CN117187383A - Application of Ccser1 and Lum in preparation of skin squamous cell carcinoma identification reagent or kit - Google Patents
Application of Ccser1 and Lum in preparation of skin squamous cell carcinoma identification reagent or kit Download PDFInfo
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Abstract
The application relates to the technical field of medical biological detection, and provides application of Ccser1 and Lum in preparation of a skin squamous cell carcinoma identification reagent or kit. The application further provides a biological agent and a detection kit for identifying the squamous cell carcinoma of the skin by utilizing quantitative PCR detection aiming at two genes Ccser1 and Lum. The application can be used as the identification basis of the skin squamous cell carcinoma, AK and normal skin to a certain extent, helps the clinician to individually judge, optimizes the diagnosis or treatment scheme of the skin squamous cell carcinoma, and improves the compliance of patients.
Description
Technical Field
The application relates to the technical field of medical biological detection, relates to application of a combined genome consisting of Ccser1 and Lum as a skin squamous cell detection marker, and further provides application of the combined genome in preparation of a skin squamous cell carcinoma detection reagent or kit.
Background
Squamous cell carcinoma of the skin (cutaneous squamous cell carcinoma, cSCC) is one of the most common tumors in non-melanoma skin cancers, and is well developed in the exposed areas of the face of the elderly and in special areas of the external genitalia, where cSCC rapidly increases at a rate of 3-10% per year worldwide. Clinically, surgery is mainly used, but the problems of easy recurrence, large tissue structure damage at special parts, multiple skin lesions or complicated basic diseases combined, limited treatment of elderly patients and the like exist.
About 65% of cSCC originates from keratosis optically (Actinic keratoses, AK), and the annual risk of progression to cSCC for each individual AK lesion is about 0.025-20%. AK is the most common precancerous skin lesion originating from epidermal keratinocytes. Histologically, they are characterized by partial allotypes of basal and acanthosis, and dysplastic keratinocytes may extend to the full layer of the epidermis (bowdisease, also known as squamous cell carcinoma in situ) or further evolve into invasive skin squamous cell carcinoma (cSCC). Genetic studies reveal the nuclear genomic features of AK and cSCC in the progression from AK to cSCC, with a high degree of complexity and heterogeneity. Although most AK cases can spontaneously regress or be effectively treated by destructive treatment, there is still no effective way to prevent their progression to cSCC. The molecular mechanism during the transition from AK to cSCC has not been fully understood so far.
The prior researches reveal that TP53, NOTCH1-2, CDKN2A and other tumor suppressor genes are found in AK and cSCC, and mutation and transcription networks thereof are fully studied; key signaling pathways involving EGFR or RAS are also involved in AK progression. Previous whole genome sequencing encompasses genomic, transcriptomic, epigenomic and proteomic analysis, with little consensus on culprit from healthy skin to cSCC. Notably, AK and cSCC are highly complex and heterogeneous at the cellular and genetic level, e.g., two different subclasses of AK and cSCC have been reported for different cell-derived differentiation stages, indicating the presence of multiple potential subgroups or subtypes. Single cell RNA sequencing (scRNA-seq) is capable of analyzing single pre-cancerous lesions or cancer cells due to the limited insight provided by batch sequencing into cell heterogeneity.
Disclosure of Invention
The present application has been made in view of the above problems, and a first object is to provide applications of ccs er1, lum genes as markers for detecting squamous cell carcinoma of the skin; a second object is to provide a biological agent that identifies photo-linear keratosis (AK) and cutaneous squamous cell carcinoma (cSCC); a third object is to provide a kit for detecting squamous cell carcinoma of the skin.
In order to achieve the above purpose, the technical scheme adopted by the application is as follows: a mouse AK-cSCC model is constructed by adopting a UV induced SKH-1 mouse, and the cSCC lesion skin, the AK lesion skin and the normal skin are simultaneously present on an experimental mouse. When an experimental sample is taken, the skin lesion is required to cover normal skin, AK pathological change skin tissue sample and cSCC pathological change skin tissue sample simultaneously, the experimental sample is analyzed and screened to obtain differential protein, and clustering analysis is carried out on the differential protein on the expression level to obtain three differential expression genes Ccser1, lum and Papln. Experimental results show that Lum gene and ccs ser1 gene are significantly expressed in the cSCC lesion skin tissue sample, compared to normal skin and AK skin; papln gene was significantly low expressed in AK skin, but was not significantly reduced in cSCC diseased skin tissue. The same results were verified for the Lum gene and ccs er1 gene in human cSCC tissue, and the same detection results were obtained.
In a first aspect of the application there is provided the use of a reagent for detecting a combined genome consisting of two genes ccs er1, lum in the preparation of a skin squamous cell carcinoma discriminating reagent or kit.
Preferably, the identification reagent is a reagent for detecting the expression amounts of two genes Ccser1 and Lum in a biological sample; the identification kit comprises reagents for detecting the expression amounts of two genes Ccser1 and Lum in a biological sample.
Further preferably, the reagent for detecting the expression levels of two genes of ccs er1 and Lum in the biological sample is selected from PCR primers having detection specificity for two genes of ccs er1 and Lum, and the primer sequences are as follows:
the biological sample is obtained from skin tissue of a subject.
In a second aspect of the present application, there is provided a biological agent for identifying photo-linear keratosis (AK) and cutaneous squamous cell carcinoma (cSCC), comprising a reverse transcription system, a primer system and an amplification system for quantitatively detecting the expression levels of two genes ccs er1 and Lum, wherein the primer system comprises PCR primers as shown in SEQ ID nos. 1 to 4.
In a third aspect of the application, a kit for detecting squamous cell carcinoma of skin is provided, the kit comprises reagents for detecting the expression levels of two genes Ccser1 and Lum in a biological sample, and specifically comprises a reverse transcription system, a primer system and an amplification system, wherein the primer system comprises PCR primers shown as SEQ ID NO. 1-4.
In a fourth aspect of the present application, there is provided a method for diagnosis of squamous cell carcinoma of skin using the above-mentioned biological detection agent or detection kit, as follows:
A. extracting DNA from skin tissue by a conventional method;
B. quantitatively detecting copy numbers of the Ccser1 gene and the Lum gene by adopting a real-time quantitative PCR technology;
C. interpretation of results
The Lum and ccs er1 genes were found to be significantly low expressed in skin squamous cell carcinoma by bioinformatics analysis; the above results suggest that two genes can be applied to the differentiation between squamous cell carcinoma of the skin and keratosis optically.
The beneficial guarantee and effect of the application are as follows:
the application screens and obtains the gene which can be used as the diagnosis marker of the skin squamous cell carcinoma, can be used as the identification basis of the skin squamous cell carcinoma, AK and normal skin to a certain extent, helps the individual judgment of clinicians, optimizes the diagnosis or treatment scheme of the skin squamous cell carcinoma, and improves the compliance of patients.
In terms of technology, the detection of two genes is essentially a quantitative PCR detection of blood genome, has the characteristics of simple operation, sensitive detection, good specificity, high repeatability and the like, and is increasingly applied to clinical examination technology nowadays. The detection laboratory can detect by only having a common quantitative PCR amplification instrument and can obtain detection results faster.
Drawings
FIG. 1 shows the skin change process of SKH-1 mice during construction of a mouse AK-cSCC model;
FIG. 2 is a schematic representation of a skin sample of a mouse model;
FIG. 3 is a technical analysis roadmap of a mouse sample according to the application;
FIG. 4 shows the results of a trusted protein screening and the results of a PCA analysis, wherein A is the results of the trusted protein screening; b, trusted protein PCA analysis;
FIG. 5 shows the results of preliminary statistical screening of differential proteins;
FIG. 6 is a graph showing the results of clustering analysis of differential proteins at the expression level;
FIG. 7 shows a differential protein correlation analysis, wherein red represents high expression, and circle size represents the intensity of correlation;
FIG. 8 shows Venn analysis of differential protein properties and commonality between groups;
FIG. 9 is a comparison of the expression of the Lum and Ccser1 genes in normal human skin, AK and cSCC, wherein A is the expression result of the Lum gene and B is the expression result of the Ccser1 gene.
Detailed Description
The present application will now be described in detail with reference to examples and drawings, but the practice of the application is not limited thereto.
The reagents and starting materials used in the present application are commercially available or may be prepared by literature procedures. The experimental procedures, which are not specified in the following examples, are generally carried out under conventional conditions or under conditions recommended by the manufacturer.
Example one mouse AK-cSCC model construction
1. Realizes the selection and feeding of mice
And (3) constructing an animal model by adopting the SKH-1 mouse as a realization mouse. SKH-1 mice are a type of hairless mice without significant specificity formed by distant line cross breeding, which were first introduced from the dermatological tumor hospital of Stanford university in 1986, and stable hairless genes were obtained by long-term mating. The mice of the strain have no or little hair throughout the body and a albino background due to genetic mutation or loss of skin and hair traits. The mouse thymus in immunity normally develops and has no immunodeficiency gene, so the mouse thymus is very suitable for constructing wound healing models, skin disease related models and the like.
The strain mice are immunized normally as C57 mice, the raising conditions are relatively less severe, but the drinking water is high-temperature sterilized water, padding, feed and mouse cages are replaced regularly, and the general omnipotent mouse feed is used. The mice lack hair protection, if the breeding environment is not SPF grade, the skin is easy to be influenced by some parasitic organisms, especially some lice and fleas, so that the experiment progress is influenced, the periodic replacement of the mouse appliance is very important, the mice skin is inspected by irradiation every time, and if the skin is subjected to non-irradiation related mechanical damage, such as bite marks and scratch marks, the importance is required.
2. Mouse AK-cSCC model construction
Preparation before irradiation: ear tags are required to be marked on each SKH-1 mouse, and marks are made, so that the skin damage progress of each mouse can be independently observed and recorded at a later stage. MED intensity at irradiation was 0.06J/cm 2 The method comprises the steps of carrying out a first treatment on the surface of the Using LS125 type UV-irradiatorMeasuring the irradiation dose of the epidermis of the mouse, and setting the dose according to an adjusting machine based on the irradiation dose; the distance from the light source is about 30cm, and the light source is arc-shaped and irradiates the walls of the cage to generate an attenuation effect, so that the mouse cannot be fixed in the cage, and the calibration is needed for adjusting the dosage each time; the ultraviolet radiometer probe simulates the back skin position of the mouse, 3-point measurement is carried out, each point is repeated for 3 times, and the total average value is taken as the skin dose of the mouse under the machine intensity.
And (3) an irradiation step: (1) Preheating is needed after each cold start of the machine, and a temperature of 1.5J/cm is generally set 2 The dosage is run out. (2) The mice are placed in the irradiation cage, and the irradiation cage is wiped by alcohol after each use, so that the permeation of four walls is ensured. (3) After 5 days of irradiation per week, the skin recovers for 2 days, slow-acting photochemical reaction is established, acute photodamage is avoided, and if obvious erythema, blisters and the like appear, the irradiation is stopped for 5-7 days as required and then the treatment is carried out. (4) Lower than MED doses (0.05, 0.055J/cm) were used for the first two weeks, respectively 2 Irradiation, skin tolerance is established. Then increase by 0.005J/cm every 2 weeks 2 Gradually destroying the tolerance established by the skin.
Selection of nodes: the back skin lesions of mice were gradually changed with increasing total dose of irradiation, see fig. 1: normal, deepening of the skin sulcus, rising of Pi Ji, occurrence of mild erythema, accompanied desquamation, single follicular keratinization on the basis of erythema, gradual development of follicular keratinization into red pimples, scabbing skin, gradual increase of rash, tendency of fusion, further development of central obvious tissue necrosis and massive scab skin coverage.
AK. Rough judgment of SCC nodes: AK: AK is more likely when extensive desquamation, erythema, and single developing papules are seen, with crusting on the papules; in general, the skin damage and the corner formation of SCC are serious, and even the SCC can be in a cauliflower shape and the skin corner shape is outwards protruded, so that the SCC is more likely to be generated.
Lesion speed: normal to AK: slower (about 16-18 weeks); AK to SCC: the development was faster (SCC appeared around 8-10 weeks, i.e. around 26 weeks of total irradiation cycle).
3. Sample material and preservation
After the mouse is anesthetized by intraperitoneal injection of chloral hydrate, the required skin lesions are excised. Considering that the late space transcriptome is performed, the lesion requires to cover normal skin, AK, and SCC lesion tissue samples at the same time, so that the strip-shaped lesion is mainly cut, see fig. 2.
When the mouse specimen is dissociated, tissue forceps and tissue forceps should be carefully used, so that the damage to the skin structure is avoided, and the deviation of the subsequent cell space positioning information is avoided. Rapidly sucking blood, tissue fluid and the like on the surface of the tissue by using gauze after dissociation, simultaneously completing sample segmentation, transferring the sequenced sample into a freezing tube, placing the frozen sample into liquid nitrogen for 5-10 minutes, and transferring the frozen sample into a-80 ℃ refrigerator for preservation; the pathological specimen is fixed in formalin fixing liquid, and the requirement of sample sampling (selection of slicing mode after waxing) is communicated with a pathological company in time.
Example two, differential Gene screening
The mouse skin tissue samples of example one were grouped according to the following table 1 and screened according to the technical route shown in fig. 3.
Table 1 grouping of mouse skin tissue samples
Firstly, screening the credible protein from the sample (figure 4A); then, principal component analysis (PCA, principal Component Analysis) is carried out by using the expression quantity of the genes, the distribution condition of the samples is observed, the relation among the samples is explored or the experimental design is verified, the PCA can show the relation among the samples from different dimensions, and as a result, the larger the sum of variances explained on each coordinate axis on the PCA diagram is, the larger the information quantity seen on the corresponding diagram is, and the more single the acting factor is; conversely, the smaller the sum of variances, the less the sample distribution seen on the picture is to represent the actual sample information.
Further, preliminary statistical screening of the differential proteins revealed that there were differential proteins between AK and normal skin samples (N), between cSCC-AK, between cSCC-N and between different normal skin samples, with the greatest differences between cSCC-N, followed by between cSCC-AK (fig. 5).
Clustering analysis is carried out on the differential protein on the expression horizontal layer on the basis of preliminary statistical screening of the differential protein, and the result is shown in figure 6; further analysis of correlation of gene expression levels between samples, and examination of experimental reliability and sample selection rationality, the closer the correlation coefficient is to 1, the higher the similarity of expression patterns between samples is, and the results are shown in fig. 7, which shows analysis results of the cSCC-N group differential proteins.
FIG. 8 shows the results of Venn analysis of the properties and commonality of the differential protein between groups, with the middle oval in-loop region being the differential protein of interest, but should be removed if present in the N8/N30 group. In combination with the screening function using Excel, three sets of differential proteins described in table 2 coexist, and specific information is as follows:
TABLE 2 differential protein display
From this, the expression change tendencies of Lum and ccs er1 were the same, and both were significantly low-expressed in ccs, and both were verified as combined genome in human skin tissue.
Example III detection of expression levels of Lum, ccser1 in squamous cell carcinoma of skin and non-cancerous tissues
1. Sample collection and detection:
collecting paraffin embedded skin tissue samples of the excision of the dermatology department and the plastic surgery outpatient department of Shanghai long-sign hospitals and Shanghai fourth people hospitals, comprising: 8 human skin squamous cell carcinoma, 8 human solar keratosis, and 8 normal healthy skin tissues (non-tumor whole surgery of foreskin, eyelid, etc. resected skin tissues). During detection, total RNA is extracted by using an AllPrep DNA/RNA FFPE kit of Qiagen company; and performing fluorescence quantitative PCR corresponding operation according to a TagMan customized mRNA detection kit reference flow.
2. Detection method
Detection was performed using designed Lum, ccs er1 mRNA primer probes, see table 3 for primer sequences. The program was run on ABI7500, cycled 40-45 times, all experiments were repeated three times and the data were presented as mean.+ -. Standard deviation using SPSS16.0 treatment. When any of the genes showed abnormal expression in the cSCC, it was judged as positive, and the detection results are shown in table 4.
TABLE 3 primer sequence summarization
TABLE 4 results of human skin squamous cell carcinoma validation
The results are also shown in fig. 9, where LUM and ccs er1 genes were significantly low expressed in 8 human skin squamous cell carcinoma (cSCC), and the expression levels in 8 human solar keratoses (AK) were not significantly different from normal skin, and could be used to identify cSCC and AK.
While the preferred embodiments of the present application have been described in detail, the present application is not limited to the embodiments, and various equivalent modifications and substitutions can be made by those skilled in the art without departing from the spirit of the present application, and these equivalent modifications and substitutions are intended to be included in the scope of the present application as defined in the appended claims.
Claims (8)
1. The application of the reagent for detecting the expression level of Ccser1 and Lum in the preparation of the skin squamous cell carcinoma identification reagent or kit.
2. The use according to claim 1, wherein the discrimination reagent is a reagent for detecting the expression levels of two genes ccs 1 and Lum in a biological sample.
3. The use according to claim 1, wherein the identification kit comprises reagents for detecting the expression levels of two genes ccs 1 and Lum in the biological sample.
4. The use according to claim 2 or 3, wherein the reagent for detecting the expression level of two genes of ccs er1 and Lum in the biological sample is selected from PCR primers with detection specificity to ccs er1 and Lum, and the primer sequences are shown in SEQ ID nos. 1 to 4.
5. Use according to claim 2 or 3, wherein the biological sample is obtained from skin tissue of a subject.
6. The biological agent for identifying the keratosis and the squamous cell carcinoma of the skin is characterized by comprising a reverse transcription system, a primer system and an amplification system, wherein the reverse transcription system, the primer system and the amplification system are used for quantitatively detecting the expression quantity of two genes Ccser1 and Lum, and the primer system comprises PCR primers shown in SEQ ID NO. 1-4.
7. A kit for identifying the keratosis and the squamous cell carcinoma of skin is characterized by comprising reagents for detecting the expression levels of two genes Ccser1 and Lum in a biological sample.
8. The kit for identifying keratosis and squamous cell carcinoma of skin of claim 7, wherein the kit comprises a reverse transcription system, a primer system and an amplification system, wherein the primer system comprises PCR primers as shown in SEQ ID No. 1-4.
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2023
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