CN116219004A - Application of exosome HOXD-AS1 AS prognosis screening of prostate cancer - Google Patents
Application of exosome HOXD-AS1 AS prognosis screening of prostate cancer Download PDFInfo
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Abstract
The invention discloses application of exosome HOXD-AS1 AS a prognosis screening for prostate cancer. The test proves that the reagent and/or the device for detecting the exosome LncRNA HOXD-AS1 can be used for assisting in diagnosing the distant metastasis of the prostate cancer and predicting the prognosis of the prostate cancer by detecting the expression quantity of the serum exosome LncRNA HOXD-AS1. Only a small amount of serum of a patient needs to be collected for detection, the wound is small, the detection is convenient, and the sample can be stably stored.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to application of exosome HOXD-AS1 AS a prognosis screening of prostate cancer.
Background
Prostate Cancer (PCa) has increased in incidence in china year by year, and has become the most common malignancy of the male genitourinary system, severely threatening the health of older men. Early prostate cancer is primarily treated by surgery, but some patients have postoperative recurrence of the tumor in situ or distant metastasis. Some patients are already late in diagnosis and have very low probability of successful surgery.
Androgen deprivation therapy (AndrogenDeprivation Therapy, ADT) or Castration therapy (Castration) is the first line treatment of metastatic prostate cancer, and although about 90% of prostate cancer patients respond well to Castration therapy (manifested as a decline in the prostate specific antigen PSA and a tumor shrink), tumors typically no longer respond to Castration therapy after 18-24 months, developing Castration resistant prostate cancer (Castration Resistance Prostate Cancer, CRPC). Poor prognosis of castration resistant prostate cancer is a major cause of death in prostate cancer patients. Novel endocrine therapies (e.g., abiraterone) and taxoids (e.g., docetaxel) are first-line medications for metastatic castration against prostate cancer, and although such medications can alleviate symptoms and reduce PSA levels, patient prognosis is poor. Therefore, there is a need to find markers for aiding diagnosis and prognosis of prostate cancer, for aiding diagnosis and clinical decision-making of prostate cancer.
In recent years, a great deal of research has shown that exosomes can be stably present in body fluids including blood, bile and urine, among which DNA, proteins, RNA are therefore ideal liquid biopsy markers. Long non-coding RNAs (lncRNA) refer to non-coding RNA subclasses with transcripts longer than 200 nucleotides in length that do not code for proteins, and are widely involved in regulating various vital activity processes of the human body at the epigenetic level. lncRNA is abnormally expressed in various malignant tumors, and plays an important role in oncogenes or oncogene inhibitors in tumorigenesis and development through the regulation of downstream target genes. Recent researches show that the exosome lncRNA in body fluid is involved in information transmission among cells and can be used as a diagnosis and prognosis marker of various tumors such as bladder cancer, prostate cancer and the like.
In summary, although the new drug treatment improves the therapeutic effect of metastatic prostate cancer, the survival of prostate cancer patients remains to be further improved. Early diagnosis of metastatic prostate cancer, accurate evaluation of metastatic burden, and reasonable follow-up treatment are important links for improving the efficacy of metastatic prostate cancer. Therefore, there is a need to develop a novel diagnostic marker as a marker for prognosis diagnosis of prostate cancer.
Disclosure of Invention
For this reason, the present invention provides the use of reagents and/or devices for detecting the exosome LncRNA HOXD-AS1 in the preparation of a product for screening or aiding in the diagnosis of a prognosis of prostate cancer.
In order to achieve the above object, the present invention provides the following technical solutions:
the embodiment of the invention also provides application of the reagent and/or the device for detecting the exosome LncRNA HOXD-AS1 in preparing a product for screening or assisting in diagnosing prostate cancer prognosis.
In one embodiment of the invention, the LncRNA HOXD-AS1 gene has a nucleotide sequence shown in SEQ ID NO: 1.
In one embodiment of the present invention, the reagent and/or device for detecting the exosome LncRNA HOXD-AS1 comprises a reagent and/or device for detecting the exosome LncRNA HOXD-AS1 by reverse transcription-polymerase chain reaction, real-time fluorescent quantitative PCR technique, transcriptome sequencing technique, in situ hybridization technique, gene chip technique or PacBio sequencing technique.
In one embodiment of the invention, the detection reagent is a reagent for detecting the expression level of the exosome LncRNA HOXD-AS1.
In one embodiment of the present invention, the reagent for detecting the expression level of LncRNA HOXD-AS1 in exosomes comprises primers for specifically amplifying LncRNA HOXD-AS1 gene and/or probes for specifically binding LncRNA HOXD-AS1 gene.
In one embodiment of the invention, the primer comprises an upstream primer: ACCTGCCTCTACTACTGCAAA;
a downstream primer: GCAAAGACAATATAAGGGCCC.
In one embodiment of the invention, the test sample of the product is blood.
The invention has the following advantages:
the test proves that the reagent and/or the device for detecting the exosome LncRNA HOXD-AS1 can be used for assisting in diagnosing the distant metastasis of the prostate cancer and predicting the prognosis of the prostate cancer by detecting the expression quantity of the serum exosome LncRNA HOXD-AS1. Only a small amount of serum of a patient needs to be collected for detection, the wound is small, the detection is convenient, and the sample can be stably stored.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below. It will be apparent to those of ordinary skill in the art that the drawings in the following description are exemplary only and that other implementations can be obtained from the extensions of the drawings provided without inventive effort.
The structures, proportions, sizes, etc. shown in the present specification are shown only for the purposes of illustration and description, and are not intended to limit the scope of the invention, which is defined by the claims, so that any structural modifications, changes in proportions, or adjustments of sizes, which do not affect the efficacy or the achievement of the present invention, should fall within the ambit of the technical disclosure.
FIG. 1 is a schematic diagram of a patient with high tumor burden, wherein the expression of the serum exosome HOXD-AS1 is significantly higher than that of a patient with low tumor burden, or a patient with localized prostate cancer according to the embodiment of the invention;
FIG. 2 is a schematic diagram showing a significant increase in expression of the serum exosome HOXD-AS1 in a patient with lymph node metastasis provided in the examples of the present invention;
FIG. 3 is a schematic representation of a significant increase in exosome HOXD-AS1 expression in patients with higher Gleason (Gleason) scores (8-10 minutes) provided by an example of the present invention;
FIG. 4 is a graph showing the correlation between higher levels of LncRNA HOXD-AS1 and shorter PSA-free progression survival provided by examples of the present invention;
FIG. 5 is a graph showing the correlation between higher levels of LncRNA HOXD-AS1 and progression-free survival provided in the examples of the present invention.
Detailed Description
Other advantages and advantages of the present invention will become apparent to those skilled in the art from the following detailed description, which, by way of illustration, is to be read in connection with certain specific embodiments, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1 preparation of serum samples
Collecting a patient blood sample, and placing the patient blood sample in a collection tube without anticoagulant; standing at room temperature for natural solidification for 30-60 min; gently scratching the blood clot along the wall of the collection tube after blood coagulation; standing at 4deg.C overnight, balancing, and centrifuging at 4deg.C and 2000rpm for 10min; the collected supernatant is serum, and about 1ml of blood can collect 300-400 mu l of serum; immediately carrying out the next operation or preserving at minus 80 ℃.
Example 2 isolation of serum exosomes
Exosomes were isolated from human serum samples using a total exosome isolation kit (for serum) according to the manufacturer's instructions (Thermo Scientific, waltham, MA USA).
The serum samples were centrifuged at 2000g for 30min at 4℃and then 200. Mu.l of serum samples were mixed with 40. Mu.l of total exosome separation reagent and incubated for 1h at 4 ℃. The mixture was then centrifuged at 10000g for 30 minutes at room temperature. The supernatant was discarded and finally the exosome particles were resuspended in 50. Mu.l, 0.2 μm filtered phosphate buffer. Immediately carrying out the next operation or preserving at-80 ℃.
Example 3 extraction of RNA
Total RNA was extracted from exosomes using TRIzol reagent (Thermo Scientific, waltham, mass. USA).
To 50. Mu.l of the exosome sample, 1ml of TRIzol reagent was added, and the mixture was mixed upside down and incubated for 5 minutes. 0.2ml of chloroform was added and mixed with shaking, and incubated for 2-3 minutes. The mixture was centrifuged at 12000g for 15 minutes at 4 ℃. The top colorless aqueous phase was aspirated and transferred to a new centrifuge tube.
To the colorless aqueous phase was added 0.5ml of isopropanol and incubated at 4℃for 10 minutes. After that, the mixture was centrifuged at 12000g at 4℃for 10 minutes, and the supernatant was discarded.
1ml of 75% ethanol was added to the centrifuge tube to fully dissolve the pellet. Shaking and mixing, centrifuging 7500g at 4deg.C for 5 min, and discarding supernatant. The precipitate was dried at room temperature for 5-10 minutes.
To the centrifuge tube 50. Mu.l of enzyme free water was added to resuspend the pellet followed by incubation in a 55-60℃water bath or heat block for 10-15 minutes. Mu.l of sample was added to the NanoDrop 2000 spectrophotometer to measure the concentration of RNA sample. The RNA can then be used directly for the next step or stored at minus 80 ℃.
EXAMPLE 4 reverse transcription of RNA
Total RNA was reverse transcribed using PrimeScript RT Master Mix reagent (Takara Biotechnology Co., ltd., daida, china).
Mu.l of PrimeScript RT Master Mix reagent, 4. Mu.l of RNA and 4. Mu.l of enzyme-free water were mixed in a new EP tube. Then, the cDNA was synthesized by reverse transcription at 37℃for 15 minutes, 85℃for 5 seconds and 4 ℃. The nucleotide sequence of LncRNA HOXD-AS1 gene is shown AS SEQ ID NO. 1.
Real-time fluorescent quantitative polymerase chain reaction (qRT-PCR) qRT-PCR was performed using TBGreen II and Mir-X miRNA qRT-PCR TB Green kit (Takara Biotechnology Co., ltd., dai of China). Table 1 shows the preparation of PCR reaction solutions (the preparation of the reaction solutions was performed on ice) according to the following components. In view of the draw error, the premix volume is formulated to be at least 10% greater than the total volume for all reactions.
TABLE 1
The primers used for qRT-PCR are as follows:
HOXD-AS1: ACCTGCCTCTACTACTGCAAA (upstream primer)
GCAAAGACAATATAAGGGCCC (downstream primer);
GAPDH: CAAGGCTGAGAACGGGAAG (upstream primer)
TGAAGACGCCAGTGGACTC (downstream primer).
Conditions employed for qRT-PCR:
the first step: cycle number 1, (95 ℃ C. For 30 seconds).
And a second step of: cycle number 40, (95 ℃ C. For 5 seconds; 60 ℃ C. For 34 seconds).
And a third step of: cycle number 1, (95 ℃ C. For 15 seconds; 60 ℃ C. For 1 minute; 95 ℃ C. For 15 seconds)
After the PCR reaction solution was prepared, a PCR reaction was performed in a Roche Light-Cycler 480system (Roche, calif., USA), and 3 wells were run for each sample and each gene.
EXAMPLE 5 detection of serum exosome HOXD-AS1 from prostate cancer patients
This example shows the detection of the serum exosome HOXD-AS1 from 130 cases (38 cases limited, 92 cases metastatic) of prostate cancer patients. The detection results are as follows:
clinical analysis data are represented in the median of the quartile range. When the population cannot be assumed to be normally distributed, the Mann-Whitney U test is applied to the individual samples. The chi-square test of Pearson was used to analyze clinical variables. The analysis results are shown in Table 2, and Table 2 shows the relationship between the expression of the serum exosome HOXD-AS1 and the clinical pathological characteristics of the prostate cancer patients.
The Spearman correlation analysis was used to determine the correlation between the two variables. The cumulative time to live was calculated using the Kaplan-Meier method and analyzed by a log rank test. The multivariate Cox proportional hazards model is used to estimate the adjusted risk ratio and 95% confidence interval and determine independent prognostic factors. P values <0.05 are considered significant. The results of the analysis are shown in Table 3, table 3 being univariate and multivariate analysis of the survival-related factors without PSA progression in the metastatic prostate cancer cohort.
TABLE 2
P <0.05 was considered significant, and the median relative expression of the serum exosome HOXD-AS1 was used AS a cut-off for the analysis.
As shown in Table 2, the present invention found that the exosome HOXD-AS1 was significantly up-regulated in the serum exosomes of patients with metastatic prostate cancer by examining the serum exosomes HOXD-AS1 of 130 patients with metastatic prostate cancer (38 cases of limitation, 92 cases of metastatic prostate cancer). Furthermore, in patients with high tumor burden (with visceral metastasis or (and) greater than 4 bone metastasis lesions and 1 outside of the pelvis and spine), serum exosome HOXD-AS1 expression was significantly higher than in patients with low tumor burden, or localized prostate cancer patients, AS shown in fig. 1.
In addition, AS shown in FIG. 2, the present invention found that the expression of the serum exosome HOXD-AS1 was significantly elevated in patients with lymph node metastasis, and significantly elevated in patients with higher Gleason (Gleason) scores (8-10 minutes) AS shown in FIG. 3. And serum exosome HOXD-AS1 levels are significantly correlated with the Gleason (Gleason) score, lymph node status and distant metastasis status of prostate cancer patients. Table 3 shows univariate and multivariate analysis of factors associated with survival without PSA progression in a metastatic prostate cancer cohort.
TABLE 3 Table 3
Univariate and multivariate analysis as shown in table 3. Cox proportional risk regression model. The variables associated with survival in the univariate analysis were used as covariates in the multivariate analysis. Important P values are shown in bold. HR >1, increased risk of death; HR <1, risk of death is reduced. The median relative expression of the serum exosome HOXD-AS1 was used AS a cut-off for the analysis.
The present invention has found that exosome HOXD-AS1 can be detected in serum of a prostate cancer patient. In patients with metastatic prostate cancer, AS shown in fig. 4 and 5, the present invention found that higher levels of serum exosomes LncRNA HOXD-AS1 were associated with shorter PSA-free progression-free survival and progression-free survival (PSA-free progression-free survival: p=0.006, hr= 2.05,1.24 to 3.38; progression-free survival: p=0.02, hr= 2.27,1.00 to 5.14). Through a COX risk model, the present invention found that serum exosome HOXD-AS1 levels and T-stage are prognostic factors for the PSA-free progression survival of metastatic prostate cancer patients.
The present invention finds that serum exosome HOXD-AS1 can be used for early diagnosis of metastatic prostate cancer, assessing metastatic burden, correlating with Gleason (Gleason) score, lymph node metastasis and distant metastasis of prostate cancer, and can be used to predict PSA-free progression survival and progression-free survival of patients with metastatic prostate cancer.
While the invention has been described in detail in the foregoing general description and specific examples, it will be apparent to those skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Sequence listing
<110> first affiliated Hospital of Kunming university of medical science
<120> application of exosome HOXD-AS1 AS prognosis screening for prostate cancer
<130> GG211023724A
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 4183
<212> DNA
<213> Artificial Sequence
<400> 1
aaagcagccc agggccgccg cccagcgccc ccgagccccg aggagagccg tcgcgaccgg 60
gggctgcctg ggcaagggcg gggcgagagg gcagaacgcc tgctctcgga cagggtgttc 120
ttaaaataca ccggaaagaa cggaggtaaa aatactggag tgtttcctcc acactggaaa 180
gagaggaggt ggggacggat atggtatttg tagggcagcg ctgtgaacaa tggagccatc 240
aaaaaggcct tctcccagcc tcctcaccaa ccacggacat tcctggccca acaagacctc 300
ctggccaggt tttgagccct gcttcccaga tctggggacc cttcacctgc ctctactact 360
gcaaatgatt ggatattcgt ctgacttggc tcttccctaa tgtgtggaac taattttgtt 420
cactctgttt catgctggac ctggcctgaa aatgggccct tatattgtct ttgcactttc 480
caacttgcca agtttctgaa agaaggacca aagtaatgcc aagaactccc agatgaagag 540
gaccttcctt agacctataa cagattcact acttgacttc agagggaagt ttcagctaat 600
tctcaggaaa aggtcaaaac aggcctgtga ctgtcccttg aatactgaag gtataaacaa 660
gcttctggct cctccaggtc aagcagagga acacttttat tagaatgaag ggttacttct 720
ttgttattca aacacacacc cacacacatc ctctgtagtc tccctaatga aaaacctcaa 780
cagatggaga gcctgagctc ccaagtccgc aggctgagaa gatgcacttc actcaagggc 840
cagtgggctg gtacagacta gggaagagaa aagggacaaa ggctgctagt ataggtaatc 900
actatctttt aatactccta acacatagaa attcgaaaaa tcttgttcag caatgttagc 960
agcctgtgag cctcggaaac taatttggct gatgtgtctc tgatagggag cttggggctg 1020
caccaagggg aaacattctc ctcctgtatt ctctcctctc tctcgtttga aagcctgtac 1080
aaagaaatgg tacaaatgga tggttaactt aatgattcca tcagaggcag atttttctct 1140
gtcaggtccc tggataaggt ggaggtctga aagccaggag acagaatatg tacttaatcg 1200
ccctttctga cctgcttacc agtgaccagg actactctcc aggcctttct tctgccactt 1260
ccccattctt cctggctagt tcagttgggt ctcagcactt caactcctgg ctggatcctg 1320
agtttttctt ggttgcaggg aaagctctca ctgtctgcat ctccttcccc acccccacct 1380
cccgttttgc agctggccat tctggctgcc tcacagctat cctgccttaa ttcatatttg 1440
attgcctgga cagaaccacc tagaatgtcc agacgactgc cacccctcag ccatgggcct 1500
gagatcctgc cctgacccct ttcttcagac cccaggagga cccacaatct ccctgatccc 1560
caccttcccc aaagtagtct ctaggaaaaa agcagagctg gctaaggcct gagacagagc 1620
catttcttcc ccttgggcaa acgaggtaga ccctcccaga gaccaatgcc ggagaggccc 1680
ggatccgagc tccctttata tggcctcccc catcacggat cccgtgggaa aacctgacac 1740
acaatggcgc ggctgctccg ggcaccctca acccagcgcc ctgcggcaag gcctggagca 1800
gcctggtggt cttggtgcgc tccctttgcc ctccccgcaa ccctctgcga gcactcgtcc 1860
caggagagag accctggcta gggtggaggt tgcctcccgc aaagggcgct gttgctgggg 1920
cctcccctcc cccattctct tgtttttctg cttctgagac tcgggaggga ggcggcaggc 1980
gcctccgcag ccgatagagc agctattgtg accttccctt gcagagcggg aacccagccg 2040
agtgtgtacc gccggcggtg gcgggggagg aggcgactgt gtgtgcaaac ctcagtcgga 2100
gagggggtgg cgctgggaaa taaaggtttg caacctcagg cagttgggcc cttcagaggg 2160
cgtttggttg tgtttagact cggttgcttt ctaacactaa ttgcaggctc aaaaggagcg 2220
gaatttattg cgaaggccaa ggcggcttgt ttataattta tgaagtttta aatggctgtg 2280
ccgcgggccc caactttctc ctgggctggc agccgcgcgg aaacgcgccc tcccccggct 2340
cgccgcccac ccccacccac gcccctcaga tgagtctcac gccaatggca ggaacagatc 2400
cgagctatta gaagtctcgg gaacctccag agatgacaga aggtggcaga tttggtccaa 2460
gccctcaccc cactcgagag gagggcgtcg gggaaagctg tggctgcctg gagagggagc 2520
ccctgatccc acacttcctt ggagcctgag tgtgagagga agctaatgtc agccgggcta 2580
gacaccgttt aaagtctaat ccatgaaacc ctaagccgca aatgacactt ggggctttgc 2640
agacacccgc agactcagca gatactcctg cggtaacaca ctgtgctgga aaacgcctgg 2700
agccatgaca cattacgtat ttttattatt atttatggta ggaagaggag atgagggaaa 2760
atcgggctga atctaagcag cctgacaatt cagctagtta atatttagcc agttaagaaa 2820
ggtggttgta atccctgcaa ggcagtgagc tctcctttga tttctctaat agcttaaaaa 2880
ataatttctt acaagacaat tagaaattct gcctatggat gcaaatttca tagaaaacaa 2940
tgttaagcct tgaaagtggg taaaatgtgc cttcgattga agtcatttat tctgttgata 3000
cacttctaca acatatatat tttaaacctg ctcagataaa gtgaaattta gcaaaaggat 3060
atcccctccc acccccaaat ctaacatgcc attttaaaaa cagcacaaag gaacaaggaa 3120
actaaatatt attttgagcc atcattactt agtgaaatac aaacatccaa atagaaacga 3180
ttgttgttcc accagaagat tagaagttaa attatttcca cacaatagga atttcacttc 3240
gtgtcatcgg ggatgtagta ggagttgcat tatttgtcaa accaaataga gatgcgtggg 3300
ctgactttgc tgaaatacaa aagaattgaa ccttatctga aggcgttttc taagtggccc 3360
ctcccataga agccgggaag ttgtccaccc ctagcagaag ccacattaat atttacaaaa 3420
tggttataaa tccttgaaga catttttgcc cattttgtat aagaaacagg agtaaacaag 3480
gtgtttatac ttgttatgtt tcattaatga aatattatta ctttacatct tggggaatgt 3540
ttgttttctt tagaaatgaa ctaatgatca ctacaaagga agaattcatg acagcatttc 3600
ctagagaaaa agcttaggta cgcgtctcca aatgatggtt tctttttcta caccttagct 3660
ttttcatagc tcaattcttc ttacaacttc tcttgtgttt tttctttgat tgtcagtttt 3720
ctttcaaagt ttattttttg ttacttaatg cagaaaagtg agctctggtc tccccagtgt 3780
aaaagtcgat tgtgtctaga aagaaggcag taaaaacagc aaagcaggcg ctgctttcaa 3840
aaagccctgc ccaaatcatc ccgcccagaa ccttcttagg tttgcatcct catttccccc 3900
tcccctaatt tcaatagctg gaaagtaatc aagtatcccg agaaccaatt tatgcactag 3960
actccaaata aataaaatat ttttttcaaa gtgtcagtct gtttcggggc cttccatggg 4020
gattgtgcga gataaaggca tagtgatctg cttgcaagtg tgcatgatta acacagatac 4080
agttccgact gggaaatttt tttattctaa atatttgttc caatagtacc agacaatttg 4140
aaatcaataa actgtggtga ggactgtggt ttttgagaaa tga 4183
Claims (7)
1. The use of a reagent and/or device for detecting exosome LncRNA HOXD-AS1 in the preparation of a product for screening or aiding in the prognosis of prostate cancer.
2. The use according to claim 1, wherein,
the gene nucleotide sequence of LncRNA HOXD-AS1 is shown AS SEQ ID NO: 1.
3. The use according to claim 1, wherein,
the reagent and/or device for detecting the exosome LncRNA HOXD-AS1 comprises a reagent and/or device for detecting the exosome LncRNA HOXD-AS1 by a reverse transcription-polymerase chain reaction, a real-time fluorescent quantitative PCR technology, a transcriptome sequencing technology, an in situ hybridization technology, a gene chip technology or a PacBio sequencing technology.
4. The use according to claim 1, wherein,
the detection reagent is a reagent for detecting the expression level of the LncRNA HOXD-AS1 of an exosome.
5. The use according to claim 4, wherein,
the reagent for detecting the expression level of the LncRNA HOXD-AS1 of the exosome comprises a primer for specifically amplifying the LncRNA HOXD-AS1 gene and/or a probe for specifically binding the LncRNA HOXD-AS1 gene.
6. The use according to claim 4, wherein,
the primer includes an upstream primer: ACCTGCCTCTACTACTGCAAA;
a downstream primer: GCAAAGACAATATAAGGGCCC.
7. The use according to any one of claims 1-6, wherein,
the test sample of the product is blood.
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