CN114672566A - Application of RFC4 gene as a diagnostic marker for cervical lesions - Google Patents

Abstract

The present application proposes the application of the RFC4 gene as a diagnostic marker for cervical lesions, and relates to the technical field of clinical medicine. The present invention can judge the degree of cervical lesions by detecting the expression level of the RFC4 gene in the cervical lesion tissue. The immunohistochemical staining of RFC4 demonstrated its high accuracy in diagnosing high-grade cervical lesions, and its diagnostic performance was superior to traditional indicators p16 ^INK4a and Ki‑67. The invention provides an effective molecular marker for diagnosing cervical lesions. The marker has high detection sensitivity and strong specificity, and has important clinical value and prospect of popularization and application.

Description

Application of RFC4 gene as a diagnostic marker for cervical lesions

technical field

The present application relates to the technical field of clinical medicine, in particular, to the application of the RFC4 gene as a diagnostic marker for cervical lesions.

Background technique

Worldwide, cervical cancer is the second most common cancer in women. There are about 600,000 new cases of cervical cancer and about 300,000 deaths worldwide each year. There are more than 130,000 new cases of cervical cancer in China every year, and China is facing a severe situation of cervical cancer prevention and treatment. Cervical cancer is mainly divided into squamous cell carcinoma and adenocarcinoma, of which squamous cell carcinoma accounts for 75%-80%. Persistent infection with high-risk HPV leads to cervical squamous intraepithelial lesion (SIL, formerly known as cervical intraepithelial neoplasia, CIN), which subsequently progresses to cervical squamous cell carcinoma (SCC).

According to the 2014 WHO classification of female genital tumors, cervical squamous intraepithelial lesions can be divided into high-grade (HSIL) and low-grade (LSIL). In clinical practice, patients with different grades of cervical lesions adopt different treatment strategies, so the accurate histological grading of cervical lesions is particularly important for the clinical treatment of patients. Due to the similarity of different cervical lesions, the repeatability of pathological diagnosis based on morphology by different pathologists is poor. Based on this, it is particularly necessary to use various biomarkers to assist in clinical differential diagnosis, distinguish people at real risk of progression, and guide subsequent treatment.

At present, two immunohistochemical markers, p16 ^INK4a and Ki-67, are commonly used in clinical practice to assist diagnosis. Among them, p16 ^INK4a immunohistochemical staining can help distinguish reactive, immature metaplasia or atrophic squamous epithelium from high-grade cervical squamous intraepithelial lesions (HSIL), which can improve the accuracy of HSIL diagnosis and the inter-observer relationship. the repeatability. However, p16 ^INK4a also has a certain positive rate in normal cervix, inflammatory tissues, etc., which makes its diagnostic specificity not high, and easily leads to misdiagnosis and overtreatment of patients. Therefore, looking for diagnostic markers of cervical lesions with high sensitivity and specificity can early detect HSIL that may progress to cervical invasive cancer, and treat it in time to reduce the possibility of cervical invasive cancer.

SUMMARY OF THE INVENTION

For solving the deficiencies in the prior art, the purpose of this application is to provide a molecular marker for the histological diagnosis of female cervical lesions, namely by detecting the expression level of the RFC4 gene in the cervical lesions, thereby evaluating the degree of cervical lesions, for Histological diagnosis of cervical lesions provides new means.

This application solves its technical problems by adopting the following technical solutions.

In this application, by searching the GEO database of the National Center for Biotechnology Information of the United States, 4 expression data sets were screened, among which GSE63514, GSE27678 and GSE7803 were used as discovery sets. We employed a multi-step bioinformatics approach to find 32, 19, and 17 key genes in the discovery set, respectively. The genes shared by at least two datasets were further screened as the final key genes, and 4 genes were obtained, namely RFC4, CEP55, AURKA and TOP2A. Normal, CIN1-3 and cervical squamous cell carcinoma tissues were collected, and immunohistochemical techniques were used to verify that the expression of key genes and proteins was positively correlated with the degree of cervical lesions. At the same time, compared with p16 ^INK4a and Ki-67 currently used in clinical practice, it was found that RFC4 has a good effect on the diagnosis of cervical high-grade squamous intraepithelial lesion (HSIL)/cervical high-grade squamous intraepithelial lesion and cervical squamous cell carcinoma (HSIL+).

The present application provides the application of the RFC4 gene and the reagent for detecting its expression in the preparation of a product for assisting the diagnosis of high-grade cervical lesions.

Further, above-mentioned reagent comprises the reagent that detects RFC4 gene mRNA expression amount or protein expression amount;

Optionally, the above-mentioned reagents include reagents required to detect the expression level of RFC4 by common detection methods such as immunoblotting, real-time quantitative PCR, in situ hybridization, gene or protein chip, high-throughput sequencing, and immunohistochemistry;

Preferably, the above-mentioned reagents are required for detecting the expression level of the RFC4 gene protein by immunohistochemistry, including antibodies that specifically bind to the RFC4 protein, and the like.

The present application also provides judgment criteria for assisting the diagnosis of high-grade cervical lesions by RFC4 immunohistochemistry.

The scoring criteria include: (1) In normal cervical tissue and intraepithelial neoplasia, the score is mainly based on the staining pattern. 0 points, no staining, only cytoplasmic or only basal and parabasal staining; 1 point, 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, 2 points and 3 points were judged as positive. (2) In invasive squamous cell carcinoma, if more than 10% of the cancer cells are positive, it is judged to be positive.

Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

1. This application confirms the correlation between the expression of RFC4 and the degree of cervical lesions at the mRNA and protein levels, and provides a new research direction for the diagnosis/screening of cervical lesions and the development of targeted therapy drugs for cervical cancer.

2. RFC4 immunohistochemical staining has good sensitivity and specificity in diagnosing HSIL and HSIL+. Compared with the traditional indicator p16 ^INK4a , its specificity was significantly increased, and there was no significant difference in sensitivity. Its diagnostic accuracy was better than that of p16 ^INK4a or Ki-67 alone, and was close to that of p16 ^INK4a + Ki-67 as a combined marker.

Description of drawings

In order to illustrate the technical solutions of the embodiments of the present application more clearly, the following drawings will briefly introduce the drawings that need to be used in the embodiments. It should be understood that the following drawings only show some embodiments of the present application, and therefore do not It should be regarded as a limitation of the scope, and for those of ordinary skill in the art, other related drawings can also be obtained according to these drawings without any creative effort.

1 is an analysis of the mRNA expression of key genes in the expression profile chip data and the correlation with disease progression in the embodiment of the application;

Among them, Figure 1A is the result of GSE63514, Figure 1B is the result of GSE27678, Figure 1C is the result of GSE7803, and Figure 1D is the result of GSE138080;

2 is a schematic diagram of the immunohistochemical scoring standard in the embodiment of the application (corresponding to Table 3);

Wherein, Figure 2A is a schematic diagram of p16 ^INK4a , Figure 2B is a schematic diagram of Ki-67, and Figure 2C is a schematic diagram of RFC4;

3 is a comparison of the positive rates of p16 ^INK4a , Ki-67, and RFC4 in different grades of cervical lesions in the embodiment of the application;

Among them, Figure 3A is the result of p16 ^INK4a , Figure 3B is the result of Ki-67, and Figure 3C is the result of RFC4;

Fig. 4 is the ROC curve of single and combined immunohistochemical markers for the diagnosis of HSIL and HSIL+ in the embodiment of the application;

Among them, FIG. 4A is the ROC curve of HSIL diagnosis, and FIG. 4B is the ROC curve of HSIL+ diagnosis.

Detailed ways

In order to make the purposes, technical solutions and advantages of the embodiments of the present application more clear, the technical solutions in the embodiments of the present application will be described clearly and completely below, and the specific embodiments do not represent limitations on the protection scope of the present invention. If the specific conditions are not indicated in the examples, it is carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used without the manufacturer's indication are conventional products that can be purchased from the market.

It should be noted that the embodiments of the present application and the features of the embodiments may be combined with each other without conflict, and the present application will be described in detail below with reference to specific embodiments.

Application of RFC4 gene as a diagnostic marker for cervical lesions.

In some embodiments of the present application, the above-mentioned cervical lesions refer to high-grade cervical lesions, including cervical high-grade squamous intraepithelial lesions and cervical squamous cell carcinoma.

The general idea of the technical solution of the present application is as follows:

In this application, by searching the GEO database of the National Center for Biotechnology Information of the United States, 4 expression data sets were screened, among which GSE63514, GSE27678 and GSE7803 were used as discovery sets. We employed a multi-step bioinformatics approach to find 32, 19, and 17 key genes in the discovery set, respectively. The genes shared by at least two datasets were further screened as the final key genes, and 4 genes were obtained, namely RFC4, CEP55, AURKA and TOP2A. Normal, CIN1-3 and cervical squamous cell carcinoma tissues were collected, and immunohistochemical techniques were used to verify that the expression of key genes and proteins was positively correlated with the degree of cervical lesions. At the same time, compared with p16 ^INK4a and Ki-67 currently used in clinical practice, it was found that RFC4 has a good effect on the diagnosis of cervical high-grade squamous intraepithelial lesion (HSIL)/cervical high-grade squamous intraepithelial lesion and cervical squamous cell carcinoma (HSIL+).

The features and properties of the present application will be described in further detail below with reference to the embodiments.

Example 1

Screening of diagnostic genes for cervical lesions

1. Data download

In the Gene Comprehensive Expression (GEO) database, "Cervical Intraepithelial Neoplasia" and ("cervical"OR"cervix")AND("neoplasm"OR"cancer"OR"tumor") were used as search methods, and the limited species was " Homo sapiens". The inclusion criteria of the chip are: (1) mRNA expression profile data; (2) the dataset contains at least normal (Normal), low-grade squamous intraepithelial lesion (LSIL), high-grade squamous intraepithelial lesion (HISL), cervical squamous Cancer (SCC) three of the four samples; (3) The total number of samples in the dataset is ≥ 25.

According to the above criteria, a total of 4 chips were screened, namely: GSE63514, GSE27678, GSE7803 and GSE138080. Among them, GSE63514, GSE27678 and GSE7803 are used as the discovery set, and GSE138080 is used as the verification set. The chip information table is shown in Table 1.

Table 1 Chip Information Table

¹ The GSE63514 dataset contains five samples of normal control, CIN1, CIN2, CIN3 and early invasive squamous cell carcinoma. In this study, CIN1 was classified as LSIL, and CIN2 and CIN3 were classified as HSIL.

² The GSE27678 dataset contains data from two chip platforms, and only the data from the HG-U133A2.0 platform was selected in this study.

2. Key Gene Screening

Defining differential genes: Differential gene analysis (LSIL vs.Normal, HSIL vs.Normal, SCC vs.Normal) was performed on each group using the R package limma. Inclusion criteria for differential genes: |Log ₂ FC|≥1 (ie, the difference fold of gene expression is more than 2 times or less than 0.5 times); p-value<0.05 after correction. Defining progressive genes: genes that are consistently up- or down-regulated (Normal<LSIL<HSIL<SCC or Normal>LSIL>HSIL>SCC). Define Gene Set 2: In each dataset, the intersection of differential genes and progressive genes. Finally, 725, 123, and 121 genes were obtained in GSE63514, GSE27678, and GSE7803, respectively. The specific results are shown in Table 2.

Table 2 Gene screening table

The protein interaction network of gene set 2 was constructed using STRING and Cytoscape, and key genes were screened by Cytohubba plugin. We select 9 topological analysis methods (Betweenness, Bottleneck, Closeness, Degree, EPC, DMNC, MNC, Radiality, and Stress) to rank nodes in the network, select the top 10 genes of each method, and after removing duplicate genes, The key gene sets that make up each dataset. A total of 32 key genes were obtained in GSE63514, 19 key genes were obtained in GSE27678, and 17 key genes were obtained in GSE7803. The key genes of the three datasets were intersected, and the genes shared by at least two datasets were screened as the final key genes in this study. Finally, four genes were obtained, namely RFC4, CEP55, AURKA and TOP2A. As shown in Figure 1, the mRNA expression of key genes in the discovery set (GSE63514, GSE27678, GSE7803) and validation set GSE138080 gradually increased with the progression of cervical lesions, suggesting that they can be used as potential diagnostic molecular markers for cervical lesions.

Example 2

Evaluation of the diagnostic efficacy of RFC4

For the four key genes found in Example 1, we performed immunohistochemical analysis on them. Considering the novelty of key genes in cervical lesions, staining pattern, diagnostic efficacy and other factors, we found that RFC4 had the best effect in diagnosing HSIL/HSIL+. The following will explain the diagnostic efficacy of RFC4 immunohistochemistry for high-grade cervical lesions and its comparison with p16 ^INK4a and Ki-67 currently used in clinical practice.

The cervical tissue used in this example comes from tissue chips (CIN1021, CIN1022) of Guilin Panspectrum Biotechnology Co., Ltd. and samples from the Obstetrics and Gynecology Department of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, including 42 cases of normal cervical tissue, 59 Cases of CIN1 tissues, 26 cases of CIN2 tissues, 42 cases of CIN3 tissues, and 61 cases of cervical squamous cell carcinoma tissues. All sections were diagnosed by three senior pathologists.

The main reagents and configuration methods used in this example are as follows:

(1) PBS buffer (pH 7.2-7.4): accurately weigh 18g NaCl, 0.832g NaH ₂ PO ₄ ·2H ₂ O, 5.496g NaH ₂ PO ₄ · 12H ₂ O on an analytical balance and add distilled water to the volume 2000mL.

(2) EDTA antigen retrieval solution: purchased from Wuhan Sevier Biological Company, 20×Tris-EDTA retrieval solution (pH 9.0), product number G1203. Mix 10 mL of 20×Tris-EDTA antigen retrieval solution (pH 9.0) with 190 mL of distilled water to obtain 1× pH 9.0 antigen retrieval solution containing 1 mM EDTA.

(3) 3% hydrogen peroxide: obtained by diluting 30% hydrogen peroxide with pure water. Add 30 mL of 30% hydrogen peroxide to 270 mL of pure water to obtain 300 mL of 3% hydrogen peroxide.

(4) 3% bovine serum albumin (BSA): purchased from Wuhan Sevier Biological Company, item number G5001-100G. When used, PBS buffer was used to prepare 3% BSA. 3% BSA can be obtained by adding 3g BSA to 100mL PBS buffer.

(5) Primary antibody: p16 ^INK4a (Company Abcam, Item No. ab108349, dilution ratio 1:100); Ki-67 (Company Immunoway, Item No. YM6189, dilution ratio 1:200); RFC4 (Company Abcam, Item No. ab156780, diluted ratio of 1:500), diluted with prepared PBS buffer.

(6) Secondary antibody: purchased from Wuhan Sewell Biological Company, goat anti-rabbit secondary antibody, product number G1213-100μL containing DAB chromogenic solution.

(7) Tween-20: purchased from Wuhan Sevier Biological Company, product number G0004, used for the preparation of PBST.

(8) PBST buffer (pH 7.2-7.4): add 500 μL of Tween-20 to 1000 mL of PBS buffer, mix well and use for later use.

(9) Secondary antibody dilution solution: Dilute the secondary antibody working stock solution with PBST buffer at pH 7.2-7.4 at a ratio of 1:200, and mix thoroughly for use.

(10) DAB chromogenic solution: add 1 mL of DAB dilution solution to every 20 μL of DAB stock solution.

(11) Neutral gum: purchased from Wuhan Sevier Biological Co., Ltd., item number WG10004160, used for coverslipping.

The specific steps of immunohistochemistry in this example are as follows:

(1) Dewaxing the paraffin sections to water: After baking the sections in a constant temperature oven at 37°C for 30 minutes, put the sections into the environmental protection dewaxing solution I for 15 minutes, the environmental protection dewaxing solution II for 15 minutes, anhydrous ethanol for 15 minutes, and anhydrous Ethanol II for 5 minutes, 95% alcohol for 5 minutes, 85% alcohol for 5 minutes, 75% alcohol for 5 minutes, and distilled water for 5 minutes. The slides were then placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time.

(2) Antigen retrieval: Place the tissue sections in a retrieval box filled with EDTA antigen retrieval buffer (pH 9.0) for antigen retrieval in a microwave oven, medium heat for 10 minutes to boiling, cease fire for 5 minutes, and then switch to medium-low heat for 10 minutes. During the process, the buffer should be prevented from over-evaporating, and the slices should not be dried. After natural cooling, the slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time.

(3) Block endogenous peroxidase: put the slices into 3% hydrogen peroxide solution, incubate them in the dark at room temperature for 25 minutes, place the glass slides in PBS (pH 7.4) and shake and wash 3 times on a decolorizing shaker for 5 minutes each time .

(4) Serum blocking: 3% BSA was added dropwise in the histochemical circle to cover the tissue evenly and completely, and the tissue was blocked for 30 minutes at room temperature.

(5) Add primary antibody: gently shake off the blocking solution, drop the primary antibody prepared with PBS on the slice, and incubate the slice at 4°C overnight in a wet box.

(7) Adding secondary antibody: The slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After the sections were slightly dried, the HRP-labeled goat anti-rabbit-mouse universal secondary antibody was added dropwise in the circle to cover the tissue, and incubated in a 37°C incubator for 30 min.

(8) DAB color development: The slides were placed in PBS (pH 7.4) and washed three times with shaking on a destaining shaker, 5 min each time. After the slices were slightly dried, the freshly prepared DAB chromogenic solution was added dropwise in the circle to completely cover the tissue, and the chromogenic time was controlled under the microscope.

(9) Counterstaining nuclei: counterstaining with hematoxylin for about 3 minutes, washing with tap water, differentiation with hematoxylin differentiation solution for several seconds, rinsing with tap water, returning to blue with hematoxylin solution, and rinsing with running water.

(10) Dehydration and sealing: put the sections into 75% alcohol for 5 minutes, 85% alcohol for 5 minutes, 95% alcohol for 5 minutes, absolute ethanol II for 5 minutes, absolute ethanol I for 5 minutes, environmental protection dewaxing solution II for 5 minutes, and environmental protection dewaxing solution for 5 minutes. Dehydrated and transparent in 1 5min, the slices were taken out and air-dried, and the slices were sealed with neutral gum.

Light microscopy, image acquisition analysis, and scoring of immunohistochemical results for p16 ^INK4a , Ki-67, and RFC4. The scoring criteria for normal cervical tissue and intraepithelial neoplasia are shown in Table 3. The scoring criteria for invasive squamous cell carcinoma are: more than 10% of cancer cells in the tissue are positive. Figure 2 is a schematic diagram of the overall scoring criteria.

Table 3 Immunohistochemical scoring scheme

Result statistics and analysis:

Using immunohistochemical technology, the stained samples were subjected to light microscopy and image acquisition analysis, and the immunohistochemical results of p16 ^INK4a , Ki-67 and RFC4 were scored, and the p16 ^INK4a , Ki-67 and RFC4 genes were detected in Expression of normal, CIN1, CIN2, CIN3 and cervical squamous cell carcinoma. The results of immunohistochemical scoring of normal cervix and intraepithelial neoplasia tissues are shown in Table 4; the histochemical schematic diagrams and positive rates of p16 ^INK4a , Ki-67 and RFC4 in all grades of lesions are shown in Figure 3. Among them, the abscissa of Figure 3 represents the lesion samples of each grade; the ordinate represents the positive rate of each index.

Table 4 Immunohistochemical scoring results

The experimental results showed that RFC4 was similar to p16 ^INK4a and Ki-67. With the progress of cervical lesions, the protein expression of RFC4 gradually increased. The positive rates of RFC4 in normal and CIN1 tissues were 4.8% and 13.6%, respectively; the positive rates of p16 ^INK4a in normal and CIN1 tissues were significantly higher than RFC4, 11.9% and 54.2%, respectively; The positive rate was equal to RFC4, and the positive rate in CIN1 was 30.5%, which was higher than RFC4. The positive rates of the three histochemical indexes were higher in CIN2+ lesions. At the same time, the comparison found that the positive rate of the three indicators in HSIL (CIN2-3) and HSIL+ (CIN2-3+SCC) was significantly higher than that in the control group (Normal+CIN1), and the difference was statistically significant, proving that RFC4 was used as the diagnosis of cervical lesions marker potential.

Further, comparing the efficacy of single and combined immunohistochemical markers for the diagnosis of HSIL and HSIL+, the results are shown in Table 5 and Figure 4. We calculated the sensitivity (Sen), specificity (Spe), positive predictive value (PPV), negative predictive value (NPV) and area under the ROC curve (AUC) to evaluate the diagnostic performance of the markers. Among them, 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 a certain accuracy when it is 0.7-0.9, and when AUC is above 0.9, it has a higher accuracy.

Table 5 Comparison of the diagnostic efficacy of single and combined immunohistochemical markers for HISL/HSIL+

Sen: Sensitivity; Spe: Specificity; 95% CI: 95% Confidence Interval; PPV: Positive Predictive Value; NPV: Negative Predictive Value; AUC: Area under the ROC curve enclosed by the coordinate axis; Ref: Control group.

As can be seen from Table 5 and Figure 4A, in the diagnosis of HSIL, although the sensitivity of RFC4 was slightly lower than that of p16 ^INK4a , there was no statistical difference (88.2% vs. 92.6%, p>0.05); while the specificity of RFC4 was was significantly higher than p16 ^INK4a , the difference was statistically significant (90.1% vs. 63.4%, p<0.05). The diagnostic accuracy of RFC4 and the combination of p16 ^INK4a + Ki-67 was similar, their AUC values were 0.89 and 0.88, respectively; while the AUC values of p16 ^INK4a and Ki-67 were relatively low, 0.78 and 0.86, respectively.

As can be seen from Table 5 and Figure 4B, in the diagnosis of HSIL+, although the sensitivity of RFC4 is lower than that of p16 ^INK4a , there is no statistical difference (91.5% vs. 95.3%, p>0.05); while the specificity of RFC4 It was significantly higher than p16 ^INK4a , and the difference was statistically significant (90.1% vs. 63.4%, p<0.05). Similarly, RFC4 has obvious advantages over p16 ^INK4a and Ki-67 alone, and the diagnostic accuracy of the combination of p16 ^INK4a + Ki-67 is similar, their AUC values are 0.91 and 0.89, respectively; the AUC values of p16 ^INK4a and Ki-67 were 0.79 and 0.87, respectively.

To sum up, compared with the existing clinically used cervical lesions diagnostic markers p16 ^INK4a and Ki-67, the present application creatively provides RFC4 as a molecular marker for the histological diagnosis of cervical lesions. The expression level of RFC4 increased with the progression of cervical lesions. The diagnostic performance of RFC4 immunohistochemistry for HSIL and HSIL+ was better than that of single p16 ^INK4a or Ki-67. The use of this marker can make up for the poor specificity of p16 ^INK4a to a certain extent, and has important clinical application value.

The above-described embodiments are some, but not all, embodiments of the present application. The detailed descriptions of the embodiments of the application are not intended to limit the scope of the application as claimed, but are merely representative of selected embodiments of the application. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative work fall within the protection scope of the present application.

Claims (2)

1. The application of RFC4 gene as a diagnostic marker for cervical lesions. 2. the application of RFC4 gene according to claim 1 as cervical lesion diagnostic marker, is characterized in that, described cervical lesion refers to cervical high-grade lesion, including cervical high-grade squamous intraepithelial lesion and cervical squamous cell carcinoma.

Images