CN103667283A - Molecular marker miR-340-5P for prostatic cancer and application of molecular marker miR-340-5P - Google Patents

Abstract

The invention provides a novel molecular marker miR-340-5P for the diagnosis of prostatic cancer and an application of the molecular marker to the preparation of a diagnostic kit for diagnosing prostatic cancer. The content of miR-340-5P in the blood serum of a prostatic cancer patient is higher than that in the blood serum of a normal person, while the content of the miR-340-5P in the blood serum of the prostatic cancer patient is returned to the normal level after the patient undergoes an operation. The invention also provides the diagnostic kit for diagnosing prostatic cancer. When used for diagnosing prostatic cancer, the molecular marker and the diagnostic kit with the molecular marker are simple in operation, convenient to obtain materials, safe and noninvasive and have the characteristics of high specificity and sensitivity and easiness for large-area screening. The molecular marker is particularly suitable for reagents applied to the fields such as high risk group screening of prostatic cancer, prostatic cancer authentication, prostatic cancer treatment condition monitoring, prostatic cancer guiding pharmacy monitoring, prostatic cancer prognosis monitoring and the like.

Description

Prostate cancer molecular marker miR-340-5P and application thereof

Technical field

The present invention relates to biological technical field, be specifically related to a kind of prostate cancer molecular marker miR-340-5P and the application in preparing diagnosing prostate cancer reagent thereof, relate in particular to prostate cancer molecular marker miR-340-5P in prostate cancer high risk population's screening, the monitoring of the evaluation of prostate cancer, prostate cancer therapy situation, the application in reagent for the field such as the monitoring of prostate cancer direction of medication usage and prostate cancer Prognosis scoveillance.

Background technology

MicroRNA(miRNA) be the non-coding strand microRNA molecules that length that a class is encoded by native gene is about 22 Nucleotide, it has multiple important regulating effect in cell.Each miRNA can have a plurality of target genes, and several miRNA also can regulate same gene.The regulating networks of this complexity both can regulate and control by a miRNA expression of a plurality of genes, also can carry out by the combination of several miRNA the expression of certain gene of finely regulating.By inference, miRNA is regulating the gene of one of trichotomy.MicroRNA plays an increasingly important role at aspects such as disease incidence Mechanism Study, early diagnosis, individualized treatment and prognosis because its feature such as conservative property, Space-time speciality, stability and tissue specificity highly makes it be better than the other biological marks such as protein, DNA fragmentation.

Patients with prostate cancer is mainly elderly men, generally at 50 years old with sequela, 95% betides 60 years old above elderly men, incidence increases with age growth constantly.In the U.S., the sickness rate of prostate cancer has surpassed lung cancer, becomes the tumour of first harm men's health.The sickness rate of Asia prostate cancers such as China are well below American-European countries, but present in recent years ascendant trend.Prostate cancer is early stage many without any symptom, even uncomfortable to some extent, is also not enough to cause patient's attention.When tumour increases compressing urethra, often obscure mutually with hyperplasia of prostate again.First patient in China approximately 80% finds distant metastasis focus, then just finds prostate cancer.Now, pathology has belonged to late period, prognosis mala.

At present, the clinical diagnosis mode of prostate cancer mainly contains following several: digital rectal examination, serum PSA (PSA) detection, the detection of endorectal ultrasonography ripple, living tissue pathologic finding etc.Digital rectal examination is method the simplest, most economical practicality, is mainly the forefinger touch prostate gland by doctor, in order to find a lot of asymptomatic patients with prostate cancer, likely obtains the chance of early diagnosis and radical cure.But aforesaid method all has some limitations.As the limitation of digital rectal examination is:, when patient's prostate gland lump is little, easily fail to pinpoint a disease in diagnosis (1); (2) the patient's prostate cancer enlargement having is not obvious, but has belonged to late period, is difficult for radical cure; (3) patient is had to certain harm that do not accommodate, when patient's rectum has illness, can not use this detection; (4) when doctors experience deficiency, fail to pinpoint a disease in diagnosis or mistaken diagnosis possibility.PSA in blood not high (not higher than 4ng/ml) under normal circumstances, when in prostate cancer and other prostatosis disease states, PSA raises, become the most responsive knurl mark of current examination prostate cancer, but also there is certain limitation in it: (1) need to get blood examination and survey, and patient is had to certain damage; (2) PSA increases and is also common in non-prostate cancer disease, as prostatitis, prostatomegaly etc., is therefore difficult for making a definite diagnosis; (3) PSA increases while making a definite diagnosis prostate cancer, and often patient has belonged to the intermediary and later stages, does not reach the object of early diagnosis.Prostate gland ultrasound examination directly perceived, not damaged easy and simple to handle, locates and qualitative sign judge the character of pathology by showing that size, number, position, density, edge, form, the form that has or not calcification and calcification, size, number, substep and halo around, the skin change etc. of lump provide: its limitation is: easily fail to pinpoint a disease in diagnosis the little cancer kitchen range of compactness (1); (2) sometimes can not provide clear and definite etiologic diagnosis; (3) thus because of its can not show the internal structure of tumour and surrounding tissue diagnostic accordance rate very low; (4) the good Malignant mass of real property that some is lacked to typical sign has higher misdiagnosis rate.Living tissue pathologic finding because it is traumatic, complicacy can not be as the means of primary dcreening operation, but its gold standard that to be prostate cancer make a definite diagnosis, general and additive method technical battery use.

Research in recent years shows, prostate cancer is closely related with miRNA, and they may participate in generation, development and the transfer of tumour, so may have corresponding effect to the detection of the pathogenesis of tumour, early diagnosis, individualized treatment, transfer and prognosis etc.More and the effect of the miRNA kind relevant to prostate cancer differs, and there are some researches show that the miRNA raising comprises miR-375 in patients with prostate cancer, miR-148a, miR-200c, miR-106a, mir-128, miR-218, miR-20a, and miR-30b etc.; The miRNA lowering comprises miR-143, miR-145, miR-199a-5p, miR-223, miR-27a, miR-152 and miR-424 etc.Although carried out some researchs in this field, all there is deficiency the accuracy of existing miRNA mark, susceptibility aspect, in clinical and research, still there is the demand of finding more accurate and sensitive prostate cancer miRNA mark.

Summary of the invention

In order to overcome above-mentioned the deficiencies in the prior art, the invention provides a kind of new prostate cancer molecular marker relevant to prostate cancer, and the application of this molecular marker in preparing diagnosing prostate cancer reagent is provided.

The molecular marker of this prostate cancer is miR-340-5P, and its nucleotides sequence is classified 5 '-UUAUAAAGCAAUGAGACUGAUU-3 ' (SEQ ID NO:1) as.In research process, contriver finds, the content of miR-340-5P in serum of patients with prostate cancer is higher than the content in normal human serum, and receiving after operation when patients with prostate cancer, the content of the miR-340-5P in its serum just falls back to the normal level the same with content in normal human serum.As can be seen here, patient exists close dependency at the miR-340-5P increasing during one's sickness and the tumour of prostate cancer.On the basis of above-mentioned discovery, the invention provides a kind of prostate cancer molecular marker, and the application of this molecular marker in preparing diagnosing prostate cancer reagent.

The present invention for the technical scheme that solves its technical problem and adopt is:

A prostate cancer molecular marker miR-340-5P, its nucleotides sequence is classified the sequence shown in SEQ ID NO.1 as, is 5 '-UUAUAAAGCAAUGAGAC UGAUU-3 '.

The application of described prostate cancer molecular marker miR-340-5P in preparing diagnosing prostate cancer reagent is, described diagnosing prostate cancer reagent passes through to detect the content of miR-340-5P in subject's serum, and this miR-340-5P content is compared diagnosing prostate cancer with normal level miR-340-5P content.

In described subject's serum, the content of miR-340-5P detects by quantifying PCR method.

Described prostate cancer molecular marker miR-340-5P is for the preparation of the diagnostic kit of diagnosing prostate cancer.

The diagnostic kit of described diagnosing prostate cancer comprises:

(1) serum total RNA extraction reagent,

(2) RNA adds polyA reagent,

(3) RT-PCR reagent,

(4) quantitative PCR reagent;

Wherein said quantitative PCR reagent comprises the specificity forward primer (being GSP external source contrast-3) of described prostate cancer molecular marker miR-340-5P, the nucleotides sequence of this specificity forward primer is classified the sequence shown in SEQ ID NO.7 as, is 5 '-UUGAGCAACGCGAACAAAUCA-3 '.

In described diagnostic kit, serum total RNA extraction reagent comprises that nucleotides sequence classifies the external source contrast-1 of SEQ ID NO.2 as, is 5 '-CAACCTCCTAGAAA GA-3 '.

In described diagnostic kit, RNA adds polyA reagent and comprises that nucleotides sequence classifies the external source contrast-2 of SEQ ID NO.3 as, is 5 '-TGAGCAACGCGAACA A-3 '.

In described diagnostic kit, RT-PCR reagent comprises that sequence is the RT-primer of SEQ ID NO.4, be 5 '-CAGTGGTATCAACGCACTCCTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ', wherein V is any in A, C and G, and N is any in A, T, C and G.

In described diagnostic kit, quantitative PCR reagent comprises that nucleotides sequence classifies the general reverse primer UPM-short-movie section of SEQ ID NO.5 as, is 5 '-CTCACAC GACTCACGACAC-3 '; Classifying the general reverse primer UPM-long segment of SEQ ID NO.6 as with nucleotides sequence, is 5 '-CTCACACGACTCACGACACC AGTGGTATCAACGCACTC-3 '.

Useful technique effect of the present invention is: the diagnosis that the present invention is prostate cancer provides a kind of new molecular marker miR-340-5P, and this molecular marker is applied to prepare in the diagnostic kit of diagnosing prostate cancer, the diagnostic kit diagnosing prostate cancer that uses this molecular marker and contain this molecular marker, simple to operate, draw materials conveniently, safety is without wound, and there is high specific, high sensitivity and the feature that is easy to a large amount of examinations, this molecular marker is particularly suitable for prostate cancer high risk population's screening, the evaluation of prostate cancer, the monitoring of prostate cancer therapy situation, the monitoring of prostate cancer direction of medication usage, with the field such as prostate cancer Prognosis scoveillance with in reagent.

Embodiment

For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention, but following embodiment is only not used in and limits the scope of the invention for the present invention is described, in the following example, NM specific experiment method, carries out according to normal experiment method conventionally.

1. the extraction of total RNA in serum or blood plasma:

Extract each 2 milliliters of the blood that 3 prostate patients perform the operation first 7 days and perform the operation latter 7 days, extract simultaneously the same period each 2 milliliters of 3 normal human bloods in contrast.Centrifugal by carrying out after above-mentioned blood coagulation, finally get the centrifuge tube without DNA and RNA pollution that 1 milliliter of upper serum is placed in 1.5 milliliters.

Use RNA that Kang Wei ShiJi Co., Ltd produces to extract test kit and in above-mentioned serum, extract total RNA, in every 250 microlitre serum, add 1 microlitre external source contrast-1(Beijing Kuang Bosheng technology company limited to provide) monitor the extraction quality of RNA in above-mentioned serum.The total RNA extracting is by being used Therm NanoDrop 2000c instrument, the concentration determination that the ratio of mensuration 260/280nm ultraviolet wavelength carries out.The sequence of described external source contrast-1 is 5 '-CAACCTCCTAGAAAGA-3 ' (SEQ ID NO:2).

2. the microRNA in detection by quantitative serum:

1) add poly(A) tail:

In the PCR pipe (VWR company produces, 200 microlitres) without RNA enzyme, preparation adds the reaction solution of A tail, and system total amount is 20 microlitres.In every 20 microlitre system reaction solutions, add the special external source contrast-2(Beijing Kuang Bosheng technology company limited of 1 microlitre to provide) monitor tailing and the reverse transcription quality of miRNA, described reaction solution system is as shown in table 1.By the PCR pipe for preparing reaction solution is housed, put into PCR instrument (Thermo), at 37 ℃, hatch 1 hour, obtain incubation reaction liquid I.The sequence of described external source contrast-2 is 5 '-TGAGCAACGCGAACAA-3 ' (SEQ ID NO:3).

Table 1

Component	Add-on (microlitre)
		External source contrast-2(20nm)	1
E.Coli polyA polymerase (takara company)	0.5
		10 times of polymerase solution of E.Coli polyA	2
Deoxidation gland sweet (10mm)	2
		RNA	x
Ultrapure water without RNA and DNA pollution	14-x
		RNA enzyme inhibitors	0.5
Total amount	20

In table, x represents that the RNA volume adding determined by the concentration of RNA, is x=500 nanogram/RNA concentration in this experiment.

2) RT-PCR obtains cDNA strand:

To through 1) add the RT-primer (Beijing Kuang Bosheng technology company limited provides) of 0.5 microlitre (0.5 nanogram/microlitre) in the reaction solution I that obtains after hatching, at 70 ℃, hatch 5 minutes, hatch and will hatch gained reaction solution II after end and put into immediately ice bath at least 2 minutes; Then according to becoming assignment system inverse transcription reaction liquid III shown in table 2; Gained inverse transcription reaction liquid III was hatched after 50 minutes at 50 ℃, at 70 ℃, be incubated 15 minutes, insulation is positioned over after finishing to be carried out coolingly in ice bath, obtain cDNA.After the cDNA obtaining can being diluted to the reverse transcription product that contains the total RNA of 1 nanogram in 1 microlitre system, carry out packing, be positioned over minus 20 degrees and preserve.The sequence of described RT-primer is 5 '-CAGTGGTATCAACGCACTCCTTTTTTTTT TTTTTTTTTTTTTTTTTTTTTVN-3 ' (SEQ ID NO:4), and wherein V is any in A, C and G, and N is any in A, T, C and G.

Table 2

3) QPCR detection by quantitative:

At 2 milliliters of EP, manage in (production of VWR company) as table 3 dosage preparation reaction solution IV; After the reaction solution IV preparing is fully put upside down and mixed, be distributed in 96 holes point end PCR plates (production of VWR company) every Kong Zhongwei 18 microlitres; Use the volley of rifle fire (Gibson company, 1-10 microlitre) to adding specificity forward primer in above-mentioned hole, (be GSP external source contrast-3, by Beijing, Kuang Bosheng technology company limited provides), the sequence of this Auele Specific Primer is 5 '-UUGAGCAACGCGAACAAAUCA-3 ' (SEQ ID NO:7), and every hole is 2 microlitres; Then to adding respectively in above-mentioned hole after 10 microlitre paraffin oils with putting into ABI 7900PCR instrument after special-purpose pad pasting (VWR company) sealing, setting program is as shown in table 4.

Table 3

The short-movie section of 10 * UPM-described in table 3 and 10 * UPM-long segment are general reverse primer, and wherein the nucleotides sequence of the general reverse primer of 10 * UPM-short-movie section is classified 5 '-CTCACACGACTCACGACAC-3 ' (SEQ ID NO:5) as; The nucleotides sequence of described 10 * UPM-long segment is classified 5 '-CTCACACGACTC ACGACACCAGTGGTATCAACGCACTC-3 ' (SEQ ID NO:6) as.

Table 4

3. adopt Array Tools 4.1.0 to carry out data analysis: with aforesaid method, can record target miRNA in sample serum and with reference to the Ct value of miRNA, according to the Ct value level of reference, try to achieve the relative content of target miRNA in serum.With aforesaid method can record that prostate cancer is preoperative, the average delta Ct value of miR-340-5P is respectively 10.50,4.25 and 3.53 in postoperative 7 days of prostate cancer and each sample Peripheral Blood of normal control, result shows that the relative content of miR-340-5P in prostate cancer peripheral blood increases than Normal group is obvious, within postoperative 7 days, is substantially returned to normal value level.Single factor Cox risk regression analysis and the demonstration of K-M survival analysis, miR-340-5P can be used as the biomarker that development occurs in prostate cancer.

4. the standardization of data:

The Ct value of the external source of take contrast-1 is reference, tries to achieve the relative content of miRNA in serum, and result shows that miR-340-5P relative content in serum before operation in patients significantly raises, and operative results is to normal level.With 2 of classics in qPCR detection ^{-Δ Ct}mode represent the level (Δ Ct is that target miRNA contrasts the poor of-1 Ct value with external source) of object miRNA in serum.

5. the horizontal diagnosing prostate cancer of object miRNA in serum:

Compare with the low levels of miR-340-5P in normal control serum, in the preoperative serum of patients with prostate cancer, the level of miR-340-5P raises more than 5 times, and difference has statistical significance; Statistical study shows after operation in patients in serum that miR-340-5P content is compared difference not statistically significant with normal control.

Sequence table

Bo Taian bio tech ltd, <110> Suzhou

<120> prostate cancer molecular marker miR-340-5P and application thereof

<130> 2012

<160> 7

<170> PatentIn version 3.5

<210> 1

<211> 22

<212> RNA

<213> Homo sapiens

<400> 1

uuauaaagca augagacuga uu 22

<210> 2

<211> 16

<212> DNA

<213> artificial sequence

<400> 2

caacctccta gaaaga 16

<210> 3

<211> 16

<212> DNA

<213> artificial sequence

<400> 3

tgagcaacgc gaacaa 16

<210> 4

<211> 52

<212> DNA

<213> artificial sequence

<220>

<221> misc_feature

<222> (52)..(52)

<223> n is a, c, g, or t

<400> 4

cagtggtatc aacgcactcc tttttttttt tttttttttt tttttttttt vn 52

<210> 5

<211> 19

<212> DNA

<213> artificial sequence

<400> 5

ctcacacgac tcacgacac 19

<210> 6

<211> 38

<212> DNA

<213> artificial sequence

<400> 6

ctcacacgac tcacgacacc agtggtatca acgcactc 38

<210> 7

<211> 21

<212> DNA

<213> artificial sequence

<400> 7

uugagcaacg cgaacaaauc a 21

Claims (10)

1. a prostate cancer molecular marker miR-340-5P, is characterized in that: the nucleotides sequence of described prostate cancer molecular marker miR-340-5P is classified the sequence shown in SEQ ID NO.1 as.

2. the application of prostate cancer molecular marker miR-340-5P according to claim 1 in preparing diagnosing prostate cancer reagent.

3. the application of prostate cancer molecular marker miR-340-5P according to claim 2 in preparing diagnosing prostate cancer reagent, it is characterized in that: described diagnosing prostate cancer reagent passes through to detect the content of miR-340-5P in subject's serum, and this miR-340-5P content is compared diagnosing prostate cancer with normal level miR-340-5P content.

4. the application of prostate cancer molecular marker miR-340-5P according to claim 3 in preparing diagnosing prostate cancer reagent, is characterized in that: in described subject's serum, the content of miR-340-5P detects by quantifying PCR method.

5. the application of prostate cancer molecular marker miR-340-5P according to claim 2 in preparing diagnosing prostate cancer reagent, is characterized in that: described prostate cancer molecular marker miR-340-5P is for the preparation of the diagnostic kit of diagnosing prostate cancer.

6. a diagnostic kit for diagnosing prostate cancer claimed in claim 5, is characterized in that: described diagnostic kit comprises:

(1) serum total RNA extraction reagent,

(2) RNA adds polyA reagent,

(3) RT-PCR reagent,

(4) quantitative PCR reagent;

Wherein said quantitative PCR reagent comprises the specificity forward primer of described prostate cancer molecular marker miR-340-5P, and the nucleotides sequence of this specificity forward primer is classified the sequence shown in SEQ ID NO.7 as.

7. the diagnostic kit of diagnosing prostate cancer according to claim 6, is characterized in that: in described diagnostic kit, serum total RNA extraction reagent comprises that nucleotides sequence classifies the external source contrast-1 of SEQ ID NO.2 as.

8. the diagnostic kit of diagnosing prostate cancer according to claim 6, is characterized in that: in described diagnostic kit, RNA adds polyA reagent and comprises that nucleotides sequence classifies the external source contrast-2 of SEQ ID NO.3 as.

9. the diagnostic kit of diagnosing prostate cancer according to claim 6, is characterized in that: in described diagnostic kit, RT-PCR reagent comprises that sequence is the RT-primer of SEQ ID NO.4.

10. the diagnostic kit of diagnosing prostate cancer according to claim 6, it is characterized in that: in described diagnostic kit, quantitative PCR reagent comprises that nucleotides sequence classifies the general reverse primer UPM-short-movie section of SEQ ID NO.5 as, and nucleotides sequence is classified the general reverse primer UPM-long segment of SEQ ID NO.6 as.