Application of Erdafitinib in upregulating lactoferrin expression in cells

Abstract

The invention provides an application of erdafitinib in up-regulating the expression of lactoferrin in cells, relates to the field of biotechnology, and proposes for the first time an application of erdafitinib in up-regulating the expression of lactoferrin in cells. Experiments have shown that erdafitinib can activate the LF promoter, significantly enhance the expression of lactoferrin in cells, and promote the synthesis of lactoferrin.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of erdafitinib in up-regulating lactoferrin expression in cells. Background Art

[0002] Lactoferrin (LF) is an important non-heme iron-binding glycoprotein in breast milk, which is mainly expressed and secreted by mammary epithelial cells. Lactoferrin not only participates in the transport of iron, but also has powerful biological functions such as broad-spectrum antibacterial, antioxidant, anti-cancer, and immune system regulation. It is considered to be a new type of antibacterial and anti-cancer drug and a food and feed additive with great development potential. Lactoferrin has broad market prospects, especially in the field of infant formula.

[0003] Erdafitinib is a fibroblast growth factor receptor (FGFR) family inhibitor used to treat patients with advanced or metastatic bladder cancer with FGFR mutations. However, there are currently no studies on the effect of Erdafitinib on promoting lactoferrin expression in breast epithelial cells.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a new use of Erdafitinib, namely, use of Erdafitinib in up-regulating the expression of lactoferrin in cells.

[0006] A second object of the present invention is to provide a method for promoting in vitro cell synthesis of lactoferrin.

[0007] The third object of the present invention is a method for promoting lactoferrin production in mammals.

[0008] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0009] In a first aspect, the present invention provides a use of erdafitinib in up-regulating the expression of lactoferrin in cells.

[0010] Furthermore, the erdafitinib upregulates the expression of lactoferrin by activating the LF promoter.

[0011] Furthermore, the cells include cells containing a lactoferrin gene controlled by a LF promoter.

[0012] Furthermore, the cells include mammary epithelial cells or host cells transfected with a lactoferrin gene expression vector containing a LF promoter.

[0013] Furthermore, the mammary epithelial cells are from any one of cattle, sheep, camel, horse, donkey, reindeer, alpaca or llama.

[0014] In a second aspect, the present invention provides a method for promoting in vitro cell synthesis of lactoferrin, comprising adding erdafitinib during in vitro cell culture to obtain lactoferrin through culture;

[0015] The concentration of Erdafitinib in the in vitro cell culture medium is ≤100nM and >0.

[0016] Furthermore, the concentration of erdafitinib in the culture medium is 1-100 nM.

[0017] Furthermore, the culturing time is at least 24 hours.

[0018] In a third aspect, the present invention provides a method for promoting lactoferrin production in mammals, comprising administering erdafitinib to an animal containing a lactoferrin gene controlled by a LF promoter.

[0019] Furthermore, the method of administering Erdafitinib includes oral administration, injection or external application.

[0020] The invention provides an application of erdafitinib in up-regulating the expression of lactoferrin in cells, and proposes for the first time an application of erdafitinib in up-regulating the expression of lactoferrin in cells. Experiments have shown that erdafitinib can activate the LF promoter, significantly enhance the expression of lactoferrin in cells, and promote the synthesis of lactoferrin. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0022] Figure 1 The specific structure of the LF promoter-copGFP expression vector provided in Example 1 of the present invention;

[0023] Figure 2 This is a picture of the fluorescent reporter cells observed under a fluorescence microscope after being treated with Erdafitinib provided in Example 1 of the present invention;

[0024] Figure 3 This is a diagram of the cell status observation under an inverted microscope after bovine mammary epithelial cells were treated with different concentrations of Erdafitinib for 24 hours as provided in Example 2 of the invention;

[0025] Figure 4 This is a statistical graph of LF expression after bovine mammary epithelial cells were treated with different concentrations of Erdafitinib for 24 hours as provided in Example 2 of the present invention;

[0026] Figure 5 This is a statistical graph of LF expression after human mammary epithelial cells were treated with different concentrations of erdafitinib for 24 hours as provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0027] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0028] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0029] In one aspect, the present invention provides the use of erdafitinib in up-regulating the expression of lactoferrin in cells.

[0030] The application of erdafitinib in upregulating the expression of lactoferrin in cells was proposed for the first time. Experiments have shown that erdafitinib can activate the LF promoter, significantly enhance the expression of lactoferrin in cells, and promote lactoferrin synthesis.

[0031] In some specific embodiments, the erdafitinib upregulates the expression of lactoferrin by activating the LF promoter.

[0032] In some specific embodiments, the cells include cells containing a lactoferrin gene controlled by a LF promoter. Erdafitinib can promote the expression of lactoferrin in cells containing a lactoferrin gene controlled by a LF promoter by activating the LF promoter.

[0033] In some specific embodiments, the cell comprises a mammary epithelial cell or a host cell transfected with a lactoferrin gene expression vector containing a LF promoter.

[0034] Erdafitinib can be applied to mammary epithelial cells or host cells transfected with a lactoferrin gene expression vector containing the LF promoter to upregulate lactoferrin expression in the cells.

[0035] In some specific embodiments, the mammary epithelial cells are from any one of cattle, sheep, camel, horse, donkey, reindeer, alpaca or llama.

[0036] According to one aspect of the present invention, there is also provided a method for promoting in vitro cell synthesis of lactoferrin, comprising adding erdafitinib during in vitro cell culture to obtain lactoferrin through culture;

[0037] The concentration of Erdafitinib in the in vitro cell culture medium is ≤100nM and >0.

[0038] In some specific embodiments, the concentration of erdafitinib in the culture medium is 1-100 nM.

[0039] The concentration of erdafitinib in the culture medium may be, but is not limited to, 1 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM or 100 nM, or any value between 1 and 100 nM.

[0040] Among them, when the concentration of Erdafitinib in the culture medium is 100nM or 1nM, the expression of lactoferrin is significantly increased. Specifically, in practical applications, Erdafitinib can be directly added to the culture medium or used as a reagent to prepare a culture medium that promotes cell lactoferrin expression.

[0041] In some specific embodiments, the culturing time is at least 24 hours.

[0042] According to another aspect of the present invention, a method for promoting lactoferrin production in mammals is provided, comprising administering erdafitinib to an animal containing a lactoferrin gene controlled by a LF promoter.

[0043] In some specific embodiments, the administration of Erdafitinib includes oral administration, injection or external application.

[0044] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0045] Cell culture

[0046] MAC-T cells: purchased from Shanghai Cell Bank, culture conditions: 90% DMEM + 10% FBS.

[0047] Bovine mammary epithelial cells (BMEC): The mammary tissue of Holstein cows from the Dachang slaughterhouse in Hebei Province was processed by enzyme digestion, and the primary bovine mammary epithelial cells were purified by differential adhesion and differential digestion methods. The culture conditions were 90% DMEM / F12 + 10% FBS. The cells were digested with 0.25% EDTA-trypsin for subculture, and digestion was terminated with complete medium.

[0048] Example 1 Verification of Erdafitinib Activating LF Promoter to Promote Lactoferrin Synthesis

[0049] 1. Vector construction and plasmid amplification

[0050] pGreenZeo lenti reporter vector, enzyme digestion to obtain linearized vector, synthesize LF promoter sequence, use homologous recombination to construct LF promoter-copGFP expression vector, transform E. coli, screen positive colonies on resistance medium, and after identification and sequencing, perform bacterial liquid expansion and extract plasmid for use. The specific structure of LF promoter-copGFP expression vector is as follows: Figure 1 As shown, Cell-specific Promoter refers to LF promoter, and the nucleotide sequence of LF promoter is shown in SEQ ID NO.1.

[0051] 2. Preparation of LF promoter-copGFP promoter reporter cells

[0052] (1) The LF promoter-copGFP expression vector plasmid was transfected into 293T cells together with the packaging vector. After 48 hours, the supernatant containing the virus was collected and the lentivirus was concentrated, purified and titered.

[0053] (2) When the MAC-T cell fusion rate reaches about 70%, virus infection is performed. After 24 hours, the culture medium is replaced with fresh complete culture medium and culture is continued in a carbon dioxide incubator.

[0054] (3) 48 hours after adding the virus, add Zeocin to screen for stable expression cell lines.

[0055] 3. Verification of Erdafitinib

[0056] pGreenZeo lenti reporter (LF promoter-copGFP) cells were cultured in 96-well plates. When the cells were attached and fused to 80%, they were treated with 1 μM erdafitinib. Cell fluorescence was observed under a fluorescence microscope after 24 hours. The specific results are shown in the figure. Figure 2As shown, by constructing a fluorescent reporter cell line (LF promoter drives copGFP expression), fluorescence microscopy observation revealed obvious fluorescence production under erdafitinib treatment.

[0057] Example 2 Erdafitinib upregulates lactoferrin expression in bovine mammary epithelial cells in vitro

[0058] The 11th generation BMECs were cultured in 6-well plates. When the cells were attached and fused to 80%, they were treated with 1 nM, 10 nM, 100 nM and 1000 nM of erdafitinib, respectively. 0.1% DMSO was used as a control group instead of erdafitinib. After 24 hours, the cells were collected and the cell status was observed under an inverted microscope. The results are shown in Figure 2. Figure 3 At the same time, RNA was extracted and RT-qPCR was performed to detect the expression of LF. The primers used were shown in Table 1, with GAPDH as the internal reference. The specific results are shown in Figure 4 shown.

[0059] Table 1 List of qPCR specific primers used

[0060]

[0061] like Figure 3 and Figure 4 As shown in the figure, compared with the control group, the LF expression levels in the 1 nM, 10 nM and 100 nM treatment groups were all higher than those in the control group, among which the 1 nM and 100 nM treatments significantly increased the LF expression levels, with the 1 nM treatment having the highest expression level. The 1000 nM treatment had no difference in expression level from that in the control group.

[0062] Example 3 Erdafitinib upregulates lactoferrin expression in human mammary epithelial cells

[0063] Referring to the method of Example 2, 1 nM, 10 nM and 100 nM of erdafitinib were respectively applied to human mammary epithelial cells (MCF-10A). After 24 hours, the cells were collected, RNA was extracted, and RT-qPCR was performed to detect the expression of LF. The primers used were shown in Table 2, and GAPDH was used as the internal reference. The specific results are shown in Figure 5 As shown. It can be seen that Erdafitinib has the effect of upregulating the expression of lactoferrin in human mammary epithelial cells.

[0064] Table 2 List of specific primers used in qPCR in human mammary epithelial cells (MCF-10A)

[0065]

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. The use of erdafitinib in upregulating lactoferrin expression in cells; The cells are mammary epithelial cells; The cells are in vitro cells; The mammary epithelial cells are from any one of humans, cattle, sheep, camels, horses, donkeys, reindeers, alpacas or llamas.

2. The use according to claim 1, characterized in that: The erdafitinib upregulates the expression of lactoferrin by activating the LF promoter; The nucleotide sequence of the LF promoter is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The cells are cells containing the lactoferrin gene controlled by the LF promoter.

4. A method for promoting in vitro cell synthesis of lactoferrin, characterized in that: It includes adding erdafitinib during in vitro cell culture to obtain lactoferrin; The concentration of erdafitinib in the in vitro cell culture medium is ≤100 nM and >0; The cells are mammary epithelial cells; The mammary epithelial cells are from any one of humans, cattle, sheep, camels, horses, donkeys, reindeers, alpacas or llamas.

5. The method according to claim 4, characterized in that The concentration of erdafitinib in the culture medium is 1-100 nM.

6. The method according to claim 4, characterized in that The culturing time is at least 24 hours.

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