Use of inpp5f protein in the preparation of a breast cancer diagnosis and / or prognosis product

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of INPP5F protein in preparation of breast cancer diagnosis and / or prognosis product. The amino acid sequence of the INPP5F protein is shown as SEQ ID NO. 1. According to the experiment of the application, the expression of the INPP5F protein in breast cancer tissue is significantly higher than that in adjacent cancer-adjacent tissue, and the INPP5F protein can be used as a biomarker for breast cancer diagnosis. Moreover, the expression of the INPP5F protein is closely related to TNM stage and survival period of breast cancer patients. The method is simple and easy to implement, the diagnosis process is safe and effective, is easy to be accepted by patients, the diagnosis standard is unified, and is less affected by personal subjective factors, which shows that the INPP5F protein has important significance for prognosis judgment of breast cancer.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of INPP5F protein in preparation of breast cancer diagnosis and / or prognosis product. BACKGROUND

[0002] Breast cancer is one of the malignant tumors seriously affecting women's health. Global cancer statistics show that the incidence and mortality of female breast cancer are in the forefront in the world, and it is one of the main causes of female cancer death. It is worth noting that the cancer statistics released by the International Cancer Research Center (IARC) and the National Cancer Center of China show that breast cancer is the most common cancer in Chinese women, and its incidence and mortality are in the forefront of female malignant tumors, seriously threatening women's health. At present, the diagnosis of breast cancer mainly depends on clinical breast examination, mammography, ultrasonic examination and magnetic resonance imaging. Exploring new breast cancer diagnosis and prognosis markers will help early detection and diagnosis of breast cancer, promote early treatment and increase the chance of successful treatment, thereby reducing the mortality rate. SUMMARY

[0003] The purpose of the present application is to solve the problems of the prior art, and to provide application of INPP5F protein in preparation of breast cancer diagnosis and / or prognosis product. The technical solutions adopted by the present application are as follows:

[0004] In a first aspect, the present application provides application of INPP5F protein in preparation of breast cancer diagnosis and / or prognosis product, wherein the amino acid sequence of the INPP5F protein is shown as SEQ ID NO. 1.

[0005] SEQ ID NO. 1:

[0006]

[0007] INPP5F protein is also known as inositol polyphosphate-5-phosphatase F, and its gene sequence is located on the long arm of chromosome 10. The protein encoded by INPP5F gene is a kind of inositol 1, 4, 5-trisphosphate (InsP3)-5-phosphatase, with a full length of 1132 amino acids. The protein contains a SAC phosphatase domain composed of 352 amino acids, which is the main region of enzyme activity. The main function of INPP5F protein is to degrade phosphatidylinositol 4, 5-bisphosphate (PIP2) and phosphatidylinositol 3, 4, 5-trisphosphate (PIP3) by removing the 5' phosphate group on the inositol ring, and then to regulate cell function.

[0008] As a further preferred embodiment, the product comprises a chip, a kit or a test paper.

[0009] As a further preferred embodiment, the chip comprises a gene chip and a protein chip.

[0010] As a further preferred embodiment, the gene chip comprises a nucleotide probe for detecting the transcription level of INPP5F gene; and the protein chip comprises a specific binding reagent of INPP5F protein.

[0011] The sample for diagnosis and / or prognosis in the present application is breast cancer tissue and / or paracancerous tissue from a breast cancer patient, a breast cancer suspected patient, a breast cancer susceptible population, a breast cancer high-risk population or a healthy population.

[0012] In a second aspect, the present application provides a kit for breast cancer diagnosis and / or prognosis, comprising a detection reagent for detecting the marker of the above-mentioned INPP5F protein; and the marker is any one of radioisotope, nucleotide chromophore, enzyme, fluorescent molecule, chemiluminescent moiety, magnetic particle and bioluminescent moiety.

[0013] As a further preferred embodiment, the radioisotope comprises 32 P, 35 S or 125 I;

[0014] The nucleotide chromophore includes pyrene, chrysene or naphthalene amide; the enzyme includes horseradish peroxidase or alkaline phosphatase; the fluorescent molecule includes fluorescein isothiocyanate, rhodamine, Cy3 or Cy5; the chemiluminescent moiety includes acridinium ester, luminol or luminol derivatives; the bioluminescent moiety includes biotin or digoxin.

[0015] As a further preferred embodiment, the detection reagent is used in a method for detecting a marker of INPP5F protein, which includes a radio method, an immune method, a fluorescence method, a flow fluorescence method, a latex turbidimetry method, a biochemical method, an enzyme method, a hybridization method, a gas chromatography-mass spectrometry method, a liquid chromatography-mass spectrometry method, a chromatography method, a chemiluminescence method, a magnetoelectric method or a photoelectric conversion method.

[0016] In a third aspect, the present application provides an application of INPP5F protein as a target in screening preparation of breast cancer drugs, wherein the amino acid sequence of the INPP5F protein is shown as SEQ ID NO. 1.

[0017] In a fourth aspect, the present application provides an application of INPP5F protein in constructing a prognosis prediction model of breast cancer, wherein the amino acid sequence of the INPP5F protein is shown as SEQ ID NO. 1.

[0018] In a fifth aspect, the present application provides an application of INPP5F protein in preparing a breast cancer tumor inhibitor, wherein the amino acid sequence of the INPP5F protein is shown as SEQ ID NO. 1.

[0019] The present application has the following beneficial effects:

[0020] (1) The present application provides that the INPP5F protein can be used for breast cancer diagnosis or prognosis. According to the experiments of the present application, the expression of INPP5F protein in breast cancer tissue is significantly higher than that in adjacent cancer tissues, which can be used as a biomarker for breast cancer diagnosis, and it is shown that the expression of INPP5F protein is closely related to the TNM stage and survival period of breast cancer patients.

[0021] (2) In the experimental process of the present application, the sample is detected by immunohistochemical staining method, and it is determined that the INPP5F protein can be used as a molecular marker for diagnosing breast cancer. It is simple and easy to operate, safe and effective in the diagnosis process, easy to be accepted by patients, the diagnostic standard is unified, and the influence of personal subjective factors is small. The INPP5F protein has important significance for the prognosis of breast cancer. BRIEF DESCRIPTION OF DRAWINGS

[0022] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some of the embodiments of the present application, and other drawings can be obtained by those of ordinary skill in the art without creative effort based on these drawings.

[0023] Figure 1 The expression of INPP5F in breast cancer and adjacent tissues is shown. Statistically significant (p<0.001);

[0024] Figure 2 The correlation between the expression of INPP5F and the prognosis of breast cancer patients is shown. p<0.001;

[0025] Figure 3 The ROC analysis result of INPP5F predicting breast cancer is shown. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only some of the embodiments of the present application, not all. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative effort fall within the scope of the present application.

[0027] Breast cancer is one of the malignant tumors that seriously affects women's health. At present, the diagnosis of breast cancer mainly relies on clinical breast examination, mammography, ultrasonic examination and magnetic resonance imaging. Exploring new breast cancer diagnosis and prognosis markers will help to early detection and diagnosis of breast cancer, promote early treatment, increase the chance of successful treatment, and thus reduce the mortality rate.

[0028] The INPP5F protein is also known as inositol polyphosphate-5-phosphatase F. Its gene sequence is located on the long arm of human chromosome 10, and it plays a potential regulatory role in various disease models. However, the expression and role of INPP5F in breast cancer are not clear.

[0029] Based on this, the application provides use of INPP5F protein in preparation of a breast cancer diagnosis and / or prognosis product, wherein the amino acid sequence of the INPP5F protein is shown as SEQ ID NO. 1. In the application, the protein expression score of the INPP5F protein in the test sample is detected by using an immunohistochemical staining method; if the protein expression score of the INPP5F protein in the test sample is greater than 4.5, the patient is judged to be a breast cancer patient or a high-risk patient of breast cancer, which is of great significance for the diagnosis and prognosis of breast cancer.

[0030] Example 1

[0031] Expression analysis of INPP5F protein in breast cancer and paracancerous tissues

[0032] 1. Tissue sample source:

[0033] The 172 breast cancer patient samples were from the tissue sample library of Shanghai Zhuohao Pharmaceutical Technology Co., Ltd., and the operation time was from January 2004 to August 2017, and the final follow-up time was April 2022. All cases were pathologically diagnosed as breast cancer, and were female, aged 31-84 years old, with a median age of 48 years old. Among the 172 samples, 130 cases contained cancer tissue and matched paracancerous tissue at a distance of 1.5 cm from the cancer, and the remaining 42 cases only had cancer tissue. The case data of the 172 patients are shown in Table 1.

[0034] Table 1 Clinical and pathological data of 172 breast cancer patients

[0035]

[0036] 2. Tissue chip production, the method is as follows:

[0037] The production of the tissue chip was completed by Shanghai Zhuohao Pharmaceutical Technology Co., Ltd. After the routine pathological section of all donor tissue blocks, HE staining was performed, and a second diagnosis was made by a pathologist, and typical pathological sites were marked on the HE section. Using a tissue chip production instrument Beecher Instruments. Inc, punch holes with a diameter of 1.5 mm in the blank wax block of the receptor wax block, then according to the marked range on the HE section, the target tissue in the donor tissue wax block was obtained at the corresponding position and placed in the array hole of the receptor wax block, and the above steps were repeated, and finally the breast cancer tissue and paracancerous tissue matching point array block BRC1601 and BRC1603 were produced. Using a paraffin sectioning machine (Leica, Germany) to continuously section with a thickness of 4 μm, the sections were attached to imported glass slides treated with anti-sheeting.

[0038] 3. Immunohistochemical staining experiment, the method is as follows:

[0039] The prepared tissue chip was subjected to immunohistochemical experiment using a two-step immunohistochemical kit from Beijing Zhongshan Golden Bridge Biotechnology Co., Ltd.

[0040] (1) Dewaxing of breast cancer tissue chip: the breast cancer tissue chip was placed on a film baking machine and baked at 65°C for two hours, and then dewaxed in a fume hood.

[0041] (2) After dewaxing, the breast cancer tissue chip was washed in tap water for 5 min, and then placed in an immunohistochemical box containing ddH2O and washed on a shaker for 5 min.

[0042] (3) Antigen repair: antigen repair solution was added to the immunohistochemical box, and then the breast cancer tissue chip was placed therein; the temperature of the constant temperature water bath was adjusted to 99°C, the immunohistochemical box containing the breast cancer tissue chip was placed in the water bath, and the real-time temperature in the immunohistochemical box was measured with a thermometer; when the temperature rose to 95°C, the timing started, and the antigen repair was performed for 18 min.

[0043] (4) After antigen repair, the immunohistochemical box was taken out of the water bath, and the breast cancer tissue chip was allowed to recover to room temperature.

[0044] (5) After the breast cancer tissue chip recovered to room temperature, PBST solution was added to the immunohistochemical box, and the breast cancer tissue chip was placed therein and washed on a shaker for three times, each for 5 min.

[0045] (6) Removal of peroxidase: the breast cancer tissue chip was taken out of the immunohistochemical box, and the liquid around the tissue was wiped clean with a water-absorbing paper; an immunohistochemical pen was used to draw a circle around the tissue, and the tissue was circled in the circle; then the breast cancer tissue chip was placed in a wet box, and 3% hydrogen peroxide solution was added dropwise on the tissue, and incubated in the dark for 10 min.

[0046] (7) After removal of peroxidase, PBST solution was added to the immunohistochemical box, and the breast cancer tissue chip was placed therein and washed on a shaker for three times, each for 10 min.

[0047] (8) Blocking: the breast cancer tissue chip was taken out of the immunohistochemical box, and the liquid on the chip was shaken off; the breast cancer tissue chip was placed in a wet box, and blocking solution was added dropwise on the tissue; the lid of the wet box was covered, and the blocking was performed at room temperature for one and a half hours.

[0048] (9) Preparation of primary antibody: INPP5F primary antibody was prepared according to a ratio of 1:200 using antibody diluent.

[0049] (10) Incubation of primary antibody: after blocking, the blocking solution on the tissue was shaken off, the breast cancer tissue chip was placed in a wet box, and the prepared primary antibody solution was added dropwise on the tissue; the lid was covered, and the incubation was performed in a 4°C refrigerator overnight.

[0050] (11) After the incubation of the first antibody is completed, take the wet box from the 4°C refrigerator, and place it at room temperature for half an hour to restore the breast cancer tissue chip to room temperature.

[0051] (12) After being restored to room temperature, add the PBST solution in the immunohistochemistry box, place the breast cancer tissue chip therein, and place it on a shaker for washing three times, each time for 10 min.

[0052] (13) Incubation of the second antibody: shake off the PBST liquid on the tissue, place the breast cancer tissue chip in the wet box, add the HRP-labeled goat anti-rabbit secondary antibody dropwise onto the tissue, cover the lid, and incubate at room temperature for one hour.

[0053] (14) After the incubation of the second antibody is completed, add the PBST solution in the immunohistochemistry box, place the breast cancer tissue chip therein, and place it on a shaker for washing three times, each time for 10 min.

[0054] (15) Preparation of DAB developing solution: prepare the DAB developing solution at a ratio of 1:20, use it in the dark, and prepare it fresh each time.

[0055] (16) DAB developing: shake off the PBST liquid on the tissue, place the breast cancer tissue chip under a microscope, and add the DAB developing solution dropwise onto the tissue for developing, when the specific coloration is obvious and the background is clean, place the breast cancer tissue chip in tap water to terminate the developing.

[0056] (17) Hematoxylin staining: place the breast cancer tissue chip in the hematoxylin staining solution for staining for 2 min 30 s, and place it in tap water for washing for 5 min.

[0057] (18) Hydrochloric acid alcohol differentiation: dip the breast cancer tissue chip in hydrochloric acid alcohol, take it out quickly, and place it in tap water for washing for 5 min.

[0058] (19) Light ammonia water counterstaining: place the breast cancer tissue chip in light ammonia water for counterstaining for 1 min 30 s, place it in tap water for washing for 5 min, and dehydrate the tissue.

[0059] (20) Tissue mounting: after the breast cancer tissue chip is dehydrated, take the chip out of the transparent liquid, add neutral resin dropwise onto the tissue, and then cover a cover glass.

[0060] 4. Quantitative analysis is as follows:

[0061] The staining results were photographed, and the positive area and staining intensity in each slice were converted into corresponding H-Score values to achieve semi-quantitative tissue staining. The H-Score score was the lowest 0 and the highest 8, and the median was 4. Grouping was performed according to the H-Score score, and the high expression group was higher than the median, and the low expression group was lower than or equal to the median.

[0062] 5. Statistical analysis as follows:

[0063] The expression of INPP5F protein in breast cancer and paracancerous tissues was analyzed by t test. The correlation between INPP5F protein expression and clinical indicators of breast cancer patients was analyzed by chi-square test. The correlation between INPP5F protein and prognosis of breast cancer patients was analyzed by Kaplan-Meier survival analysis and log-rank statistical test for single factor analysis of survival period. p<0.05 has statistical difference.

[0064] 6. The experimental results are as follows:

[0065] (1) Analysis of INPP5F protein expression in breast cancer and paracancerous tissues.

[0066] The results of immunohistochemical analysis are shown in Figure 1 , the expression of INPP5F protein in breast cancer tissue is significantly higher than that in paracancerous tissue, p <0.001.

[0067] (2) Correlation between INPP5F protein and clinical indicators of breast cancer patients.

[0068] The breast cancer patients were grouped according to age or TNM stage and other factors, and the correlation between different factors and the expression of INPP5F protein was analyzed by chi-square test. The results are shown in Table 2, the expression of INPP5F protein in breast cancer has no obvious statistical relationship with the age of the patient, p>0.05; and is closely related to the TNM stage of the patient, p<0.001.

[0069] Table 2 Correlation between expression of INPP5F protein and clinical indicators of breast cancer patients

[0070]

[0071] (3) The follow-up of breast cancer patients is as follows: the operation time is from January 2004 to August 2017, and the final follow-up time is April 2022. Single factor analysis of survival period was performed by Kalplan-Meier survival analysis and log-rank statistical test, and the results are shown in Figure 2As shown, breast cancer patients with low expression of INPP5F in cancer tissues have longer overall survival, p<0.001, and their 5-year survival rate is significantly higher than that of breast cancer patients with high expression of INPP5F. This result suggests that detecting INPP5F is of great significance for the prognosis of breast cancer.

[0072] Example 2

[0073] Verification of INPP5F protein in the diagnosis of breast cancer

[0074] S1, tissue sample source, as follows:

[0075] 105 breast cancer patient cancer tissue samples and 105 cancer-adjacent tissue samples were obtained from the tissue sample library of Shanghai Xisuo Biotechnology Co., Ltd.

[0076] S2, tissue chip preparation, as follows:

[0077] The preparation of the tissue chip was completed by Shanghai Xisuo Biotechnology Co., Ltd. The tissue chip preparation instrument TMAGrand Master was used to punch the target tissue on the receptor wax block and place it into the receptor wax block array hole with a hole diameter of 1.5 mm. The above steps were repeated to finally prepare the dot array block HBreD120CS01 and HBreD090CS01 of breast cancer tissue and cancer-adjacent tissue. A paraffin sectioning machine (Leica, Germany) was used to continuously section with a thickness of 4 pm, and the sections were attached to imported glass slides treated with anti-sheeting.

[0078] 3, immunohistochemical staining experiment, as follows:

[0079] A two-step immunohistochemical kit from Beijing Zhongshanjingqiao Biotechnology Co., Ltd. was used for immunohistochemical experiments on the prepared tissue chip.

[0080] (1) Breast cancer tissue chip deparaffinization: Place the breast cancer tissue chip on the film oven, bake at 65°C for two hours, and deparaffinize the tissue chip in a fume hood.

[0081] (2) After deparaffination, wash the breast cancer tissue chip in tap water for 5 min; then place it in an immunohistochemical box containing ddH2O and shake it for 5 min.

[0082] (3) Antigen repair: Add antigen repair solution to the immunohistochemical box, then place the breast cancer tissue chip in it; adjust the temperature of the constant temperature water bath to 99°C, place the immunohistochemical box containing the breast cancer tissue chip in the water bath, and measure the real-time temperature in the immunohistochemical box with a thermometer. When the temperature rises to 95°C, start timing, and antigen repair for 18 min.

[0083] (4) After the antigen repair is completed, the immunohistochemical box is taken out of the water bath, and the breast cancer tissue chip is restored to room temperature.

[0084] (5) After the breast cancer tissue chip is restored to room temperature, PBST solution is added to the immunohistochemical box, the breast cancer tissue chip is placed in it, and is washed on a shaking table for three times, 5 min each time.

[0085] (6) Peroxidase removal: the breast cancer tissue chip is taken out of the immunohistochemical box, the liquid around the tissue is wiped clean with a water-absorbing paper, the tissue is circled with an immunohistochemical pen, and the breast cancer tissue chip is placed in a wet box, 3% hydrogen peroxide solution is added dropwise on the tissue, and incubation is performed in the dark for 10 min.

[0086] (7) After the peroxidase removal is completed, PBST solution is added to the immunohistochemical box, the breast cancer tissue chip is placed in it, and is washed on a shaking table for three times, 10 min each time.

[0087] (8) Blocking: the breast cancer tissue chip is taken out of the immunohistochemical box, the liquid on the chip is shaken off, the breast cancer tissue chip is placed in a wet box, blocking solution is added dropwise on the tissue, the lid of the wet box is covered, and blocking is performed at room temperature for one and a half hours.

[0088] (9) Preparation of primary antibody: INPP5F primary antibody is prepared according to a ratio of 1:200 by using an antibody diluent.

[0089] (10) Incubation of primary antibody: after the blocking is completed, the blocking solution on the tissue is shaken off, the breast cancer tissue chip is placed in a wet box, the prepared primary antibody solution is added dropwise on the tissue, the lid is covered, and incubation is performed in a 4°C refrigerator overnight.

[0090] (11) After the incubation of the primary antibody is completed, the wet box is taken out of the 4°C refrigerator, and the breast cancer tissue chip is restored to room temperature for half an hour.

[0091] (12) After the room temperature is restored, PBST solution is added to the immunohistochemical box, the breast cancer tissue chip is placed in it, and is washed on a shaking table for three times, 10 min each time.

[0092] (13) Incubation of secondary antibody: the PBST liquid on the tissue is shaken off, the breast cancer tissue chip is placed in a wet box, HRP-labeled goat anti-rabbit secondary antibody is added dropwise on the tissue, the lid is covered, and incubation is performed at room temperature for one hour.

[0093] (14) After the incubation of the secondary antibody is completed, PBST solution is added to the immunohistochemical box, the breast cancer tissue chip is placed in it, and is washed on a shaking table for three times, 10 min each time.

[0094] (15) Preparation of DAB color developing solution: DAB color developing solution was prepared at a ratio of 1:20, and was used in the dark. It was prepared and used immediately.

[0095] (16) DAB color development: The PBST liquid on the clean tissue was spun dry. The breast cancer tissue chip was placed under a microscope, and DAB color developing solution was added dropwise to the tissue for color development. When the specific color was obvious and the background was clean, the breast cancer tissue chip was placed in tap water to terminate the color development.

[0096] (17) Hematoxylin staining: The breast cancer tissue chip was placed in hematoxylin staining solution for 2 min 30 s, and then washed in tap water for 5 min.

[0097] (18) Hydrochloric acid alcohol differentiation: The breast cancer tissue chip was dipped in hydrochloric acid alcohol, quickly taken out, and washed in tap water for 5 min.

[0098] (19) Light ammonia water counterbluing: The breast cancer tissue chip was placed in light ammonia water for counterbluing for 1 min 30 s, washed in tap water for 5 min, and dehydrated.

[0099] (20) Tissue mounting: After the breast cancer tissue chip was dehydrated, the chip was taken out of the transparent liquid, neutral resin was added dropwise to the tissue, and then a cover glass was placed on top.

[0100] 4. Quantitative analysis, as follows:

[0101] The staining results were photographed, and the positive area and staining intensity in each slice were converted into corresponding H-Score values to achieve semi-quantitative tissue staining. The H-Score score was the lowest at 0 and the highest at 8, with a median of 4. The H-Score score was grouped, with the high expression group being higher than the median and the low expression group being lower than or equal to the median.

[0102] 5. Statistical analysis, as follows:

[0103] The diagnostic efficiency of the protein expression intensity value of INPP5F protein for breast cancer was analyzed using ROC curve.

[0104] 6. Experimental results, as follows:

[0105] AUC is the area under the ROC curve, which is the most commonly used parameter to evaluate the characteristics of the ROC curve and is also an important test accuracy index. AUC is calculated by Figure 3It can be seen that the area under the curve AUC is 0.930, the 95% confidence interval is 0.894~0.967, p<0.001, indicating that the INPP5F protein can be used as a biomarker for the diagnosis of breast cancer. When the immunohistochemical score H-Score of the INPP5F protein is 4.5, the sensitivity is 92.4% and the specificity is 87.6%. When individual detection is performed, when the immunohistochemical score H-Score of the INPP5F protein is greater than 4.5, the breast cancer patient is judged, and the accuracy is 89.5%.

[0106] The embodiments of the present application are described above in combination with the drawings, and the principles and implementation modes of the present application are described herein by applying specific examples. The above description of the embodiments is only used to help understand the core idea of the present application, but the present application is not limited to the above specific implementation modes. The above specific implementation modes are only illustrative and not restrictive. Those skilled in the art can make many forms without departing from the scope of the present application under the inspiration of the present application, and all forms belong to the protection of the present application.

Claims

1. The application of a reagent for detecting INPP5F protein expression levels in the preparation of breast cancer diagnostic and / or prognostic products, characterized in that, The amino acid sequence of the INPP5F protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The products include chips, reagent kits, or test strips.

3. The application according to claim 2, characterized in that, The chips include gene chips and protein chips.

4. The application according to claim 3, characterized in that, The gene chip includes nucleotide probes for detecting the transcriptional level of the INPP5F gene; the protein chip includes a specific binding reagent for the INPP5F protein.

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