CN110184269A

CN110184269A - LOC105377068 diagnoses new application

Info

Publication number: CN110184269A
Application number: CN201910472046.6A
Authority: CN
Inventors: 杨承刚; 向常娟
Original assignee: Gu'an Bojian Biotechnology Co Ltd
Current assignee: Gu'an Bojian Biotechnology Co Ltd
Priority date: 2019-05-31
Filing date: 2019-05-31
Publication date: 2019-08-30

Abstract

The invention discloses the molecular markers that can be used for diagnosing Male Osteoporosis.The molecular marker is LOC105377068.Pass through the level of LOC105377068 in detection subject's blood, it can be determined that whether subject suffers from osteoporosis or judge whether subject suffers from the risk of osteoporosis.The product that diagnoses Male Osteoporosis can be developed according to LOC105377068 through detection LOC105377068 content, which can clinically promote the use of.

Description

LOC105377068 diagnoses new application

Technical field

The invention belongs to fields of biomedicine, are related to the diagnostic uses of non-coding RNA, and in particular to LOC105377068 exists Diagnostic uses in Male Osteoporosis.

Background technique

The disease incidence of Male Osteoporosis (osteoporosis in men) is significantly less than postclimacteric women, therefore with Toward to Male Osteoporosis far away from the attention to women.Research achievement in recent years shows that the male of nonage compares female Though property can reach higher Peak Bone Mass, the initial time of bone loss is also obviously later than women, and men and women's both sexes have one Increase the bone loss process of phase, i.e. senile osteoporosis (II type) with the age.Male Osteoporosis and women sclerotin Osteoporosis has many similarities, but still has apparent sex difference in terms of teiology, pathology and clear ground disease.65 years old The osteoporosis of the generally existing varying degree of above male, severe complication are fracture.

In the U.S., the incidence of male's hip fracture greater than 65 years old is 4 ‰ -5 ‰, and the women incidence at corresponding age is 8‰-10‰.Hip fracture incidence in Northern Europe is male: female=1: 2, in southern Europe and other areas, and the incidence of hip fracture It is relatively low in both sexes.Worldwide in the patient of all hip fractures, about 30% is male, nineteen ninety 51 Ten thousand, it is contemplated that the number to male's hip fracture in 2025 will be added to 1,160,000 people, and this ascendant trend will more dash forward in Asia Out.

Cooper report 50 years old or more male is 13.1% in hip, centrum and fracture of forearm danger level in life, and Women is 39.7%.Neill report, the male through European 19 36, country 50-79 years old crowd's vertebral fractures in 15570, center Illness rate is 20%.Illness rate of the China in relation to Male Osteoporosis, according to District of Shanghai Xu Shang loyalty, 60 years old or more male It is 13.9%；Beijing area Wu Qing is reported as 13.4% (lumbar vertebrae)；Shen Huiliang is reported as 23.8% (lumbar vertebrae).

Osteoporotic fracture causes the forfeiture and life danger of viability, the death rate of Poor report fracture to patient It is higher than women with disease incidence male.

Identical as women osteoporosis, common x line is still the necessary means for diagnosing osteoporosis and related fracture.It is first The bone density for showing as backbone and pelvis lowers.Centrum cortical bone is thinning, and row bone trabecula is reduced or disappeared, and stringer bone is small Beam is relatively obvious, and structure disappears in centrum when serious；Centrum becomes flat, and lower edge indent, intervertenral space are broadening thereon, and centrum is in concave-concave Shape is often compressed because of microtrauma wedge shaped.Visible bone trabecula attenuates on long bone, quantity is reduced, cortical bone is thinning and is occurred Lamination.The dotted of several millimeters of sizes for being dispersed in distribution can occur on the basis of diffusivity bone density lowers when serious Clear zone, clear border or fuzzy, easy mistaken diagnosis are destruction of bone.But x-ray plain film is to the sensibility of diagnosis of osteoporosis and accurate Property it is low, the early diagnosis of osteoporosis is helped little.Therefore it urgently develops a kind of in osteoporosis early stage i.e. diagnosable bone The method of matter osteoporosis.

Summary of the invention

A technical problem to be solved by this invention is provided and a kind of is dredged with male's sclerotin for the status of the prior art Loose disease diagnoses relevant long-chain non-coding RNA marker.

Another technical problem to be solved by this invention is to provide a kind of detection method of above-mentioned lncRNA marker, should Detection method is not directly used for the Clinics and Practices of disease.

It is male in preparation diagnosis that another technical problem to be solved by this invention is to provide a kind of above-mentioned lncRNA marker Application in the product of property osteoporosis.

The present invention is in order to solve the above technical problems, adopt the technical scheme that

One aspect of the present invention, provide it is a kind of diagnose relevant isolated long-chain non-coding RNA to Male Osteoporosis, The long-chain non-coding RNA is LOC105377068.

In another preferred example, the LncRNA is isolated from the blood and/or tissue of people or non-human mammal.

In another preferred example, the blood is blood plasma and/or serum.

In another preferred example, the blood does not include haemocyte.

In another preferred example, the non-human mammal is mouse, rat, rabbit, pig, ox, sheep etc..

In another preferred example, the LncRNA is isolated from human blood.

Second aspect of the present invention, provides a kind of isolated polynucleotides, and the polynucleotides can be transcribed by people's cell The long-chain non-coding RNA of first aspect present invention.

Third aspect present invention provides a kind of carrier, and the carrier contains the polynucleotides of second aspect of the present invention, or Express the long-chain non-coding RNA of first aspect present invention.

Fourth aspect present invention, provides a kind of reagent, and the reagent is the long-chain for being able to detect first aspect present invention The reagent of non-coding RNA expression quantity.

Further, the reagent including the use of SYBR Green, TaqMan probe, molecular beacon, double cross probe or is answered The PCR amplification primer used when closing long-chain non-coding RNA expression quantity described in probe in detecting.

In a specific embodiment of the present invention, the primer sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.

Fifth aspect present invention, provides a kind of chip of Diagnosis of osteoporosis, the chip include solid phase carrier with And and the oligonucleotide probe that is orderly fixed on the solid phase carrier, the oligonucleotide probe specifically correspond to this The long-chain non-coding RNA of invention first aspect.

Sixth aspect present invention provides the purposes of the chip of fifth aspect present invention, is used to prepare diagnosis male's sclerotin The kit of osteoporosis.

Seventh aspect present invention, provides a kind of kit, and the long-chain that the kit contains first aspect present invention is non- The chip of the reagent or fifth aspect present invention of coding RNA or fourth aspect present invention.

Further, the following ingredients that kit of the invention can also be required comprising RT-PCR reaction: PCR buffer, 10mM dNTPs, Hot start Taq enzyme, ddH₂O。

Further, kit of the invention can also be comprising the cDNA that the reverse transcription of long-chain non-coding RNA obtains, as detection The positive template of above-mentioned primer feasibility.

Eighth aspect present invention provides the long-chain non-coding RNA or fourth aspect present invention of first aspect present invention The purposes of reagent is used to prepare the chip or kit of diagnosis Male Osteoporosis.

The application method of kit of the invention has following record:

(1) LncRNA of first aspect present invention is always extracted in the sample of self-test object；

(2) LncRNA content is detected, and result is compared with the LncRNA content in normal population, if sample The content L1 of LncRNA in this is substantially less than the LncRNA content L0 of normal population, then illustrates that the test object is dredged with sclerotin Loose or probability with osteoporosis is high.

In another preferred example, described be substantially less than refers to L0/L1 >=1.5, it is preferable that >=2.0, it is highly preferred that >=5.

In another preferred example, the test object include the normal male for not suffering from osteoporosis, it is doubtful with sclerotin Loose male patient or the male patient for making a definite diagnosis osteoporosis.

In the context of the present invention, " Diagnosis of osteoporosis " includes judging whether subject has suffered from osteoporosis Disease judges that subject whether there is the risk with osteoporosis.

As used herein, term " LncRNA ", " long-chain non-coding RNA ", " Long non-coding RNA " meaning phase Together, be used interchangeably, all refer to it is a kind of by rna plymerase ii transcription, do not encode albumen, general length be greater than the RNA piece of 200bp Section.

As used herein, term " separation " refers to that substance is separated from its primal environment (if it is natural object Matter, primal environment are natural surroundings).As under the native state in active somatic cell polynucleotide and polypeptide be not separate Purifying, but same polynucleotide or polypeptide in native state in other substances with existing for such as from separating, then is separation Purifying.

It is, of course, also possible to according in database relative to LncRNA sequence or its complementary series, according to routine techniques (oligonucleotides that can such as combine with LncRNA is prepared as probe, and further for synthesis LncRNA or its detection reagent At chip), or expression vector is prepared into using its cDNA sequence, and be transcribed into LncRNA.

Polynucleotides construction

Provided people LncRNA sequence according to the present invention, can be designed can be processed to influence after being imported into accordingly MRNA expression LncRNA polynucleotides construction namely the polynucleotides construction can raise in vivo accordingly The amount of LncRNA.Therefore, the present invention provides a kind of isolated polynucleotides (construction), the polynucleotides (construction) LncRNA can be transcribed by people's cell.

In general, the polynucleotides construction is located on expression vector.Therefore, the invention also includes a kind of carrier, it Contain the LncRNA or the polynucleotides construction.The expression vector usually also contains promoter, replicates Point and/or marker gene etc..

Method well-known to those having ordinary skill in the art can be used to construct the required expression vector of the present invention.These methods include Recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination technology etc..The expression vector preferably includes one or more Selected marker, to provide the phenotypic character for selecting the host cell of conversion, such as kalamycin, gentamicin, tide Mycin, amicillin resistance.

Chip

LncRNA chip of expression spectrum usually contains up to several hundred a probes, covers a variety of RNA, homologous mutually using DNA double chain The principle of benefit detects the content of contained various RNA in sample in full-length genome level.Therefore, test sample can be treated in the same time The transcriptional level of RNA in this within the scope of full-length genome is detected.

Using LncRNA sequence of the present invention, corresponding LncRNA detection chip can also be prepared, and then studies it The regulative mode of express spectra and LncRNA.

Specifically, can LncRNA according to the present invention, design suitable probe, be fixed on solid phase carrier, shape At " oligonucleotide arrays "." oligonucleotide arrays " refer to (addressable i.e. with distinctive with addressable point The position that address is characterized) array, each addressable point is containing a coupled characteristic oligonucleotides.According to It needs, oligonucleotide arrays can be divided into multiple sub- battle arrays.

The various common used materials in genetic chip field, such as, but not limited to nylon membrane can be used in the solid phase carrier, through work Property group (such as aldehyde radical, amino) slide or silicon wafer, unmodified slide, plastic sheet etc. for modifying.

The conventional manufacturing method of biochip known in the art can be used in the preparation of the LncRNA chip.For example, If solid phase carrier is gone here and there using modification slide or silicon wafer, 5 ' ends of probe containing amido modified poly- dT, can be by few nucleosides Acid probe is configured to solution, then uses point sample instrument that its point on modification slide or silicon wafer, is arranged in scheduled sequence or battle array Column, are then fixed by standing overnight, so that it may obtain miRNA chip of the invention.If nucleic acid without amido modified, Preparation method can also refer to: " the gene diagnosis technology-on-radiation operation manual " of Wang Shenwu chief editor；J.L.erisi, V.R.Iyer,P.O.BROWN.Exploring the metabolic and genetic control of geneexpression on a genomic scale.Science,1997；278:680 and Ma Li people, it is raw that Jiang Zhonghua edits The Beijing object chip: Chemical Industry Press, 2000,1-130.

Kit

Kit of the present invention can be used for detecting the expression of LncRNA described in blood.Preferably, the reagent Also containing the marker for labeled RNA sample, and substrate corresponding with the marker in box.Preferably, of the invention LncRNA contained in the kit can be used for carrying out positive control.In addition, may also include in the kit For various reagents needed for extracting RNA, PCR, hybridization, colour developing etc., including but not limited to: extract, amplification liquid, hybridization solution, Enzyme, comparison liquid, developing solution, washing lotion, antibody etc..In addition, may also include operation instructions and/or chip figure in the kit As analysis software.

Detailed description of the invention

Fig. 1 shows the statistical chart of the differential expression situation using QPCR detection LOC105377068.

Specific embodiment

The present invention is described in further detail with reference to the accompanying drawings and examples.Following embodiment is merely to illustrate this It invents rather than limits the scope of the invention.Test method without specific conditions in embodiment, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring HarborLaboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.

Embodiment 1 screens differential expression molecule

1, case is collected

Selection osteoporosis in aged males patient 5, the age 62-79 years old；Diagnosis of osteoporosis is referring to Aged in China Learn the standard that the osteoporosis committee formulates, i.e., is set to position bone density value lower than two standard deviations of this area peak reduction Osteoporosis.Healthy geriatric male 6 (healthy control group) of same period selection, the age 61-78 years old.Selected object excludes glycosuria Disease, Primary hyperparathyroidism and hyperthyroidism, hypercortisolism, hepatic and renal function are bad, are addicted to drink, seek Supporting bad, rheumatoid arthritis etc. influences the disease of bone metabolism；Androgen, calcitonin, a large amount of calcium agent, two are not applied in half a year The drug of phosphonate and other influences bone metabolism.Two groups of ages, height, weight and diet, daily exercise etc. have Comparativity.

2, blood Total RNAs extraction

Total serum IgE is extracted using the RNA extracts kit of Promega company.Specific step is as follows:

1) it takes 1ml to collect the whole blood in heparin or the processed test tube of EDTA, puts into sterile centrifugation tube；

2) 3000rpm is centrifuged 5min, carefully siphons away supernatant at the top of sample；

3) plus 1ml haemocyte lysate, careful inhale are put 4-5 times, sediment are resuspended, 3000rpm is centrifuged 5min；

4) step 3 is repeated twice；

5) cell precipitate is avoided, nearly all supernatant is carefully siphoned away, only retains 100 μ l supernatants；Check BME It is added in RNA lysate, then plus 175 μ l RNA lysates are into cell, and resuspension and lytic cell are put in suction；

6) add 350 μ l RNA dilutions, overturn mixing 3-4 times, 13000g is centrifuged 10min at room temperature, by limpid lysate It is transferred in a sterile centrifugation tube；

7) 200 μ l, 95% ethyl alcohol is added into cleared lysate, puts 3-4 times with liquid-transfering gun suction with mixing；By this mixture It is transferred in centrifugal column assembly, 13000g is centrifuged 1min；

8) centrifugal column is taken down from centrifugal column assembly, discards the liquid in collecting pipe, centrifugal column is installed onto collecting pipe On, add 600 μ l RNA cleaning solutions in centrifugal column assembly, 13000g is centrifuged 1min；

9) liquid in collecting pipe is discarded, centrifugal column is installed on collecting pipe, the freshly prepared DNase of 50 μ l is incubated for Mixture is applied directly on the film in centrifugal column；

10) it is incubated for 15min at room temperature, 200 μ l DNA enzymatic stop buffers, 13000g centrifugation are added into centrifugal column 1min；

11) 600 μ l RNA cleaning solutions are added, 13000g is centrifuged 1min；

12) collecting pipe is emptied, 250 μ l RNA cleaning solutions (ethyl alcohol has been added), high speed centrifugation 2min are added into centrifugal column；

13) centrifugal column is transferred on elution pipe from collecting pipe, 100 μ l nuclease-free waters is added on film, by centrifugal column Assembly puts centrifuge into and makes the lid facing outwards for eluting pipe, and 13000g is centrifuged 1min, abandons centrifugal column, covers and fill RNA's Elution pipe, is stored in -70 DEG C.

3, RNA quality testing

Use the concentration and purity of NanoDropND-1000 type ultraviolet specrophotometer measurement total serum IgE.

4, total serum IgE integrity mensuration' is observed under ultraviolet transmission light through 1% denaturing formaldehyde agarose gel electrophoresis, detects RNA Integrality.

5, Agilent2100 measures RIN value.

LncRNA sequencing requires: sample requirements: >=200ng；Sample concentration: C >=20ng/ μ L；Sample purity: RIN >= 7.0,28S/18S >=1.0.

6, rRNA is removed

The long-chain non-coding RNA of intracellular a part (> 24%) is all the absence of traditional poly A tail, therefore using removal The mode of rRNA, which builds library, can obtain more comprehensive lncRNA information.

7, fragmentation RNA

Illumina platform is sequenced for short sequence fragment, and the mRNA and lncRNA that removal rRNA is obtained are complete RNA sequence, average length may reach several kb, it is therefore desirable to be interrupted at random to it.It, can be by RNA using metal ion Random fracture at 200bp or so small fragment.

8, reversion synthesis cDNA

Under the action of reverse transcriptase, using random primer, one chain cDNA of synthesis is inverted by template of mRNA, carries out two chains When synthesis, dTTP is replaced with dUTP in dNTPs reagent, making base in the second chain of cDNA includes A/U/C/G.

9, adaptor is connected

The cDNA structure of double-strand is cohesive end, and End Repair Mix is added and is mended into flat end, then at 3 ' ends End adds an A base, for connecting the connector of Y-shaped.

10, bis- chain of UNG enzymic digestion cDNA

Before PCR amplification, the second chain of cDNA is digested with UNG enzyme, to make in library only comprising the first chain of cDNA.

11, machine is sequenced on Illumina Hiseq2500

Illumina Hiseq2500 microarray dataset carries out 2*150bp sequencing.

12, bioinformatic analysis

It is as follows that sequencing data obtains later rawdata analytic process:

(1) base of quality < 20 is fallen to the 5 ' of reads and 3 ' Duan Jinhang trim, trim with cutadapt, and deletes N Reads greater than 10%；

(2) tophat is compared onto reference genome.Reference genome version used be GRCh38.p7, fasta and Gff file download is from NCBI；

(3) cuffquant quantifies the expression quantity and normalization output of lncRNA；

(4) cuffdiff compares control group with the differential expression of disease group lncRNA.

13, result

Significant difference lncRNA screening conditions: FDR < 0.01.Following result is obtained with the above standard screening: with normal healthy controls It compares, differential expression LncRNA is 359 in patients with osteoporosis blood, wherein raising is 214, downward is 145 It is a.

2 large sample of embodiment verifies the differential expression LncRNA filtered out

1, sample collection

Patients with osteoporosis 32 are collected according to the method for embodiment 1, and men's health compares 30.

2, blood total serum IgE is extracted

Step is the same as embodiment 1.

3, it is verified on rna level

(1) reverse transcription: according to the RNA concentration measured, each sample quantifies the RNA of 1000ng for reverse transcription.Using big Even the DDR037A reverse transcription reagent box of precious biology, the system (being shown in Table 1) of 20 μ l are added to going in enzyme EP pipe for 200ul, are centrifuged After be placed in PCR instrument, temperature condition is set as: 37 DEG C of 15min, 85 DEG C of 5sec.After by cDNA dilute 10 times, -20 DEG C preservation It is spare.

1 reverse transcription system of table

(2) real-time quantitative PCR

Give birth to work biosynthesis primer in Shanghai.

LOC105377068

Upstream primer: 5 '-CTGATAGTCATAAGTAGCATAGC-3 ' (SEQ ID NO.1)；

Downstream primer: 5 '-CCATAATCCACAGCAACAATA-3 ' (SEQ ID NO.2).

β-actin

Upstream primer: 5 '-CGCGAGAAGATGCCCAGATC-3 ' (SEQ ID NO.3)；

Downstream primer: 5 '-TCACCGGAGTCCATCACGA-3 ' (SEQ ID NO.4).

It is detected, Dongyang spinning company using the real-time PCR (AB StepOne Plus) of American AB I company (TOYOBO) the SYBRGreen dyestuff incorporation methods detection of company, the cDNA that reverse transcription obtains dilutes ten times, using 20 μ l as reactant It is (being shown in Table 2).Amplification condition are as follows: 95 DEG C of initial denaturation 60sec, later 95 DEG C of denaturation 15sec, 55 DEG C of extension 60sec, totally 40 are followed Ring obtains the Ct value of target gene and internal reference.

2 PCR system of table

Reagent	Usage amount
		cDNA	2μl
SYBR green	10μl
		Dye	0.4μl
Upstream primer (10 μM)	1μl
		Downstream primer (10 μM)	1μl
Sterilize distilled water	To 20 μ l

As a result relative quantification method, formula 2 are used^-△△ctIt calculates.

4, result

The results show that having 26 in 32 Male Osteoporosis patients compared with the average level of 30 healthy males LOC105377068 level is substantially less than normal healthy controls in blood samples of patients.Statistical result as shown in Figure 1, compared with healthy male, LOC105377068 level is substantially reduced in Male Osteoporosis blood samples of patients, and difference has statistical significance (P < 0.05).

The explanation of above-described embodiment is only intended to understand method and its core concept of the invention.It should be pointed out that for this For the those of ordinary skill in field, without departing from the principle of the present invention, several improvement can also be carried out to the present invention And modification, these improvement and modification will also be fallen into the protection scope of the claims in the present invention.

Sequence table

<110>Guan County Bo Jian Bioisystech Co., Ltd

<120>LOC105377068 diagnoses new application

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 23

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 1

ctgatagtca taagtagcat agc 23

<210> 2

<211> 21

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 2

ccataatcca cagcaacaat a 21

<210> 3

<211> 20

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 3

cgcgagaaga tgcccagatc 20

<210> 4

<211> 19

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 4

tcaccggagt ccatcacga 19

Claims

1. a kind of isolated long-chain non-coding RNA, which is characterized in that the long-chain non-coding RNA is LOC105377068.

2. a kind of isolated polynucleotides, which is characterized in that the polynucleotides can be transcribed into described in claim 1 by people's cell Long-chain non-coding RNA.

3. a kind of carrier, which is characterized in that the carrier contains polynucleotides as claimed in claim 2, or expression claim 1 The long-chain non-coding RNA.

4. a kind of reagent, which is characterized in that the reagent is to be able to detect long-chain non-coding RNA expression described in claim 1 The reagent of amount.

5. a kind of chip of Diagnosis of osteoporosis, which is characterized in that the chip includes solid phase carrier and and orderly consolidates The oligonucleotide probe being scheduled on the solid phase carrier, the oligonucleotide probe specifically correspond to described in claim 1 Long-chain non-coding RNA.

6. the purposes of chip described in claim 5, which is characterized in that be used to prepare the reagent of diagnosis Male Osteoporosis Box.

7. a kind of kit, which is characterized in that the kit contains long-chain non-coding RNA described in claim 1 or right It is required that chip described in reagent described in 4 or claim 5.

8. the purposes of long-chain non-coding RNA described in claim 1 or reagent as claimed in claim 4, which is characterized in that use In the chip or kit of preparation diagnosis Male Osteoporosis.

9. purposes according to claim 8, which is characterized in that the reagent is visited including the use of SYBR Green, TaqMan Needle, molecular beacon, double cross probe or combined probe detect the pcr amplification primer used when the long-chain non-coding RNA expression quantity Object.

10. purposes according to claim 9, which is characterized in that the primer sequence such as SEQ ID NO.1 and SEQ ID Shown in NO.2.