CN117551676A - Biomarker for detecting prostate cancer, kit and application - Google Patents
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Abstract
The invention provides a biomarker for detecting prostate cancer, a kit and application thereof, belonging to the technical field of molecular biology detection, wherein the nucleotide sequence of the biomarker is shown as SEQ ID No.1, and the biomarker is a binding fragment of Argonaute protein and 3' tRF-ArgCCT complex. Meanwhile, the probe obtained by the optimal design of the invention has only 15 bases, and has higher sensitivity and specificity.
Description
Technical Field
The invention belongs to the technical field of molecular biology detection, and particularly relates to a biomarker for detecting prostate cancer, a kit and application thereof.
Background
The 2016 Nature Genetics journal reported tRNA as a highly abundant, ubiquitous, passively involved mRNA decoder and protein translation element. trnas significantly affect biological processes and disease progression by binding their anticodons to the codons of mRNA. Meanwhile, the high abundance of tRNA in body fluid makes it a biomarker for clinical application, which can be used in the detection of tumor proliferation and metastasis. A review in 2019 panjiang et al, journal CurrentMedicinal Chemistry, reported that trnas were sheared under the action of specific nucleases into trfs and tirnas, which belong to a class of small non-coding RNAs, collectively referred to as tsrnas. tRF is classified into tRF-1, tRF-2, tRF-3, tRF-5 and i-tRF; the tiRNAs are classified into 5 'tiRNAs and 3' tiRNAs. tRF and tiRNA play important roles in inhibiting protein synthesis, regulating gene expression, initiating viral reverse transcriptase, and regulating DNA damage response, and can be considered as functional units of trnas. Although tRNA plays an important role in tumor proliferation and metastasis, its insufficient in vivo stability limits its biological role.
In 2023 Nina Kirstein et al reported on science Advances that the Argonaute (AGO) protein in the body is a large family of proteins, including AGO1-4.AGO proteins comprise mainly four domains: n, PAZ, MID and PIWI. Wherein N charges the tRNA, MID binds to the 5 'end of the tRNA, PAZ binds to the 3' end of the tRNA, and PIWI has ribonuclease H activity.
Disclosure of Invention
In view of the above, the present invention aims to provide a biomarker, a kit and an application for detecting prostate cancer, wherein the biomarker is a binding fragment of Argonaute protein and 3' tRF-ArgCCT complex, and the biomarker is more valuable than single detection tRNA fragment by detecting the binding fragment of Argonaute protein and 3' tRF-ArgCCT complex, namely the binding condition of Argonaute protein and 3' tRF-ArgCCT complex, and can display the level of important complex in the tumor proliferation and metastasis process.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the invention provides a biomarker for detecting prostate cancer, and the nucleotide sequence of the biomarker is shown as SEQ ID No. 1.
The invention also provides application of the reagent for detecting the biomarker in preparation of a kit for detecting the prostate cancer.
Preferably, the kit comprises a probe for detecting the biomarker, and the nucleotide sequence of the probe is shown as SEQ ID No. 2.
The invention also provides a kit for detecting the prostate cancer, which comprises a probe for detecting the biomarker, wherein the nucleotide sequence of the probe is shown as SEQ ID No. 2.
Compared with the prior art, the invention has the following beneficial effects:
the invention can judge the proliferation level of tumor cells by detecting the increase or decrease of the content of the binding fragment of Argonaute protein and 3'tRF-ArgCCT complex, namely the binding condition of Argonaute protein and 3' tRF-ArgCCT complex. And the stability of the 3'tRF-ArgCCT can be increased after the Argonaute protein is combined with the 3' tRF-ArgCCT, compared with the existing method for simply detecting the 3'tRF-ArgCCT in blood, the method for detecting the proliferation state of the Argonaute protein and the 3' tRF-ArgCCT in the blood can more accurately prompt the proliferation state of tumors in a human body, and has practical significance.
The probe for detecting the Argonaute protein and the 3' tRF-ArgCCT complex is optimized to 15 bases, and has higher sensitivity and specificity as shown in SEQ ID No. 2.
Drawings
FIG. 1 is a scan of a tissue chip of the binding of Argonaute protein to tRNA in normal human blood;
FIG. 2 is a scan of a tissue chip of Argonaute protein binding to tRNA in blood of a prostate cancer patient;
FIG. 3 is a hierarchical cluster heatmap of the binding of enriched different Argonaute proteins to tRNA; the level of binding of enriched Argonaute protein to tRNA is indicated by the upper left red-blue scale; the top dendrogram shows the relative proximity of enrichment profiles between samples; LYJ represents normal human, LDK represents prostate cancer patient.
Detailed Description
The invention provides a biomarker for detecting prostate cancer, and the nucleotide sequence of the biomarker is shown as SEQ ID No. 1.
In the present invention, the nucleotide sequence of the biomarker is: GGGCGGGCGCCAAAAGGCGCCCGCCCGTGGTGCATTGGCCATATCCTA CTATACGTATCA (SEQ ID No. 1).
The invention also provides application of the reagent for detecting the biomarker in preparation of a kit for detecting the prostate cancer.
Preferably, the kit comprises a probe for detecting the biomarker, and the nucleotide sequence of the probe is shown as SEQ ID No. 2.
The invention also provides a kit for detecting the prostate cancer, which comprises a probe for detecting the biomarker, wherein the nucleotide sequence of the probe is shown as SEQ ID No. 2.
In the present invention, the nucleotide sequence of the probe is: GTGGTGCATTGGCCA (SEQ ID No. 2).
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 detection of Argonaute protein and 3' tRF-ArgCCT Complex in blood sample 1.1 lysed tissue
10ml of blood of prostate cancer patients and healthy physical examination patients, which are confirmed by auxiliary hospitals of Yangzhou university, are extracted, placed in a blood sampling tube, centrifuged at 4 ℃ for 20min at 8000r/min, the supernatant is discarded, 1ml of RIPA lysate (purchased from Simer Feishan technology Co., ltd., product No. 89900) is added, fully and uniformly mixed, ice bath is carried out for 5min until the solution is clear, centrifugation is carried out at 8000r/min for 20min at 4 ℃, the supernatant is taken to a new blood sampling tube, and placed on ice, thus obtaining the supernatant.
1.2 preparation of Argonaute protein
Taking 50 μl of supernatant, rotating at 4deg.C for 3 hr, washing with 1ml NET Buffer (purchased from Shanghai such as Ji Biotechnology development Co., ltd.) for 4 times each for 10min, washing with PBS Buffer after 1 time, and mixing completely; after resuspension with 1ml PBS buffer, 300. Mu.l of the supernatant was removed by centrifugation, 30. Mu.l of lysate and 5 Xloading solution (both from Takara Shuzo Co., ltd.) were added, and incubation was performed at 95℃for 10min, followed by running. The specific glue running step comprises the following steps: taking 100g of agarose in a 500mL triangular flask, adding TAE buffer (purchased from Simer Feishul technology Co.) and heating in a microwave oven to dissolve the agarose completely; nucleic acid dye (purchased from zemoeimer technology) was added and shaken well. Pouring the dissolving liquid into a glue box inserted into the comb, waiting until the glue is solidified; lightly placing the gel into an electrophoresis tank, and completely immersing the gel by using TAE buffer solution; finally, taking 5 mu l of the prepared solution to fill the gel hole, opening the switch of the electrophoresis tank, and closing the electrophoresis tank after 30 min.
1.3 dyeing and bleaching
Cutting gel after gel running into gel strips with the size of 1×1×0.5cm, placing into a test tube, adding decolorizing solution (purchased from Shanghai ze leaf Biotechnology Co., ltd.) into the test tube, shaking for 30min, placing into new decolorizing solution, shaking for 30min until the color of the gel strips becomes light to colorless, and obtaining decolorized gel strips.
1.4 protein reductive alkylation
Adding 500 μl acetonitrile into the decolorized adhesive tape, shrinking, whitening, centrifuging, and removing all liquid after 10min; adding 5mmol/LTECP (tetrachlorophenol, purchased from Sigma Aldrich trade Co., ltd.) and passing through the adhesive tape, incubating at 55deg.C for 10min, cooling to room temperature, adding 10mmol/L indoleacetic acid to double the volume of liquid in the test tube, reacting for 20min in the absence of light, and alkylating the reduced disulfide bond; all liquids were discarded, 500. Mu.l acetonitrile was added to the gel strip, and after 10min the gel strip was contracted, all liquids were removed to obtain a gel strip containing the reduced alkylated protein.
1.5 pancreatin digestion
Adding 50 μl trypsin solution (1 μg trypsin is added into 100 μl ABC buffer solution during preparation) into the adhesive tape containing the protein subjected to reductive alkylation, standing at 4deg.C for 30min until the adhesive tape is completely expanded (10-20 μl trypsin solution is added if not completely expanded), standing at 4deg.C for 80min until the trypsin solution is completely saturated with the adhesive tape, adding 20 μl ABC buffer solution (purchased from Simer Feishan technologies Co.), enzyme-cutting at 37deg.C overnight, centrifuging at 4deg.C for 30min at 8000r/min, and collecting precipitate.
1.6 preparation of cell sections
Fixing the collected precipitate with 95% ethanol for 24 hr, making into cell wax block, slicing into slices with thickness of 3-5 μm, baking at 65deg.C for 2 hr, and dewaxing to obtain cell slice.
1.7 pretreatment
At room temperature, the cell slice is soaked in 100% ethanol for 5min and repeated; taking out the slices, and sequentially placing the slices in 80%, 75% gradient ethanol and deionized water for soaking for 3min respectively; soaking in boiled penetrating agent (purchased from Shanghai ze leaf Biotechnology Co., ltd.) for 30min, taking out, and soaking the slices in preheated pepsin working solution at 37deg.C for digestion for 10min; taking out, soaking in 2 XSSC buffer (available from Shanghai Charpy technology Co., ltd.) and rinsing for 5min each time; taking out, and sequentially soaking in 70%, 85% and 100% gradient ethanol for 3min respectively; taking out, airing at room temperature, and obtaining the pretreated cell slice.
1.8 detection
Tissue chip scanning and signal processing are processed by Agilent Feature Extraction Software (version 11.0.1.1), and 60 nucleotide fragments are found at the joint of the 3' tRF-ArgCCT and Argonaute protein, specifically GGGCGGGCGCCAAAAGGCGCCCGCCCGTGGTGCATTGGCCATATCCTA CTATACGTATCA (SEQ ID No. 1), so that the detection sensitivity is improved while the positive result detection is ensured in the real detection process. Therefore, we used the 15 nucleotide core sequence GTGGTGCATTGGCCA (SEQ ID No. 2) as a detection probe.
This nucleotide sequence GTGGTGCATTGGCCA (SEQ ID No. 1) was supplied to the Bodhisattva bioengineering Co., ltd (https:// www.boster.com.cn) from which a finished fluorescent in situ hybridization probe was made.
At the same time, a scan of a tissue chip in the blood of a normal human that binds to Argonaute protein and tRNA in the blood of a prostate cancer patient suggests that Argonaute protein and tRNA complex are differentially expressed.
1.9 hybridization
Standing the fluorescent in-situ hybridization probe for 5min at room temperature, mixing the probe upside down, centrifuging for 2min at 8000r/min, taking 10 μl of supernatant liquid to drop in a hybridization area (namely a liquid center area on a glass slide), immediately covering a cover glass with the same size as the tissue, and sealing the edge by using rubber glue; the slide was placed on a hybridization apparatus, co-denatured at 85℃for 5min, and hybridized overnight at 37℃to obtain hybridized sections.
2.0 washing, counterstaining and viewing
Removing rubber on the hybridized sections, immersing the sections in 2 XSSC buffer (available from Shanghai Inset technology Co., ltd.) for about 5s, and gently removing the cover sheet with forceps; the sections were immersed in 2 XSSC buffer (available from Shanghai Charpy technologies Co., ltd.) preheated at 37℃for 40min; taking out the slices, immersing in 2 XSSC buffer (available from Shanghai Ind. Co., ltd.) preheated to 60℃for 10min, and washing in 0.1% NP-40/2 XSSC solution (available from Shanghai Ind. Co., ltd.) for 5min; taking out the slices, and soaking in 75% ethanol for 3min; 10 μl DAPI counterstain (purchased from Shanghai Inset technology Co., ltd., fluorescent dye) was dropped onto the hybridization area (i.e., the liquid center area on the slide glass) and immediately covered with a cover slip; after standing in the dark for 10min, the mixture was observed with a fluorescence microscope. The results are shown in FIGS. 1-2.
In the figure, the fluorescent spots are labeled with two fluorescent groups, cy5 and Cy3, cy5 is red, cy3 is green, and both fluorescent group signals are yellow when output.
Because the Argonaute protein and the 3'tRF-ArgCCT complex have the effect of promoting the proliferation of the prostate cancer cells, if the fluorescence in situ hybridization detection value is reduced in the second detection based on the fluorescence in situ hybridization detection value in the blood of the first organism, the increase of the Argonaute protein and the 3' tRF-ArgCCT complex represents the increase of the proliferation level of the prostate cancer cells, and the like; if the fluorescence in situ hybridization detection value is increased at the time of the second detection, the decrease of Argonaute protein and 3' tRF-ArgCCT complex is indicated, representing the decrease of the proliferation level of the prostate cancer cells, and so on.
Because the 3'tRF-ArgCCT in blood is easy to degrade, and the Argonaute protein and the 3' tRF-ArgCCT complex are more stable and can reflect biological effects, compared with the existing method for simply detecting the content of the 3'tRF-ArgCCT in blood, the method for detecting the Argonaute protein and the 3' tRF-ArgCCT complex in blood has more practical significance.
Meanwhile, the probe for detecting the Argonaute protein and the 3' tRF-ArgCCT complex is optimized to 15 bases, and has higher sensitivity and specificity as shown in SEQ ID No. 2.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (4)
1. A biomarker for detecting prostate cancer, which is characterized in that the nucleotide sequence of the biomarker is shown as SEQ ID No. 1.
2. Use of a reagent for detecting a biomarker according to claim 1 in the manufacture of a kit for detecting prostate cancer.
3. The use according to claim 2, wherein the kit comprises a probe for detecting the biomarker of claim 1, the nucleotide sequence of the probe being shown in SEQ ID No. 2.
4.A kit for detecting prostate cancer, comprising a probe for detecting the biomarker of claim 1, wherein the nucleotide sequence of the probe is shown in SEQ ID No. 2.
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2023
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