Application of RFWD3 gene as esophageal cancer treatment target

Abstract

The application discloses application of an RFWD3 gene as an esophageal cancer treatment target point. The application first constructs an RFWD3 gene knockdown virus, detects the proliferation and apoptosis of esophageal cancer cells after the RFWD3 gene is knocked down at a cell level, and the result shows that knocking down the RFWD3 has the effect of inhibiting the proliferation of esophageal cancer cells and promoting the apoptosis of esophageal cancer cells. Further, the application verifies that knocking down the RFWD3 can effectively inhibit the proliferation of tumor cells and the growth process of tumors by constructing an esophageal cancer animal model in vivo. Therefore, the application provides a new target point for treating esophageal cancer, and is expected to screen and prepare a new drug for effectively treating esophageal cancer aiming at the target point.

Description

Technical Field

[0001] The present invention relates to the application of RFWD3 gene as a therapeutic target for esophageal cancer and belongs to the technical field of biomedicine. Background Art

[0002] Ubiquitination is a highly conserved intracellular protein post-translational modification pathway that determines the fate of proteins by coupling the small protein ubiquitin (Ub) to lysine residues of protein substrates (Xu P, et al. Cell 2009), controlling multiple biological functions such as cell proliferation, cell cycle progression, transcription, and apoptosis (MOONEY, et al. J Dent Res 2021).

[0003] RFWD3 (Ring finger and WD repeat domain 3) is a RING finger protein that acts as an E3 ubiquitin ligase, targeting substrate proteins for ubiquitination and degradation, thereby increasing substrate protein turnover. Previous studies have shown that RFWD3 is involved in the occurrence and progression of various tumors. For example, high expression of RFWD3 is associated with the occurrence and development of gastric cancer and may be an important adverse prognostic factor for gastric cancer (Jia J, et al. Physiol. Biophys 2020); knockdown of the RFWD3 gene inhibits the development of colorectal cancer in vitro and in vivo (Xu F, et al. Front Cell Dev Biol 2021); RFWD3 plays a tumor promoter role in the occurrence and development of bladder cancer (Jiang P, et al. Histol Histopathol 2023); knockdown of RFWD3 can inhibit the colony formation activity of non-small cell lung cancer cells (Zhang Y, et al. Front Med. 2020); RFWD3 is highly expressed in pancreatic cancer and may coordinate genes in the DNA repair process (Zhu Y, et al. Chin Med J (Eng) 2022); RFWD3 regulates the proliferation and metastasis of hepatocellular carcinoma through the Wnt / β-catenin signaling pathway (Liang RP, et al. World J Gastroenterol 2022). However, there are currently no reports on the function of RFWD3 in esophageal cancer. Summary of the Invention

[0004] The purpose of the present invention is to provide a new potential therapeutic target for esophageal cancer.

[0005] In order to achieve the above object, the present invention provides the use of RFWD3 gene and / or its expression product in screening and preparing drugs for treating esophageal cancer.

[0006] Preferably, the active ingredient of the drug includes an agent that inhibits the expression and / or activity of the RFWD3 gene and / or its expression product.

[0007] Preferably, the active ingredient of the drug is at least one of siRNA, shRNA, RNAi plasmid vector and siRNA lentivirus that specifically inhibits the expression of RFWD3 gene.

[0008] Compared with the prior art, the present invention has the following beneficial effects:

[0009] The present invention verifies at the cellular level that knocking down RFWD3 has the effect of inhibiting the proliferation of esophageal cancer cells and promoting the apoptosis of esophageal cancer cells. Furthermore, the present invention constructs an esophageal cancer animal model and verifies in the animal body that knocking down RFWD3 can effectively inhibit the proliferation of tumor cells and the growth process of the tumor. Therefore, the present invention provides a new target for the treatment of esophageal cancer, and it is expected to screen and prepare new drugs that are effective in treating esophageal cancer by targeting this target. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 The expression of RFWD3 gene in esophageal cancer cells TE-1 and ECA109 was detected by RT-PCR.

[0011] Figure 2 The expression of RFWD3 gene in esophageal cancer cells TE-1 and ECA109 was detected by RT-PCR.

[0012] Figure 3 The results of celigo detection of cell proliferation;

[0013] Figure 4 This is the result of MTT assay for cell proliferation after RFWD3-siRNA lentivirus knockdown.

[0014] Figure 5 This is the result of MTT assay for tumor cell proliferation after RFWD3-siRNA lentivirus 1 knockdown;

[0015] Figure 6 Flow cytometry was used to detect apoptosis in the experimental and control groups after tumor cells were infected with RFWD3-siRNA lentivirus 1;

[0016] Figure 7 Flow cytometry was used to detect apoptosis in the experimental and control groups after tumor cells were infected with RFWD3-siRNA lentivirus 2;

[0017] Figure 8To detect the apoptosis of tumor cells in experimental and control groups after infection with RFWD3-siRNA lentivirus 1 for caspase3-7;

[0018] Figure 9 To detect the apoptosis of tumor cells in experimental and control groups after infection with RFWD3-siRNA lentivirus 2 for caspase3-7;

[0019] Figure 10 The results of tumor size and volume test;

[0020] Figure 11 In vivo fluorescence photography shows the tumor size and metastasis in each group of mice. DETAILED DESCRIPTION

[0021] To make the present invention more clearly understood, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0022] The present invention detected the high expression of RFWD3 gene in esophageal cancer through tissue chips of 167 esophageal cancer case specimens and adjacent control tissues, and found that the expression of RFWD3 gene was positively correlated with the occurrence and development of esophageal cancer. The higher the TM stage, the stronger the expression abundance of the gene. Bioinformatics mining of the TCGA public database further found that the expression of RFWD3 was consistent with the chip data. Compared with adjacent tissues, the expression of RFWD3 in esophageal cancer was higher. The above results indicate that RFWD3 gene is highly expressed in esophageal cancer, and silencing the expression of RFWD3 gene may be used for the treatment of esophageal cancer. Therefore, in the following examples of the present invention, by constructing RFWD3 knockdown virus, the therapeutic effect of RFWD3 gene in esophageal cancer is verified at the in vivo and in vitro levels.

[0023] Example 1

[0024] 1. Construction of RFWD3 knockdown virus

[0025] 1) Identification of knockdown targets: Based on RNA interference sequence design principles, multiple 19-21 nt RNA interference target sequences were designed using the RFWD3 gene as a template. After evaluation using design software, the following two sequences were selected as interference sequences: Target 1: CAGAGAATGATGGCAACAT (SEQ ID NO: 1); Target 2: ATGGTTCAATTCTGGTATA (SEQ ID NO: 2); and negative control siRNA target sequence: 5'-TTCTCCGAACGTGTCACGT-3' (SEQ ID NO: 3). Short-shRNA interference sequences were designed based on the selected target sequences, and appropriate restriction endonuclease sites were added to complete vector construction. In addition, a TTTTT termination signal was added to the 3' end of the forward strand, and a sequence complementary to the termination signal was added to the 5' end of the reverse strand. After completion of the design, the single-stranded DNA oligo was synthesized by Generics. The synthesized single-stranded DNA oligo powder was dissolved in annealing buffer (final concentration 20 μM) and incubated at 90°C for 15 minutes. After cooling to room temperature, a double-stranded DNA with sticky ends was formed.

[0026] 2) Preparation of linearized vector: Prepare a 50 μl reaction system according to the NEB instructions, and use AgeI and EcoRI to double-digest the GV115 vector to linearize it. RNA interference lentiviral vector construction: Prepare a 20 μl reaction system according to the Fermentas T4 DNA Ligase instructions, and connect the double-stranded DNA oligo to the linearized vector. Transform the ligation product into E. coli competent cells. Pick clones and culture them, and select the correct clones by sequencing. Transfer the correctly sequenced bacterial solution to 150 ml LB liquid culture medium containing Amp antibiotics and culture on a shaker at 37°C overnight. Extract the RNAi plasmid DNA using Qiagen's plasmid extraction kit and prepare a 100 ng / μl storage solution. Among them, the plasmid enzyme digestion reaction system is shown in Table 1, the vector DNA and double-stranded DNA Oligo ligation reaction system is shown in Table 2, the PCR reaction system is shown in Table 3, and the PCR reaction system procedure is shown in Table 4.

[0027] Table 1 Plasmid enzyme digestion reaction system

[0028] Reagents Volume (μl) pGCSIL-GFP plasmid (1 μg / μl) 2.0 10×buffer 5.0 100×BSA 0.5 Age I (10U / μl) 1.0 EcoRI (10U / μl) 1.0 <![CDATA[dd H2O]]> 40.5 Total 50.0

[0029] Table 2 Vector DNA and double-stranded DNA Oligo ligation reaction system

[0030] Reagents Positive control (μl) Self-ligation control (μl) Connectome (μl) Linearized vector DNA (100 ng / μl) 1.0 1.0 1.0 Annealed double-stranded DNA oligo (100 ng / μl) 1.0 - 1.0 10×T4 phage DNA ligase buffer 1.0 1.0 1.0 T4 phage DNA ligase 1.0 1.0 1.0 <![CDATA[dd H2O]]> 16.0 17.0 16.0 Total 20.0 20.0 20.0

[0031] Table 3 PCR reaction system

[0032]

[0033]

[0034] Table 4 PCR reaction system program settings

[0035]

[0036] 3) Virus packaging: 24 h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and the cell density was adjusted to 1.5×10 5 Cells were seeded at 100 μg / ml in a 6-well plate and cultured in a 37°C, 5% CO2 incubator. Cells were ready for transfection when the cell density reached 70%-80%. Two hours before transfection, the original culture medium was aspirated and 1.5 ml of fresh complete culture medium was added. Following the instructions for the Sigma-Aldrich MISSION Lentiviral Packaging Mix kit, 20 μl of Packing Mix (PVM), 12 μl of PEI, and 400 μl of serum-free DMEM were added to a sterile centrifuge tube. 20 μl of the extracted plasmid DNA was added to the PVM / PEI / DMEM mixture. The transfection mixture was incubated at room temperature for 15 minutes, then transferred to culture medium containing human embryonic kidney 293T cells and cultured in a 37°C, 5% CO2 incubator for 16 hours. The culture medium containing the transfection mixture was discarded, the cells were washed with PBS, 2 ml of complete culture medium was added, and the cells were cultured for a further 48 hours. The cell supernatant was collected and purified and concentrated using a Centricon Plus-20 centrifugal ultrafiltration device (Millipore) as follows: (1) centrifuged at 4000g for 10 min at 4°C to remove cell debris; (2) filtered the supernatant through a 0.45 μm filter into a 40 ml ultracentrifuge tube; (3) centrifuged at 4000g for 10-15 min to the required virus concentration volume; (4) after centrifugation, the filter cup was separated from the filtrate collection cup below, the filter cup was inverted on the sample collection cup, and centrifuged for 2 min at a centrifugal force not exceeding 1000g; (5) the centrifuge cup was removed from the sample collection cup, and the sample collection cup contained the virus concentrate. The virus concentrate was aliquoted and stored at -80°C; the relevant information about the knockdown virus constructed with target 1 and target 2 in this example is shown in Table 5:

[0037] Table 5

[0038]

[0039]

[0040]

[0041] 2. Functional changes after RFWD3 knockdown virus infection of TE-1 and ECA109 esophageal cancer cells

[0042] This example uses real-time fluorescence quantitative RT-PCR to detect gene silencing efficiency. The experimental process is as follows:

[0043] Cancer cells in the logarithmic growth phase were digested with trypsin and prepared into a cell suspension (cell number was about 5×10 4 Cells were inoculated with 100 μg / ml of culture medium into 6-well plates and cultured until the cell confluency reached approximately 30%. Based on the multiplicity of infection (MOI), an appropriate amount of the virus prepared in Example 1 was added. After 24 hours of culture, the medium was replaced. After 5 days of infection, the cells were harvested. Total RNA was extracted according to the Invitrogen Trizol operating instructions. RNA was reverse transcribed to obtain cDNA according to the Promega M-MLV operating instructions (reaction at 42°C for 1 hour, followed by a 10-minute water bath at 70°C to inactivate the reverse transcriptase). The reverse transcription system is shown in Table 7, the real-time PCR reaction system is shown in Table 8, and the primer sequences are shown in Table 9.

[0044] Table 7 Reverse transcription reaction system

[0045] Reagents Volume (μl) 5×RT buffer 4.0 10mM dNTPs 2.0 RNasin 0.5 M-MLV-RTase 1.0 <![CDATA[DEPC H2O]]> 3.5 Total 11.0

[0046] Table 8 Real-time PCR reaction system

[0047] Reagents Volume (μl) SYBR premix ex taq: 10.0 Upstream primer (2.5 μM): 0.5 Downstream primer (2.5 μM): 0.5 cDNA 1.0 <![CDATA[ddH2O]]> 8.0 Total 20.0

[0048] The protocol was set to a two-step real-time PCR: initial denaturation at 95°C for 15 seconds; subsequent denaturation steps at 95°C for 5 seconds; and annealing and extension at 60°C for 30 seconds, for a total of 45 cycles. The absorbance was read during each extension step. After PCR, denaturation was performed at 95°C for 1 minute, followed by cooling to 55°C to allow full binding of the DNA duplex. The temperature was increased by 0.5°C from 55°C to 95°C, held for 4 seconds, while absorbance was read to generate a melting curve. A 2- ΔΔCt The expression abundance of genes after infection was calculated by the analysis method. Cells infected with the control virus were used as controls. After RFWD3 knockdown virus infected esophageal cancer cell TE-1 and ECA109 cells, the expression of RFWD3 in the shRFWD3 group was significantly decreased compared with the shCtrl group. The test results are shown in Table 9 and Figures 1 to 4 shown.

[0049] Table 9 Detection primers and results

[0050]

[0051]

[0052] Example 2 Detecting the ability of lentivirus to inhibit tumor growth at the cellular level

[0053] Tumor cells in the logarithmic growth phase were digested with trypsin and prepared into a cell suspension (cell number was about 1×10 5 / ml) inoculated into 6-well plates and cultured until the cell confluence reached about 30%. According to the multiplicity of infection, an appropriate amount of virus was added. After 16 hours of culture, the culture medium was replaced. After the infection time reached 3 days, the cells in the logarithmic growth phase of each experimental group were collected. The cells were resuspended in complete medium to form a cell suspension (1.5×10 4 / ml), inoculated into 96-well plates at a cell density of approximately 1500 cells / well. Each group had 3 replicates, 100 μl per well. After paving the plates, they were placed in a 37°C, 5% CO2 incubator for culture. Starting from the second day after paving the plates, the plates were read once a day using a Celigo instrument (Nexcelom), and the readings were continued for 5 days. By adjusting the input parameters of Celigo, the number of cells with green fluorescence in each scanned plate was accurately calculated, and the data was statistically plotted to draw a cell proliferation curve. The Celigo test results are shown in Tables 10 and Figure 3 MTT test results are shown in Figures 4-5 shown.

[0054] Table 10

[0055]

[0056] Example 3: Detection of apoptosis levels in tumor cells infected with lentivirus

[0057] After trypsin digestion of cells in the logarithmic growth phase, the cells were resuspended in complete medium to form a cell suspension; centrifuged at 1300 rpm for 5 minutes, the supernatant was discarded, and the cell pellet was washed with 4°C pre-cooled D-Hanks (pH = 7.2-7.4); the cell pellet was washed once with 1× binding buffer, centrifuged at 1300 rpm for 3 minutes, and the cells were collected; the cell pellet was resuspended in 200 μL 1× binding buffer; 10 μL Annexin V-APC was added for staining, and the suspension was kept in the dark at room temperature for 10-15 minutes; 400-800 μL 1× binding buffer was added depending on the cell number, and the apoptosis of the experimental and control groups after tumor cells were infected with lentivirus was detected by flow cytometry. The results are shown in Tables 11 and Figures 6-7 shown.

[0058] Table 11

[0059]

[0060] Example 4: Detection of invasion level of tumor cells infected with lentivirus

[0061] Place the invasion chamber in an incubator and allow it to reach room temperature. Sterilize forceps with 70% ethanol and use them to handle the transwell chamber. Add 300 μl of warm serum-free medium to the chamber and leave it at room temperature for 1 to 2 hours to allow the ECM layer (Extracellular Matrix) to rehydrate. Prepare 1.0×10 6 / ml (using serum-free medium) cell suspension; after rehydration in step 3, carefully remove the medium from the chamber; add 500μl of culture medium containing 10% FBS to the lower chamber; add 300μl of the cell suspension prepared in step 4 to each chamber; culture in a tissue culture incubator for 48h; gently remove non-invasive cells with a cotton swab; add 500μl of staining solution to the empty wells of the plate; soak the chamber in the staining solution for 20min to stain the invading cells on the lower surface of the membrane; soak the chamber in a large cup of water and rinse several times. Dry the chamber in air; photograph the membrane under a microscope; dissolve the membrane in acetic acid and detect the OD value, the results are as follows Figures 8-9 shown.

[0062] Example 5: Tumorigenicity of Lentivirus-Infected Tumor Cells in Vivo

[0063] Tumor cells in the logarithmic growth phase were digested with trypsin and prepared into a cell suspension (cell number was about 5×10 4 / ml) were inoculated into 6-well plates and cultured until the cell confluence reached about 30%. According to the multiplicity of infection, an appropriate amount of virus was added. After 24 hours of culture, the culture medium was replaced. After the infection time reached 5 days, the experimental group and control group cells in the logarithmic growth phase were collected. The cells were resuspended in complete culture medium to form a cell suspension. The cell suspension (1×10 7 cells / mouse) were injected into the right axilla of 4-week-old female BALB / c nude mice. The experimental group was injected with tumor cells infected with lentivirus, and the control group was injected with tumor cells infected with control lentivirus, with 10 nude mice in each group. After injection, the nude mice were raised until the tumor was visible to the naked eye (one week), and then the volume and weight of the tumor were measured using a vernier caliper and a precision balance. Tumor size and volume changes over time are shown in Figure 2. Figure 10 In vivo fluorescence imaging showed the tumor size and metastasis in each group of mice, as shown in Figure 11 shown.

[0064] The experimental results showed that the tumorigenicity of the experimental group's tumor cells was much lower than that of the control group. Lentiviral knockdown of RFWD3 could inhibit tumor cell proliferation in vivo.

[0065] The above description is only a preferred embodiment of the present invention and does not constitute any formal or substantial limitation to the present invention. It should be noted that ordinary technicians in this technical field can make several improvements and supplements without departing from the present invention, and these improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. Use of an agent for inhibiting RFWD3 gene expression in the preparation of a drug for treating esophageal cancer, characterized in that: The agent for inhibiting RFWD3 gene expression is at least one of siRNA, shRNA, RNAi plasmid vector and siRNA lentivirus that specifically inhibits RFWD3 gene expression, and the target sequence of the siRNA, shRNA, RNAi plasmid vector and siRNA lentivirus is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. Application of RFWD3 gene as a detection target in screening drugs for the treatment of esophageal cancer.

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