Application of TFRC inhibitors in the preparation of drugs for preventing and / or treating endometrial cancer

Abstract

The present invention provides the use of a TFRC inhibitor in the preparation of a drug for preventing and / or treating endometrial cancer, and belongs to the technical field of endometrial cancer prevention and treatment. The present invention proposes for the first time that there is a negative correlation between the expression of PTEN and TFRC proteins in endometrial cancer, and that TFRC protein or its encoding gene can be used as a target for preventing and / or treating endometrial cancer. The present invention proposes for the first time that inhibiting TFRC gene expression or inhibiting TFRC protein activity can effectively prevent and treat PTEN-deficient endometrial cancer. The shRNA and siRNA provided by the present invention can effectively inhibit the expression of TFRC gene or protein, and thus can play a medicinal effect of effectively preventing and treating PTEN-deficient endometrial cancer.

Description

Technical Field

[0001] The present invention belongs to the technical field of endometrial cancer prevention and treatment, and specifically relates to the use of a TFRC inhibitor in the preparation of a drug for preventing and / or treating endometrial cancer. Background Art

[0002] Endometrial cancer (EC) is an epithelial malignant tumor that occurs in the endometrium and is one of the three most common malignant tumors of the female reproductive tract. Total hysterectomy + bilateral oophorectomy and lymph node assessment is the most basic surgical method for patients with lesions confined to the uterus, supplemented by comprehensive treatments such as radiotherapy, chemotherapy, and hormones. Although the 5-year overall survival rate of patients without metastasis can reach 60-88% after surgery, and the risk of recurrence of advanced tumors is 40%-70%, the median survival of EC relapsed patients does not exceed 12 months, so there is an urgent need to find new treatment methods to improve the prognosis of EC patients.

[0003] Inactivation of PTEN is common in human cancers, and is most common in endometrial cancer, with a mutation rate of up to 60%. It is also the gene with the highest mutation rate in endometrial cancer. Endometrial-specific PTEN-deficient mice develop tumors at 1 month of age. As a lipid phosphatase, PTEN antagonizes PI3K function, dephosphorylates PIP3 to PIP2, prevents PIP3 accumulation, prevents AKT activation, and keeps cells in a quiescent state. PTEN functional loss leads to PIP3 accumulation, followed by uncontrolled hyperactivity of the AKT-mTOR signaling pathway, promoting cell proliferation, survival, migration, and even tumorigenesis. Therefore, the PI3K / PTEN-AKT-mTOR signaling pathway is a therapeutic target for endometrial cancer. However, in clinical trials of PI3K, AKT, and mTOR inhibitors for the treatment of endometrial cancer, problems such as low efficacy, drug tolerance, and high drug toxicity have occurred to varying degrees. Therefore, it is necessary to develop drugs for endometrial cancer targeting PTEN loss.

[0004] Transferrin Receptor (TFRC) is the main iron uptake protein on the cell surface. It transports iron into cells through receptor-mediated endocytosis of diferric transferrin to maintain intracellular iron homeostasis. Cancer cells often show elevated TFRC expression due to their high iron demand for proliferation. For example, TFRC-mediated iron uptake promotes colon tumorigenesis, and IGF2BP2 promotes colorectal cancer progression by upregulating TFRC expression and enhancing iron metabolism. However, the relationship between TFRC and PTEN, as well as its role in endometrial cancer, is still unclear. Exploring the relationship provides an experimental basis for the treatment of endometrial cancer with PTEN deficiency. Summary of the invention

[0005] In view of this, the object of the present invention is to provide the use of TFRC protein or its encoding gene as a target in the preparation of a drug for preventing and / or treating endometrial cancer.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides the use of TFRC protein or its encoding gene as a target in the preparation of a drug for preventing and / or treating endometrial cancer.

[0008] The present invention also provides the use of the TFRC inhibitor in the preparation of a drug for preventing and / or treating endometrial cancer.

[0009] Preferably, the TFRC inhibitor comprises any one of the following: (1) shRNA targeting the TFRC gene; (2) siRNA targeting the TFRC gene; (3) an agent that inhibits the transcriptional activity of the TFRC gene; (4) an agent that inhibits the transcriptional level of TFRC mRNA; (5) an agent that promotes the degradation of TFRC mRNA; (6) an agent that specifically recognizes the guide nucleic acid of the TFRC gene and cleaves it to reduce the expression level of TFRC; (7) an agent for partially or completely knocking out the TFRC gene; (8) an agent that inhibits the activity of the TFRC protein.

[0010] Preferably, the shRNA includes shRNA1 and shRNA2, the nucleotide sequence of the shRNA1 is shown as SEQ ID NO.1, and the nucleotide sequence of the shRNA2 is shown as SEQ ID NO.2.

[0011] Preferably, the siRNA includes siRNA1 and siRNA2, the nucleotide sequence of the siRNA1 is shown as SEQ ID NO.3, and the nucleotide sequence of the siRNA2 is shown as SEQ ID NO.4.

[0012] Preferably, the endometrial cancer is PTEN-deficient endometrial cancer.

[0013] The present invention also provides a drug capable of preventing and / or treating endometrial cancer, wherein the drug can inhibit the expression of TFRC gene or protein.

[0014] Preferably, the drug comprises shRNA and / or siRNA that inhibits TFRC gene expression; the shRNA comprises shRNA1 and shRNA2, the nucleotide sequence of the shRNA1 is shown in SEQ ID NO.1, and the nucleotide sequence of the shRNA2 is shown in SEQ ID NO.2.

[0015] Preferably, the siRNA includes siRNA1 and siRNA2, the nucleotide sequence of the siRNA1 is shown as SEQ ID NO.3, and the nucleotide sequence of the siRNA2 is shown as SEQ ID NO.4.

[0016] Preferably, the dosage form of the drug includes oral solution, injection, tablet, pill, dispersion, capsule or granule.

[0017] Beneficial effects of the present invention:

[0018] The present invention proposes for the first time that there is a negative correlation between the expression of PTEN and TFRC proteins in endometrial cancer, and that TFRC protein or its encoding gene can be used as a target for preventing and / or treating endometrial cancer. The present invention proposes for the first time that inhibiting the expression of TFRC gene or inhibiting the activity of TFRC protein can effectively prevent and treat PTEN-deficient endometrial cancer. The shRNA and siRNA provided by the present invention can effectively inhibit the expression of TFRC gene or protein, and thus can play a medicinal effect of effectively preventing and treating PTEN-deficient endometrial cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The results of analyzing the relationship between PTEN and TFRC protein expression in endometrial cancer using the cBioPortal database, where A is the result of analyzing the relationship between PTEN and TFRC protein expression in endometrial cancer using the TCGA, Firehose Legacy data; B is the result of analyzing the relationship between PTEN and TFRC protein expression in endometrial cancer using the TCGA, PanCancer Atlas data;

[0020] Figure 2 The result is that PTEN deficiency increases the expression level of TFRC protein, where A is Pten + / + MEFs and Pten - / - The results of TFRC protein expression in MEFs; B is the results of TFRC protein expression after overexpression of PTEN in PTEN-deficient endometrial cancer cell lines ISHIKAWA and RL95-2, where vector is the non-overexpression treatment group and PTEN is the PTEN overexpression group; C is the results of TFRC protein expression after PTEN knockdown in KLE and HEC-1-B groups, where NC indicates the group without PTEN knockdown, 1 indicates the sh-human PTEN-1 knockdown group, and 2 indicates the sh-human PTEN-2 knockdown group;

[0021] Figure 3 The results of knocking down TFRC to inhibit the proliferation of PTEN-deficient cells, where A is PTEN-deficient MEF cells ( Pten - / -Results of shRNA knockdown of TFRC in MEFs, where NC represents the non-knockdown group, 1 represents the shRNA1 knockdown group, and 2 represents the shRNA2 knockdown group; B represents Pten - / - The results of free iron level detection in MEFs cells with TFRC knockdown, where the left figure is the flow cytometry results, and the right figure is the statistical results of relative fluorescence in each group; C is Pten - / - Results of knocking down TFRC by shRNA in MEFs cells; D is Pten - / - Results of cell clone formation ability of MEFs cells with shRNA knockdown of TFRC; E is Pten - / - The oxygen consumption rate results of MEFs cells after shRNA knockdown of TFRC; F is the result of siRNA knockdown of TFRC in ISHIKAWA cells, where NC represents the non-knockdown group, 1 represents the siRNA1 knockdown group, and 2 represents the siRNA2 knockdown group; G is the free iron level detection result of ISHIKAWA cells with TFRC knockdown, where the left figure is the flow cytometry result, and the right figure is the statistical result of the relative fluorescence quantity of each group; H is the CCK8 result of ISHIKAWA cells with siRNA knockdown of TFRC; I is the cell clone formation ability result of ISHIKAWA cells with siRNA knockdown of TFRC; J is the oxygen consumption rate result of ISHIKAWA cells with siRNA knockdown of TFRC; *** indicates p < 0.001. DETAILED DESCRIPTION

[0022] The present invention provides the use of TFRC protein or its encoding gene as a target in the preparation of a drug for preventing and / or treating endometrial cancer.

[0023] The present invention uses the cBioPortal database (https: / / www.cbioportal.org / ) to analyze the correlation between PTEN and TFRC protein expressions in endometrial cancer. The results show that PTEN and TFRC protein expressions are negatively correlated, indicating that TFRC protein or its encoding gene can be used as a target for preventing and / or treating PTEN-deficient endometrial cancer.

[0024] The present invention also provides the use of the TFRC inhibitor in the preparation of a drug for preventing and / or treating endometrial cancer.

[0025] In the present invention, the TFRC inhibitor preferably includes any one of the following: (1) shRNA targeting the TFRC gene; (2) siRNA targeting the TFRC gene; (3) an agent that inhibits the transcriptional activity of the TFRC gene; (4) an agent that inhibits the transcriptional level of TFRC mRNA; (5) an agent that promotes the degradation of TFRC mRNA; (6) an agent that specifically recognizes the guide nucleic acid of the TFRC gene and cleaves it to reduce the expression level of TFRC; (7) an agent for partially or completely knocking out the TFRC gene; (8) an agent that inhibits the activity of the TFRC protein.

[0026] In the present invention, the shRNA preferably includes shRNA1 and shRNA2, the nucleotide sequence of the shRNA1 is 5'-GCAATTGGTGTCCTCATATAC-3' (SEQ ID NO.1), and the nucleotide sequence of the shRNA2 is 5'-CGTTGAATTGAACCTGGACTA-3' (SEQ ID NO.2).

[0027] In the present invention, the siRNA preferably includes siRNA1 and siRNA2, the nucleotide sequence of the siRNA1 is 5'-CAUUCGUGGUGAUCAAUUATT-3' (SEQ ID NO. 3), and the nucleotide sequence of the siRNA2 is 5'-UGUUGAAUUGAACCUGGACUATT-3' (SEQ ID NO. 4).

[0028] In the present invention, the endometrial cancer is preferably PTEN-deficient endometrial cancer.

[0029] The present invention also provides a drug capable of preventing and / or treating endometrial cancer, wherein the drug can inhibit the expression of TFRC gene or protein.

[0030] In the present invention, the drug preferably includes shRNA and / or siRNA that inhibits TFRC gene expression; the shRNA includes shRNA1 and shRNA2, and the nucleotide sequences of the shRNA1 and shRNA2 are shown as SEQ ID NO.1 and SEQ ID NO.2, respectively. In the present invention, the siRNA includes siRNA1 and siRNA2, and the nucleotide sequences of the siRNA1 and siRNA2 are shown as SEQ ID NO.3 and SEQ ID NO.4, respectively. In the present invention, the dosage form of the drug preferably includes oral solution, injection, tablet, pill, dispersant, capsule or granule. In the present invention, the drug preferably also includes pharmaceutical excipients. The present invention does not specifically limit the specific types of pharmaceutical excipients, which depends on the specific drug dosage form.

[0031] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0032] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0033] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0034] Example 1

[0035] The cBioPortal database (https: / / www.cbioportal.org / ) was used to analyze the correlation between PTEN and TFRC protein expression in endometrial cancer. Figure 1 As shown,

[0036] In 440 endometrial cancer proteomic samples from TCGA, Firehose Legacy, PTEN and TFRC protein expressions were negatively correlated (see Figure 1 A in Figure 2). TCGA, 432 endometrial cancer proteomic samples in the Pan-Cancer Atlas, PTEN and TFRC protein expressions were negatively correlated (see Figure 1 B in the figure).

[0037] Example 2

[0038] PTEN deficiency increases TFRC protein expression levels

[0039] 1. Experimental Materials

[0040] Pten + / + MEFs (mouse embryonic fibroblasts) and Pten - / - The source of MEFs was the same as the article mTOR-dependent upregulation of xCT blocks melanin synthesis and promotes tumorigenesis. They were cultured in a humidified incubator at 37°C and 5% CO2.

[0041] The sources and culture conditions of endometrial cancer cell lines are shown in Table 1 below:

[0042] Table 1 Sources and culture conditions of endometrial cancer cell lines

[0043]

[0044] Lentivirus-mediated shRNA vector targeting human PTEN, lentiviral plasmid for human PTEN overexpression, and infection enhancement solution HiTransG A or HiTransG P were purchased from GeneCare Biotechnology Co., Ltd. (Shanghai, China).

[0045] Antibodies: PTEN antibody was purchased from CST, TFRC antibody was purchased from Abcame, and GAPDH antibody was purchased from Abclonal.

[0046] 2. Experimental methods

[0047] (1) Construction of PTEN knockdown or overexpression endometrial cancer cell lines

[0048] Transduce cells with a confluency of 30% with 20-40 mol of lentivirus mixed with 40 μl of HiTransG A or HiTransG P. After transduction, puromycin selection was applied to establish stably transfected cell lines. Successful gene disruption or overexpression was verified by immunoblotting of the corresponding target protein.

[0049] The shRNA sequence used in the lentiviral-mediated shRNA vector targeting human PTEN is as follows:

[0050] sh-Human PTEN-1: 5'-AGGCGCTATGTGTATTATTAT-3' (SEQ ID NO. 5).

[0051] sh-Human PTEN-2: 5'-CCACAGCTAGAACTTATCAAA-3' (SEQ ID NO. 6).

[0052] (2) Western blot detection of PTEN protein expression in expanded cultured monoclonal cells

[0053] Cells were lysed with RIPA lysis buffer and protein was quantified by BCA method. Proteins were separated on NuPAGE 10% Bis-Tris gel and transferred to nitrocellulose membrane (Merck Millipore, Darmstadt, Germany). The membrane was blocked for 1 hour and 10 minutes and then incubated with primary antibody overnight at 4°C. After washing three times with TBST (10 mM Tris-HCl (pH 8), 150 mM NaCl, 0.05% Tween20), the membrane was incubated with secondary antibody for 2 hours at room temperature. After a final three washes with TBST, the membrane was developed using LI-COROdyssey infrared scanner.

[0054] 3. Experimental results

[0055] Western Blot results showed that compared with WT MEFs ( Pten + / + Compared with PTEN-deficient MEFs, Pten - / - TFRC expression levels were increased in MEFs (see Figure 2 A in the figure); Compared with PTEN-deficient endometrial cancer cells ISHIKAWA and RL95-2 (vector), the PTEN overexpression group (PTEN) showed a significant decrease in PTEN protein expression, and TFRC protein expression was inhibited after PTEN overexpression (see Figure 2 B); Compared with PTEN wild-type (shPTEN-NC) cells, the PTEN expression level in the knockdown group (sh PTEN-1, sh PTEN-2) was reduced, and TFRC protein expression was promoted after PTEN knockdown (see Figure 2 C in.

[0056] Example 3

[0057] Knockdown of TFRC inhibits proliferation of PTEN-deficient cells

[0058] 1. Experimental Materials

[0059] TFRC lentivirus-mediated shRNA and HiTransG A were purchased from Jikai Biotechnology Co., Ltd. (Shanghai, China), siRNA was purchased from Jiman Biotechnology Co., Ltd. (Shanghai, China), FerroOrange (F374, Tongren Chemical), CCK8 (Yisheng Biotechnology Co., Ltd.), Lipofectamine™ RNAiMax (thermo), Trypan blue (Beijing Zhongshan Biotechnology Co., Ltd.), crystal violet (Beijing Solebao Co., Ltd.), and the sources and culture conditions of each endometrial cancer cell line are shown in Table 1 above.

[0060] 2. Experimental methods

[0061] (1) Construction of TFRC knockdown cell line

[0062] 20 mol of lentivirus and 40 μl of HiTransGA were mixed to transduce PTEN-deficient MEF cells with a confluence of 30% ( Pten - / - MEFs). Three days after transduction, puromycin was used for selection for 2 days to establish a stably transfected cell line. Immunoblotting of the corresponding target protein was used to verify whether the knockdown was successful. The sequence of the TFRC lentivirus-mediated shRNA was as follows (after the design was completed, it was synthesized by Jikai Biotechnology Co., Ltd.):

[0063] sh-mouse Tf-1: 5'-GCAATTGGTGTCCTCATATAC-3' (SEQ ID NO. 1).

[0064] sh-mouse Tf -2: 5'-CGTTGAATTGAACCTGGACTA-3' (SEQ ID NO. 2).

[0065] Human siRNA for knocking down TFRC (as shown below) was designed and synthesized by Yoshiman Biotechnology Co., Ltd. (Shanghai, China) to achieve human TFRC knockdown:

[0066] si-people TFRC -1: 5'-CAUUCGUGGUGAUCAAUUATT-3' (SEQ ID NO. 3).

[0067] si-people TFRC -2: 5'-UGUUGAAUUGAACCUGGACUATT-3' (SEQ ID NO.4). Transfected into ISHIKAWA cells using Lipofectamine™ RNAiMax. Specific experimental steps: Inoculate cells into 6-well plates 1 day in advance so that the density reaches about 30-50% during transfection. Before transfection, replace the cell culture medium with fresh serum-free medium; prepare solution A: 150μL opti-MEM + 5μL Lipofectamine™ RNAiMax; prepare solution B: 150μL opti-MEM + 10μL 20μM siRNA; mix solution A and solution B and place them for 5 minutes respectively; mix solution A and solution B and place them for 20 minutes; add the mixture of A and B to the cell culture medium and shake well; replace with fresh complete culture medium after 6-8 hours, and continue to culture the cells for 48 hours before conducting subsequent experiments. Verify whether the knockdown is successful by immunoblotting of the corresponding target protein.

[0068] (2) Detection of intracellular free iron levels

[0069] Knockdown of TFRC Pten - / -MEFs and ISHIKAWA cells were seeded in 6-well plates at appropriate density and measured after the cells adhered to the wall; the fluorescent probe FerroOrange was used to detect the level of free iron ions in the cells. FerroOrange emits red fluorescence after binding to iron ions. FerroOrange was first diluted with HBSS to a final concentration of 1 μM. For flow cytometry analysis, the cells were digested and harvested and washed twice with HBSS. The cells were incubated with 1 μM FerroOrange for 30 minutes and then analyzed on a BD Accuri C6 Plus (Franklin Lakes, NJ, USA).

[0070] (3) Cell proliferation assay (CCK8)

[0071] Knockdown of TFRC Pten - / - MEFs and ISHIKAWA cells were digested to obtain cell suspension. After counting the cells with trypan blue, the dilution method of the cell suspension was calculated to make the number of cells in each well of the 96-well plate reach a density of 1000-3000 / 200μL. The cells were cultured in an incubator. After a certain period of time (0, 24, 48, and 72 hours), 20μL of CCK8 reagent was added to each well and incubated for another 2 hours. The cell count was measured. OD 450 , and according to OD 450 Results The cell proliferation was calculated.

[0072] (4) Clone formation experiment

[0073] Cell plating: knockdown TFRC Pten - / - MEFs and ISHIKAWA cells were digested to obtain cell suspension, and the cells were counted with trypan blue. Cell suspension dilution method: cells were inoculated at a density of 300-1000 cells / well and shaken to make the cells evenly adhere to the wall.

[0074] Cell culture: Place the cells in an incubator and continue to culture them, changing the culture medium every 3 days. After about 6-10 days, observe the size of the cell clones under a microscope. When the number of single cell clones reaches about 60, fix them and perform crystal violet staining.

[0075] Crystal violet staining: discard the cell culture medium, wash the cells 2-3 times with pre-cooled PBS, discard the PBS, add 1mL of 4% paraformaldehyde fixative to each well, and incubate on a shaker at room temperature for 15-30 min; discard the fixative, slowly wash the cells 3 times with PBS, add 1mL of 0.1% crystal violet stain to each well, and incubate on a shaker at room temperature for 30min or longer until clear clones are visible to the naked eye. Finally, slowly wash the culture dish with tap water until the floating color disappears, and take pictures after drying.

[0076] (5) Hippocampal XFe24 detection of cellular oxygen consumption rate

[0077] OCR analysis was performed using a Seahorse Xfe 24 flux analyzer (Agilent). For this assay, 10,000 cells were seeded in a 24-well microplate. Before measurement, the cell culture medium was replaced with XF assay medium supplemented with pyruvate, glucose, and glutamine (pH 7.4). OCR was measured at baseline, followed by consecutive injections of 1 μM oligomycin, 2 μM FCCP, and 0.5 μM rotenone / antimycin. Data were normalized to cell number.

[0078] 3. Experimental results

[0079] Depend on Figure 3 As shown in A, the shRNA1 and shRNA2 provided by the present invention can indeed knock down PTEN-deficient MEF cells ( Pten - / - Knockdown of TFRC leads to a decrease in the level of labile iron pool in cells (see Figure 3 B in the figure), the cell proliferation capacity is weakened (see Figure 3 C in the figure), the cell colony-forming ability is reduced (see Figure 3 D) and decreased oxygen consumption rate (OCR) levels (see Figure 3 E in.

[0080] Depend on Figure 3 As shown in F, the siRNA provided by the present invention can indeed knock down the expression of TFRC in PTEN-deficient ISHIKAWA cells. Knocking down TFRC leads to a decrease in the level of unstable iron pool in cells (see Figure 3 G in), the cell proliferation ability is weakened (see Figure 3 H in the cell), the ability of cell colony formation is reduced (see Figure 3 I) and oxygen consumption rate (OCR) levels decreased (see Figure 3 in J).

[0081] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of a TFRC inhibitor in the preparation of a drug for preventing and / or treating endometrial cancer, characterized in that: The TFRC inhibitor includes siRNA targeting the TFRC gene; the siRNA includes siRNA1 and siRNA2, the nucleotide sequence of the siRNA1 is shown in SEQ ID NO.3, and the nucleotide sequence of the siRNA2 is shown in SEQ ID NO.4; the endometrial cancer is PTEN-deficient endometrial cancer.

2. A drug capable of preventing and / or treating endometrial cancer, characterized in that: The drug can inhibit the expression of TFRC gene or protein; the drug includes siRNA that inhibits the expression of TFRC gene; the siRNA includes siRNA1 and siRNA2, the nucleotide sequence of siRNA1 is shown in SEQ ID NO.3, and the nucleotide sequence of siRNA2 is shown in SEQ ID NO.

4.

3. The drug according to claim 2, characterized in that The dosage form of the drug includes oral solution, injection, tablet, pill, dispersant, capsule or granule.

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