CN110106249A - Application of the long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker - Google Patents
Application of the long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker Download PDFInfo
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Abstract
A kind of application the invention discloses long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker, the present invention has expression in sufferers of osteoporosis face bone tissue and normal person's bone tissue by detection discovery LncRNA NONHSAG039951.2, and the expression in sufferers of osteoporosis face bone tissue is all remarkably higher than normal person, difference has statistical significance, prompts feasibility and clinical value of the LncRNA NONHSAG039951.2 as diagnosing osteoporosis marker.Long-chain non-coding RNA NONHSAG039951.2 provided by the invention can be used as diagnosing osteoporosis marker, reach the purposes of osteoporosis early diagnosis, early treatment.
Description
Technical field
The invention belongs to field of biomedicine, specially a kind of long-chain non-coding RNA NONHSAG039951.2 is as bone
The application of the loose diagnosis marker of matter.
Background technique
Osteoporosis is a kind of characterized by bone amount is reduced and bone micro-structure destroys, and leads to the increase of bone brittleness and Yi Fasheng
A kind of complicated, multifactor systemic chronic bone diseases of fracture.Aggravation of the China with ageing phenomenon, osteoporosis
The number of the infected of disease increases year by year, this brings whole body ostalgia, fracture, height to patient and becomes the pain such as short and harm, while
Heavy economy and living burden are brought to society and family.
Osteoporosis early stage incidence of occult, atypical clinical manifestations will rely on imageological examination and auxiliary serum/urine
Biochemical index inspection could be found, and there are certain limitations, and then lead to osteoporosis Newly diagnosed rate not
It is high.If do not paid attention to, fracturing repeatedly, which will lead to, disables, lethal serious consequence.Therefore, the height of early detection osteoporosis
Danger crowd, and carry out intervention processing, prevent osteoporosis, it is to improve the elderly population general level of the health to prevent to fracture
And quality of life, reduce one of the important measures of the elderly's death rate.Clinically the early diagnosis of osteoporosis still lacks at present
Specific marker.This discovery finds highly sensitive and high specific bone to the diagnosis and survival rate for improving osteoporosis
The loose diagnosis molecular marked compound of matter has great clinical meaning.
Only has 2% DNA encoding the protein in the gene of the mankind, most gene transcribes the RNA to form non-coding
(ncRNA).Wherein long-chain non-coding RNA (LncRNA) is that a kind of length is greater than 200 nucleotide, lacks special Complete Open and read
Frame, the RNA without protein coding function.LncRNA takes part in genomic imprinting, transcription control, post-transcriptional control, albumen function
The important link being adjustable during equal signal transductions.Existing research shows the occurrence and development and prevention and treatment of LncRNA and human diseases
There are close association, such as disease of immune system, cardiovascular disease and tumour etc..LncRNA is considered taking part in point of osteoblast
Change, LncRNA is prompted to play key player in osteoporosis.However, at present both at home and abroad to osteoporosis LncRNA's
The research of expression spectrum signature and biological function is but rarely reported.Sclerotin is dredged from LncRNA level using high throughput sequencing technologies
Loose disease carries out going deep into anatomy, supports strong scientific research is provided for the molecular diagnosis of disease.
The present invention is directed to pass through high-throughput platform, osteoblast in patients with postmenopausal osteoporosis is formed and osteoclastic thin
The regulatory mechanism that born of the same parents absorb LncRNA in stable state is studied.Compare patients with osteoporosis and the comparable normal person of age-sex
The LncRNA of differential expression in group further clarifies regulation of the LncRNA of significant difference expression in osteoporosis forming process
Mechanism, target gene, the biological function and its importance of the target gene of analysis LncRNA regulation further investigate osteoporosis
Pathogenic mechanism, to develop noninvasive prediction, diagnosis type biomarker and new therapy target provide basic science foundation.
Summary of the invention
The purpose of the present invention is to clinically, the detection of osteoporosis still lacks highly sensitive and high specific at present
Early diagnosis index, thus provide LncRNA NONHSAG039951.2 for as diagnosis of osteoporosis marker
Purposes.
To achieve the goals above, technical solution provided by the invention are as follows:
Detect the LncRNA of (PBMC) of bone density normal person and sufferers of osteoporosis face peripheral blood mononuclear cells；
The gene expression amount for calculating cell line, determines the difference between bone density normal person and sufferers of osteoporosis face
LncRNA, and detect expression quantity of the LncRNA NONHSAG039951.2 in bone tissue, it was demonstrated that LncRNA
NONHSAG039951.2 high expression in sufferers of osteoporosis face bone tissue；
The differentiation of human marrow mesenchymal stem cell induced osteogenesis, observes variation of the difference LncRNA in osteogenetic process,
Confirm that difference LncRNA NONHSAG039951.2 is related with bon e formation, LncRNA NONHSAG039951.2 is as osteoporosis
Diagnosis marker.
LncRNA NONHSAG039951.2 is for the purposes as diagnosing osteoporosis marker, the long-chain non-coding
The nucleic acid sequence of RNA NONHSAG039951.2 are as follows:
ATATTAACCGTTTTCAAGCATCCAAAGGCGAAATGCTTCATCTCTGGGATGGAGTAGAGGAGTAGCCT
CTGCGGTTGAGTTGCAGTCGTGGGTGCAAGaagcagcgctgaagcagagtgcagccccggaatagggcgggatgga
atggcctgggcagttcagaaacccgcgaggagtccagggcggaacagagggaagaaggccatttggagatagaagc
agggattggagtgatgcaactggaagccaagaaacgccgcagaatgccaggagccagcagaagctaagaaaaggca
aag。
Beneficial effects of the present invention: long-chain non-coding RNA NONHSAG039951.2 provided by the invention can be used as sclerotin
Loose diagnosis marker, reaches the purposes of osteoporosis early diagnosis, early treatment, and long-chain genetic regulation by non-coding RNAs mechanism is that sclerotin is dredged
A frontier in loose study of pathogenesis, LncRNA NONHSAG039951.2 is in sufferers of osteoporosis face bone tissue and normally
There is expression in people's bone tissue, and the expression in sufferers of osteoporosis face sclerotin is all remarkably higher than normal person, difference has
Statistical significance prompts feasibility and clinical application of the LncRNA NONHSAG039951.2 as diagnosing osteoporosis marker
Value.
Detailed description of the invention
Fig. 1 is Technology Roadmap of the invention；
Expression quantity (log10 (the exp+1)) Heatmap of Fig. 2 potential difference different LncRNA and mRNA schemes；
Fig. 3 is calcium Alizarin red staining osteoblast；
Fig. 4 is tpm Distribution value of the difference LncRNA NONHSAG039951.2 in osteoporosis and normal two crowds；
Fig. 5 is expression quantity of the difference LncRNA NONHSAG039951.2 in four periods (D0, D3, D7 and D14) of cell
Distribution.
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.
Application of the long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker: finding and sclerotin
Loose relevant LncRNA simultaneously predicts its function, and in osteoporosis, the expression of certain specific LncRNA can occur
Change, the variation of this expression can be total to table with variation LncRNA by finding as the marker of osteoporosis screening
It reaches and the encoding gene of difference, to speculate the function of LncRNA, specifically, three phases of the present invention are carried out:
1st stage collected sample, high-flux sequence；
In 2nd stage, data filtering, sample are quantitative, calculate differential expression and the microRNA target prediction of difference LncRNA；
3rd stage, candidate targets functional annotation, and screening LncRNA, LncRNA and mRNA qPCR and cell function are tested
Card.
The detailed process in the 1st stage includes: (collecting sample, high-flux sequence)
Step 1.1 collects 60 45-70 years old section menopausal womens and passes through Dx dual intensity ray detection bone density (hip joint and waist
Vertebra), value≤- 2.5 T are classified as osteoporosis group, T value >=- 1 it is classified as normal group of bone amount, bone metabolism index P1NP,
CTX value is as reference.
Step 1.2 separates PBMC cell, extracts total serum IgE, builds library and high-flux sequence, machine under data；
(1) blood sample of post menopausal 30 patients with osteoporosis and 30 normal females is collected；
(2) blood is separated, is divided into the blood plasma on upper layer and the haemocyte of lower layer.
(3) peripheral blood mononuclear cells (peripheral blood monouclear is isolated from the haemocyte of lower layer
Cell, PBMCs), total serum IgE removes rRNA, to retain all non-coding RNAs to greatest extent；Obtained RNA is interrupted at random
As short-movie section, then the RNA being had no progeny using piece is template, with hexabasic base random primer (random hexamers) synthesis cDNA the
One chain, and buffer, dNTPs, RNase H and DNA polymerase I synthesis the second chain of cDNA is added, by QiaQuick
PCR kit purifies and the elution of EB buffer is added to repair through end, add base A, adds sequence measuring joints, then passes through UNG
(Uracil-N-Glycosylase) enzyme degradation Article 2 chain, then clip size selection is carried out with agarose gel electrophoresis, it carries out
PCR amplification；
(4) library 150bp PE built up carries out high-flux sequence on BGISEQ500；
The detailed process in the 2nd stage includes: that (data processing, sample is quantitative, calculates differential expression LncRNA, difference
LncRNA microRNA target prediction)
The original reads pretreatment of the lower machine of step 2.1
Lower machine data are passed through into SOAP (parameter: 32-v of-m 0-x, 1000-s, 28-l, 5-r 2) and ribosomes number
It is compared according to library, removes rRNA data in sequence.Then remove again connector pollution, low quality reads (mass value Q≤5
Base number accounts for 10% or more of whole read), the reads of base containing N, obtain clean data, each sample, which generates, is no less than 6G
clean data。
Step 2.2 sample is quantitative and calculates differential expression
By the clean data of each sample by hisat2 compare back hg19 (parameter: -- phred64--
1) 1-X of sensitive--no-discordant--no-mixed-I, 1000-N, then passes through obtained bam file
Htseq-count (parameter: 10-m union of-r name-s no-a) calculates the raw read of each transcript and gene
count.Read count in 80% sample >=0 gene is filtered out, then uses DESeq2, EdgeR and limma simultaneously
Calculate the group difference gene that osteoporosis group and bone amount are normally organized.In addition by clean data by bowtie2 (parameter :-
q--phred64--sensitive--dpad 0--gbar 99999999--mp 1,1--np 1--score-min L,0,-
1) 4-k of 0.1-I 1-X 1000--no-mixed--no-discordant-p, 200-N is compared onto RNA sequence, pass through
Rsem calculates the TPM value and FPKM value of each gene.The group that osteoporosis group and bone amount are normally organized is calculated separately by DEGseq
Between difference.
Step 2.3 difference LncRNA microRNA target prediction
It is calculated to predict the complementary combination between antisense LncRNA and mRNA according to its composite nalysis using RNAplex
Minimum free energy predicts best base-pair relationships.Meanwhile calculate each LncRNA and mRNA Pearson correlation coefficient and
Its significance, related coefficient absolute value are more than or equal to LncRNA-mRNA of the 0.6 and p-value value less than 0.05 to judgement
For candidate LncRNA and its target gene pair.Additionally, it is believed that the protein coding gene correlation that the function of LncRNA is adjacent thereto, position
There may be intersection with other of promoter or co-expression gene cis-acting elements in the LncRNA of coding albumen upstream and downstream.Meter
LncRNA and the relative position of mRNA on chromosome are calculated, the LncRNA of the upstream mRNA 10k and downstream 20k, existing can be used as
The target gene of LncRNA candidate.
The detailed process in the 3rd stage includes: that target gene function annotation and candidate gene qPCR and cell function are verified
Step 3.1 target gene function annotation
Due to obtained difference LncRNA negligible amounts, the side such as literature search, Genecards, KEGG access is used in combination
Formula searches the correlation function that LncRNA corresponds to target gene.
Step 3.2 is selected differential expression significant mRNA and LncRNA between group (between preferred gene area or include sub-district) and is carried out
QPCR verifying and cell function verifying.
The technology of the present invention approach is shown in Fig. 1:
(1) it tests: collecting the blood of 30 pairs of patients with osteoporosis and normal control, extract RNA respectively, carry out high-throughput
After LncRNA builds library and sequencing sample extraction total serum IgE, total serum IgE removes rRNA, to retain all LncRNA to greatest extent；
Obtained mRNA is interrupted as short-movie section at random, then the mRNA being had no progeny using piece is template, with hexabasic base random primer (random
Hexamers the first chain of cDNA) is synthesized, and buffer, dNTPs, RNase H and DNA polymerase I synthesis cDNA is added
Second chain purifies by QiaQuick PCR kit and the elution of EB buffer is added to repair through end, add base A, sequencing is added to connect
Head then by UNG (Uracil-N-Glycosylase) enzyme degradation Article 2 chain, then carries out segment with agarose gel electrophoresis
Size selection, carries out PCR amplification, and the sequencing library finally built up is with carrying out high-flux sequence.
(2) information analysis: impurity is removed to lower machine data, removal rRNA obtains clean data, then utilizes clean
Data constructs transcript, the library non-coding RNA annotation, protein pool comparison, CPC (Coding Potential
Calculator) prediction, expression quantity statistics etc..It finally finds out to express in patient and check sample in conjunction with the clinical information of sample and show
The LncRNA of difference is write, and functional analysis is carried out to it.
(3) experimental cell system high-flux sequence is verified: by inducing human mesenchymal stem cells (BMSC) skeletonization, calcium is alizarin
Plain red colouring proves osteogenic induction skeletonization.High-flux sequence analyzes (0 day, 3 days, 7 days, 14 days) LncRNA content during skeletonization
Variation.BMSC cell is cultivated using the fetal calf serum (gibico) of α-MEM culture medium and 10%, osteogenic induction liquid (α-MEM is added
+ 10%FBS+0.2mM ascorbic acid+100nM dexamethasone+10mM sodium β-glycerophosphate) induced osteogenesis, and temporally point is collected
Cell extraction total serum IgE total serum IgE removes rRNA, to retain all non-coding RNAs to greatest extent；Obtained RNA is interrupted at random
As short-movie section, then the RNA being had no progeny using piece is template, with hexabasic base random primer (random hexamers) synthesis cDNA the
One chain, and buffer, dNTPs, RNase H and DNA polymerase I synthesis the second chain of cDNA is added, by QiaQuick
PCR kit purifies and the elution of EB buffer is added to repair through end, add base A, adds sequence measuring joints, then passes through UNG
(Uracil-N-Glycosylase) enzyme degradation Article 2 chain, then clip size selection is carried out with agarose gel electrophoresis, it carries out
PCR amplification；The library 150bp PE built up carries out high-flux sequence on BGISEQ500；
The calculating of the gene expression amount of cell line (data processing, sample are quantitative)
The original reads pretreatment of the lower machine of step 1
Lower machine data are passed through into SOAP (parameter: 32-v of-m 0-x, 1000-s, 28-l, 5-r 2) and ribosomes number
It is compared according to library, removes rRNA data in sequence.Then remove again connector pollution, low quality reads (mass value Q≤5
Base number accounts for 10% or more of whole read), the reads of base containing N, obtain clean data, each sample, which generates, is no less than 50M
clean reads。
Step 2.2 sample is quantitative and calculates differential expression
The clean data of each sample is passed through into bowtie2 (parameter :-q--phred64--sensitive--dpad
0--gbar 99999999--mp 1,1--np 1--score-min L,0,-0.1 -I 1-X 1000--no-mixed--no-
1) 4-k of discordant-p, 200-N is compared onto mRNA and LncRNA sequence, calculate each encoding gene by rsem
With the TPM value of LncRNA.
In summary: the present invention is detection bone density normal person and sufferers of osteoporosis face peripheral blood mononuclear cells first
(PBMC) LncRNA collects the blood of 30 patients with osteoporosis blood samples and 30 normal females in above-described embodiment
Liquid sample in contrast group；Detect (PBMC's) of bone density normal person and sufferers of osteoporosis face peripheral blood mononuclear cells
LncRNA；Then the gene expression amount for calculating cell line, determines the difference between bone density normal person and sufferers of osteoporosis face
LncRNA, and detect expression quantity of the LncRNA NONHSAG039951.2 in bone tissue, it was demonstrated that LncRNA
NONHSAG039951.2 high expression in sufferers of osteoporosis face bone tissue；Last human marrow mesenchymal stem cell induced osteogenesis
Differentiation, observes variation of the difference LncRNA in osteogenetic process, it was demonstrated that difference LncRNA NONHSAG039951.2 and bone
Formed related, LncRNA NONHSAG039951.2 is as diagnosing osteoporosis marker.
Expression quantity (log10 (the exp+1)) Heatmap of Fig. 2 difference lncRNA and mRNA scheme, and right ordinate scale is in Fig. 2
Long-chain RNA title, shade indicates expression quantity level in Fig. 2；Fig. 3 is calcium Alizarin red staining osteoblast；Fig. 4 is difference
Tpm Distribution value of the LncRNA NONHSAG039951.2 in osteoporosis and normal two crowds；Fig. 5 is difference LncRNA
NONHSAG039951.2 is distributed in the expression quantity of four periods (D0, D3, D7 and D14) of cell.This result confirms difference LncRNA
NONHSAG039951.2 high expression quantity in osteoporosis people's bone tissue, and difference LncRNA NONHSAG039951.2 with
Bon e formation is related, and difference has statistical significance, and difference LncRNA NONHSAG039951.2 is prompted to can be used as diagnosing osteoporosis
Marker.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Sequence table
<110>Shenzhen people's hospital；Shenzhen Hua Da life science institute
<120>application of the long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker
<141> 2019-05-09
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 299
<212> DNA
<213> Homo sapiens
<400> 1
atattaaccg ttttcaagca tccaaaggcg aaatgcttca tctctgggat ggagtagagg 60
agtagcctct gcggttgagt tgcagtcgtg ggtgcaagaa gcagcgctga agcagagtgc 120
agccccggaa tagggcggga tggaatggcc tgggcagttc agaaacccgc gaggagtcca 180
gggcggaaca gagggaagaa ggccatttgg agatagaagc agggattgga gtgatgcaac 240
tggaagccaa gaaacgccgc agaatgccag gagccagcag aagctaagaa aaggcaaag 299
Claims (3)
1. application of the long-chain non-coding RNA NONHSAG039951.2 as diagnosing osteoporosis marker, which is characterized in that packet
Include following steps:
Detect the LncRNA of (PBMC) of bone density normal person and sufferers of osteoporosis face peripheral blood mononuclear cells；
The gene expression amount for calculating cell line, determines the difference LncRNA between bone density normal person and sufferers of osteoporosis face, and
Detect expression quantity of the difference LncRNA NONHSAG039951.2 in bone tissue, it was demonstrated that LncRNA NONHSAG039951.2 exists
High expression in sufferers of osteoporosis face bone tissue；
The differentiation of human marrow mesenchymal stem cell induced osteogenesis, observes variation of the difference LncRNA in osteogenetic process, it was demonstrated that
Difference LncRNA NONHSAG039951.2 is related with bon e formation, it was demonstrated that LncRNA NONHSAG039951.2 is as osteoporosis
Diagnosis marker.
2. application as described in claim 1, it is characterised in that: detection bone density normal person and sufferers of osteoporosis face peripheral blood list
The LncRNA of (PBMC) of nucleus, including collect sample, high pass measurement, comprising the following steps:
S21, collect 30 sufferers of osteoporosis face and 30 bone density normal females blood sample, wherein sufferers of osteoporosis face and
Bone density normal person is 45-70 years old section menopausal women；
S22, above-mentioned blood is separated, is divided into the blood plasma on upper layer and the haemocyte of lower layer, is isolated in the haemocyte of lower layer
Peripheral blood mononuclear cells (PBMC), total serum IgE removes rRNA, to retain all non-coding RNAs to greatest extent；
S23, non-coding RNA build library by transcription, enzyme degradation, agarose gel electrophoresis and PCR amplification；
S24, the library 150bp PE built up carry out high-flux sequence on BGISEQ500.
3. application as described in claim 1, it is characterised in that: the gene expression amount for calculating cell line determines that bone density is normal
Difference LncRNA between people and sufferers of osteoporosis face, and difference LncRNA NONHSAG039951.2 is detected in bone tissue
Expression quantity, it was demonstrated that LncRNA NONHSAG039951.2 high expression in sufferers of osteoporosis face bone tissue, comprising the following steps:
The original reads pretreatment of S41, lower machine, lower machine data are compared by SOAP and ribosomes database, are removed in sequence
Then rRNA data remove connector pollution, low quality reads, the reads of base containing N again, obtain clean data, each
Sample, which generates, is no less than 50M clean data；
S42, sample are quantitative and calculate differential expression, by the clean data of each sample by bowtie2 compare to mRNA with
In LncRNA sequence, each encoding gene and the TPM value of LncRNA are calculated by rsem, sclerotin is calculated separately by DEGseq and is dredged
The group difference that pine group and bone amount are normally organized；
The microRNA target prediction of S43, difference LncRNA, the difference LncRNA microRNA target prediction uses RNAplex, to predict antisense
Complementary combination between LncRNA and mRNA.
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