Application of CLEC1B gene expression promoter in the preparation of anti-tumor drugs

Abstract

The present invention relates to an anti-tumor drug and its use. The drug includes a CLEC1B expression promoter that can increase the expression of CLEC1B nucleic acid or protein. The CLEC1B expression promoter is CLEC1B-3FLAG-Neo, whose nucleotide sequence has at least 90% sequence homology with SEQ ID NO: 1. The CLEC1B expression promoter of the present invention has a key regulatory effect on the occurrence and development of tumors, can induce apoptosis of liver cancer cells and increase the sensitivity of liver cancer cells to sorafenib, helps intervene in the occurrence and development of tumors, and provides an experimental basis for the development of anti-tumor drugs and reducing drug resistance.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of a CLEC1B expression promoter in anti-tumor treatment. Background Art

[0002] Hepatocellular carcinoma (HCC), a malignant tumor of the liver, can be divided into two main categories: primary and secondary. Due to its insidious nature and rapid progression, HCC is often diagnosed at an advanced stage, resulting in low survival rates and high mortality rates. It is currently the second leading cause of cancer death worldwide. The complex tumor microenvironment contributes to the high heterogeneity of HCC. Tumor bleeding is common in solid tumors, including HCC, and is closely related to the microenvironment. Tumor bleeding indicates a rapid deterioration of the tumor immune microenvironment. In particular, altered tumor microenvironmental changes, such as low expression of platelet activation and aggregation genes, are associated with poor prognosis. These changes can promote tumor growth, invasion, and metastasis, in part by activating the NF-κB pathway, often leading to a poor prognosis and placing a heavy burden on individuals and society. Although various treatment options, including systemic therapy with small molecule targeted drugs, have effectively prolonged survival, approximately 25% of HCC patients harbor potentially targetable mutations that remain untapped, necessitating the development of appropriate new diagnostic markers.

[0003] C-type lectin domain family 1, member B (CLEC1B), located on chromosome 12 within the Dectin-1 gene cluster, is a hallmark gene highly associated with tumor progression. It is secreted by activated platelets surrounding tumors. CLEC1B has been shown to bind to the surface of colorectal cancer cells and inhibit platelet aggregation and tumor metastasis. CLEC1B-deficient dendritic cells have been shown to be unable to enter the Gp381-mediated lymphatic system, resulting in reduced T cell priming and antigen responses within lymph nodes. CLEC-2, a C-type lectin-like receptor encoded by CLEC1B, mediates lymphovascular separation, tumor-induced platelet aggregation, and immune responses. Furthermore, CLEC1B genotype is associated with significantly reduced CLEC-2 plasma levels, suggesting that CLEC1B may be a promising biomarker for tumor progression. Summary of the Invention

[0004] To solve the above technical problems, the present invention includes the following aspects:

[0005] The present invention provides an anti-tumor drug, which comprises a CLEC1B expression promoter capable of increasing the expression of CLEC1B nucleic acid or protein.

[0006] Preferably, the CLEC1B expression promoter is CLEC1B-3FLAG-Neo, which is obtained by linking a CLEC1B expression nucleic acid sequence to a FLAG tag and then inserting the FLAG tag into a Neo expression vector.

[0007] Preferably, the tumor is a hepatocellular carcinoma.

[0008] More preferably, the hepatocellular carcinoma is the SMMC-7721 hepatocellular carcinoma cell line.

[0009] The second aspect of the present invention is to provide a use of a CLEC1B expression promoter in the preparation of an anti-tumor drug.

[0010] Preferably, the CLEC1B expression promoter is CLEC1B-3FLAG-Neo.

[0011] Preferably, the tumor is a hepatocellular carcinoma.

[0012] More preferably, the hepatocellular carcinoma is the SMMC-7721 hepatocellular carcinoma cell line.

[0013] Preferably, the hepatocellular tumor is a sorafenib-resistant hepatocellular tumor.

[0014] The third aspect of the present invention is to provide an anti-tumor pharmaceutical composition, which comprises a CLEC1B expression promoter and one or more drugs for treating liver cancer.

[0015] Preferably, the CLEC1B expression promoter is CLEC1B-3FLAG-Neo.

[0016] Preferably, the drug for treating liver cancer is sorafenib.

[0017] Preferably, the tumor is a hepatocellular carcinoma.

[0018] More preferably, the hepatocellular carcinoma is the SMMC-7721 hepatocellular carcinoma cell line.

[0019] The fourth aspect of the present invention is to provide a use of a pharmaceutical composition in the preparation of an anti-tumor drug, wherein the pharmaceutical composition comprises a CLEC1B expression promoter and one or more drugs for treating liver cancer.

[0020] Preferably, the CLEC1B expression promoter is CLEC1B-3FLAG-Neo.

[0021] Preferably, the drug for treating liver cancer is sorafenib.

[0022] Preferably, the tumor is a hepatocellular carcinoma.

[0023] More preferably, the hepatocellular carcinoma is the SMMC-7721 hepatocellular carcinoma cell line.

[0024] Preferably, the hepatocellular tumor is a sorafenib-resistant hepatocellular tumor.

[0025] The beneficial effects produced by the present invention are:

[0026] The inventors of this application unexpectedly discovered that gene interference in liver cancer cells can increase CLEC1B expression, significantly inducing apoptosis in these cells. These results demonstrate that CLEC1B expression plays a key regulatory role in the development and progression of liver cancer and other tumors, providing an experimental basis for the development of novel anticancer targets and drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Figure 1 is the RT-qPCR result showing low expression of CLEC1B in liver cancer (A) and the Western Blot result showing the effect of CLEC1B expression promoter on CLEC1B protein in liver cancer cell line SMMC-7721 (B);

[0028] Figure 2 This is a bar graph showing the cell viability of the control and CLEC1B-overexpressing liver cancer cell line SMMC-7721 after the addition of sorafenib;

[0029] Figure 3 Figures 1 and 2 are flow cytometry (A) and quantitative images (B) of apoptosis in the control and CLEC1B-overexpressing liver cancer cell line SMMC-7721 after addition of sorafenib.

[0030] Figure 4 Flow cytometry (A) and quantitative graph (B) of the effect of sorafenib on intracellular reactive oxygen species (DCF-DA) in the control and CLEC1B-overexpressing liver cancer cell line SMMC-7721;

[0031] Figure 5 Figure 1 is a flow cytometric graph (A) and quantitative graph (B) showing the effects of sorafenib on mitochondrial reactive oxygen species (mitoSOX) in the control and CLEC1B-overexpressing liver cancer cell lines SMMC-7721. DETAILED DESCRIPTION

[0032] The present invention will be described in further detail below with reference to the embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0033] Experimental Example 1: Effect of CLEC1B promoter on apoptosis of liver cancer cell line SMMC-7721

[0034] 1. Analysis of CLEC1B mRNA expression levels in normal hepatocytes and various hepatocellular carcinoma cell lines

[0035] Cell pellets of Huh7, HCC-LM3, MHCC-97H, SMMC-7721 hepatoma cell lines and LO2 normal human hepatocytes were collected by centrifugation, washed with PBS buffer, and then RNA was extracted as follows:

[0036] (1) Add 1 mL RNAiso and lyse on ice for 5 min; (2) Vortex on a vortex shaker for 20 s, then let it stand at 4°C for 30 s; (3) Add 1 / 5 volume of pre-cooled chloroform of RNAiso plus to the above lysate, and invert the mixed solution until it turns milky white; (4) Let it stand at room temperature for 5 min; (5) Centrifuge at 12,000 × g at 4°C for 15 min. At this time, the homogenate is divided into three layers, namely: colorless supernatant (containing RNA), middle white protein layer (mostly DNA) and colored lower organic phase; (6) Pipette the supernatant and transfer it to another new centrifuge tube (do not aspirate the white middle layer), add pre-cooled isopropanol of the same volume as RNAiso Plus to the supernatant (to precipitate RNA), invert the centrifuge tube to mix thoroughly, and let it stand at room temperature for 10 min; (7) Centrifuge at 12,000 × g at 4°C for 10 min; (8) Discard the supernatant and add the same volume of RNAiso plus. Wash the RNA precipitate with 75% ethanol plus the volume of ethanol, gently invert the tube to wash the wall of the centrifuge tube, centrifuge at 7,500 × g, 4℃ for 5 min, and discard the supernatant; (9) Dry the precipitate at room temperature for 10 min after opening the test tube, then add an appropriate amount of enzyme-free water preheated at (60-70)℃ to dissolve the RNA precipitate, and then mix it repeatedly on a vortex oscillator; use the NanoDrop2000 ultra-micro spectrophotometer to extract the RNA sample, select Nucleic Acid on the main screen, use 1.5 μL of enzyme-free water to zero on the survey table, put down the upper arm and press Blank Simple type, select RNA-40 mode, enter the sample name in the Simple ID position, and then add 1.5 μL of mixed RNA solution on the survey table, and record the RNA concentration and OD 260 / 280 value (1.7-2.1).

[0037] Prepare the reaction mixture according to the ingredients in Table 1. Prepare the Master Mix based on the number of reactions + 2, then aliquot into each reaction tube. Add 1 μg of RNA and finally add RNase-free water to make a 10 μL system. Place the reaction tube in a reverse transcription analyzer (S1000 Thermal Cycler) and follow the cycle (42°C, 2 min). After the cycle is complete, quickly place the reaction tube on ice and proceed to the next step.

[0038] Table 1 Removal of genomic DNA

[0039] Reagents volume 5 × gDNA Eraser Buffer 2.0 μL gDNA Eraser 1.0 μL Total RNA Calculate the required RNA volume based on 1 μg RNA <![CDATA[RNase Free ddH2O]]> Up to 10 μL .

[0040] Prepare the PCR reaction solution according to the following components: mix TB Green Premix Ex Taq II and DNA template, mix PCRForward Primer, PCR Reverse Primer and sterile water, and prepare the total number of reactions + 2. Then distribute it to each reaction well, that is, prepare 25 μL of the reaction mixture of the system. Then, perform centrifugation and place it in a fluorescent quantitative PCR instrument (CFX96). Finally, amplify according to the standard two-step PCR amplification procedure.

[0041] Table 2 Fluorescence quantitative PCR substances

[0042] Reagents volume TB Green Premix Ex Taq II (Tli RNaseH Plus (2 ×) 12.5 μL PCR Forward Primer (10 μM) 1 μL PCR Reverse Primer (10 μM) 1 μL DNA template (< 100 ng) 2 μL <![CDATA[RNase Free ddH2O]]> 8.5 μL Total 25 μL .

[0043] Standard two-step PCR amplification procedure: sample volume: 25 μL; Step 1: 95°C, 30 s; Step 2: PCR reaction, GOTO: 39 (40 cycles); 95°C, 5 s; 60°C, 30 s; Step 3: Melt Curve.

[0044] The results are as follows Figure 1 As shown, CLEC1B expression in LO2 normal hepatocytes is significantly higher than in liver cancer cell lines, by 5-fold or more. This indicates the great potential and research prospects of CLEC1B as a marker in liver cancer. Therefore, we subsequently constructed CLEC1B-3FLAG-Neo, which specifically recognizes CLEC1B. After large-scale data analysis and experimental verification, it was found that it does not target any known human, mouse, or rat genes.

[0045] 2. Construction of CLEC1B-3FLAG-Neo

[0046] (1) Enzyme digestion of empty vector plvx-IRES-NEO

[0047] ① Enzyme digestion system: first add water, buffer, plasmid, and finally add enzyme. The specific formula is as follows:

[0048] Reagents volume 10 × Qickcut buffer 10 μL plasmids 6-7 μg XBa-I 5 μL <![CDATA[ddH2O]]> Up to 100 μL .

[0049] ② Instant centrifugation; ③ Procedure: Set the metal heater to 37°C for 15 minutes; ④ Electrophoresis identification (pre-enzyme digestion control is required); ⑤ Recover the product from the kit; ⑥ Measure the concentration.

[0050] (2) Seamless cloning (10 μL) system

[0051] Reagents volume Enzyme-digested empty vector (plvx-NEO) 100 ng Amplified fragments 30 ng Seamless cloning enzyme 1 μL Seamless Buffer Cloning 2 μL <![CDATA[ddH2O]]> Up to 10 μL .

[0052] Program: Set the metal heater to 37°C for 30 min.

[0053] (3) Conversion

[0054] ① Immediately remove 100 μL competent cells, place on ice, and immediately add all the ligation products; ② Place on ice for 30 min; ③ 42°C, 90 s; ④ Place on ice for 5 min; ⑤ Add 1 mL of incubated LB; ⑥ 37°C, 250 rpm, shake for 1 hour; ⑦ Take half of the plate (Amp + ), 37℃ overnight.

[0055] (4) Take several single colonies and streak them on a plate

[0056] Divide the plate into several small grids and streak different single colonies in the grids. At the same time as streaking, streak an empty grid with bacteria as a positive control.

[0057] (5) PCR amplification and identification (20 μL system, control system)

[0058] Reagents volume pLVX-NEO identification 0.2 μL IRES reverse 0.2 μL 2 × Taq enzyme 10 μL template That is, the bacteria grown from a single colony, gently tap the colony with a small gun tip and mix it in the reaction tube <![CDATA[ddH2O]]> Up to 20 μL .

[0059] Escherichia coli were transformed with the plasmids pLVX-neo and CLEC1B-3FLAG-Neo. Ampicillin plates were coated with lysates, and single colonies were picked, amplified, and plasmids were extracted. Lentivirus packaging was performed using 293T cells using the ipofectamine 3000 transfection kit (Invitrogen). Exponentially growing 293T cells were plated in 60 mm dishes with 3 mL of DMEM medium containing 10% fetal bovine serum and without double-stranded antibodies, allowing the cells to adhere to the wells to a maximum of 70% of the well area. Two enzyme-free 1.5 mL EP tubes were added: 300 μL of MEM and 13 μL of Lipo3000 to one tube, and 300 μL of MEM, 13 μL of p3000, 5 μg of PSPAX2, 2.5 μg of PMD2G, and 6 μg of the target plasmid to the other tube. Both tubes were mixed thoroughly, then mixed together, and allowed to stand at room temperature for 5 minutes. Aspirate 400 μL of the antibody-free medium from the dish to a final volume of 2 mL. Add the rested mixture dropwise to the 293T cell culture medium. Incubate in a 37°C CO2 incubator. After 8-10 hours, add 2 mL of antibody-free DMEM medium. After 36-48 hours, remove the first batch of viral supernatant and store in a centrifuge tube at 4°C. Simultaneously, add 4 mL of antibody-free DMEM medium. After 24-36 hours, collect the second batch of viral supernatant. Finally, filter both batches of virus through a 0.45 μm filter and store at -80°C.

[0060] 3. Construction of SMMC-7721 cell line overexpressing CLEC1B

[0061] SMMC-7721 cells were seeded at an appropriate density in 24-well plates. 400 μL of the corresponding packaged pLVX-Neo and CLEC1B-3FLAG-Neo viruses and 100 μL of dual-antibody-free DMEM medium were added and incubated at 37°C in a CO2 incubator. After 8 hours, the medium was replaced with fresh 10% complete DMEM medium. SMMC-7721 cell lines were selected using complete medium containing G418 (1000 μg / mL) to identify drug-resistant cell lines.

[0062] SMMC-7721 control cells and CLEC1B-overexpressing cell pellets were collected in 1.5 mL pre-gel tubes and lysed with 50 μL of RIPA lysis buffer to extract total protein. Protein samples were prepared at a concentration of 2 μg / μL with loading buffer and heated at 96°C for 10 minutes to denature the protein. A 10 μL sample was electrophoresed on a 12% polyacrylamide gel and transferred to a membrane. The membranes were blocked with 5% skim milk for 60 minutes, washed with TBST for 10 minutes, and incubated with primary antibodies against FLAG and β-actin at 4°C for 8 hours. The membranes were washed three times with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies for 60 minutes. After washing with TBST for 10 minutes, the membranes were visualized with ECL chemiluminescence solution.

[0063] β-Actin antibody is a good internal control for Western Blot. Internal control, or internal reference, generally refers to proteins encoded by housekeeping genes in mammalian cell expression. Their expression is relatively constant across tissues and cells and is often used as a reference when detecting changes in protein expression levels. β-Actin is a commonly used protein internal control (cytoskeleton protein). Compared to the empty lentiviral control (pLVX-neo), the expression level of CLEC1B protein in SMMC-7721 cells successfully transfected with the CLEC1B-3FLAG-Neo sequence was significantly increased several times (see Figure 1 B). Western blot results demonstrated that the SMMC-7721 stable cell line overexpressing the CLEC1B gene was successfully constructed.

[0064] 4. Effect of sorafenib on cell viability of CLEC1B-overexpressing hepatocellular carcinoma cell line SMMC-7721

[0065] Sorafenib is currently the only approved first-line drug for the treatment of advanced liver cancer, but clinical trials have shown that liver cancer patients develop resistance to the drug within only 6 months of sorafenib treatment. Sorafenib resistance in liver cancer patients is one of the problems that need to be urgently addressed in clinical practice. The SMMC-7721 cell line that stably overexpresses CLEC1B, which was successfully verified by Western Blot, was seeded in a 96-well plate at the same seeding density (5000 cells / well). Three replicate wells were set up in each group, and two independent repeated experiments were performed. The cells were cultured in a cell culture incubator at 37°C and 5% CO2 for 12 hours. After the cells adhered to the wall, sorafenib drugs (0, 5, 10 μM) were added. After 24 hours of culture, 8 μL of CCK8 was added to each well for 2 hours to detect the cell viability. The results are as follows: Figure 2This cell model showed that overexpression of the CLEC1B gene could effectively inhibit the viability of human liver cancer cell lines and increase their sensitivity to sorafenib.

[0066] Control and CLEC1B-overexpressing SMMC-7721 cells were seeded at appropriate densities in 6-well plates and treated with certain concentrations of sorafenib (0 and 10 μM). After 24 h of treatment, the cells were collected and stained with the Annexin V-FITC / PI apoptosis kit (Lianke Biotechnology Co., Ltd., Hangzhou) for 10 min. Finally, flow cytometry (Beckman, USA) was used for detection.

[0067] Annexin V-FITC is a membrane-attached protein V labeled with fluorescein that has a high affinity for phosphotidylserine (PS). PS is a component of the cell membrane, so Annexin V-FITC is used to detect normal cells (living cells) in the early stages of apoptosis. The fluorescent dye PI (propidium iodide) is an analog of ethidium bromide that releases red fluorescence after being embedded in double-stranded DNA and is used to detect apoptotic cells. Therefore, based on the fluorescence intensity of the two fluorescent probes, the cells are divided into four quadrants: Q1 (necrotic cells), Q2 (late apoptotic cells), Q3 (early apoptotic cells), and Q4 (living cells). After the addition of sorafenib, liver cancer cells with overexpression of CLEC1B showed a significant increase in late apoptotic cells, up to 58%, compared with the control (24.3%) (see Appendix). Figure 3 The above results indicate that overexpression of CLEC1B in liver cancer cell lines can promote sorafenib-induced apoptosis of liver cancer cell lines and significantly increase the sensitivity of liver cancer cells to sorafenib, which is expected to solve the problem of sorafenib resistance in clinical practice.

[0068] Experimental Example 2: Effect of CLEC1B promoter on oxidative stress in liver cancer cell line SMMC7721

[0069] Sorafenib can induce ferroptosis and oxidative stress in liver cancer cell lines. Control and CLEC1B-overexpressing liver cancer cells were seeded in 6-well plates. After the cells were fully attached, they were treated with sorafenib (10 μM) for 24 hours, washed twice with PBS, and 500 μL of DCF-DA (5 μM) and mitoSOX (4 μM) were added respectively. The cells were incubated in a cell culture incubator at 37°C and 5% CO2 for 30 minutes. The cells were washed twice with PBS, collected, resuspended in 500 μL of Hanks' medium, and then tested on the flow cytometer. The fluorescence intensity of the cells was detected by flow cytometry. The flow cytometric graphs and fluorescence intensity quantification graphs of DCF-DA and mitoSOX are shown in the figure. Figure 4 and 5 shown.

[0070] DCF-DA is a fluorescent dye used to quantify intracellular reactive oxygen species (ROS) levels, and mitoSOX is a fluorescent dye used to quantify mitochondrial ROS levels. Flow cytometry results showed that sorafenib significantly induced oxidative stress in liver cancer cells, with elevated levels of both intracellular and mitochondrial ROS. Compared to control cells, the fluorescence intensities of DCF-DA and mitoSOX increased significantly after sorafenib treatment in cell lines overexpressing CLEC1B. This suggests that overexpression of CLEC1B in liver cancer cell lines can promote sorafenib-induced oxidative stress and increase sensitivity to sorafenib.

[0071] Since the current chemotherapy drugs for liver cancer are ineffective and often cause drug resistance, using gene regulation methods to increase the expression of CLEC1B at the transcriptional level and combining it with other treatment methods can achieve better anti-cancer effects.

[0072] The present invention can also be applied to the diagnosis and drug development targets of other systemic tumors. Any diagnosis based on the detection of CLEC1B expression and any treatment of tumors by increasing CLEC1B expression are within the scope of protection of the present invention.

[0073] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a pharmaceutical composition in preparing a drug for treating liver cancer, the pharmaceutical composition comprising a CLEC1B expression promoter and sorafenib, wherein the CLEC1B expression promoter is CLEC1B-3FLAG-Neo, which is obtained by attaching a FLAG tag to a CLEC1B expression nucleic acid sequence and inserting it into a Neo expression vector.

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