CN116004816A - Application of IRF7 in skin fibroma - Google Patents

Abstract

The invention relates to medical application of IRF7, in particular to application of IRF7 in skin fibroma. The invention provides a method for preparing a detection reagent for the expression level of IRF7 to assist in diagnosing skin fibromatosis diseases, and also provides application of an IRF7 inhibitor in preparing medicines for treating skin fibromatosis. Provides a simpler and more effective clinical index of the skin fibroma, and provides an early basis for the occurrence of the skin fibroma. The IRF7 plays an important role in the pathogenesis of the skin fibroma, and can be used as a novel molecular marker and a drug target for diagnosing the skin fibroma.

Description

Application of IRF7 in skin fibroma

Technical Field

The invention relates to medical application of IRF7, in particular to application of IRF7 in skin fibroma.

Background

Dermal Fibromas (DF), also known as benign fibrocytomas (benign fibrous histiocytoma, BFH), are a benign tumor in the dermis caused by focal hyperplasia of fibroblasts or tissue cells, a common skin lesion, accounting for about 3% of skin lesion specimens received in a skin pathology laboratory. The mechanism of its occurrence is not clear and is thought to be a reactive fibrous hyperplasia caused by minor skin lesions. The disease is well developed in young and middle-aged people, is seen in any part of the body by women, but is seen in limbs, often appears as a single brown skin nodule, and is mainly composed of fibroblasts and histopathological cells. Typical dermal fibroids are easy to diagnose, but because of their wide histological lineages and varying manifestations, this presents a certain difficulty in diagnosis. The treatment method of the disease is mainly surgical treatment and conservative treatment.

IRF7 (interferon regulatory factor 7) is a member of the interferon regulatory factor family and plays an important role in immune system diseases. IRF7 interacts with Smad3 to mediate TGF- β signaling in collagen production. IRF7 knockout mice are less susceptible to bleomycin induction to produce fibrotic skin than wild type mice. However, IRF7 has not been studied in terms of skin wound healing and skin fibrogenesis.

There is an urgent need in the art to find diagnostic markers for cutaneous fibroids, and to apply marker inhibitors to cutaneous fibroid medicines, which are technical problems that need to be solved.

Disclosure of Invention

The invention aims at providing medical application of the active fragment for interfering with IRF7 expression or inhibiting IRF7 expression to prevent or treat skin fibroids.

In order to achieve the above purpose, the present invention provides the following technical solutions.

The application of a detection reagent for the expression level of IRF7 in preparing a product for assisting in diagnosing skin fibroma.

Furthermore, the skin fibroma product takes IRF7 gene or IRF7 protein as a diagnostic marker.

Further, the IRF7 expression level is up-regulated in skin fibroids.

Further, the product is a chip, a preparation or a kit.

Use of an IRF7 inhibitor in the manufacture of a medicament for the treatment of cutaneous fibroids.

Further, the IRF7 inhibitor inhibits human fibroblast differentiation, and the IRF7 inhibitor inhibits the occurrence and development of skin fibroids by inhibiting IRF7 gene expression.

Furthermore, the IRF7 inhibitor comprises an shRNA interference target sequence, and the nucleotide sequence of the shRNA is shown as SEQ ID NO. 2.

Further, the medicament is in any pharmaceutically acceptable dosage form.

Further, the drug is in any pharmaceutically acceptable dose.

Compared with the prior art, the invention has the beneficial effects.

The inventors found through a number of experiments that IRF7 expression was significantly up-regulated in human dermal fibroid skin tissue.

Cytologic levels have found that inhibition of IRF7 expression can inhibit fibroblast differentiation and thus skin fibromatosis.

The results indicate that IRF7 plays an important regulatory role in the course of dermal fibroids.

Drawings

FIG. 1 is the IRF7 mRNA expression levels in normal human skin and skin fibromatous tissue.

FIG. 2 is protein expression levels of IRF7 in normal human skin and skin fibromatous tissue.

FIG. 3 is a graph of mRNA expression of IRF7 over-expression, collagen type I (collage I, col 1), collagen type III (collage III, col 3), and fibronectin (Fn).

FIG. 4 is a graph showing protein expression of IRF7 over-expression, collagen type I (collage I, col 1), collagen type III (collage III, col 3), and fibronectin (Fn).

FIG. 5 is a graph of mRNA expression of knock-down IRF7, collagen type I (collage I, col 1), collagen type III (collage III, col 3), and fibronectin (Fn).

FIG. 6 is a graph of protein expression of knock-down IRF7, collagen type I (collage I, col 1), collagen type III (collage III, col 3), and fibronectin (Fn).

Detailed Description

Example 1.

1. Total RNA extraction and qRT-PCR in skin tissue or cells.

1.1, total RNA extraction in tissues.

1ml of Trizol lysate was added to the Ep tube containing the tissue, and the tissue was thoroughly lysed to a uniform state using a low temperature tissue homogenizer. The tissue was left on ice for 10min waiting for further lysis to be sufficient. 200. Mu.L of chloroform was added to the EP tube, and the mixture was stirred thoroughly with a vortex machine for 15s to mix the liquid, and the mixture was left to stand on ice for 10min. And centrifuging at 12000g for 10min by using a low-temperature centrifuge, taking the transparent liquid at the upper layer into a new EP pipe, adding equal volume of isopropanol, mixing uniformly upside down, and standing on ice for 10min. After that, the mixture was centrifuged at 12000g for 10 minutes using a low-temperature centrifuge. The supernatant was discarded, 1ml of 75% ethanol was added to wash the precipitate, and the precipitate was centrifuged at 7000g for 5min using a cryocentrifuge, and the supernatant was discarded. The ethanol was blotted using filter paper and placed in a fume hood to volatilize the residual liquid. Subsequent experiments were performed using aqueous precipitation of the desrnase.

1.2, RNA quantification, reverse transcription, real-time fluorescence quantitative PCR.

Quantification of RNA: the absorbance of the RNA at 260mm and 280mm is detected by a Nanodrop 2000 spectrophotometer to obtain the ratio, if A260/A280 is 1.8-2.0, the purity of the RNA is better, and the measured concentration of the RNA can be used for subsequent experiments.

Reverse transcription of RNA: the RNA was reverse transcribed into cDNA, and the procedure was as follows.

Table 1 Degenomic DNA reaction System.

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Reaction conditions: heating at 42deg.C for 2 min; cooling to 4 ℃. Reaction volume: 10 mu L.

TABLE 2 cDNA synthesis reaction system.

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Reaction conditions: 15min at 37 ℃; 5. 5s at 85 ℃; cooling to 4 ℃. Reaction volume: 20 mu L

After completion of reverse transcription, real-time fluorescent quantitative PCR was performed, and the reaction system was as follows.

Table 3 PCR reaction system.

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Reaction conditions: first stage, pre-denaturation: 95 ℃ for 5min; and in the second stage, the cyclic reaction: 95 ℃ for 15s;60 ℃,45s (40 cycles); third stage, dissolution profile: 95 ℃ for 15s;60 ℃,60s,95 ℃ and 15s.

TABLE 4 PCR primer sequences.

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Conclusion of the test: as shown in fig. 1, IRF7 mRNA was significantly increased in the skin tissue of patients with cutaneous fibroids.

2. The Western blot method is used for detecting the expression condition of irf7 in the cell fibroma.

Western-Blot experiments: the experimental group is skin fibroma tissue, and the control group is normal skin.

Protein extraction and quantification: the sample to be tested is placed on ice at 1:100 a mixed solution of PMSF and RIPA was prepared, and the mixed solution was added as appropriate according to the amount and size of cells and tissues. The tissue was crushed using a private tissue mill and ultrasonication, and placed on ice for 30min at low temperature. Samples were centrifuged at 15000g for 25min at 4 ℃. The supernatant was removed and the sample name was marked in a new EP tube. Preparing and detecting total protein concentration according to BCA kit, preparing protein mixed solution according to proportion, adding 6xloadingbuffer, mixing, heating at 95deg.C for 5min, and performing subsequent experiment or preserving at-20deg.C.

And (3) glue preparation: concentrated glue and separating glue are prepared according to the instruction of the kit, and the separating glue is firstly added into a glass plate groove rapidly, and a deionized water pressure Ji Jiaoshang layer plane is used. About 20 minutes, a clear boundary between the colloid and the water surface is seen, the upper water layer is discarded, and the residual liquid is sucked by using filter paper. Adding concentrated glue into the groove, horizontally inserting the comb along the groove wall from left to right, and extruding redundant bubbles. After the colloid is completely solidified, the subsequent stage experiment is carried out.

Electrophoresis: and (3) placing the prepared gel into an electrophoresis tank, using 1x electrophoresis liquid, pulling out a comb, adding a corresponding volume according to the required protein amount, and adding marker mark positions at the front end and the rear end. The voltage of the concentrated gel is initially 80V, and then the concentrated gel is converted into 100V for separation until bromophenol blue runs out of the bottom of the separation gel.

Transfer printing: taking out the gel after electrophoresis, transferring by using a 1x transfer liquid and adopting a wet transfer method, and assembling a wet transfer device in one-to-one correspondence with the black glue and white film, and fixing the wet transfer device in a transfer groove. The transfer tank is cooled by using ice during transfer according to the constant pressure of 90V for about 1 h.

Incubation resistance: the transferred PVDF membrane was taken out, washed 3 times with TBST, the membrane was cut out according to the position of the target band, incubated in an incubation box with the antibody of the target gene, and incubated overnight at 4℃in a shaker.

Secondary antibody incubation and chemiluminescence: recovering primary antibody every other day, cleaning the membrane on a shaking table for 3 times by TBST for 10min each time, and preparing corresponding secondary antibody and incubating for 1-2h at room temperature. The membranes were washed 3 times with TBST for 10min each on a shaker. And (3) performing exposure imaging by using the luminous liquid, and analyzing after preserving the pictures. The expression condition of the protein is expressed by adopting relative optical density, GAPDH is taken as an internal reference, and the experimental result is ensured to be independently repeated for more than 3 times.

A test conclusion; as shown in fig. 2, the protein expression of irf7 in human skin fibromatous tissue was significantly increased.

Example 2.

1. Cell transfection.

Human skin fibroblasts (HSDFs) were seeded into 6-well plates (about 1X 10 per well) ⁵ Individual cells) and cultured to an appropriate density prior to transfection. The IRF7 CDS sequence is shown as SEQ ID NO. 1.

SEQ ID NO.1 sequence: IRF7 CDS (Human, nm_ 004031):

5-ATGCCAGTCCCCGAGCGCCCTGCAGCCGGCCCTGACTCTCCGCGGCCGGGCACCCGCAGGGCAGCCCCACGCGTGCTGTTCGGAGAGTGGCTCCTTGGAGAGATCAGCAGCGGCTGCTATGAGGGGCTGCAGTGGCTGGACGAGGCCCGCACCTGTTTCCGCGTGCCCTGGAAGCACTTCGCGCGCAAGGACCTGAGCGAGGCCGACGCGCGCATCTTCAAGGCCTGGGCTGTGGCCCGCGGCAGGTGGCCGCCTAGCAGCAGGGGAGGTGGCCCGCCCCCCGAGGCTGAGACTGCGGAGCGCGCCGGCTGGAAAACCAACTTCCGCTGCGCACTGCGCAGCACGCGTCGCTTCGTGATGCTGCGGGATAACTCGGGGGACCCGGCCGACCCGCACAAGGTGTACGCGCTCAGCCGGGAGCTGTGCTGGCGAGAAGGCCCAGGCACGGACCAGACTGAGGCAGAGGCCCCCGCAGCTGTCCCACCACCACAGGGTGGGCCCCCAGGGCCATTCCTGGCACACACACATGCTGGACTCCAAGCCCCAGGCCCCCTCCCTGCCCCAGCTGGTGACAAGGGGGACCTCCTGCTCCAGGCAGTGCAACAGAGCTGCCTGGCAGACCATCTGCTGACAGCGTCATGGGGGGCAGATCCAGTCCCAACCAAGGCTCCTGGAGAGGGACAAGAAGGGCTTCCCCTGACTGGGGCCTGTGCTGGAGGCCCAGGGCTCCCTGCTGGGGAGCTGTACGGGTGGGCAGTAGAGACGACCCCCAGCCCCGGGCCCCAGCCCGCGGCACTAACGACAGGCGAGGCCGCGGCCCCAGAGTCCCCGCACCAGGCAGAGCCGTACCTGTCACCCTCCCCAAGCGCCTGCACCGCGGTGCAAGAGCCCAGCCCAGGGGCGCTGGACGTGACCATCATGTACAAGGGCCGCACGGTGCTGCAGAAGGTGGTGGGACACCCGAGCTGCACGTTCCTATACGGCCCCCCAGACCCAGCTGTCCGGGCCACAGACCCCCAGCAGGTAGCATTCCCCAGCCCTGCCGAGCTCCCGGACCAGAAGCAGCTGCGCTACACGGAGGAACTGCTGCGGCACGTGGCCCCTGGGTTGCACCTGGAGCTTCGGGGGCCACAGCTGTGGGCCCGGCGCATGGGCAAGTGCAAGGTGTACTGGGAGGTGGGCGGACCCCCAGGCTCCGCCAGCCCCTCCACCCCAGCCTGCCTGCTGCCTCGGAACTGTGACACCCCCATCTTCGACTTCAGAGTCTTCTTCCAAGAGCTGGTGGAATTCCGGGCACGGCAGCGCCGTGGCTCCCCACGCTATACCATCTACCTGGGCTTCGGGCAGGACCTGTCAGCTGGGAGGCCCAAGGAGAAGAGCCTGGTCCTGGTGAAGCTGGAACCCTGGCTGTGCCGAGTGCACCTAGAGGGCACGCAGCGTGAGGGTGTGTCTTCCCTGGATAGCAGCAGCCTCAGCCTCTGCCTGTCCAGCGCCAACAGCCTCTATGACGACATCGAGTGCTTCCTTATGGAGCTGGAGCAGCCCGCC-3’。

IRF7 overexpression plasmid and control (IRF 7& pcDNA) constructs were biosynthesized from jean. HSFs were transfected with 1.8. Mu.g plasmid per well in 2 mL medium using JetPRIME reagent transfection. The cells were harvested after 6 hours of liquid exchange and cumulative culture for 48 hours.

The effect of IRF7 overexpression on the expression of markers type I collagen (collage I, col 1), type III collagen (collage III, col 3) and fibronectin (Fn) was detected by fluorescent Real-time PCR (Real-time PCR) method.

Total RNA in skin fibroblasts of a test group and a control group are extracted, the influence of IRF7 expression on the formation process of cell fibromas of human fibroblasts is compared, the test group is obtained by transfecting IRF7 over-expression plasmids by Human Skin Fibroblasts (HSFs), and the control group is obtained by transfecting blank controls by Human Skin Fibroblasts (HSFs).

The procedure was as in example 1.

The results are shown in FIG. 3: after IRF7 is overexpressed, the transcript levels of collagen type I (collage I, col 1), collagen type III (collage III, col 3) and fibronectin (Fn) are significantly higher than in the control group, and IRF7 overexpression increases skin fibromatosis.

3. Western blot detects the effect of IRF7 overexpression on expression of markers type I collagen (collage I, col 1), type III collagen (collage III, col 3) and fibronectin (Fn).

Protein extraction and quantification: samples of the experimental group, which was Human Skin Fibroblasts (HSFs) transfected with IRF7 overexpression plasmid, and the control group, which was Human Skin Fibroblasts (HSFs) transfected with a blank control, were placed on ice.

The procedure was as in example 1.

The results are shown in FIG. 4: the expression of various extracellular matrices, including collagen type I (collgenin, col 1), collagen type III (Col 3) and fibronectin (Fn), in skin fibroblasts, in which overexpression was interfering, was increased, indicating an increased viability of the fibroblasts to differentiate into myofibroblasts, in comparison to the control group, in positive correlation with skin fibromas and tissue fibrosis. In vitro experiments indicate that IRF7 overexpression can increase dermal fibromatosis.

Example 3.

1. Establishment of IRF7 low expression skin fibroblast model.

Knocking down plasmids and controlling by shRNA interference technology, wherein shRNA interferes with a target sequence: SEQ ID NO.2: IRF7 shRNA (Human): GCTGGACGTGACCATCATGTA. The siRNA used in the shRNA interference experiment was purchased from Liaoning-type hundred-Hao biosynthesis, and the specific steps are as follows.

HSFs were transfected with 1.8. Mu.g plasmid per well in 2 mL medium using JetPRIME reagent transfection. The cells were harvested after 6 hours of liquid exchange and cumulative culture for 48 hours.

The effect of IRF7 low expression on the expression of markers type I collagen (collage I, col 1), type III collagen (collage III, col 3) and fibronectin (Fn) was detected by a fluorescent Real-time PCR (Real-time PCR) method.

Total RNA in skin fibroblasts of a test group and a control group are extracted, the influence of IRF7 expression on the formation process of cell fibromas of human fibroblasts is compared, the test group is obtained by transfecting IRF7 knockdown plasmids by Human Skin Fibroblasts (HSFs), and the control group is obtained by transfecting blank controls by Human Skin Fibroblasts (HSFs).

The procedure was as in example 1.

The results are shown in FIG. 5: after IRF7 knockdown, transcript levels of type I collagen (collageeni, col 1), type III collagen (collageeni, col 3) and fibronectin (Fn) were significantly lower than in the control group, and inhibition of IRF7 expression reduced skin fibromatosis.

3. Western blot detects the effect of IRF7 low expression on expression of markers type I collagen (collage I, col 1), type III collagen (collage III, col 3) and fibronectin (Fn).

Protein extraction and quantification: samples of the experimental group and the control group were placed on ice, the experimental group was Human Skin Fibroblasts (HSFs) transfected with IRF7 knockdown plasmid, and the control group was Human Skin Fibroblasts (HSFs) transfected with blank control.

The procedure was as in example 1.

The results are shown in FIG. 6: in shRNA-interfered skin fibroblasts, expression of various extracellular matrices, including collagen type I (collagenI, col 1), collagen type III (collagenIII, col 3) and fibronectin (Fn), was decreased in positive correlation with skin fibroma and tissue fibrosis, compared to the control group, indicating decreased viability of the fibroblasts to differentiate into myofibroblasts. In vitro experiments show that inhibiting IRF7 expression can inhibit skin fibromatosis.

Claims (9)

1. Use of a detection reagent for the expression level of irf7 for the preparation of a product for the assisted diagnosis of cutaneous fibroids.
2. 2. Use of a reagent for detecting the expression level of IRF7 according to claim 1 for the preparation of a product for aiding in the diagnosis of cutaneous fibroids, wherein said cutaneous fibroid product uses the IRF7 gene or IRF7 protein as a diagnostic marker.
3. 3. The use according to claim 1, wherein the IRF7 expression level is up-regulated in cutaneous fibroids.
4. 4. The use according to claim 1, wherein the product is a chip, a formulation or a kit.
5. Use of an irf7 inhibitor for the preparation of a medicament for the treatment of cutaneous fibroids.
6. 6. The use according to claim 5, wherein the IRF7 inhibitor inhibits human fibroblast differentiation, and wherein the IRF7 inhibitor inhibits the occurrence and progression of cutaneous fibroids by inhibiting IRF7 gene expression.
7. 7. The use according to claim 5, wherein the IRF7 inhibitor comprises an shRNA interfering target sequence, the nucleotide sequence of the shRNA being shown in SEQ ID No. 2.
8. 8. The use according to claim 5, wherein the medicament is in any pharmaceutically acceptable dosage form.
9. 9. The use according to claim 5, wherein the medicament is in any pharmaceutically acceptable dose.

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