CN117604107B - Use of FOXO6 and SNX4 in combination for diagnosing breast cancer - Google Patents
Use of FOXO6 and SNX4 in combination for diagnosing breast cancer Download PDFInfo
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Abstract
The invention belongs to the field of biotechnology and medical diagnosis, and particularly relates to application of a combination of FOXO6 and SNX4 in diagnosing breast cancer. The present invention has found that FOXO6 and SNX4 can be used in combination for diagnosing breast cancer, in particular HER2 positive breast cancer. The inventors have also unexpectedly found that FOXO6 and SNX4 both participate in PI3K/AKT signaling pathways that play an important role in the development of breast cancer and are functionally in a mutually redundant relationship, so that FOXO6 and SNX4 need to be used in combination to diagnose breast cancer with high specificity.
Description
Technical Field
The invention belongs to the field of biotechnology and medical diagnosis, and particularly relates to application of a combination of FOXO6 and SNX4 in diagnosing breast cancer.
Background
Breast cancer is one of the most common malignant tumors in the female population, with an incidence of 23% of all tumors. New cases worldwide reach 160 ten thousand per year, and are the second most dying cancer for women worldwide.
There is growing evidence that various factors (i.e., genetic and environmental factors) may be associated with the occurrence and progression of breast cancer. Early diagnosis of breast cancer patients is one of the important aspects of breast cancer treatment. Among the various diagnostic platforms, imaging techniques are the primary diagnostic means that can provide valuable data to breast cancer patients. Furthermore, the use of biochemical biomarkers, such as proteins, DNA, mRNA and microRNA, can be used as a new diagnostic and therapeutic tool for breast cancer patients. With the development of immunology and molecular biology, tumor-associated protein markers show increasingly important clinical value in diagnosis and treatment of breast cancer, and become indispensable biological indexes for assisting diagnosis, observing curative effects and judging prognosis.
A plurality of tumor markers which can be used for diagnosing breast cancer, classifying pathology and clinical stage and judging prognosis and curative effect are found clinically, but the diagnostic efficacy of the currently commonly used (CA 153, CA125 and CEA) breast cancer markers is not ideal, and a specific tumor marker has higher sensitivity and specificity for diagnosing breast cancer.
Circulating tumor cells are a subset of tumor cells that shed from a primary tumor or metastatic tumor and are released into the blood circulation. Recent studies have found that, on the one hand, circulating tumor cells may appear in the peripheral blood of patients very early in tumorigenesis, which aids in early diagnosis of cancer. On the other hand, these circulating tumor cells can also be used to predict prognosis in cancer patients, and the discovery of circulating tumor cells often predicts recurrence or metastasis of a tumor, which also suggests poor prognosis in patients. How to use circulating tumor cells for diagnosis or prognosis of cancer, especially specific cancers such as breast cancer, is also an important direction in our future in the search of circulating tumor cell lines. A great benefit of using circulating tumor cells for diagnosis or prognosis is that it can effectively replace tumor biopsies, which is a good surrogate indicator for those patients who cannot take a pathological tissue biopsy, and can help clinicians to dynamically monitor and determine the biological characteristics of cancer in real time. However, due to the scarcity of circulating tumor cells, the use thereof as a means of diagnosing cancer, particularly specific cancers such as breast cancer, presents challenges, and not all cancer-related markers can be detected in circulating tumor cells.
Therefore, it is of great clinical value to find new markers relevant for breast cancer diagnosis, in particular biomarkers suitable for diagnosis by means of circulating tumor cells.
Disclosure of Invention
In order to solve the problems, the present invention finds that FOXO6 and SNX4 are expressed higher in breast cancer, especially HER2 positive breast cancer tissues than normal tissues by bioinformatics methods. Further, high expression of FOXO6 and SNX4 in breast cancer was verified by clinically collected tissue samples, and diagnostic efficacy of individual diagnosis and combined diagnosis thereof was evaluated. In addition, the difference and gene function of FOXO6 and SNX4 in breast cancer cells and normal breast cells were also verified by cell experiments. Compared with normal breast cells MCF10A, FOXO6 and SNX4 are abnormally and highly expressed in HER2 positive breast cancer cells BT474, and the high expression of the FOXO6 and the SNX4 has the effect of obviously promoting tumor progress and migration invasion, namely the FOXO6 and the SNX4 can be used as oncogenes to participate in the occurrence and the development of breast cancer. The inventors have also unexpectedly found that FOXO6 and SNX4 are both involved in phosphatidylinositol 3-kinase (PI 3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway (PAM signaling pathway) that play an important role in the development of breast carcinogenesis and are functionally in a mutually redundant relationship. Thus, FOXO6 and SNX4 can be used in combination to diagnose breast cancer, especially HER2 positive breast cancer, whereas diagnosis alone is not effective.
As used herein, NYAP2 is an abbreviation for neuronal tyrosine phosphorylated phosphoinositide 3 kinase adaptor 2 (neuronal tyrosine-phosphorylated phosphoinositide-3-kinase adaptor 2, NYAP 2), with NCBI Gene ID 57624.
As used herein, SNX33 is a short for sorting microtubule-associated protein 33 (SNX 33), with NCBI Gene ID 257364.
In particular, in a first aspect, the invention provides a biomarker for diagnosing breast cancer, the biomarker comprising FOXO6 and SNX4, the breast cancer being HER2 positive breast cancer.
In a second aspect, the invention provides the use of FOXO6 and SNX4 in combination as biomarkers for diagnosing breast cancer, which is HER2 positive breast cancer.
In a third aspect, the invention provides a kit for diagnosing breast cancer, the kit comprising reagents for detecting expression of FOXO6 and SNX4 in a sample of a subject, the breast cancer being HER2 positive breast cancer.
In some embodiments, the subject comprises a mammal, preferably a primate mammal, more preferably a human.
In some embodiments, the sample comprises a clinical biological sample of a subject, including but not limited to one or more of serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, tumor tissue, circulating tumor cells, circulating tumor DNA, or exosomes. Preferably, the clinical biological sample is a circulating tumor cell.
In a fourth aspect, the invention provides the use of an agent that detects expression of FOXO6 and SNX4 in a sample from a subject in the manufacture of a kit for diagnosing breast cancer.
In some embodiments, the breast cancer is HER2 positive breast cancer.
In some embodiments, the subject comprises a mammal, preferably a primate mammal, more preferably a human.
In some embodiments, the sample comprises a clinical biological sample of a subject, including but not limited to one or more of serum, plasma, whole blood, secretions, cotton swabs, pus, body fluids, tissues, organs, paraffin sections, tumor tissue, circulating tumor cells, circulating tumor DNA, or exosomes. Preferably, the clinical biological sample is a circulating tumor cell.
As used herein, the reagents for detecting expression of FOXO6 and SNX4 in a subject sample are not particularly limited and are well known and readily available to those of skill in the art for detecting expression of FOXO6 and SNX4 at mRNA or protein levels in a subject sample. For example, reagents for detecting expression of FOXO6 and SNX4 in a subject sample may include corresponding reagents for real-time fluorescent quantitative PCR, enzyme-linked immunosorbent assay (ELISA), protein/peptide fragment chip detection, chemiluminescence, immunoblotting, microbead immunodetection, microfluidic immunization.
The beneficial effects of the invention are that
The present invention has found that FOXO6 and SNX4 can be used in combination for diagnosing breast cancer, in particular HER2 positive breast cancer. The inventors have also unexpectedly found that FOXO6 and SNX4 both participate in PI3K/AKT signaling pathways that play an important role in the development of breast cancer and are functionally in a mutually redundant relationship, so that FOXO6 and SNX4 need to be used in combination to diagnose breast cancer with high specificity. In addition, the present invention also finds that diagnosis and prognosis of breast cancer can be performed by harvesting circulating tumor cells from a subject and detecting expression levels of FOXO6 and SNX4 therein.
Drawings
FIG. 1 is a graph showing analysis of the results of expression of FOXO6 and SNX4 in breast cancer and paracancestral tissues.
Figure 2 shows analysis of FOXO6 and SNX4 expression results in clinical samples of breast cancer and paracancestral tissue.
Figure 3 shows ROC curve analysis of FOXO6 and SNX4, alone and in combination, in breast cancer patients and healthy people.
Figure 4 shows the expression levels of FOXO6 and SNX4 in circulating tumor cells of breast cancer patients.
Fig. 5 shows the analysis of the expression differences of FOXO6 and SNX4 in normal breast cells MCF10A and breast cancer cells BT 474.
Figure 6 shows the analysis of the experimental results of the effect of FOXO6 and SNX4 on the biological behaviour of breast cancer cells. Panel A shows the silencing effect of interfering siRNA on FOXO6 and SNX4 at the mRNA level. Panels B and C are graphs of BT474 cell migration and invasion results analysis using Transwell experiments after single and combined silencing of FOXO6 and SNX 4.
Figure 7 shows the effect on PI3K/AKT signaling activation in BT474 cells after single and combined silencing of FOXO6 and SNX 4.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Example 1: analysis of FOXO6 and SNX4 Gene expression in TCGA database
Collecting 513 cases of HER2 positive breast cancer tissues and 98 cases of FOXO6 and SNX4 expression profile data of the tissues beside the cancer from a TCGA database, performing quality control and homogenization by adopting an edge method, and performing difference analysis of the FOXO6 and the SNX4 in the HER2 positive breast cancer tissues and the tissues beside the cancer by adopting the edge method; and drawing a dot-shaped graph.
The expression levels of FOXO6 and SNX4 are shown in fig. 1, with significantly up-regulated expression in breast cancer tissues compared to control FOXO6 and SNX 4.
Example 2: analysis of diagnostic value of FOXO6 and SNX4 genes
48 clinical HER2 positive breast cancer tissue samples and 42 paracancerous normal tissue samples were collected,
mRNA levels of FOXO6 and SNX4 were detected by RT-qPCR method by extracting mRNA from the HER2 positive breast cancer tissue sample and the paracancerous normal tissue sample, respectively, using TRIzol (15596018, invitrogen). The results are depicted in fig. 2, which demonstrates that FOXO6 and SNX4 expression is significantly up-regulated in breast cancer tissues.
Independent diagnosis and combined diagnosis experimental results of FOXO6 and SNX4 were analyzed using a subject work curve (ROC). The results are shown in fig. 3, which shows that FOXO6 (sensitivity 66.67%, specificity 76.19%) and SNX4 (sensitivity 90.48%, specificity 40.48%) mRNA expression has a general independent diagnosis effect on HER2 positive breast cancer, but the combined diagnosis effect is very good, the area AUC (area under the ROC curve) = 0.9291 under ROC curve, the sensitivity can reach 80.95%, and the specificity can reach 97.62%. Thus, FOXO6 and SNX4 can be used in combination to diagnose HER2 positive breast cancer.
Example 3: detection of FOXO6 and SNX4 expression levels in circulating tumor cells of breast cancer patients
1) Extracting 10mL of venous blood of a breast cancer patient in an ACD anticoagulation tube, and conventionally centrifuging and separating plasma for later use;
2) Enrichment and separation of CTC cells in plasma comprises the following specific steps: extracting single cell layer from blood plasma by adding sample density separating liquid (Cytelligen) into blood plasma, and removing immune cellsCD45 in single cell layer extracted by magnetic bead pair + Immune cells are removed, and CTC in a single cell layer is concentrated and enriched through differential enrichment;
3) The enriched CTC cells were harvested by centrifugation and 1ml of RNA lysate was added to the enzyme-free EP tube; 200ul of chloroform is added into an EP tube, vigorously oscillated for 15 seconds, and kept still at room temperature for 3 minutes, and repeated for 3 times; centrifuging at 12000 Xg and 4 ℃ for 15min; adding the upper water phase into a new enzyme-free EP pipe, adding equal volume of isopropanol into the EP pipe, reversing, mixing uniformly, and standing for 10min; centrifuging at 12000 Xg and 4 ℃ for 15min; the EP tube liquid was discarded, 1ml of 75% ethanol was added, and the EP tube was shaken; centrifuging at 12000 Xg and 4 ℃ for 5min; discarding the supernatant, and standing at room temperature for drying; adding a proper amount of DEPC water to dissolve RNA; the purity and concentration of RNA were measured and expression of FOXO6 and SNX4 in CTC cells was measured by RT-qPCR and compared to expression of FOXO6 and SNX4 in cells harvested from normal breast tissue, as shown in fig. 4, which demonstrates high expression of FOXO6 and SNX4 in CTC cells of breast cancer patients.
Example 4: verification of the effect of FOXO6 and SNX4 on invasion and migration functions of breast cancer cells
HER2 positive breast cancer cell line BT474 was cultured in RPMI-1640 medium (100 u.ml containing 10% fetal bovine serum -1 Penicillin and 0.1 mg.mL -1 Streptomycin), at 37 ℃,5% co 2 Culturing in a constant temperature incubator, culturing normal mammary gland cell line MCF10A in F12 medium containing 5% horse serum, 20ng/ml EGF, 0.5 μg/ml hydrocortisone, 10 μg/ml insulin, 1% non-essential amino acid, and double antibody at 37deg.C and 5% CO 2 Culturing in a constant temperature incubator.
After the adherent cells are digested and collected, 1ml of RNA lysate is added to be absorbed into an enzyme-free EP tube; 200ul of chloroform is added into an EP tube, vigorously oscillated for 15 seconds, and kept still at room temperature for 3 minutes, and repeated for 3 times; centrifuging at 12000 Xg and 4 ℃ for 15min; adding the upper water phase into a new enzyme-free EP pipe, adding equal volume of isopropanol into the EP pipe, reversing, mixing uniformly, and standing for 10min; centrifuging at 12000 Xg and 4 ℃ for 15min; the EP tube liquid was discarded, 1ml of 75% ethanol was added, and the EP tube was shaken; centrifuging at 12000 Xg and 4 ℃ for 5min; discarding the supernatant, and standing at room temperature for drying; adding a proper amount of DEPC water to dissolve RNA; the purity and concentration of RNA was detected and expression of FOXO6 and SNX4 in breast and cancer cells was detected by RT-qPCR. The results are shown in fig. 5, which shows that FOXO6 and SNX4 are significantly higher in breast cancer cell line BT474 than normal breast cell line MCF 10A.
The expression of FOXO6 and SNX4 in breast cancer cells was interfered with by siRNA (siRNA sequence: siNC:5'-UUCUCCGAACGUGUCACGUUCAUACTT-3' (SEQ ID No. 1), siFOXO6:5'-CGUUACGUGCCCUACUUCAAGGAUATT-3' (SEQ ID No. 2), siSNX4:5'-CGAACUGGAAGAAAUGCCAUGAACATT-3' (SEQ ID No. 3)), and then examined by Transwell cell migration and invasion experiments. The results are shown in fig. 6, which shows that after the FOXO6 and SNX4 genes are jointly interfered, the migration capacity and invasion capacity of the breast cancer cell line BT474 are obviously reduced, compared with the control group, the difference is statistically significant, and when the interference is singly interfered, the difference is not obvious.
Example 5: FOXO6 and SNX4 are involved in PI3K/AKT signaling pathway and are functionally in redundant relation to each other
And respectively collecting MCF10A cells and BT474 cells in logarithmic growth phase and corresponding cells which interfere the expression of FOXO6 and SNX4 through siRNA, extracting protein, and detecting the activation condition of PI3K/AKT signal path through a western blot experiment. Specifically, the extracted proteins were separated by 10% (w/w) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto nitrocellulose membranes and detected with the corresponding antibodies. First, 20. Mu.L of protein sample and 5. Mu.L of protein label were added to lanes on an acrylamide gel, then electrophoresis was started, the conditions for electrophoresis on a concentrated gel were 80V for 30 minutes, and the conditions for separation of the gel were 100V for 1 hour. Next, the protein was transferred to NC membrane for 1 hour at 100V. Thereafter, the membranes were immersed in TBST solution containing 5% skim milk for 2 hours, and then incubated with the corresponding primary antibodies overnight at 4 ℃): including p-AKT (Ser 473), AKT (pan), GAPDH. The membranes were incubated with the relevant secondary antibodies for 1 hour at room temperature after washing 3 times with TBST for 10 minutes each. Finally, the labeled protein is detected by enhancing the development solution (ECL). The results are shown in FIG. 7. From fig. 7, it can be seen that Akt is phosphorylated in HER2 positive breast cancer cells, and the phosphorylation level of Akt in cells can be significantly inhibited after the expression of FOXO6 and SNX4 is simultaneously interfered, while the effect is not obvious when the FOXO6 and SNX4 are interfered alone, which proves that FOXO6 and SNX4 participate in PI3K/Akt signaling pathway in HER2 positive breast cancer cells, and are in a functionally redundant relationship.
In summary, by detecting expression of both FOXO6 and SNX4 in a subject's breast tissue, it is possible to determine whether the subject has breast cancer or whether the subject is at risk of having breast cancer.
It should be noted that the description of the present invention and the accompanying drawings illustrate preferred embodiments of the present invention, but the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein, which are not to be construed as additional limitations of the invention, but are provided for a more thorough understanding of the present invention. The above-described features are further combined with each other to form various embodiments not listed above, and are considered to be the scope of the present invention described in the specification; further, modifications and variations of the present invention may be apparent to those skilled in the art in light of the foregoing teachings, and all such modifications and variations are intended to be included within the scope of this invention as defined in the appended claims.
Claims (2)
1. Use of an agent that detects the combined expression of FOXO6 and SNX4 in a sample of a subject in the manufacture of a tool for diagnosing breast cancer, which is HER2 positive breast cancer.
2. The use of claim 1, wherein the sample comprises a clinical biological sample of a subject,
the clinical biological sample is one or more of serum, plasma, whole blood, pus, organs, tumor tissues, circulating tumor cells, circulating tumor DNA or exosomes.
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