Applications of CRABP2 protein

Abstract

The present invention relates to the use of a reagent or reagent set for detecting CRABP2 protein in the preparation of a reagent or kit for diagnosing the micropapillary subtype of lung adenocarcinoma. The semi-dry spot detection kit of the present invention has a sensitivity of 61.8% and a specificity of 79.4% in diagnosing the micropapillary subtype of lung adenocarcinoma. Furthermore, the semi-dry spot detection kit does not increase the intraoperative freezing time (total duration is 25 minutes and 30 seconds) or the patient's financial burden.

Description

Technical Field

[0001] The present invention relates to the field of diagnosis of micropapillary subtypes of lung adenocarcinoma, and in particular to the use of a reagent or reagent group for detecting CRABP2 protein in preparing a reagent or kit for diagnosing micropapillary subtypes of lung adenocarcinoma. Background Art

[0002] In surgical practice over the past five years, the prognosis after sublobar resection for early-stage invasive lung cancer varies widely. Among the five pathological subtypes of lung adenocarcinoma (lepidic, acinar, papillary, solid, and micropapillary), micropapillary and solid subtypes have a significantly poorer prognosis and are not suitable for sublobar resection. The more detailed and accurate pathological information provided by updated frozen pathology provides stronger evidence-based medical support for the selection of individualized minimally invasive surgical procedures for pulmonary nodules.

[0003] Currently, frozen pathology can better identify solid subtypes (sensitivity 72.6%, specificity 95.8%) and visceral pleural invasion (accuracy 95.5%, specificity 100%), but there are still differences in the sensitivity and accuracy of intraoperative frozen pathology in distinguishing micropapillary subtypes and trans-air cavity dissemination.

[0004] Studies on frozen pathology diagnosis of lung adenocarcinoma are almost exclusively single-center, retrospective studies with small sample sizes. These studies cannot serve as high-level clinical evidence for frozen pathology guidance in surgical selection for early-stage lung cancer. Based on multiple current retrospective studies, the accuracy and diagnostic consistency of frozen pathology in identifying lung adenocarcinoma subtypes (particularly micropapillary) have yet to reach satisfactory levels. In 2015, Humberto et al. conducted a study on the sensitivity and specificity of detecting various subtypes of stage I lung adenocarcinoma in 112 frozen samples. The specificity of micropapillary morphology in diagnosis was as high as 98.1%, while the sensitivity was only 13.3%, indicating a high rate of missed diagnosis. In June of the same year, Travis et al. conducted similar work, finding a sensitivity of 37% and a specificity of 94% for detecting micropapillary subtypes in 361 frozen samples of stage I lung adenocarcinoma. These results were similar, with low sensitivity and high specificity. Therefore, there is an urgent need to improve the sensitivity of identifying micropapillary subtypes in lung adenocarcinoma. Summary of the Invention

[0005] The purpose of the present invention is to address the deficiencies in the prior art and provide a reagent or reagent group for detecting CRABP2 protein for use in preparing a reagent or kit for diagnosing micropapillary subtypes of lung adenocarcinoma.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Provided is the use of a reagent or reagent group for detecting CRABP2 protein in preparing a reagent or kit for diagnosing micropapillary subtype of lung adenocarcinoma.

[0008] Preferably, the reagent for detecting CRABP2 protein comprises: a first antibody for specifically binding to the CRABP2 protein.

[0009] Preferably, the reagent set for detecting CRABP2 protein comprises: a first antibody for specifically binding to the CRABP2 protein and a second antibody for specifically binding to the first antibody.

[0010] Preferably, the second antibody is EnVision reagent, HRP / R.

[0011] Preferably, the reagent set for detecting CRABP2 protein further comprises: a color developing agent for detecting the second antibody.

[0012] Preferably, the color developer is a Vector VIP substrate Kit color developer.

[0013] Preferably, the kit for diagnosing the micropapillary subtype of lung adenocarcinoma is a semi-dry spot detection kit.

[0014] The present invention adopts the above technical solution, which has the following technical effects compared with the prior art:

[0015] The semi-dry spot detection kit of the present invention has a sensitivity of 61.8% and a specificity of 79.4% in diagnosing the micropapillary subtype of lung adenocarcinoma; at the same time, it neither increases the freezing time during surgery nor increases the financial burden on patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is a bar graph of the protein quantitative difference results in Example 1 of the present invention;

[0017] Figure 2 This is a volcano plot of differential expression between the experimental group and the control group in Example 1 of the present invention. The horizontal axis 0 is used as the boundary in the figure. The dark labels on the left are actually blue labels, the dark labels on the right are actually red labels, and the remaining light labels are gray labels;

[0018] Figure 3 This is another bar graph of protein quantitative difference results in Example 1 of the present invention;

[0019] Figure 4 The expression of the four differentially expressed proteins in lung adenocarcinoma using immunohistochemistry in Example 1 of the present invention is shown in FIG. Figure 4 A is MIP50% lung adenocarcinoma, HE high magnification; Figure 4 B shows strong positive expression of CRABP2, medium magnification, EnVision method; Figure 4 C is MIP60% lung adenocarcinoma, HE high magnification; Figure 4D is negative expression of CILP, medium magnification, EnVision method; Figure 4 E is MIP50% lung adenocarcinoma, HE high magnification; Figure 4 F, negative expression of PCB, medium magnification, EnVision method; Figure 4 G is MIP70% lung adenocarcinoma, HE high magnification; Figure 4 H, negative expression of B4GALT5, medium magnification, EnVision method;

[0020] Figure 5 The relationship between CRABP2 expression and MIP in Example 1 of the present invention;

[0021] Figure 6 This is the relationship between CRABP2 expression and prognosis in Example 1 of the present invention. DETAILED DESCRIPTION

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] It should be noted that, in the absence of conflict, the embodiments of the present invention and the features in the embodiments may be combined with each other.

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but they are not intended to limit the present invention.

[0025] Example 1

[0026] Fresh lung cancer tissues were stored in liquid nitrogen at -80°C. Three groups of micropapillary-predominant lung adenocarcinomas were selected as experimental groups, and three groups of adherent-predominant lung adenocarcinomas were selected as control groups.

[0027] The analysis process of this embodiment mainly includes two stages: mass spectrometry experiment and data analysis. The mass spectrometry analysis process includes steps such as protein extraction, peptide digestion, chromatographic fractionation, liquid chromatography-tandem mass spectrometry (LC-MS / MS) data acquisition, and database retrieval. Samples were lysed using the SDT (4% (w / v) SDS, 100 mM Tris / HCl pH 7.6, 0.1 M DTT) method to extract proteins, and then the BCA method was used for protein quantification. An appropriate amount of protein from each sample was digested with trypsin using the Filter aided proteome preparation (FASP) method, and the peptides were desalted using a C18 cartridge. After lyophilization, the peptides were reconstituted in 40 μL of 0.1% formic acid solution, and the peptides were quantified (OD280). Each sample was separated using the Easy nLC HPLC system at a nanoliter flow rate.

[0028] The statistical results of the number of spectra, peptides and proteins obtained in this example are as follows: Figure 1 As shown in the figure, the expression fold change (FC)>2.0 times (up-regulated greater than 2.0 times or down-regulated less than 0.5 times) and P<0.05 were defined as significantly differentially expressed proteins; to compare the significant differences of proteins between groups, the proteins in the comparison groups were plotted using the expression fold difference (Foldchange) and P value (T-test) as the two factors as the standard to draw a volcano plot, in which the significantly down-regulated proteins were marked in blue (FC<0.5 and p<0.05), the significantly up-regulated proteins were marked in red (FC>2.0 and p<0.05), and the proteins with no difference were gray, as shown in the figure. Figure 2 As shown;

[0029] like Figure 3 As shown in the figure, the screening of significant differential proteins found that the experimental group upregulated 170 proteins and downregulated 106 proteins, with 4 proteins upregulated and 4 proteins downregulated most significantly. Among them, the 4 significantly upregulated proteins were cellular retinoic acid binding protein 2 (CRABP2), cartilage intermediate layer protein 1 (CILP), pyruvate carboxylase (PC) and β-1,4-galactosyltransferase 5 (BGALT5).

[0030] Example 2

[0031] 147 cases of lung adenocarcinoma were selected, and their basic information is shown in Table 1:

[0032] Table 1

[0033]

[0034]

[0035] The expression of the four most significantly upregulated proteins in micropapillaries using immunohistochemistry is shown in Table 2:

[0036] Table 2

[0037]

[0038] Note: MIP: micropapillary type.

[0039] Among them, CRABP2 was highly expressed in micropapillaries (p<0.0001), while the expression of CILP, PCB, and B4GALT5 in micropapillaries was not statistically significant; the typical figures of the four most significantly upregulated differentially expressed proteins are as follows: Figure 4 As shown; CRABP2 protein is significantly expressed in micropapillary lung adenocarcinoma;

[0040] The expression of CRABP2 protein in lung adenocarcinoma is shown in Table 1. High CRABP2 protein expression was significantly associated with male gender, larger tumor size, pathological T stage, STAS, major histological subtype, and recurrence status (p < 0.05). CRABP2 expression was 100% in micropapillary-predominant adenocarcinomas. The H-score cutoff value of 110 categorized CRABP2 expression as high or low. As micropapillary content increased, the CRABP2 H-score also increased, indicating that CRABP2 expression was highly correlated with micropapillary content. Figure 5 As shown in Figure 2, high expression of CRABP2 is significantly associated with poor prognosis of patients, and patients with high expression of CRABP2 have a worse prognosis, as shown in Figure 2. Figure 6 And as shown in Table 3:

[0041] Table 3

[0042]

[0043] Example 3

[0044] This embodiment provides a method for diagnosing the micropapillary subtype of lung adenocarcinoma using a semi-dry spot detection kit for CRABP2 protein, comprising the following steps:

[0045] S1. Receive specimen (1 min), perform gross description (1 min), cut lung tissue into book-like sections, dissect the tumor at 1 cm intervals, and obtain frozen fresh tissue along the largest surface of the tumor, 2 cm × 1 cm × 1 cm (2 min), add frozen embedding medium (OCT), and place on a cryostat (30 s).

[0046] S2, 10 μm serial sections were cut three times (1 min), the sections were rinsed twice with PBS (30 s), the sections were scraped into a pre-cooled 1.5 mL centrifuge tube (30 s), and lysed with RIPA lysis buffer for 5 min;

[0047] S3. Cut the NC membrane into 10 cm × 5 cm size (1 min), make two wells with a diameter of 1 cm (1 min), add 20 μL of the product obtained in step S2 to each well, blow dry with cold air (30 s), and then soak in PBS (containing 0.05% Tween 20) (1 min);

[0048] S4. Add the primary antibody (antibody to CRABP2, clone 14256(B), Abcam) to both wells and incubate for 3 minutes, followed by washing with PBS (containing 0.05% Tween 20) for 3 minutes. Add the secondary antibody (EnVision reagent (HRP / R)) to one well and incubate for 2 minutes, followed by washing with PBS (containing 0.05% Tween 20) for 2 minutes. Then, add the Vector VIP substrate Kit colorimetric reagent to both wells (30 seconds).

[0049] S5. If the wells where the second antibody is added appear purple, micropapillary components are present in the surface specimen. The greater the difference in purple between the two wells, the higher the content of micropapillary components.

[0050] Example 4

[0051] In 118 prospective lung adenocarcinoma cases, the method described in Example 3 was used to diagnose the micropapillary subtype of lung adenocarcinoma, with a sensitivity of 61.8% and a specificity of 79.4%. The improved frozen pathology diagnostic method of the present inventors was used to diagnose the micropapillary subtype of lung adenocarcinoma, with a sensitivity of 58.2% and a specificity of 87.3%. When the method described in Example 3 was combined with the improved frozen pathology diagnostic method of the present inventors for diagnosing the micropapillary subtype of lung adenocarcinoma, the sensitivity was 70.9% and the specificity was 90.5%, as shown in Table 4:

[0052] Table 4

[0053]

[0054] In summary, the semi-dry spot detection kit of the present invention has a sensitivity of 61.8% and a specificity of 79.4% in diagnosing the micropapillary subtype of lung adenocarcinoma; at the same time, it neither increases the intraoperative freezing time (total time is 25 minutes and 30 seconds) nor increases the financial burden on patients.

[0055] The above description is only a preferred embodiment of the present invention and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the description and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Use of a reagent or reagent set for detecting CRABP2 protein in the preparation of a reagent or kit for diagnosing the micropapillary subtype of lung adenocarcinoma.

2. The use according to claim 1, characterized in that The reagent for detecting CRABP2 protein includes: a first antibody for specifically binding to the CRABP2 protein.

3. The use according to claim 1, characterized in that The reagent set for detecting CRABP2 protein includes: a first antibody for specifically binding to the CRABP2 protein and a second antibody for specifically binding to the first antibody.

4. The use according to claim 3, characterized in that The secondary antibody was EnVision reagent.

5. The use according to claim 3, characterized in that The reagent set for detecting CRABP2 protein further includes: a color developing agent for detecting the second antibody.

6. The use according to claim 5, characterized in that The color developer is Vector VIP substrate Kit color developer.

7. The use according to claim 1, characterized in that The kit for diagnosing the micropapillary subtype of lung adenocarcinoma is a semi-dry spot detection kit.

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