CN114672566A - Application of RFC4 gene as cervical lesion diagnosis marker - Google Patents

Application of RFC4 gene as cervical lesion diagnosis marker Download PDF

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CN114672566A
CN114672566A CN202210386086.0A CN202210386086A CN114672566A CN 114672566 A CN114672566 A CN 114672566A CN 202210386086 A CN202210386086 A CN 202210386086A CN 114672566 A CN114672566 A CN 114672566A
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cervical
rfc4
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ink4a
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李双
马丁
张兼伟
孟思露
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Tongji Medical College of Huazhong University of Science and Technology
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Abstract

The application provides an application of RFC4 gene as a cervical lesion diagnosis marker, and relates to the technical field of clinical medicine. According to the invention, the degree of cervical lesion can be judged by detecting the expression level of the RFC4 gene in the cervical lesion tissue. The immunohistochemical staining of RFC4 proves that the diagnosis accuracy of the high-grade cervical lesions is high, and the diagnosis efficiency is superior to the traditional index p16INK4aAnd Ki-67. The invention provides an effective molecular marker for cervical lesion diagnosis, which has high detection sensitivity and strong specificity and has important clinical value and popularization and application prospects.

Description

Application of RFC4 gene as cervical lesion diagnosis marker
Technical Field
The application relates to the technical field of clinical medicine, in particular to application of RFC4 gene as a cervical lesion diagnosis marker.
Background
Worldwide, cervical cancer is the second most common tumor in women. Worldwide, there are about 60 million new cases of cervical cancer and about 30 million deaths per year. More than 13 ten thousand new cases of cervical cancer occur each year in China, and face a severe cervical cancer prevention and treatment situation. Cervical cancer is mainly classified into squamous carcinoma and adenocarcinoma, wherein squamous carcinoma accounts for 75% -80%. Persistent infection with high-risk HPV types leads to cervical squamous intraepithelial lesions (SIL, a formerly known as cervical intraepithelial neoplasia, CIN), which in turn progress to cervical Squamous Cell Carcinoma (SCC).
According to the 2014 WHO classification of female genital tumors suggests that cervical squamous intraepithelial lesions can be classified as high grade (HSIL) and low grade (LSIL). In clinical practice, different treatment strategies are adopted for cervical lesion patients of different grades, so that accurate histological grading of cervical lesions is particularly important for clinical treatment of patients. Due to certain similarity of different cervical lesions, the pathological diagnosis repeatability of different pathologists based on morphology is poor. Based on the method, various biomarkers are used for assisting clinical differential diagnosis, really differentiating people with progress risks and guiding subsequent treatment particularly necessary.
Currently, p16 is commonly used clinicallyINK4aKi-67, two immunohistochemical markers to aid diagnosis. Wherein, p16INK4aImmunohistochemical staining can help identify reactive, immature or atrophic squamous epithelium and high-grade cervical squamous intraepithelial lesions (HSIL), and can improve the accuracy of HSIL diagnosis and reproducibility among observers. However, p16INK4aThe positive rate exists in normal cervix uteri, inflammatory tissues and the like, so that the diagnosis specificity is not high, and misdiagnosis and over-treatment of patients are easily caused. Therefore, the cervical lesion diagnostic marker with high sensitivity and specificity is searched, HSIL which is possibly developed into cervical invasive carcinoma can be found as soon as possible, and the cervical invasive carcinoma is treated in time, so that the possibility of occurrence of the cervical invasive carcinoma is reduced.
Disclosure of Invention
In order to solve the defects existing in the prior art, the application aims to provide a molecular marker for histological diagnosis of female cervical lesions, namely, the expression level of RFC4 gene in cervical lesions is detected, so that the cervical lesion degree is evaluated, and a new means is provided for histological diagnosis of cervical lesions.
The technical problem to be solved by the application is solved by adopting the following technical scheme.
The method searches a GEO database of the national center for Biotechnology information of the United states4 expression data sets were selected, with GSE63514, GSE27678 and GSE7803 as discovery sets. We used a multi-step bioinformatics approach to find 32, 19 and 17 key genes in the discovery set, respectively. Further screening of genes common to at least two datasets as final key genes resulted in 4 genes, RFC4, CEP55, AURKA and TOP2A, respectively. Normal CIN1-3 and cervical squamous carcinoma tissues are collected, and the immunohistochemical technology is utilized to verify that the expression quantity of key gene protein is positively correlated with the cervical lesion degree. At the same time, the compound is similar to the currently clinically used p16INK4aCompared with Ki-67, RFC4 was found to be effective in diagnosing cervical high-grade squamous intraepithelial lesions (HSIL)/cervical high-grade squamous intraepithelial lesions and cervical squamous cell carcinoma (HSIL +).
The application provides an RFC4 gene and application of a detection reagent thereof in preparation of a product for assisting diagnosis of high-grade cervical lesions.
Further, the reagent comprises a reagent for detecting the mRNA expression quantity or the protein expression quantity of the RFC4 gene;
optionally, the reagent comprises a reagent required for detecting the expression level of RFC4 by common detection methods such as immunoblotting, real-time quantitative PCR, in-situ hybridization, gene or protein chips, high-throughput sequencing, immunohistochemistry and the like;
preferably, the reagent is a reagent required for detecting the expression level of the RFC4 gene protein through immunohistochemistry, and comprises an antibody specifically binding to the RFC4 protein and the like.
The application also provides a judgment standard for assisting diagnosis of cervical high-level lesion diagnosis through RFC4 immunohistochemistry.
The scoring criteria included: (1) scoring is based primarily on staining patterns in normal cervical tissue and intraepithelial neoplasia. Score 0, no staining, staining only of the cytoplasm or only of the basal and accessory basal layers; score 1, weak nuclear staining with or without cytoplasmic staining; 2 points, moderate nuclear staining with or without cytoplasmic staining; 3 points, strong nuclear staining with or without cytoplasmic staining. Among them, scores 2 and 3 were judged as positive. (2) In invasive squamous cell carcinoma, more than 10% of the tissues are positive for cancer cells, and the tissues are judged to be positive.
Compared with the prior art, the embodiment of the application has at least the following advantages or beneficial effects:
1. the application proves the correlation between the expression of RFC4 and the cervical lesion degree in the mRNA and protein level, and provides a new research direction for cervical lesion diagnosis/screening and cervical cancer targeted therapy drug development.
RFC4 immunohistochemical staining was good in sensitivity and specificity in diagnosing HSIL and HSIL +. And the conventional index p16INK4aCompared with the traditional Chinese medicine, the specificity is obviously increased, and the sensitivity is not obviously different. The diagnosis accuracy is better than that of the single use of p16INK4aOr Ki-67 with p16INK4a+ Ki-67 as a combination marker was used with close accuracy.
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In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present application and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained from the drawings without inventive effort.
FIG. 1 is the correlation analysis of mRNA expression level of key genes in expression profile chip data and disease progression in the present application example;
wherein FIG. 1A shows the GSE63514 result, FIG. 1B shows the GSE27678 result, FIG. 1C shows the GSE7803 result, and FIG. 1D shows the GSE138080 result;
FIG. 2 is a diagram illustrating immunohistochemical scoring criteria in the examples of the present application (corresponding to Table 3);
wherein, FIG. 2A is p16INK4aFIG. 2B is a schematic representation of Ki-67, FIG. 2C is a schematic representation of RFC 4;
FIG. 3 shows p16 in this exampleINK4aKi-67, RFC4 in different levels of cervical lesion tissue positive rate comparison;
wherein, FIG. 3A is p16INK4aThe results are shown in FIG. 3B as Ki-67 results and FIG. 3C as RFC4 results;
FIG. 4 is a ROC curve for diagnosis of HSIL and HSIL + by single and combined immunohistochemical markers in the examples of the present application;
wherein, FIG. 4A is ROC curve of HSIL diagnosis, and FIG. 4B is ROC curve of HSIL + diagnosis.
Detailed Description
In order to make the purpose, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below, and the specific embodiments do not represent a limitation on the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
It should be noted that the embodiments and features of the embodiments in the present application can be combined with each other without conflict, and the present application will be described in detail with reference to specific embodiments.
Application of RFC4 gene as cervical lesion diagnosis marker.
In some embodiments of the present application, the cervical lesion refers to cervical high-grade lesions, including cervical high-grade squamous intraepithelial lesions and cervical squamous carcinoma.
The technical scheme of the application has the following general idea:
the application screens out 4 expression data sets by searching a GEO database of the national center for biotechnology information, wherein GSE63514, GSE27678 and GSE7803 are used as discovery sets. We used a multi-step bioinformatics approach to find 32, 19 and 17 key genes in the discovery set, respectively. Further screening of genes common to at least two datasets as final key genes resulted in 4 genes, RFC4, CEP55, AURKA and TOP2A, respectively. Normal CIN1-3 and cervical squamous carcinoma tissues are collected, and the immunohistochemical technology is utilized to verify that the expression quantity of key gene protein is positively correlated with the cervical lesion degree. At the same time, the compound is similar to the currently clinically used p16INK4aCompared with Ki-67, RFC4 was found to be effective in diagnosing cervical high-grade squamous intraepithelial lesions (HSIL)/cervical high-grade squamous intraepithelial lesions and cervical squamous cell carcinoma (HSIL +).
The features and properties of the present application are described in further detail below with reference to examples.
Example 1
Screening of cervical lesions diagnostic Gene
1. Data download
In the Gene expression integration (GEO) database, "Central Intra genetic Neopalasia" AND ("Central" OR "Central") AND ("Neoplast" OR "cancer" OR "tumor") were used as search queries, AND the restriction species was "Homo sapiens". The inclusion criteria for the chip were: (1) mRNA expression profile data; (2) the data set contains at least three of four samples of Normal (Normal), low grade squamous intraepithelial lesions (LSIL), high grade squamous intraepithelial lesions (HISL), cervical squamous carcinoma (SCC); (3) the total number of the data set samples is more than or equal to 25.
According to the standard, screening out 4 chips, respectively: GSE63514, GSE27678, GSE7803, and GSE 138080. GSE63514, GSE27678 and GSE7803 are used as discovery sets, GSE138080 is used as verification sets, and chip information is shown in Table 1.
TABLE 1 chip information Table
Figure BDA0003595026930000061
1The GSE63514 dataset contains five samples of normal controls, CIN1, CIN2, CIN3, and early invasive squamous cell carcinoma, with CIN1 being assigned as LSIL and CIN2 and CIN3 being assigned as HSIL in this study.
2The GSE27678 data set contains data for both chip platforms, only the HG-U133A2.0 platform was selected in this study.
2. Key gene screening
Defining the differential genes: differential gene analysis (LSIL vs. normal, HSIL vs. normal, SCC vs. normal) was performed on each group using R-package limma. Inclusion criteria for differential genes: log |2FC | ≧ 1 (i.e., the fold difference of gene expression is more than 2 times or less than 0.5 times); corrected p-value<0.05. Defining progressive genes: genes that are continuously up-or down-regulated (Normal)<LSIL<HSIL<SCC or Normal>LSIL>HSIL>SCC). Define Gene set 2: intersection of differential genes with progressive genes in each dataset. Finally, 725, 123 and 121 genes were obtained in GSE63514, GSE27678 and GSE7803, respectively, and the specific results are shown in Table 2.
TABLE 2 Gene screening Table
Figure BDA0003595026930000071
The protein interaction network of the gene set 2 is constructed by using STRING and Cytoscape, and key genes are screened by using the Cytohubba plug-in. We select 9 topology analysis methods (Betwenness, Bottleneck, Closense, Degree, EPC, DMNC, MNC, Radiality, and Stress) to rank the nodes in the network, select the genes of 10 top ranks of each method, and form the key gene set of each data set after removing the repeated genes. 32 key genes are obtained in total in GSE63514, 19 key genes are obtained in total in GSE27678, and 17 key genes are obtained in total in GSE 7803. The intersection of the key genes of the three data sets is selected, and the genes shared by at least two data sets are screened as the final key genes of the research. The final 4 genes were RFC4, CEP55, AURKA and TOP 2A. As shown in FIG. 1, the mRNA expression levels of the key genes in the discovery set (GSE63514, GSE27678 and GSE7803) and the verification set GSE138080 are gradually increased along with the progress of cervical lesions, which indicates that the key genes can be used as potential molecular markers for diagnosing cervical lesions.
Example 2
Assessment of diagnostic efficacy of RFC4
We performed immunohistochemical analysis on the 4 key genes found in example 1. Considering the novelty of key genes in cervical lesions, staining pattern, diagnostic efficacy and other factors, we found that RFC4 was most effective for diagnosing HSIL/HSIL +. The following diagnosis efficacy of RFC4 immunohistochemistry on high-grade cervical lesions and p16 in clinical use at presentINK4aAnd Ki-67 comparison.
The cervical tissue used in this example was derived from Guilin pan-Biotechnology Limited tissue chips (CIN1021, CIN1022) and samples from the gynecological specimen bank of the affiliated college of Tongji medical university, including 42 normal cervical tissues, 59 CIN1 tissues, 26 CIN2 tissues, 42 CIN3 tissues, and 61 cervical squamous carcinoma tissues. All sections were diagnosed by three qualified pathologists.
The main reagents and the preparation method used in this example are as follows:
(1) PBS buffer (pH7.2-7.4): accurately weighing 18g of NaCl and 0.832g of NaH on an analytical balance2PO4·2H2O、5.496g NaH2PO4·12H2And adding distilled water to the volume of 2000 mL.
(2) EDTA antigen retrieval solution: purchased from Wuhan Severe Biometrics, 20 × Tris-EDTA repair solution (pH 9.0), cat # G1203. When in use, 20 XTris-EDTA antigen retrieval solution (pH 9.0) is mixed with 190mL of distilled water uniformly every 10mL to obtain 1 XpH 9.0 antigen retrieval solution containing 1mM EDTA.
(3) 3% of hydrogen peroxide: diluting 30% hydrogen peroxide with pure water. 30mL of 30% hydrogen peroxide is added to 270mL of pure water to obtain 300mL of 3% hydrogen peroxide.
(4) 3% Bovine Serum Albumin (BSA): purchased from Wuhan Seville, Inc., cat # G5001-100G. When used, the BSA was prepared at 3% using PBS buffer. 3g BSA was added to 100mL PBS buffer to obtain 3% BSA.
(5) A first antibody: p16INK4a(company Abcam, cat # ab108349 with a dilution ratio of 1: 100); ki-67 (company Immunoway, cat. No. YM6189, dilution ratio 1: 200); RFC4 (Abcam, Cat ab156780, 1:500 dilution) was diluted with PBS buffer.
(6) Secondary antibody: purchased from Wuhan Seville Bio Inc., goat anti-rabbit secondary antibody, cat # G1213-100 μ L DAB-containing color development solution.
(7) Tween-20: purchased from Wuhan Seville Bio Inc., cat # G0004, for formulation of PBST.
(8) PBST buffer (pH 7.2-7.4): 1000mL of PBS buffer solution is added with 500. mu.L of Tween-20, and the mixture is fully mixed for standby.
(9) Secondary antibody diluent: diluting the secondary antibody working stock solution and the PBST buffer solution with the pH value of 7.2-7.4 according to the proportion of 1:200, and fully and uniformly mixing for later use.
(10) DAB color development liquid: every 20 mu L of DAB stock solution is added with 1mL of DAB diluent.
(11) Neutral gum: purchased from Wuhan Severe Bio Inc., cat # WG10004160 for coverslips.
The specific steps of immunohistochemistry in this example are as follows:
(1) paraffin section dewaxing to water: and baking the slices in a constant-temperature oven at 37 ℃ for 30min, and then sequentially putting the slices into an environment-friendly dewaxing liquid I for 15min, an environment-friendly dewaxing liquid II for 15min, absolute ethyl alcohol I for 5min, absolute ethyl alcohol II for 5min, 95% ethyl alcohol for 5min, 85% ethyl alcohol for 5min, 75% ethyl alcohol for 5min and distilled water for 5 min. The slides were then washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time.
(2) Antigen retrieval: placing the tissue slices in a repairing box filled with EDTA antigen repairing buffer solution (pH 9.0) in a microwave oven for antigen repairing, boiling with middle fire for 10min, stopping fire for 5min, maintaining the temperature, and turning to middle and low fire for 10min to prevent excessive evaporation of the buffer solution. After natural cooling, the slides were washed 3 times in PBS (pH7.4) on a destaining shaker for 5min each.
(3) Blocking endogenous peroxidase: the sections were placed in 3% hydrogen peroxide solution, incubated for 25min at room temperature in the dark, and the slides were washed 3 times 5min each time in PBS (pH7.4) with shaking on a destaining shaker.
(4) Serum blocking: 3% BSA was added dropwise to the assembly ring to uniformly and completely cover the tissue, and the assembly ring was sealed at room temperature for 30 min.
(5) Adding a primary antibody: the blocking solution was gently spun off, primary antibody prepared with PBS was added dropwise to the slices, and the slices were incubated overnight at 4 ℃ in a wet box.
(7) Adding a secondary antibody: slides were washed 3 times in PBS (pH7.4) with shaking on a destaining shaker for 5min each time. After the section is slightly dried, HRP marked goat anti-rabbit universal secondary antibody covering tissues are dripped into the circle, and the incubation is carried out in an incubator at 37 ℃ for 30 min.
(8) DAB color development: slides were washed 3 times for 5min in PBS (pH7.4) with shaking on a destaining shaker. After the section is slightly dried, a DAB color developing solution which is prepared freshly is dripped into the ring to completely cover the tissue, the color developing time is controlled under a microscope, the positive color is brown yellow, and the section is washed by tap water to stop color development.
(9) Counterstaining cell nuclei: counter-staining with hematoxylin for about 3min, washing with tap water, differentiating with hematoxylin differentiation solution for several seconds, washing with tap water, returning the hematoxylin to blue, and washing with running water.
(10) Dewatering and sealing: and (3) putting the slices into 75% alcohol for 5min, 85% alcohol for 5min, 95% alcohol for 5min, absolute alcohol II for 5min, absolute alcohol I for 5min, environment-friendly dewaxing liquid II for 5min and environment-friendly dewaxing liquid I for 5min in sequence, dehydrating and transparentizing, taking out the slices, slightly drying, and sealing with neutral gum.
Optical microscopy, image acquisition and analysis, for p16INK4aThe immunohistochemical results of Ki-67 and RFC4 were scored. The scoring criteria for normal cervical tissue and intraepithelial neoplasia are shown in table 3, and the scoring criteria for invasive squamous cell carcinoma are: greater than 10% of the cancer cells in the tissue were positive, and figure 2 is a graphical representation of the overall scoring criteria.
TABLE 3 immunohistochemical Scoring protocol
Figure BDA0003595026930000111
Figure BDA0003595026930000121
And (3) result statistics and analysis:
performing optical microscopy and image acquisition analysis on the stained sample by using immunohistochemical technology to obtain p16INK4aImmunohistochemical results of Ki-67 and RFC4 were scored and tested for p16INK4aKi-67 and RFC4 genes were expressed in normal, CIN1, CIN2, CIN3 and cervical squamous cell carcinoma tissues, respectively. The results of immunohistochemical scoring of normal cervical and intraepithelial neoplasia tissues are shown in table 4; in each grade of the lesion, p16INK4aThe schematic diagrams of Ki-67 and RFC4 are shown in FIG. 3. Wherein the abscissa of FIG. 3 represents the lesion of each gradeA sample; the ordinate represents the positive rate of each index.
TABLE 4 immunohistochemical Scoring results
Figure BDA0003595026930000122
Experimental results show that RFC4 and p16INK4aKi-67, the protein expression of RFC4 increased gradually with the progression of cervical lesions. The positive rates of RFC4 in normal and CIN1 tissues are 4.8% and 13.6% respectively; p16INK4aThe positive rate in normal and CIN1 tissues is obviously increased compared with RFC4, and is respectively 11.9 percent and 54.2 percent; ki-67 has a positive rate in normal tissues equal to RFC4, and a positive rate in CIN1 of 30.5% higher than RFC 4. The three histochemical indexes have higher positive rates in CIN2+ lesions. Meanwhile, the positive rates of the three indexes in HSIL (CIN2-3) and HSIL + (CIN2-3+ SCC) are obviously higher than those in a control group (Normal + CIN1) through comparison, the difference has statistical significance, and the potential of RFC4 as a cervical lesion diagnostic marker is proved.
Further, the efficacy of single and combined immunohistochemical markers for diagnosis of HSIL and HSIL + was compared, and the results are shown in table 5 and fig. 4. We calculated sensitivity (Sen), specificity (Spe), Positive Predictive Value (PPV), Negative Predictive Value (NPV) and area under the ROC curve (AUC) to assess the diagnostic efficacy of the marker. Where the AUC value is used to indicate the accuracy of the prediction. When AUC > 0.5, the closer the AUC is to 1, the better the diagnostic effect. AUC has certain accuracy at 0.7-0.9, and AUC has higher accuracy at 0.9 or above.
TABLE 5 comparison of diagnostic potency of Single and Combined immunohistochemical markers on HISL/HSIL +
Figure BDA0003595026930000131
Figure BDA0003595026930000141
Sen: sensitivity; spe: specificity; 95% CI: a 95% confidence interval; PPV: positive predictive value; NPV: negative predictive value; AUC: the area enclosed by the ROC curve and the coordinate axis; ref: and (4) a control group.
As can be seen from Table 5 and FIG. 4A, in HSIL diagnosis, although the sensitivity of RFC4 is slightly lower than that of p16INK4aBut with no statistical difference (88.2% vs. 92.6%, p)>0.05); the specificity of RFC4 is significantly higher than that of p16INK4aThe difference was statistically significant (90.1% vs. 63.4%, p)<0.05). RFC4 and p16INK4aThe diagnostic accuracy of the + Ki-67 combination was close, with AUC values of 0.89 and 0.88, respectively; and p16INK4aAnd Ki-67 were relatively low, 0.78 and 0.86, respectively.
As can be seen from Table 5 and FIG. 4B, in the diagnosis of HSIL +, although the sensitivity of RFC4 is lower than that of p16INK4aBut with no statistical difference (91.5% vs. 95.3%, p)>0.05); the specificity of RFC4 is significantly higher than that of p16INK4aThe difference was statistically significant (90.1% vs. 63.4%, p)<0.05). Likewise, RFC4 is relative to a single p16INK4aAnd Ki-67, with p16INK4aThe combined diagnostic accuracy of + Ki-67 was approximated, with AUC values of 0.91 and 0.89, respectively; p16INK4aAnd Ki-67 have AUC values of 0.79 and 0.87, respectively.
In conclusion, the cervical lesion diagnosis marker p16 is compared with the existing clinical cervical lesion diagnosis marker p16INK4aIn contrast to Ki-67, the present application inventively provides RFC4 as a molecular marker for histological diagnosis of cervical lesions. The expression level of RFC4 is increased with the progress of cervical lesion. RFC4 immunohistochemistry on HSIL and HSIL + diagnostic efficacy is better than single p16INK4aOr Ki-67. The marker can compensate p16 to a certain degreeINK4aThe specificity is poor, and the method has important clinical application value.
The embodiments described above are some, but not all embodiments of the present application. The detailed description of the embodiments of the present application is not intended to limit the scope of the claimed application, but is merely representative of selected embodiments of the application. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.

Claims (2)

  1. Application of RFC4 gene as cervical lesion diagnosis marker.
  2. 2. The use of the RFC4 gene as defined in claim 1, as a diagnostic marker for cervical lesions, wherein the cervical lesions refer to high-grade cervical lesions, including high-grade squamous intraepithelial lesions and squamous carcinoma of the cervix.
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Citations (3)

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