CN117551675A - Biomarker, molecular probe and kit for detecting prostate cancer - Google Patents
Biomarker, molecular probe and kit for detecting prostate cancer Download PDFInfo
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Abstract
The invention relates to the technical field of molecular biology detection, in particular to a biomarker, a molecular probe and a kit for detecting prostate cancer. 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 judges the proliferation level of the prostate cancer cells by detecting the relative expression level of Argonaute protein and 3' tRF-mtProTGG complex in blood. The 3'tRF-mtProTGG in blood is easy to degrade, and the complex of Argonaute protein and 3' tRF-mtProTGG is more stable, so that the biological effect of the complex can be reflected. The invention also provides a molecular probe which has higher sensitivity and specificity.
Description
Technical Field
The invention relates to the technical field of molecular biology detection, in particular to a biomarker, a molecular probe and a kit for detecting prostate cancer.
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 Current Medicinal 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.
Disclosure of Invention
The invention aims to provide a biomarker for detecting prostate cancer, a molecular probe and a kit, wherein the biomarker has higher stability, and the molecular probe has higher sensitivity and specificity.
In order to achieve the above object, 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.
Preferably, the nucleotide sequence of the biomarker includes a nucleotide sequence encoding an Argonaute protein and a nucleotide sequence encoding a 3' tRF-mtProTGG.
The invention provides application of the biomarker in preparation of a kit for detecting prostate cancer.
The invention also provides a kit for detecting the prostate cancer, which comprises a reagent for detecting the biomarker.
The invention also provides a molecular probe for detecting the prostate cancer, and the nucleotide sequence of the molecular probe is shown as SEQ ID NO. 2;
the nucleotide sequence of the molecular probe is 27 th to 60 th positions of the biomarker nucleotide sequence.
The invention also provides application of the molecular probe in preparing a kit for detecting prostate cancer.
The invention also provides a kit for detecting the prostate cancer, which comprises the molecular probe.
The invention has the beneficial effects that:
the invention provides a biomarker, a molecular probe and a kit for detecting prostate cancer. Compared with the existing method for simply detecting the 3' tRF-mtProTGG content in blood, the method has more practical significance. The 3'tRF-mtProTGG in blood is easy to degrade, and the complex of Argonaute protein and 3' tRF-mtProTGG is more stable, so that the biological effect of the complex can be reflected. Meanwhile, the probe for detecting the complex of Argonaute protein and 3' tRF-mtProTGG is optimized to 34 nucleotide GGGTCAGAGAAAAAGTCTTTAACTCCACCATTAG sequence, so that the probe has higher sensitivity and specificity.
Drawings
FIG. 1 shows the expression of Argonaute protein and tRNA complex in blood of a healthy person;
FIG. 2 shows the expression of Argonaute protein and tRNA complex in blood of a prostate cancer patient;
FIG. 3 shows the differential expression of Argonaute protein and tRNA complex in blood of healthy people and prostate cancer patients.
Detailed Description
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 screening of complexes
1 prostate patient and 1 healthy volunteer were diagnosed in the auxiliary hospital of Yangzhou university of 2023, and 10mL of blood was drawn from each of the prostate patient and healthy volunteer, and sent to a digital spectrum (Shanghai) biotechnology limited test sample for tRNAu-argonaute protein complex (contract number 2304008) (see FIGS. 1-2). FIG. 1 shows the expression of the Argonaute protein-tRNA complex in blood of a healthy person, and FIG. 2 shows the expression of the Argonaute protein-tRNA complex in blood of a prostate cancer patient.
Analysis of the differences in the expression of Argonaute protein and tRNA complex in healthy and prostate cancer patients gave 508 up-regulated and 418 down-regulated (as shown in FIG. 3), on which the complex of the Argonaute protein with the greatest difference in up-regulated expression, namely the complex of Argonaute protein and 3' tRF-mtProTGG, was manually screened.
Example 2 preparation of molecular probes
The junction of the 3' tRF-mtProTGG and Argonaute protein has 60 nucleotide fragments, and the 60 nucleotide fragments are used as biomarkers, specifically SEQ ID NO.1: GGGCGGGCGCCAAAAGGCGCCCGCCCGGGTCAGAGAAAAAGTCTTTA ACTCCACCATTAG, the 27 th to 60 th nucleotide sequence (34 th nucleotide sequence) is used as a detection probe, and the sequence of the detection probe is SEQ ID NO.2: GGGTCAGAGAAAAAGTCTTTAACTCCACCATTAG. This nucleotide sequence GGGTCAGAGAAAAAGTCTTTAACTCCACCATTAG was supplied to the Windd bioengineering Co., ltd (https:// www.boster.com.cn) and was made by this company as a fluorescent in situ hybridization probe.
Example 3 detection of Argonaute protein and 3' tRF-mtProTGG Complex
(1) Lysing tissue
(1) Extracting 10ml of blood of a prostate cancer patient, placing in a blood collection tube, and centrifuging at 4deg.C and 8000r/min for 20min to obtain precipitate and supernatant;
(2) after discarding the supernatant, 1ml of RIPA lysate (product No. 89900 from Siemens Fei Co.)
Fully and uniformly mixing, carrying out ice bath ultrasonic treatment for 5min, and clarifying the solution;
(3) centrifuging at 4deg.C and 8000r/min for 20min, and collecting supernatant to obtain new blood collection tube.
(2) Argonaute protein preparation
(1) Taking 50 μl of the supernatant in the step (1) (3), and incubating at 4deg.C for 3 hr;
(2) washing with 1ml NETbuffer solution (Shanghai such as Ji Biotechnology development Co., ltd.) for 4 times, washing with PBS buffer solution for 1 time, and mixing each time after washing;
(3) re-suspending with 1ml PBS buffer, taking 700 μl of RNA, measuring concentration, and reversing;
(4) centrifuging 300 μl of the solution obtained in the step (3), removing supernatant, adding 30 μl of lysate and 15 μl of 5×loading solution (both purchased from Takara doctor materials technology (Beijing) Co., ltd.) to the supernatant, incubating at 95deg.C for 10min, performing agarose gel electrophoresis on the solution after incubation, firstly taking a certain amount of agarose in a 500ml triangular flask, adding TAE buffer (purchased from Simer Feisher technology Co., ltd.), and heating in a microwave oven to dissolve the agarose completely; then add nucleic acid dye (purchased from zemoeimer technology company) and shake well. Pouring the dissolving liquid into a glue box inserted into the comb, and waiting until the glue is solidified; then lightly placing the glue into an electrophoresis tank, and completely immersing the glue 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 30min.
(3) Dyeing and decolorizing
(1) The colloid prepared in the step (2) was stained using coomassie brilliant blue staining kit (purchased from Shanghai department biotechnology Co., ltd.). Cutting the dyed colloid, placing the cut colloid in decolorizing solution (purchased from Shanghai ze leaf Biotechnology Co., ltd.) which is not passed through the colloid, and oscillating for 30min.
(2) Repeating the operation for 1 time by changing the decoloring liquid until the color of the adhesive tape becomes light to colorless, and obtaining the decoloring adhesive tape.
(4) Reduction and alkylation
(1) Adding 500 μl of acetonitrile into the decolored adhesive tape obtained in the step (3), soaking for 10min, centrifuging at 8000rpm at 27deg.C for 20min after the adhesive tape is contracted and whitened, and collecting precipitate.
(2) To the precipitate in step (1) was added 10. Mu.l of 5mmol/LTECP (tetrachlorophenol), which was left over the precipitate and incubated at 55℃for 10min.
(3) After cooling to 27℃10. Mu.l 10mmol/L indoleacetic acid was added to double the volume of the liquid in the tube and reacted in the dark for 20min.
(4) All the liquid was discarded, 500. Mu.l acetonitrile was added to the precipitate for reaction for 10min, and after shrinkage of the precipitate, the mixture was centrifuged at 8000rpm for 20min at 27℃and then all the liquid was removed.
(5) Pancreatin digestion
Add 50. Mu.l trypsin solution (1. Mu.g trypsin in 100. Mu.l ABC buffer) to the gel strip, place at 4deg.C for 30min, allow the gel strip to swell completely, place at 4deg.C for 90min, allow the trypsin solution to saturate completely with gel strip, add 20. Mu.l ABC buffer, and cleave overnight at 37deg.C. Centrifuging at 4deg.C and 8000r/min for 30min, and collecting precipitate.
(6) Cell slice preparation
The collected precipitate is fixed for 24 hours by using 95 percent ethanol, and the cell wax block is manufactured by a standardized dehydration and wax dipping process after the fixation. Slicing on a slicing machine with the thickness of 3-5 mu m, immediately baking slices at 65 ℃ for 2 hours, and dewaxing after baking the slices to obtain the slices.
(7) Pretreatment of
Immersing the slice prepared in the step (6) in 100% ethanol at room temperature for 5min, and repeating the steps; taking out the slices, and respectively immersing the slices in 80%, 75% gradient ethanol and deionized water for 3min; immersing in boiled permeabilizer for 30min; then immersing the slices into preheated pepsin working solution at 37 ℃ for digestion for 10min; taking out the slices, soaking in 2 XSSC buffer (purchased from Shanghai Inset technology Co., ltd.) and rinsing for 2 times, each time for 5min; taking out the slices, and respectively immersing the slices in 70%, 85% and 100% gradient ethanol for 3min; taking out the slice, and airing at room temperature to obtain the processed slice.
(8) Hybridization
Taking out the fluorescent in situ hybridization probe described in the example 2, standing at 25 ℃ for 5min, mixing the probe upside down, centrifuging at 8000r/min for 2min, and dripping 10 μl onto the treated slice described in the step (7). Immediately covering a cover glass which is consistent with the tissue size, and sealing the edge by using rubber glue; the slide was placed on a hybridization apparatus and co-denatured at 85℃for 5min and hybridized overnight at 37 ℃.
(9) Washing, counterstaining and observation
Taking out the hybridized slice, removing rubber on the cover slice, immersing the slice in 2 XSSC buffer (purchased from Shanghai Inset technology Co., ltd.) for 5s, and gently removing the cover slice 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 the slices in a 2 XSSC buffer (purchased from Shanghai Ind. Technology Co., ltd.) preheated at 60℃for 10min, and then immersing the slices in a 0.1% NP-40/2 XSSC solution (purchased from Shanghai Ind. Technology Co., ltd.) for 5min; taking out the slices, immersing the slices in 75% ethanol for 3min; mu.l of DAPI counterstain (purchased from Shanghai Inset technology Co., ltd.) was dropped onto the sections and immediately covered with a coverslip; after standing in the dark for 10min, the mixture was observed with a fluorescence microscope.
The Argonaute protein and 3' tRF-mtProTGG complex content in normal human blood was measured as described above.
Example 4 determination of results of Argonaute protein and 3' tRF-mtProTGG Complex
Because the Argonaute protein and 3'tRF-mtProTGG complex has the effect of promoting proliferation of the prostate cancer cells, the fluorescence in situ hybridization detection value is reduced when the detection is carried out for the second time and above based on the fluorescence in situ hybridization detection value in the first time, which indicates that the Argonaute protein and 3' tRF-mtProTGG complex is increased and the proliferation level of the prostate cancer cells is improved; the increase in fluorescence in situ hybridization assay values upon the second and subsequent assays indicated a decrease in the Argonaute protein and 3' tRF-mtProTGG complex and a decrease in the proliferation level of prostate cancer cells.
In summary, the invention provides a biomarker for detecting prostate cancer, a molecular probe and a kit, wherein the biomarker has higher stability, and the molecular probe has higher sensitivity and specificity.
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 (7)
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. The biomarker of claim 1, wherein the nucleotide sequence of the biomarker comprises a nucleotide sequence encoding an Argonaute protein and a nucleotide sequence encoding a 3' trf-mtProTGG.
3. Use of a biomarker according to claim 1 or 2 in the manufacture of a kit for detecting prostate cancer.
4. A kit for detecting prostate cancer, comprising reagents for detecting the biomarker of any one of claims 1 to 2.
5. A molecular probe for detecting prostate cancer, which is characterized in that the nucleotide sequence of the molecular probe is shown as SEQ ID NO. 2;
the nucleotide sequence of the molecular probe is 27-60 positions of the biomarker nucleotide sequence of claim 1.
6. The use of the molecular probe according to claim 5 for preparing a kit for detecting prostate cancer.
7. A kit for detecting prostate cancer, comprising the molecular probe of claim 5.
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| CN202311502832.9A CN117551675A (en) | 2023-11-10 | 2023-11-10 | Biomarker, molecular probe and kit for detecting prostate cancer |
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