0197] First, the packaged lentivirus of HIF-3α-3 overexpression vector was collected and used to infect bladder cancer organoids from three patients. After lentiviral infection, puromycin was used to screen the organoids with stable overexpression of HIF-3α-3. OE The three organoids in the group and the vector group were photographed and recorded under the microscope under white light every other day, and HIF-3α-3 OE The morphology of the organoids in the group was poor, and the morphology of the individual organoid spheres was not smooth and more scattered ( Figure 5 Middle A).

[0198] In addition, the statistical results also showed that HIF-3α-3 OE The number and size of bladder cancer organoids in the group were significantly lower than those in the Vector group ( Figure 5 (B, C) Thus, HIF-3α-3 also functions as a tumor suppressor gene in the in vitro 3D model.

[0199] In summary, the results of the above in vitro experiments confirmed from multiple aspects and dimensions that HIF-3α-3 inhibits the proliferation of bladder cancer cells, which may be an important way for HIF-3α-3 to participate in intervening in the development of bladder cancer.

[0200] 13.4 HIF-3α-3 inhibits the development of bladder cancer in vivo

[0201] In order to further evaluate the therapeutic effect of HIF-3α-3 in vivo, a mouse xenograft tumor model was established. BIU87 cells were infected with HIF-3α-3 overexpression lentivirus and negative empty vector control lentivirus to establish a cell line that stably and highly expresses HIF-3α-3 and can be used in animal experiments. Nude mice are inbred mice that are hairless, naked, and without thymus. They lack T cells and have poor immunity. They must be kept in a barrier system and are mainly used for experimental research in basic medicine and clinical medicine such as oncology, toxicology, and immunology. In this experiment, HIF-3α-3 was overexpressed in BIU87 cells. OE BIU87 cells in the two groups were injected into the subcutaneous tissues of BALB / c nude mice to induce tumor formation. The tumors were removed 4 weeks later. OE The subcutaneous tumor volume of mice in group A was smaller than that in group B ( Figure 6 In addition, HIF-3α-3 OE The weight of subcutaneous tumor in group mice was lower ( Figure 6 (B, C) The removed tumor tissue was sliced ​​and then subjected to H&E staining and immunohistological examination for PCNA and Ki-67 positive staining. The immunohistochemical results showed that HIF-3α-3 OE The PCNA and Ki-67 levels in the tumor sections of the group were significantly lower than those in the Vector group. The results of the tumor-bearing nude mouse model showed that HIF-3α-3 could inhibit the development of bladder cancer in vivo ( Figure 6 Middle D).

[0202] 13.5 HIF-3α-3 is involved in the regulation of hypoxia in bladder cancer

[0203] First, whole-genome RNA was extracted from cells in the HIF-3α-3 overexpression group and the control group. Transcriptome sequencing of these RNA samples was performed and gene ontology analysis was performed using bioinformatics analysis methods. The sequencing and analysis results showed that the overexpression of HIF-3α-3 in bladder cancer was positively correlated with the regulation of cellular humoral levels, neutrophil-mediated immunity, and response to decreased oxygen levels ( Figure 7 ).

[0204] HIF-3α-3 was taken after lentivirus infection OEWhole-genome total RNA was extracted from T24 cells in the two groups, and common eukaryotic cell transcriptome sequencing and GO function enrichment analysis were performed.

[0205] Therefore, we established a hypoxic model by chemically activating a bladder cancer cell line in vitro and first determined the regulation of endogenous HIF-3α-3 expression in 5637 and T24 cells. Similar to most solid tumors, HIF-1α is generally considered a key regulatory molecule in the hypoxic tumor microenvironment.

[0206] After 24 hours of hypoxia treatment of 5637 and T24 wild-type cells with CoCl2, cell proteins were extracted and immunoblotting was performed to detect changes in HIF-3α-3 protein levels when the two bladder cancer cells were exposed to hypoxia. The experimental results showed that in the wild-type bladder cancer cell lines 5637 and T24 cells, while the expression level of HIF-1α protein increased, the expression level of HIF-3α-3 protein showed a downward trend ( Figure 8 ).

[0207] Wild-type 5637 cells and T24 cells were plated in 12-well plates. After the cells adhered, they were treated with oxygen-consuming agents containing different concentrations of CoCl2 for 24 hours. The total protein of the cells in each treatment group was extracted, and the changes in the expression levels of HIF-1α and HIF-3α-3 were detected by protein immunoblotting.

[0208] The CCK-8 assay was further used to detect the cell viability of 5637 cells and T24 cells in the HIF-3α-3 stable overexpression group and the empty vector control group under normoxic and hypoxic conditions. The results of the CCK-8 assay showed that HIF-3α-3 could further reduce the viability of bladder cancer cells under hypoxic conditions ( Figure 9 A, B, C, D).

[0209] Finally, the EDU experiment was also used to detect the changes in cell proliferation of the two cell lines under hypoxic conditions. Figure 10 As shown in A and B, the proliferation of 5637 cells and T24 cells in the HIF-3α-3 overexpression group was significantly inhibited under hypoxic conditions. The corresponding statistical results are shown in Figure 10 C and D in the middle.

[0210] 13.6 HIF-3α-3 overexpression promotes HIF-1α protein degradation

[0211] The role of HIF-1α in the progression of bladder cancer has rarely been reported. Using the TCGA bladder cancer cohort and transcriptome sequencing data and clinical annotations, the Kaplan-Meier method was used to analyze the clinical prognostic relevance of the HIF-1α gene. This database cohort includes 433 RNA sequencing samples. The results of the analysis showed that in this cohort, there was a significant statistical difference between the high expression group and the low expression group of the HIF-1α gene in the survival prognosis analysis of bladder cancer patients, that is, the P value was 0.025. It can be seen that when the HIF-1α gene is expressed at a low level in bladder cancer, the patient's survival prognosis is better. Therefore, we believe that HIF-1α can be used as a prognostic biomarker molecule for bladder cancer ( Figure 11 Middle A).

[0212] The previously constructed T24 cell lines of the HIF-3α-3 stable overexpression group and the empty vector control group were further treated with normoxia and hypoxia, respectively. Then, proteins of the two groups of cells were extracted after 24 hours of normoxia and hypoxia culture. The results of Western blotting showed that the expression level of HIF-1α protein in the HIF-3α-3 overexpression group was significantly lower than that in the negative control group under hypoxia ( Figure 11 Middle B).

[0213] Further research was conducted on the cell viability of bladder cancer cell T24 cells that overexpress HIF-3α-3 after treatment with the specific HIF-1α inhibitor LW6 (i.e., inhibiting the expression level of HIF-1α). It was found that LW6 could offset the inhibitory effect of HIF-3α-3 on cell proliferation (e.g., Figure 11 In conclusion, HIF-3α-3 inhibits the proliferation of bladder cancer cells by reducing the expression of HIF-1α.

[0214] Generally speaking, protein levels are affected by protein translation efficiency and stability. First, we treated T24 cells with the tripeptide aldehyde inhibitor MG132 (a specific inhibitor of the 26S proteasome) under hypoxic conditions to verify whether HIF-3α-3 regulates the synthesis of HIF-1α protein. The results of protein immunoblotting experiments showed that when the MG132 treatment time continued to increase, the expression of HIF-1α protein showed an upward trend. HIF-3α-3 overexpression was not affected by it (the level of HIF-3α-3 overexpression protein did not change with treatment time). Therefore, it is believed that the effect of HIF-3α-3-mediated low protein expression level of HIF-1α is not through intervention in the protein translation process ( Figure 12 ).

[0215] Ubiquitination and degradation are the main pathways that affect the stability of HIF-1α protein, involving a series of complex protein members, including proline hydroxylase (PHD) hydroxylating specific proline sites, the recognition function of phosphorylated VHL, and the degradation process of E3 ubiquitin ligase linking hydroxylated proteins to recruit proteasomes. Similarly, the study used cycloheximide (CHX) to inhibit protein synthesis to analyze the degradation half-life of HIF-1α protein. As expected, the half-life of HIF-1α protein in the HIF-3α-3 high expression group was shorter than that in the control group ( Figure 13 At the same time, the hydroxylation level of HIF-1α was detected after MG132 treatment of cells under hypoxia. The results showed that OH-HIF-1α protein was generally accumulated in the cells of the HIF-3α-3 overexpression group ( Figure 15 In summary, it is believed that HIF-3α-3 inhibits the stability of HIF-1α protein by increasing the hydroxylation level of HIF-1α.

[0216] In the absence of oxygen, 5 μM CHX protein synthesis inhibitor was added for 0 h, 2 h, 4 h, 6 h, and 8 h, and the total protein of each group of cells was extracted to detect HIF-3α-3 OE The differences in the protein degradation processes of T24 cell stably transfected lines after the synthesis of HIF-3α-3, HIF-1α, and PHD3 proteins was blocked between the vector group and the vector group.

[0217] 13.7 Transcriptional Regulation of HIF-3α-3

[0218] Gene set enrichment analysis of RNA-seq data revealed that the expression regulation process of RNA polymerase II promoter for hypoxia response transcriptional regulation was significantly upregulated when HIF-3α-3 was overexpressed ( Figure 14 As shown in the heat map, HIF-3α-3 overexpressing cells under hypoxic conditions participate in transcriptional regulation, upregulating the gene expression levels of multiple molecules, including PHD3, EPAS1, PHD1, UBC, etc., and downregulating the expression levels of VHL, VEFGA, PHD2, and NOTCH1 ( Figure 14 Middle B). Proline hydroxylase domain proteins are key oxygen sensors and inhibitors controlling HIF-1α stability. Based on transcriptome sequencing results, PHD3 is hypothesized to be a molecular mediator of the functional interaction between HIF-3α-3 and HIF-1α.

[0219] Therefore, we first verified the protein and mRNA levels of PHD3 in HIF-3α-3 overexpressing cells. The experimental results showed that under both normoxic and hypoxic conditions, the protein and mRNA expression levels of PHD3 in HIF-3α-3 overexpressing cells were significantly increased ( Figure 15 Most studies have shown that hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitors (PHIs) have revolutionized cancer treatment strategies. HIF-PHI MK8617 was used to treat HIF-3α-3 overexpressing and hypoxic negative cells in a dose-dependent manner. Due to the decrease in PHD3 protein levels, CCK-8 results showed that the MK8617 treatment group abolished the anti-proliferative effect of HIF-3α-3 overexpression (e.g., Figure 15 (C, D).

[0220] To further explore the transcriptional activation function of HIF-3α-3, we planned to measure the relative luciferase activity produced by the PHD3 luciferase reporter gene through a dual-luciferase reporter gene experiment. The promoter region of PHD3 was connected to the corresponding luciferase gene to construct a reporter gene plasmid, a HIF-3α-3 overexpression vector plasmid or a control empty vector plasmid and a Renilla luciferase expression plasmid (as an internal reference plasmid) and co-transfected into 293T cells. The transfected 293T cells were incubated under normoxic and hypoxic conditions for 48 hours. The results of fluorescence detection showed that the PHD3 luciferase activity of the HIF-3α-3 overexpression group was significantly higher than that of the control group under hypoxia, but not under normoxic conditions (such as Figure 16 Therefore, it is believed that HIF-3α-3 may directly bind to the PHD3 promoter region and drive the transcription of the luciferase reporter gene.

[0221] In addition, in the GEPIA dataset, Pearson correlation analysis of the gene expression of HIF-3α and PHD3 revealed that the expression levels of the two genes showed a moderate positive correlation (e.g. Figure 16 This is consistent with the upregulation of PHD3 gene expression when HIF-3α-3 is overexpressed in bladder cancer samples detected in this example, suggesting that HIF-3α-3 is involved in regulating the transcription process of PHD3.

[0222] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Use of the HIF-3α-3 gene or the protein encoded by the HIF-3α-3 gene in the preparation of a drug for preventing and / or treating bladder cancer.

2. The application according to claim 1, characterized in that The HIF-3α-3 gene includes at least one of the following forms: A. HIF-3α-3 gene with a nucleotide sequence as shown in SEQ ID NO: 1; B. HIF-3α-3 gene encoding the protein with the amino acid sequence shown in SEQ ID NO: 2; C. The amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, only the codons are optimized to form the HIF-3α-3 gene; D. A genetically engineered product comprising at least one HIF-3α-3 gene selected from A, B, and C.

3. The application according to claim 2, characterized in that: The genetic engineering product includes at least one of the following: a recombinant expression vector, a recombinant strain and a recombinant virus.

4. The application according to claim 1, characterized in that The protein encoded by the HIF-3α-3 gene includes at least one of the following:

1. HIF-3α-3 protein with the amino acid sequence shown in SEQ ID NO: 2; II. HIF-3α-3 protein that has been chemically modified based on item I but whose biological function remains unchanged.

5. Use of an agent for promoting the expression of HIF-3α-3 gene or protein encoded by HIF-3α-3 gene in the preparation of a drug for preventing and / or treating bladder cancer.

6. The application according to claim 5, characterized in that The agents include molecules that enhance the transcriptional function of the HIF-3α-3 gene.

7. Use of a PHD3 protein, a gene encoding a PHD3 protein, or an agent that activates a PHD3 gene promoter in the preparation of a drug for preventing and / or treating bladder cancer.

8. The application according to claim 7, characterized in that: The PHD3 protein includes at least one of the following: S1. PHD3 protein with the amino acid sequence shown in SEQ ID NO: 3; S2. PHD3 protein chemically modified based on item S1 without changing its biological function.

9. The application according to claim 7, characterized in that: The gene encoding the PHD3 protein includes at least one of the following forms: A. PHD3 gene with the nucleotide sequence shown in SEQ ID NO: 4; B. a PHD3 gene encoding a PHD3 protein having an amino acid sequence as shown in SEQ ID NO: 3; C. The amino acid sequence of the encoded HIF-3α-3 protein remains unchanged, only the PHD3 gene is codon-optimized; D. A genetically engineered product comprising at least one PHD3 gene from A, B, and C.

10. The use according to claim 7, characterized in that: The reagent for activating the PHD3 gene promoter includes the protein encoded by the HIF-3α-3 gene.

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