CN107828779B - Prostate cancer specific exosome, lncRNA, preparation method and application thereof - Google Patents
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Abstract
The invention discloses a preparation method of a prostate cancer specific exosome. The invention also discloses a prostate cancer specific exosome and application thereof. The invention also discloses a preparation method of the long-chain non-coding RNA SAP30L-AS1 and SChLAP1 in the prostate cancer exosome. The invention also discloses application of the reagent of the expression levels of the long-chain non-coding RNA SAP30L-AS1 and the SChLAP1 in the prostate cancer exosome in preparing a reagent for diagnosing the prostate cancer. The invention also discloses a prostate cancer diagnostic kit. The invention can successfully separate the prostate cancer specific exosome in the plasma of a prostate cancer patient and detect the long-chain non-coding RNA SAP30L-AS1 and SChLAP 1. The invention has obviously improved diagnosis efficiency, sensitivity and specificity.
Description
Technical Field
The invention belongs to the field of biotechnology and tumor molecular biotechnology, and particularly relates to a prostate cancer specific exosome, lncRNA, a preparation method and application thereof, in particular to a kit for separating tumor-derived exosome in plasma of a prostate cancer patient by using a method of coupling double antibodies with immunomagnetic beads, and taking lncRNA SAP30L-AS1 and lncRNA SChLAP1 thereof AS prostate cancer diagnosis markers and for early diagnosis of prostate cancer.
Background
According to the latest analysis of cancer statistics, prostate cancer (PCa) is one of the leading causes of cancer death in men worldwide, in 2016, new cases of prostate cancer in the United states account for about 21% of the number of newly diagnosed cancer patients, and the incidence of prostate cancer in our country is also rapidly rising. The primary treatment for prostate cancer at early stages is castration, but progresses to Castration Resistant Prostate Cancer (CRPC) at later stages with median survival only about 2 to 3 months. Currently, plasma quantitative detection of Prostate Specific Antigen (PSA) allows diagnosis of a large number of prostate cancer patients, but PSA, due to its prostate tissue specificity, is elevated in certain benign conditions such as Benign Prostatic Hyperplasia (BPH) and prostatitis, resulting in a decrease in specificity, and false positives cause unnecessary invasive biopsy and over-treatment of the patient. Therefore, the search for molecular markers for early diagnosis of prostate cancer is the focus of prostate cancer research.
Exosomes are vesicles of 30 to 100nm that are secreted by various cells and can be transferred among cells of various tissues, are present in various body fluids such as blood, urine, saliva, and milk, and contain lipids, specific proteins derived from donor cells, and non-coding RNAs and DNAs such as longnon-coding RNAs and micrornas. Long non-coding RNA (lncRNA) is a transcript greater than 200 nucleotides in length that has no protein coding ability. With the progress of cancer transcriptome research, lncRNA is found to play an important role in human gene transcription, post-transcriptional regulation, cell growth, proliferation, differentiation and epigenetics, has complex biological functions, and is closely related to the occurrence, development, diagnosis and treatment of diseases. The exosome can provide protection for extracellular nucleic acid and can be used as an effective carrier to transfer the exosome into a target cell, and the exosome derived from the tumor plays an important role in the transmission of the genetic information of the tumor due to high RNA content, regulates and controls the cell function and the tumor microenvironment, and influences the occurrence, development and metastasis of the tumor. Previous studies have shown that lncRNA-21A may affect cell proliferation by regulating the expression of centromere protein F (CENP-F) in breast cancer, and the invasion capacity of cancer cells is reduced after the breast cancer cells phagocytose exosomes rich in lncRNA-21A; in liver cancer, exosome-derived lncRNA ROR can promote the survival of liver cancer cells in an ischemic environment and also plays an important role in resistance to chemotherapy; in gastric cancer, lnc00152 is mainly present in exosomes in plasma, and lnc00152 is more diagnostic than carcinoembryonic antigen and cai 99, which are traditional biological markers. Therefore, the specific lncRNA marker in the exosome derived from the tumor cell can become a high-specificity index for tumor diagnosis and detection, can overcome the interference of non-detection substances in the detection process of directly extracting RNA from blood, can suggest the tumor development condition and the invasion degree, and makes the diagnosis and follow-up based on exosome easier by detecting the condition of exosome in body fluid, particularly peripheral blood.
The exosome is usually separated by ultracentrifugation, magnetic bead immunocapture, or precipitation. Ultracentrifugation is time and labor consuming, requiring a large number of initial samples; the magnetic bead immunocapture method has high specificity and convenient operation, but the current method based on the marker (CD63 and CD9 protein) of the exosome cannot reflect the specificity of the tumor; the precipitation method has short time and strong feasibility, but has poor specificity of the exosome and low purity.
At present, the diagnosis of prostate cancer mainly depends on clinical signs, screening of digital rectal examination and PSA detection, ultrasonic imaging, prostate puncture biopsy and the like, and invasive examination undoubtedly brings pain to patients, and risks of tissue heterogeneity, infection and the like exist. The analysis of disease by blood-based liquid biopsy methods has become a popular trend in recent years, and currently mainly involves the detection of plasma free DNA, tumor platelets, exosomes and circulating tumor cells. With the intensive research on the nucleic acid components in the exosome, the gate of taking lncRNA in the exosome as a brand-new marker for tumor diagnosis and treatment is opened, but the prior art also lacks the technical application of lncRNA in the exosome specific to prostate cancer tumor to prostate cancer diagnosis.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to solve the technical problem of providing a preparation method of a prostate cancer specific exosome.
The invention also aims to solve the technical problem of providing a prostate cancer specific exosome.
The invention also aims to solve the technical problem of providing a preparation method of long-chain non-coding RNASAP30L-AS1 and SChLAP1 in the prostate cancer exosome.
The invention also aims to solve the technical problem of providing the application of a reagent with long-chain non-coding RNASAP30L-AS1 and SChLAP1 expression levels in prostate cancer specific exosomes in preparing a reagent for diagnosing prostate cancer.
The invention finally solves the technical problem of providing a prostate cancer diagnosis kit.
The technical scheme is as follows: in order to solve the technical problems, the invention provides a preparation method of a prostate cancer specific exosome, which comprises the following steps:
1) blood treatment: centrifuging the plasma, and taking the supernatant for later use;
2) separation of total plasma exosomes: extracting total plasma exosomes according to the Invitrogen (TM) plasma exosome extraction kit instructions;
3) preparing double-antibody coupling immunomagnetic beads: washing the magnetic beads with a phosphate buffer solution, and then resuspending the magnetic beads with the phosphate buffer solution to obtain a suspension; adding EpCAM and PSMA antibodies into the suspension for incubation; separating the magnetic beads by using a magnetic frame after incubation, cleaning the magnetic beads by using a phosphate buffer solution, and resuspending the separated magnetic beads by using the phosphate buffer solution to obtain a suspension for later use; the magnetic frame is used for separating magnetic particles, and the separation method comprises the steps of placing an EP (EP) tube on the magnetic frame, standing for 2-3min, and clarifying liquid after magnetic beads are completely attached to the wall;
4) and (3) magnetic bead specific capture: separating the suspension obtained in the step 3) by using a magnetic frame to obtain double-antibody coupled immunomagnetic beads, and adding the total plasma exosomes obtained in the step 2) into the double-antibody coupled immunomagnetic beads for incubation;
5) preparation of prostate cancer specific exosomes: and after incubation is finished, washing the mixture by using a phosphate buffer solution, and separating the magnetic beads by using a magnetic frame again to obtain the magnetic bead-diabody-plasma prostate cancer specific exosome compound, namely the prostate cancer specific exosome.
Wherein the magnetic beads are streptomycin affinity modified magnetic beads, and the diameter of the magnetic beads is 2.8 mu m.
Wherein the EpCAM and PSMA antibodies are biotin-labeled EpCAM and PSMA antibodies.
The invention also provides the prostate cancer specific exosome prepared by the method.
The invention also discloses a preparation method of the long-chain non-coding RNA SAP30L-AS1 and SChLAP1 in the prostate cancer exosome, which comprises the following steps:
1) extraction of exosome RNA: the exosome RNA is extracted according to the method of the steps required by the RNAiso Blood reagent instruction manual of TaKaRa company;
2) synthesizing cDNA through reverse transcription of exosome RNA: reverse transcription is carried out on RNA in the exosome in the step 1) by adopting a TOYOBO kit ReverTra Ace qPCR RT kit to synthesize cDNA;
3) amplification of long non-coding RNA: using TOYOBO
The Green Realtime PCR MasterMix reagent is subjected to real-time fluorescent quantitative PCR amplification by taking the cDNA reverse transcribed in the step 2) AS a template according to the operation steps specified in the instruction book to obtain long-chain non-coding RNA SAP30L-AS1 and SChLAP 1.
Wherein, the amplification primer of the real-time fluorescence quantitative PCR is as follows:
SAP30L-AS1 forward primer: 5'-TGAATGGGCTCACCTGTTCC-3'
SAP30L-AS1 reverse primer: 5'-AGGTCCGGAAGGGAGACTTT-3'
Forward primer of SChLAP 1: 5'-TGGACACAATTTCAAGTCCTCA-3'
Reverse primer of SChLAP 1: 5'-CATGGTGAAAGTGCCTTATACA-3' are provided.
The invention also comprises the application of the reagent of the expression quantity of the long-chain non-coding RNA SAP30L-AS1 and the SChLAP1 in the exosome in preparing the reagent for diagnosing the prostatic cancer.
Wherein, the reagent for detecting the expression quantity of the long-chain non-coding RNA SAP30L-AS1 and the SChLAP1 is a real-time fluorescent quantitative PCR detection reagent.
The real-time fluorescent quantitative PCR detection reagent comprises a detection primer which is designed and synthesized according to the nucleotide sequences of long-chain non-coding RNA SAP30L-AS1 and SChLAP1 and is specifically used for real-time fluorescent quantitative PCR:
SAP30L-AS1 forward primer: 5'-TGAATGGGCTCACCTGTTCC-3'
SAP30L-AS1 reverse primer: 5'-AGGTCCGGAAGGGAGACTTT-3'
Forward primer of SChLAP 1: 5'-TGGACACAATTTCAAGTCCTCA-3'
Reverse primer of SChLAP 1: 5'-CATGGTGAAAGTGCCTTATACA-3' are provided.
The invention also comprises a prostate cancer diagnosis kit, wherein the kit comprises the double-antibody coupling immunomagnetic beads and the detection primer of the real-time fluorescence quantitative PCR.
The detection kit also comprises a total exosome extraction reagent, a reverse transcription reagent and an RT-qPCR reagent.
Has the advantages that: compared with the prior art, the invention has the following advantages: the invention can successfully separate the plasma specific exosome of the prostate cancer patient and detect the long-chain non-coding RNA SAP30L-AS1 and SChLAP 1. The long-chain non-coding RNA SAP30L-AS1 and SChLAP1 in the prostate cancer patient are obviously increased, and the long-chain non-coding RNA SAP30L-AS1 is obviously increased in the prostate cancer patient at the early stage and is related to the progression of the prostate cancer; the long-chain non-coding RNA SChLAP1 is related to the Gleason score of the patient, can reflect the development of the disease, and can effectively distinguish the patients with prostatic hyperplasia and prostatic cancer in the diagnosis gray zone with the PSA concentration of 4-10 ng. The AUC (area underserve) value of the prostate cancer jointly diagnosed by the two long-chain non-coding RNAs is 0.922 (95% CI: 0.852-0.992), the AUC value of the prostate cancer jointly diagnosed by the classical marker PSA is 0.972 (95% CI: 0.918-0.99) and 0.952 (95% CI: 0.887-0.99), the sensitivity and the specificity are obviously improved, and therefore, the long-chain non-coding RNAs can be used as a marker for judging the diagnosis and the progress of the prostate cancer.
Drawings
FIG. 1 is the isolation and identification of plasma prostate cancer specific exosomes of the present invention; A. morphology and size of total exosomes in plasma as observed by transmission electron microscopy (Bar 200 nm); B. detecting the total exosome particle size and particle size distribution of plasma by Dynamic Light Scattering (DLS); western Blot to detect the expression of the exosome marker proteins CD63 and TSG101 in total plasma exosomes; detecting the expression of a prostate cancer specific exosome CD63 in plasma of a normal human and a prostate cancer patient separated by double antibody coupled immunomagnetic beads by Western Blot; E. expression conditions of long-chain non-coding RNA SAP30L-AS1 and SChLAP1 in the total plasma exosomes and the prostate cancer specific exosomes separated by a double-antibody coupling immunomagnetic bead method; n represents Normal control group, namely Normal, B represents prostatic hyperplasia group, namely BPH, P represents prostatic cancer patient group, namely PCa; N1-N4 represent the test results of different samples of a normal control group, B1-B3 represent the test results of different samples of a prostate hyperplasia group, and the test results of different samples of a P1-P5 prostate cancer patient group;
FIG. 2 real-time fluorescent quantitative PCR product detection of SAP30L-AS1 and SChLAP1 involved in the present invention; the sizes of the obtained products are respectively about 159bp and 88bp, and are consistent with the size of the product fragment predicted when the primer is designed; lane 1 is DNA maker: lowladder, the molecular weight is 20-700 bp; lanes 2-5 are fluorescent quantitative PCR product fragments of SChLAP 1; lanes 6-9 are fluorescent quantitative PCR product fragments of SAP30L-AS 1;
FIG. 3 shows the expression levels of SAP30L-AS1 and SChLAP1 in the plasma prostate cancer-specific exosomes of the present invention; Normal-Normal group; BPH-prostatic hyperplasia group; PCa-prostate cancer group; relative expression 2-ΔΔCtRepresenting, comparison between groups by non-parametric test, P<0.05 represents that the difference is statistically significant; a: table of target gene SAP30L-AS1 in plasma prostate cancer specific exosomesTo the situation; b: expression of target gene SChLAP1 in plasma prostate cancer specific exosome;
FIG. 4 ROC plot of the objective genes SAP30L-AS1 and SChLAP1 in the plasma prostate cancer specific exosomes of the present invention for diagnosing prostate cancer; Sensitivity-Sensitivity, specificity-specificity; a: ROC curve for SAP30L-AS1 in plasma prostate cancer specific exosomes; b: ROC curve for SChLAP1 in plasma prostate cancer specific exosomes for prostate cancer; c: ROC curves for combined diagnosis of prostate cancer with SAP30L-AS1 and SChLAP1 in plasma prostate cancer specific exosomes; ROC curve of combined diagnosis of SAP30L-AS1 and PSA in plasma prostate cancer specific exosomes; e: ROC curves for combined diagnosis of SChLAP1 and PSA in plasma prostate cancer specific exosomes;
FIG. 5 is a graph of analysis of the relationship between the objective genes SAP30L-AS1 and SChLAP1 and clinical pathological parameters in the plasma prostate cancer-specific exosomes of the present invention; a: the relation analysis chart of SAP30L-AS1 in plasma prostate cancer specific exosome and T (the condition of tumor primary focus), wherein T represents the condition of tumor primary focus in the tumor stage, T2 represents that the tumor is limited in the prostate, T3 and T4 represent the conditions of breaking through the prostate and invading other tissues, T2 represents non-invasion, T3+ T4 represents invasion, and the disease progress is explained; b: map of SChLAP1 versus Gleason score (biological behavior and prognosis) in plasma prostate cancer specific exosomes; c: expression of SChLAP1 in plasma prostate cancer-specific exosomes at blood PSA concentrations in the detection grey zone of 4-10 ng/ml; when the PSA is 4-10ng/ml, the BPH and PCa can be distinguished;
when the Gleason score is less than 7, the cancer is equivalent to high-differentiation adenocarcinoma, and when the Gleason score is more than or equal to 7, the cancer is equivalent to low-differentiation adenocarcinoma, the prognosis of the disease can be reflected, and the lower the differentiation is, the higher the malignancy is.
Detailed Description
For a further understanding of the invention, its features and advantages are further described by reference to the embodiments and the drawings. This example is merely illustrative of the present invention and does not limit the remainder of the disclosure in any way.
Example 1:
plasma samples were collected for a total of 110 cases, 34 histopathologically confirmed prostate cancer patient plasmas, 46 prostate hyperplasia patient plasmas and 30 healthy control patient plasmas. All plasma samples were obtained from the university of wuhan, central and south hospital, with hemolysis, lipemia and other abnormal sera excluded.
The specific detection steps are as follows:
1. blood sample pretreatment and preservation
Collecting venous blood of the above subjects in EDTA anticoagulation tubes respectively, standing, sucking upper plasma in EP tubes, centrifuging at room temperature of 2000g for 20min, collecting supernatant in new EP tubes, centrifuging at room temperature of 10000g for 20min, sucking supernatant in enzyme-free EP tubes, and storing at-80 deg.C for use.
2. Plasma Total exosome extraction and storage (Total exosome isolation Kit (cat # 4484450, Invitrogen)
If the plasma sample is in a frozen state, a water bath at 37 ℃ is needed, and the plasma sample is placed on ice for standby after being unfrozen. Taking 400 mu l of a plasma sample, adding 200 mu l of PBS, fully mixing, adding 20 mu l of proteinase K, incubating for 10min at 37 ℃, adding 120 mu l of exosome precipitation Reagent, fully mixing, incubating for 30min at 4 ℃, centrifuging for 5min at 10000g at room temperature, discarding supernatant, centrifuging for 30s at 10000g, sucking out residual liquid, obtaining a precipitate as total plasma exosomes, adding 30 mu l of PBS, re-suspending, and storing at-80 ℃.
3. Transmission electron microscope observation of total exosome form of plasma
Dropping 1 drop of plasma total exosome suspension on a carbon-containing copper net, drying, adding 1 drop of 2% phosphotungstic acid, standing for 5min, removing redundant liquid by using filter paper, adding 1 drop of PBS, rinsing for 1min, removing redundant liquid by using filter paper, cleaning for 3 times totally, and drying. The morphology of total exosomes in plasma was observed by transmission electron microscopy at 220KV voltage, and the results are shown in fig. 1A.
4. Dynamic light scattering study
For the research on the particle size distribution of the exosome, a dynamic light scattering method is adopted for detection. Here, an experiment was performed using a Zetasizer Nano-ZS90 instrument, malvern, england, with an excitation light wavelength λ of 532 nm. The total plasma exosome samples were diluted to appropriate optical signal detection levels (i.e., ratio of 1: 50) with PBS, and detected after mixing, with the detection results shown in fig. 1B.
5. Western Blot detection of exosome marker protein
Plasma total exosomes were lysed with cell lysis buffer RIPA, lysed on ice for 30min, centrifuged at 12000g for 20min, the supernatant was collected, protein concentration was determined by BCA method, then 1 μ g of protein was taken, separated by 12% SDS-PAGE, wet-transferred to PVDF membrane, PVDF membrane was blocked with 5% skim milk, incubated with anti-CD 63 and TSG101 rabbit polyclonal antibody (1:1000 dilution) overnight at 4 ℃, TBST washed, incubated with horseradish peroxidase-labeled goat anti-rabbit IgG for 2h at room temperature, treated with persistent chemiluminescent substrate reagent and exposed to light, as shown in fig. 1C.
6. Prostate cancer specific exosome capture by double antibody coupled magnetic beads
1) Streptavidin-modified Dynabeads TMM-280Streptavidin 2.8 μm magnetic beads were vortexed uniformly by a vortexer, 20 μ l of the magnetic beads were pipetted and added to 200 μ l of phosphate buffer (1 Xphosphate buffer, pH 7.4), and the mixture was washed by suspension, and the magnetic beads were separated by a magnetic frame and washed three times in total. 200. mu.l of phosphate buffer (1 Xphosphate buffer, pH 7.4) was added for resuspension;
2) adding 4 μ l of biotinylated anti-human EpCAM monoclonal antibody (R & DSystems, USA) and biotinylated anti-human PSMA monoclonal antibody (BioLegend, USA) into the suspension obtained in the step 1), mixing by inversion, and incubating for 2h (the mixing angle is 0 DEG, the speed is 30rpm) in MX-RD-Pro rotary mixer; after incubation, the beads were separated with a magnetic frame, washed with 400. mu.l of phosphate buffer (1 Xphosphate buffer, pH 7.4), and the separated beads were resuspended in 200. mu.l of phosphate buffer (1 Xphosphate buffer, pH 7.4) for further use;
3) separating the magnetic beads in the step 2) by using a magnetic frame, adding the extracted total plasma exosomes into the magnetic beads separated in the step 2), uniformly mixing, and incubating overnight (the parameter is mixing angle 0 DEG and speed is 30rpm) on a MX-RD-Pro rotary uniform mixing instrument under the condition of 4 ℃. After the incubation is finished, separating the magnetic beads by using a magnetic frame, washing the magnetic beads for three times by using 400 mu l of phosphate buffer (1 x phosphate buffer, the pH value is 7.4), and separating to obtain the magnetic bead-diabody-plasma prostate cancer specific exosome compound.
7. Western Blot detection of prostate cancer specific exosome CD63 expression
2 normal control groups and 5 prostate cancer group plasma samples were taken, prostate cancer specific exosomes were obtained by the method of step 6, and detection was performed by the method of step 5, and the results are shown in fig. 1D.
8. Plasma exosome RNA extraction and preservation
1) Adding 500 μ l of RNAlso Blood (TaKaRa) into the plasma exosome (suspension), blowing to crack, and standing for 5 min;
2) adding 100 μ l chloroform, covering the EP tube cover tightly, shaking for 15s, and standing at room temperature for 10 min;
3) centrifuging at 12000g for 15min at 4 deg.C, and collecting supernatant to new enzyme-free EP tube;
4) adding isopropanol with the same volume, tightly covering an EP tube cover, and oscillating for 15s and incubating for 10min at room temperature;
5) centrifuging at 12000g for 10min at 4 ℃, and removing supernatant;
6) adding 1ml of 75% ethanol prepared by RNase free water, reversing and mixing evenly;
7) centrifuging at 12000g for 5min, and removing supernatant;
8) repeating steps 6) and 7) once;
9) centrifuging at 12000g for 30s at 4 ℃, and carefully sucking out residual liquid in an EP tube;
10) after air-drying at room temperature, 10. mu.l of RNase free water in a water bath at 65 ℃ was added, and after sufficient dissolution, the total RNA concentration and purity were measured and stored at-80 ℃.
9. cDNA Synthesis (ReverTra Ace qPCR RT kit, TOYOBO)
1) The total RNA of step 8 was denatured at 65 ℃ for 5min and immediately cooled on ice (to destroy the higher structure of RNA);
2) taking total RNA as a template and Primer mix (oligo dT and Random Primer, which can be directly used for reverse transcription of mRNA and non-coding RNA) as a reverse transcription Primer, the reaction system is as follows:
3) the experiment was carried out under the following temperature conditions: 15min at 37 ℃; at 98 ℃ for 5 min; storing at-20 deg.C for use.
10. Real-time fluorescent quantitative PCR (
Green Realtime PCR Master Mix，TOYOBO)
The reaction system is as follows:
the reaction conditions are as follows: at 95 ℃ for 1 min; (95 ℃, 15 s; 58.8 ℃, 20 s; 72 ℃, 45 s). times.40 cycles; fluorescence signals were collected at 72 ℃ for 2min, set at 72 ℃ for 45 s.
The primer sequences are as follows:
SAP30L-AS1 forward primer: 5'-TGAATGGGCTCACCTGTTCC-3'
SAP30L-AS1 reverse primer: 5'-AGGTCCGGAAGGGAGACTTT-3'
Forward primer of SChLAP 1: 5'-TGGACACAATTTCAAGTCCTCA-3'
Reverse primer of SChLAP 1: 5'-CATGGTGAAAGTGCCTTATACA-3'
The real-time fluorescent quantitative PCR products of SAP30L-AS1 and SChLAP1 were identified by 3% agarose (Biowest, Spain) gel electrophoresis, respectively, the electrophoretogram of which is shown in FIG. 2; the sizes of the obtained products are respectively about 159bp and 88bp, and are consistent with the sizes of the product fragments predicted when the primers are designed.
Analysis of the results of this example:
1) western Blot analysis for detecting expression level of prostate cancer specific exosome CD63
As shown in fig. 1D, when the method for capturing prostate cancer-specific exosomes is performed by using diabody-coupled magnetic beads, the expression of CD63 in the normal control group is significantly lower than that in the prostate cancer group, which indicates that prostate cancer-specific exosomes can be captured in the normal control group, whereas prostate cancer-specific exosomes can be captured in the prostate cancer group, and the capturing method is effective.
2) Analysis of expression levels of the target genes SAP30L-AS1 and SChLAP1
As shown in the results of FIG. 1E, the expression level obtained by real-time fluorescence quantitative PCR shows that the expression of the target genes SAP30L-AS1 and SChLAP1 is significantly different between total plasma exosomes and prostate cancer specific exosomes, and the expression is enriched. By using 2-ΔΔCtAnalyzing quantitative data of two target genes, wherein the delta Ct is the Ct value of the target gene-the Ct of the reference gene, the delta Ct is the delta Ct value of the target gene-the delta Ct of a control group gene, the Ct value of the target gene is the Ct values of the target genes SAP30L-AS1 and SChLAP1 detected by a real-time fluorescence quantitative technology, and the result is shown in figure 3, and the result is Normal-Normal human group; BPH-prostatic hyperplasia group; PCa-prostate cancer group; the expression levels of the target genes SAP30L-AS1 and SChLAP1 in prostate cancer specific exosomes of prostate cancer patients are higher than those of healthy control groups, and the difference is significant; compared with the patients with prostatic hyperplasia, the medicine also has significant difference.
3) ROC diagnostic efficacy analysis:
ROC analysis of the diagnostic value of target genes SAP30L-AS1 and SChLAP1 in exosomes in prostate cancer; the results are shown in fig. 4 and table 1, the diagnosis efficacy of SChLAP1 in exosomes is good, and the sensitivity is high; when the two LncRNAs are jointly diagnosed, the diagnostic efficiency is obviously higher than that of the single LncRNA, and the sensitivity and the specificity are both higher; when the two are respectively combined with PSA for diagnosis, the diagnosis efficiency, sensitivity and specificity are all obviously improved, which shows that the combination of blood PSA concentration detection and SAP30L-AS1 and SChLAP1 detection can better diagnose the prostate cancer. PSA, a classic diagnostic marker for prostate cancer, has a high false positive rate and also increases levels during prostate hyperplasia, thus leading to the development of over-treatment. The SAP30L-AS1 and SChLAP1 molecules of the invention can optimize the diagnosis of prostate cancer and have a differential diagnosis of BPH and PCa at the same time.
TABLE 1 sensitivity, specificity of various diagnostics
4) Relationship analysis of LncRNA and clinical and pathological parameters:
the relationship between SAP30L-AS1 and SChLAP1 and age, PSA level, T (in the case of primary tumor foci), N (in the case of regional lymph node involvement), M (in the case of distant metastasis) and Gleason scores in the TNM staging were analyzed by non-parametric tests, and FIG. 5 shows the results of the analysis of differences in expression using 2-ΔΔCtShowing Relative expression, T showing the condition of the primary focus of the tumor; the results show that SAP30L-AS1 is negatively associated with T of prostate cancer, with SAP30L-AS1 expression being lower AS the tumor primary foci progress; and the SChLAP1 is positively correlated with the Gleason score of the prostate cancer, can effectively distinguish the prostate hyperplasia from the prostate cancer when the PSA diagnoses the gray area, and has obviously increased expression in cancer tissues.
Example 2 preparation of Long-chain non-coding RNASAP30L-AS1 and SChLAP1 kit for diagnosis of prostate cancer patients
1. Real-time fluorescent quantitative PCR specific primer sequence
SAP30L-AS1 forward primer: 5'-TGAATGGGCTCACCTGTTCC-3'
SAP30L-AS1 reverse primer: 5'-AGGTCCGGAAGGGAGACTTT-3'
Forward primer of SChLAP 1: 5'-TGGACACAATTTCAAGTCCTCA-3'
Reverse primer of SChLAP 1: 5'-CATGGTGAAAGTGCCTTATACA-3'
2. Double antibody-coupled immunomagnetic beads prepared in example 1
3. Total exosome extraction reagent
4. Reverse transcription reagent
5. RT-qPCR reagent
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
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Claims (6)
1. A method for detecting the expression levels of long non-coding RNA SAP30L-AS1 and SChLAP1 in prostate cancer exosomes for non-disease diagnosis or treatment purposes, which comprises the following steps: 1) extracting exosome RNA; 2) synthesizing cDNA by reverse transcription of exosome RNA; 3) amplification of Long non-coding RNA SAP30L-AS1 and SChLAP 1: the reverse transcription cDNA is used AS a template to carry out real-time fluorescent quantitative PCR amplification respectively to obtain long-chain non-coding RNA SAP30L-AS1 and SChLAP 1.
2. The detection method according to claim 1, wherein the amplification primers of the real-time fluorescence quantitative PCR are:
SAP30L-AS1 forward primer: 5'-TGAATGGGCTCACCTGTTCC-3'
SAP30L-AS1 reverse primer: 5'-AGGTCCGGAAGGGAGACTTT-3'
Forward primer of SChLAP 1: 5'-TGGACACAATTTCAAGTCCTCA-3'
Reverse primer of SChLAP 1: 5'-CATGGTGAAAGTGCCTTATACA-3' are provided.
3. The use of the reagent of claim 1 for detecting the expression levels of long non-coding RNA SAP30L-AS1 and SChLAP1 in prostate cancer exosomes for preparing a diagnostic reagent for prostate cancer.
4. The use of claim 3, wherein the reagent for detecting the expression levels of the long non-coding RNA SAP30L-AS1 and the SChLAP1 is a real-time fluorescent quantitative PCR detection reagent.
5. A prostate cancer diagnostic kit, comprising a diabody-coupled immunomagnetic bead and the real-time fluorescent quantitative PCR amplification primer of claim 2;
the preparation method of the double-antibody coupled immunomagnetic beads comprises the following steps: washing the magnetic beads with a phosphate buffer solution, and then resuspending the magnetic beads with the phosphate buffer solution to obtain a suspension; adding EpCAM and PSMA antibodies into the suspension for incubation; separating the magnetic beads by using a magnetic frame after incubation, cleaning the magnetic beads by using a phosphate buffer solution, and resuspending the separated magnetic beads by using the phosphate buffer solution to obtain a suspension for later use; separating the suspension by a magnetic frame to obtain the double antibody coupling immunomagnetic beads.
6. The prostate cancer diagnostic kit according to claim 5, wherein the detection kit further comprises a total exosome extraction reagent, a reverse transcription reagent and a RT-qPCR reagent.
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