circscaper applications, psoriasis diagnostic / or therapeutic formulations

Abstract

This invention discloses the application of circSCAPER and a diagnostic / or therapeutic agent for psoriasis. Belonging to the field of psoriasis diagnostic and therapeutic technology, qRT-PCR detected abnormally high expression of circSCAPER in psoriatic lesions, suggesting that circSCAPER holds promise as a diagnostic marker for psoriasis. Due to the excellent silencing effect of siRNA, this invention, after ensuring that circSCAPER was suppressed, conducted CCK8 and apoptosis experiments on HaCaT keratinocyte cell lines with silenced circSCAPER. Compared to the NC group, the proliferation rate of cells in the siRNA group was significantly slower, and the proportion of late-stage apoptotic cells was significantly increased, indicating that silencing circSCAPER inhibited keratinocyte proliferation. Therefore, inhibiting circSCAPER can treat psoriasis, possessing profound clinical significance and important prospects for widespread application.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of psoriasis diagnosis and treatment, and particularly relates to application of a reagent for detecting circSCAPER content in preparation of a psoriasis diagnosis preparation, and application of a reagent for inhibiting expression of circSCAPER in preparation of a psoriasis treatment preparation, and corresponding diagnosis and treatment preparations. BACKGROUND

[0002] Psoriasis is a common chronic inflammatory skin disease in clinic, and its etiology and pathogenesis have not been completely elucidated. Among them, 97.06% of psoriasis patients show psoriasis vulgaris, and the clinical manifestations are scaly erythema mainly occurring on the scalp, trunk and limbs. The disease has a prolonged course and a high recurrence rate, which brings great burden to the patients in both spirit and economy. The excessive proliferation and parakeratosis of keratinocytes, inflammatory cell infiltration and neovascularization in the dermis are the significant features of the histopathology of psoriasis vulgaris. Studies have shown that the appearance of characteristic lesions of psoriasis vulgaris is related to abnormal proliferation and apoptosis of local keratinocytes.

[0003] Keratinocytes are the main component cells of the epidermis, which are differentiated from the basal cells of the epidermis. During the migration to the stratum corneum, they gradually differentiate and mature, and finally fall off from the skin surface. This process is precisely and complexly regulated. In the psoriasis lesion area, the growth kinetics of keratinocytes shows a special disorder, which is manifested as accelerated proliferation of epidermal basal cells, shortened mitotic cycle, shortened epidermal transit time from 28 days to 3-4 days, delayed differentiation process of keratinocytes, delayed apoptosis process, and finally overkeratinization, parakeratosis, granular layer disappearance and acanthosis.

[0004] In recent years, a new class of non-coding RNA, circular RNA (circRNA), has been confirmed to regulate physiological and pathological processes such as embryonic development, cell differentiation, immune response and inflammatory response, and participate in the occurrence and development of human diseases. CircRNA is a kind of non-coding RNA with covalent closed loop structure widely expressed in prokaryotic and eukaryotic cells. CircRNA has unique biological characteristics: (1) stability. Because circRNA has no 5' cap structure and 3' poly(A) tail, and is in a closed covalent loop structure, it is not easy to be degraded by exonuclease, and is more stable than linear RNA. (2) High abundance. CircRNA is highly expressed in mammals. At present, more than one million circRNAs have been found in human tissues, and the expression level of some circRNAs is even more than 10 times that of their linear isomers. (3) Specificity. CircRNA shows tissue-specific function under physiological and pathological conditions. Due to these biological characteristics of circRNA, it is considered to be a more potential gene expression regulation molecule than linear RNA. Therefore, in-depth research on circRNA in diseases is of great significance to elucidate the pathogenesis of psoriasis and discover new therapeutic targets. SUMMARY

[0005] Therefore, the purpose of the present application is to solve the above technical problems, provide the application of the reagent for detecting the content of circSCAPER in the preparation of psoriasis diagnosis preparation, and the application of the reagent for inhibiting the expression of circSCAPER in the preparation of psoriasis treatment preparation, and the corresponding diagnosis and treatment preparation.

[0006] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0007] The present application provides the application of the reagent for detecting the expression amount of circSCAPER in the preparation of product for diagnosing psoriasis; the sequence of circSCAPER is shown as SEQ NO. 1.

[0008] Further, the reagent for detecting the expression amount of circSCAPER comprises a qRT-PCR detection reagent for the circSCAPER circularization site,

[0009] The qRT-PCR primer sequence is

[0010] The upstream primer is 5'-ATTGCTAGATCAACGACGCAGG-3', as shown in SEQ NO. 2;

[0011] The downstream primer is 5'-ATCCTTCAGCATCTTTCCGTTCT-3', as shown in SEQ NO. 3.

[0012] The application further provides a psoriasis diagnostic kit, comprising: the reagent for detecting the expression amount of circSCAPER.

[0013] The application provides application of the reagent for inhibiting expression of circSCAPER in preparation of a psoriasis treatment preparation, wherein the sequence of the circSCAPER is shown as SEQ NO. 1.

[0014] Further, the reagent for inhibiting expression of the circular RNA circSCAPER comprises siRNA.

[0015] Further, the siRNA is as follows:

[0016] a sense strand (5'-3') GAAGCUAAGAACGGAAAGATT, shown as SEQ NO. 4.

[0017] an antisense strand (5'-3') UCUUUCCGUUCUUAGCUUCTT, shown as SEQ NO. 5.

[0018] The reagent for inhibiting circSCAPER comprises a negative control:

[0019] a sense strand (5'-3') UUCUCCGAACGUGUCACGUTT, shown as SEQ NO. 6;

[0020] an antisense strand (5'-3') ACGUGACACGUUCGGAGAATT, shown as SEQ NO. 7.

[0021] The application provides a psoriasis treatment preparation, comprising the reagent for inhibiting expression of the circular RNA circSCAPER.

[0022] However, the application is not limited to the siRNA and the negative control provided above.

[0023] The psoriasis treatment preparation further comprises reagents required for transfection of the siRNA.

[0024] The application provides the reagent for inhibiting expression of circSCAPER in preparation of a preparation for slowing down proliferation of keratinocytes and promoting apoptosis of keratinocytes, wherein the sequence of the circSCAPER is shown as SEQ NO. 1.

[0025] The application further provides a preparation for slowing down proliferation of keratinocytes, comprising the reagent for inhibiting expression of the circular RNA circSCAPER.

[0026] The sequence of the circular RNA circSCAPER is as follows:

[0027]

[0028] The present application detects that circSCAPER is abnormally highly expressed in psoriasis lesions by qRT-PCR technology, and inhibiting the expression of circSCAPER in keratinocytes can inhibit cell proliferation and promote cell apoptosis. The above findings suggest that circSCAPER is expected to be a diagnostic marker and a new therapeutic target for psoriasis. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 For qRT-PCR detection of the expression level of circSCAPER in the epidermis of psoriasis lesions and normal epidermis;

[0030] The expression level of circSCAPER was analyzed with the reference gene RPLP0 as the reference, and the normal epidermis was standardized to 1. The two-sided Mann-Whitney U test was used, and p<0.05 was statistically significant, ****p<0.0001.

[0031] RPLP0 as a reference

[0032] Upstream primer: 5'-TGGTCATCCAGCAGGTGTTCGA-3', as shown in SEQ NO. 8;

[0033] Downstream primer: 5'-ACAGACACTGGCAACATTGCGG-3', as shown in SEQ NO. 9;

[0034] Figure 2 For qRT-PCR technology to detect the silencing efficiency of si-circSCAPER in HaCaT cells; The expression level of circSCAPER was analyzed with GAPDH as the reference, and the negative control group was standardized to 1. The two-sided unpaired t test was used, and p<0.05 was statistically significant, ***p<0.001.

[0035] GAPDH as a reference

[0036] Upstream primer: 5'-ACCACAGTCCATGCCATCACT-3', as shown in SEQ NO. 10;

[0037] Downstream primer: 5'-TGACCTTGCCCACAGCCTT-3', as shown in SEQ NO. 11;

[0038] Figure 3 For the effect of silencing circSCAPER in vitro on the CCK8 detection value of HaCaT cells; *p<0.05, **p<0.01.

[0039] Figure 4The effect of silencing circSCAPER on the proportion of HaCaT cell apoptosis in vitro; A. flow cytometry graph, B. apoptosis cell proportion histogram, *p<0.05. DETAILED DESCRIPTION

[0040] The following examples are intended to further illustrate the present application and are not intended to limit the same.

[0041] Example 1: Expression of circSCAPER in psoriasis tissues and cells

[0042] 1. Twenty-three psoriasis lesion tissues and 16 normal skin tissues were collected from the Department of Dermatology, the Second Xiangya Hospital of Central South University. After truen epidermal separation, the epidermis tissues were retained to explore the expression difference of circSCAPER in the epidermis tissues of the two groups of samples. The providers of the lesion tissues were diagnosed with psoriasis vulgaris by histopathological examination in our hospital, and were excluded from other skin diseases such as systemic lupus erythematosus, atopic dermatitis, eczema, and neurodermatitis. The skin of the affected area had not been externally applied with glucocorticoids, retinoids, vitamin D3 derivatives, and calcineurin inhibitors in the past two weeks. The providers of normal skin tissues were excluded from skin diseases such as psoriasis, systemic lupus erythematosus, atopic dermatitis, eczema, and neurodermatitis. The collection of all experimental tissue samples was authorized by the Ethics Committee of the Second Xiangya Hospital of Central South University and the consent of the specimen donors was obtained.

[0043] 2. Extraction of tissue / cell RNA

[0044] The TRIZOL method was used to extract tissue RNA manually. The whole process was strictly enzyme-free operation, and low temperature was maintained.

[0045] (1) Personnel and environmental preparation: the experimental personnel wore disposable masks and gloves, sprayed and wiped the experimental table with 0.1% exogenous RNAase inhibitor, and used experimental equipment such as pipette gun, enzyme-free tip head, 1.5 mL enzyme-free EP tube, 2 mL enzyme-free grinding tube, etc.

[0046] (2) Experimental material preparation: the high-speed low-temperature centrifuge was cooled to 4°C, and chloroform, isopropyl alcohol, anhydrous ethanol, and enzyme-free water were inserted into the ice box or placed in the 4°C refrigerator for precooling. TRIZOL was balanced to room temperature.

[0047] (3) Collect the tissue powder or cell precipitate after liquid nitrogen grinding into 1 mL TRIZOL, and lyse at room temperature for 10 minutes.

[0048] (4) Add 200 μL of chloroform to each tube, shake vigorously for more than 15 seconds, and stand at room temperature for 5-10 minutes. 4°C, 12,000g, centrifuge for 15 min. During the centrifugation waiting period, new enzyme-free 1.5 mL EP tubes can be prepared and labeled.

[0049] (5) Centrifugation is completed, the EP tube is gently removed from the centrifugal hole and inserted into the EP tube rack placed in the ice box, and the EP tube is not shaken to avoid damaging the liquid layer. Observation shows that the liquid in the tube is divided into three layers: the upper layer is the colorless aqueous phase layer, which is the RNA layer; the middle layer is the white film-like layer, which is the protein layer; and the lowermost layer is the pink organic phase layer, which mainly contains organic matter such as phenol red and chloroform. 200 μL of the colorless liquid in the upper layer is slowly sucked into the enzyme-free 1.5 mL EP tube marked with a marker, avoiding touching the white protein layer and the lower pink liquid, and can be sucked three times to obtain 400-500 μL of the upper aqueous phase liquid.

[0050] (6) Add an equal volume of pre-cooled isopropanol (about 500 μL) to each tube, tightly cap the tube, invert the EP tube, gently mix, and stand at room temperature for 10 minutes (it can also be placed at -20°C for 30 minutes or -80°C overnight incubation).

[0051] (7) 4°C, 12,000g, centrifugation for 10 min. During the centrifugation, prepare 75% ethanol (dehydrated ethanol: enzyme-free water = 3:1), and insert into the ice box for pre-cooling after preparation.

[0052] (8) After centrifugation, carefully discard the supernatant, add 1 mL of the prepared 75% ethanol to each tube, tightly cap the tube, invert the EP tube, and tap the tube wall to make the RNA adhering to the bottom of the tube float in the liquid. This washing is more complete, which can reduce organic pollution.

[0053] (9) 4°C, 7,500g, centrifugation for 5 min.

[0054] (10) After centrifugation, a porcelain white gelatin-like precipitate is visible at the bottom or side wall of the tube, which is the RNA. After pouring out the liquid in the tube, centrifuge at 4°C, 7,500g for 5 min.

[0055] (11) Carefully suck the residual liquid in the tube with a pipette until it is completely sucked out, and place the EP tube in the EP tube rack to dry naturally. When the precipitate is translucent, add an appropriate amount of enzyme-free water according to the size of the precipitate, and place it in the 4°C refrigerator for half an hour to fully dissolve.

[0056] (12) Blow and mix the RNA solution, use Nanodrop 2000 ultramicro spectrophotometer to detect the RNA concentration and OD value, and the RNA sample can be stored at -80°C or directly start the subsequent experiment.

[0057] 3. mRNA reverse transcription to synthesize cDNA

[0058] The reverse transcription kit Prime Script RT Reagent Kit for RT-PCR (Perfect Real Time) developed by Japan TaKaRa Company is used. TMThis experiment was performed by using RT reagent Kit with gDNA Eraser(RR047) which contains two steps of genomic DNA removal and cDNA synthesis. The operation was performed according to the instructions of the kit.

[0059] (1) Personnel and environmental preparation: The experimental personnel wore disposable masks and gloves, sprayed and wiped the experimental table with 0.1% exogenous RNAase inhibitor, and used pipettes, enzyme-free tip heads, 1.5mL enzyme-free EP tubes, enzyme-free PCR tubes and other experimental equipment.

[0060] (2) Experimental article preparation: The required reagents in the kit RR047 were taken out from-20℃, and after thawing, the tube wall was knocked, the liquid in the tube was mixed, and after spotting, it was inserted into the ice box for use.

[0061] (3) Removal of genomic DNA: The reaction solution was prepared in an enzyme-free PCR tube according to the system shown in Table 1 below.

[0062] Table 1 Genomic DNA removal reaction system

[0063]

[0064] (4) Mix the above-mentioned reagents and sample RNA, and after spotting, place it in the PCR instrument, and perform the reaction according to the following program:

[0065] ① 42℃, 2min; ② 4℃, ∞.

[0066] (5) Reverse transcription reaction: The reverse transcription reaction solution was prepared according to the system shown in Table 2 below.

[0067] Table 2 Reverse transcription reaction system

[0068]

[0069]

[0070] (6) Mix the above-mentioned reagents and sample RNA, and after spotting, place it in the PCR instrument, and perform the reaction according to the following program:

[0071] ① 37℃, 15min; ② 85℃, 5sec; ③ 4℃, ∞.

[0072] (7) After the reverse transcription was completed, 20μL of cDNA stock solution was obtained, diluted 4 times by adding 60μL of enzyme-free water, and stored at-20℃.

[0073] 4、Real-time fluorescent quantitative PCR reaction

[0074] TB Green Premix Ex Taq from Japan TaKaRa Company was used. Premix Ex TaqTM II(Tli RNaseH Plus) kit and refer to the kit instruction for operation. Table 3 is a preparation system of the PCR reaction solution.

[0075] Table 3 Real-time fluorescent quantitative PCR reaction system

[0076]

[0077] (1) Preparation of experimental materials: 2x TB green Premix Ex Taq II, upstream primer, downstream primer and cDNA were taken out from -20℃, and after thawing, the tube wall was knocked, the liquid in the tube was mixed, and then was inserted into an ice box for standby after being spotted away.

[0078] (2) For the same amplification gene, a mixed solution composed of 2x TB green Premix Ex Taq II, upstream primer, downstream primer and enzyme-free water was prepared according to the amount of reaction number + 1, and then was mixed. After mixing, 9 μL per hole was added to the hole of the PCR reaction plate, and finally the corresponding template was added.

[0079] (3) After the PCR reaction plate was completely sealed with a sealing film, it was centrifuged at 2,500 rpm for 20 sec.

[0080] (4) The PCR reaction plate was placed in the corresponding module of the Light Cycler 96 fluorescent quantitative PCR instrument, and the reaction program was set as shown in Table 4. After amplification, the expression of the target gene was detected.

[0081] Table 4 Real-time fluorescent quantitative PCR amplification reaction conditions

[0082]

[0083] Upstream primer: 5'-ATTGCTAGATCAACGACGCAGG-3', as shown in SEQ NO. 2;

[0084] Downstream primer: 5'-ATCCTTCAGCATCTTTCCGTTCT-3', as shown in SEQ NO. 3.

[0085] (5) Relative quantitative analysis: After each cycle of denaturation, the instrument automatically records the average fluorescence value of the last 10% of the cycle time, which represents the PCR yield at the end of the last cycle, and accumulates in turn. After all reactions are completed, the fluorescence intensity values of all reaction wells are obtained, and the amplification curve graph is automatically generated and converted into data for analysis. We set the threshold value in the exponential growth range of the curve, and according to the cycle number Ct value experienced by each reaction well when the fluorescence intensity reaches the threshold value, we carry out relative quantitative analysis. The calculation formula is: relative expression = 2^-ΔΔCt (wherein, ΔCt = Ct value of target gene - Ct value of internal reference gene, ΔΔCt = experimental group ΔCt - average value of control group ΔCt).

[0086] The results show that the expression level of circSCAPER in the epidermis of psoriasis lesions is significantly higher than that in normal skin epidermis. Figure 1

[0087] Example 2: Effect detection of silencing circSCAPER expression in keratinocyte cell lines

[0088] Specific siRNA is designed for the circSCAPER circularization site, which is transfected into HaCaT cells using Lipofectamine TM RNAiMAX liposome, and the fresh complete culture medium is replaced 6 hours after transfection. After 48 hours of transfection, the cells are collected, and the expression level of circSCAPER in HaCaT cells transfected with si-circSCAPER and its corresponding negative control is detected by real-time fluorescent quantitative PCR technology. The siRNA is as follows:

[0089] Sense strand (5'-3') GAAGCUAAGAACGGAAAGATT, as shown in SEQ NO. 4.

[0090] Antisense strand (5'-3') UCUUUCCGUUCUUAGCUUCTT, as shown in SEQ NO. 5.

[0091] The reagent for inhibiting circSCAPER includes a negative control:

[0092] Sense strand (5'-3') UUCUCCGAACGUGUCACGUTT, as shown in SEQ NO. 6,

[0093] Antisense strand (5'-3') ACGUGACACGUUCGGAGAATT, as shown in SEQ NO. 7.

[0094] ​Results: Compared with the control group (the control group is the negative control described above), si-circSCAPER can significantly reduce the expression of circSCAPER in HaCaT cells, and the silencing efficiency of si-circSCAPER on circSCAPER is greater than 50%( Figure 2 ).

[0095] Example 3: Silencing circSCAPER in vitro inhibits the proliferation of keratinocytes

[0096] Lipofectamine TM RNAiMAX liposome was used to transfect si-circSCAPER and its corresponding negative control into HaCaT cells to silence the expression of circSCAPER. The original culture medium in the cell plate was aspirated at four time points before transfection, 24h, 48h and 72h after transfection, and fresh complete culture medium containing 10% CCK8 solution was added. The cell plate was incubated in a 37°C cell incubator, and the absorbance value at 450nm was detected after 40 minutes. The detection results show that before transfection, there is no significant difference in CCK8 detection value between the two groups of cells, indicating that the cell plate is uniform and the initial number of the two groups of cells is consistent. From 48h after transfection, compared with the control group, the CCK8 detection value of si-circSCAPER group was significantly reduced, and this difference was maintained until 72h after transfection( Figure 3 ), that is, silencing circSCAPER can inhibit the proliferation of keratinocytes, indicating that circSCAPER has a promoting effect on the proliferation of keratinocytes.

[0097] Example 4: Silencing circSCAPER in vitro promotes apoptosis of keratinocytes

[0098] Lipofectamine TM RNAiMAX liposome was used to transfect si-circSCAPER and its corresponding negative control into HaCaT cells to silence the expression of circSCAPER. After 48 hours of transfection, the cells were collected, and flow cytometry was used to detect the apoptosis level of HaCaT cells. The reagent used in this experiment is Annexin V-FITC / PI Apoptosis Detection Kit from Shanghai Yisheng Company, and the operation is carried out according to the instructions.

[0099] (1) HaCaT cells were inoculated into a 12-well plate, and Lipofectamine TM RNAiMAX liposome was used to transfect si-circSCAPER and its corresponding negative control into HaCaT cells to silence the expression of circSCAPER, and the detection was carried out after 48h of continuous culture.

[0100] (2) Trypsinize the cells without EDTA, collect the cells into flow tubes, take part of the cells from the sample tube as FITC single positive tube, PI single positive tube, Blank tube, 300g, 4°C centrifugation for 5min.

[0101] (3) Wash the cells with pre-cooled PBS twice, 300g, 4°C centrifugation for 5min.

[0102] (4) Aspirate PBS, add 100μL 1×Binding Buffer to resuspend the cells.

[0103] (5) Add 5μL Annexin V-FITC to the sample tube and FITC single positive tube, mix well, avoid light, react at room temperature for 10min.

[0104] (6) Add 10μL PI Staining Solution to the sample tube and PI single positive tube, mix well, avoid light, react at room temperature for 10min.

[0105] (7) Add 400μL 1×Binding Buffer, mix well and place on ice, detect within 1 hour by flow cytometry.

[0106] (8) Apoptotic cell ratio analysis: copy flow data, adjust compensation value with FlowJo software, take FITC as abscissa and PI as ordinate, divide the cells into four quadrants with the two quadrants as boundary, the lower left quadrant is live cells, the lower right quadrant is early apoptotic cells, and the upper right quadrant is late apoptotic cells. The results show that compared with the control group, the proportion of late apoptotic cells in the si-circSCAPER group is significantly increased ( Figure 4 ), that is, silencing circSCAPER can promote the apoptosis of keratinocytes, indicating that circSCAPER has an inhibitory effect on the apoptosis of keratinocytes.​

Claims

1. Use of an agent for inhibiting expression of circSCAPER in the preparation of a psoriasis treatment preparation, wherein the sequence of the circSCAPER is shown as SEQ NO. 1; and the agent for inhibiting expression of circSCAPER is siRNA. The sequence of the siRNA is as follows: Sense strand 5'-3' GAAGCUAAGAACGGAAAGATT; Antisense strand 5'-3' UCUUUCCGUUCUUAGCUUCTT.

2. Use according to claim 1, characterized in that, The agent for inhibiting expression of circSCAPER slows down the proliferation of keratinocytes and promotes the apoptosis of keratinocytes.

Images