CN106399309B - A kind of long non-coding RNA and its application in diagnosis/treatment T2DM - Google Patents
A kind of long non-coding RNA and its application in diagnosis/treatment T2DM Download PDFInfo
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Abstract
The invention belongs to genetic engineering field, in particular to application of the Lincpint in preparation prediction T2DM and target drug treatment；The downward of Lincpint is related with islet beta cell function damage in T2DM mouse, the Lincpint of low expression level may take part in the pathogenic process of T2DM, and the expression by changing Lincpint has an impact the synthesis and secretion of the apoptosis, insulin of beta Cell of islet.
Description
Technical field
The invention belongs to genetic engineering field, in particular to a kind of long non-coding RNA and its in diagnosis/treatment T2DM
Using.
Background technique
Diabetes are to seriously affect the chronic disease of population health, and raised trend year by year is presented in disease incidence, not only to a
Body brings body and spiritual damage, returns personal, country and brings financial burden.The institutional framework and pancreas islet of pancreas complexity are raw
Reason function and the diabetes morbidity clinically remained high make the research to islet function particularly important, islet function
Maintenance is one of the research emphasis of current diabetes.
More and more report displays, lncRNA participate in the development of islet cells, function maintenance, diabetes and its complication
Occurrence and development.There is seminar by finding to Human islet's beta cell transcription group analysis, identifies 1128 in people's pancreas islet
LncRNA, and there is tissue specificity, while also found that the expression of long-chain in pancreas islet has stringent Space-time speciality, it participates in
The differentiation and maturation process of pancreas islet beta cell, research find that some lncRNA can be by the regulation of glucose and in people
Abnormal expression in the pancreas islet of T2DM, HI-LNC25 can regulate and control GLIS3, and GLIS3 is the transcription factor in islet cells, contain T2DM
Susceptibility loci prompts HI-LNC25 that islet function is being maintained to play an important role.Same seminar finds lncRNA β in the recent period
Linc1 maintains islet function by regulation islet specificity transcription factor, and beta cell development defect can be caused after knockout
And adult mice impaired glucose tolerance.In addition, have whole-genome association (Genome-wide association study,
GWAS the susceptibility loci that a large amount of non-coding regions contain T2DM) is found, as lncRNA ANRIL and KCNQ1OT1 contain T2DM
Susceptibility loci.Unconventionality expression is had found in Nonobese diabetes (non-obese diabetic, NOD) mouse islets
LncRNA causes beta Apoptosis after overexpression.GWAS analysis finds that PVT1 is significant related to diabetic nephropathy, and PVT1
It can increase the pathogenetic probability of diabetogenous nephrosis.This seminar finds that Tug1 and Meg3 are in mice pancreatic tissue in early-stage study
Relatively high expression is expressed in T1DM and T2DM mouse islets and is lowered, and is influenced insulin synthesis by regulative transcription factor and is divided
It secretes.Result above prompts the function maintenance and the occurrence and development of diabetes of lncRNA participation adult beta cell.However, lncRNA
The molecular mechanism acted in beta cell will also be explored further.
In view of importance of the lncRNAs in T2DM, we have probed into lncRNA Lincpint, have passed through quantitative PCR first
Expression of the Lincpint in mice pancreatic, the heart, liver, spleen, lung, flesh, kidney is had detected, while being detected in db/db mouse islets
Expression.Then we are in vivo and levels in vitro lowers the expression of Lincpint with RNA conflicting mode, and use real-time
The technical research such as quantitative PCR technique, flow cytometry, Western blot, RIA, ELISA Lincpint is in mouse islets function
In effect, it is intended to Primary Study Lincpint is to the synthesis of pancreas islet beta cell Proliferation, apoptosis and insulin and secretion function
The effect of energy studies Lincpint effect played in T2DM mouse beta cellular damage, and then enriches and improve pancreas islet
The gene regulatory network of function prompts lncRNA may be as the new molecular target of diagnosing and treating diabetes.
Summary of the invention
The object of the present invention is to provide for diagnosing T2DM (T2DM) incidence or as treatment T2DM drug target
LncRNALincpint, gene are located at 6A3.3, total length 1714bp, and nucleotides sequence is classified as SEQ ID NO:1,
SEQ ID NO:1:
ATGATGGACA TGATATAATG AAACAACATT GTGGAGAGGA AAGCATTAGG GGAGCCCACG
GCTACAAAAC AAGTGAGAAG AGGTGGGAGA AAGAGAAACT ACGCCACCTC CTCTGCAGCC GAGCGCACGC
AGCAGCCTGG CGTGACAAGT GGGCGACGCC AGGGACAGAG AGTGGGGGTC CTCCGCCCGC TCCGGGGACC
CTGTCGCCCA CCGTGGTGCG CTGAGCAACT TTTAGCCGGC AGCCCCGCAC CTGTCTCAGA GTCCCCATCG
CTCCCCACCA ACCATCATGG GAAAATTCTA GAAGAATACG CTCTCGGAAT CTACGTGCGC ATCATTTTCC
TCCTCACTCC AGAGGAAGGA ACCAGAAAAG AGAGCAAAGC GGTGTAGTGT TCAGCCTCAA GAACCCCCGT
GCATCCCAGG GATGGCGCCC TCTACAGGAG GGTCCTTGCC TCACTTTCGG GAGTCTCTTA ACTGCATCTC
GTTGATTCCT AGCGTTAGAA GAACTGAGCT AACAGGAGTC TGCCACCTCT CTGCCTTGCTGATGCACCCA
GGCAGGGCTC ACTGGTTTGC AGGAGTACAT GACCACAGAT CAGAGCCATT GGCTGCACAG ATACCTGACT
CTGGGCTCTC AGGAAGACTC AGGCAGAGAT GGGGAGTCGG CCTGCAAGCT GGGACGCTAG AGCTCACGTG
CAGTTCTCCA TTTAGCTAGG AGGTTTCCAG GGCAACCACC GAAGCAGTAA TTAAAGATGA AGAGCTAAAA
GGTAAGTACT TCCAAACCTG AATCCTGAAG GAGGTGGTCC GCAGGCTGGT GTCACAGTGT GCACTCTTAT
TCACTTAACA ATTAGGATGT TCATGCTCTG CTTTGATTTG TATCTTTTCA CGCGCGCACG TATTCTTGTA
TGTACGTACA CACCACACAC ACACACACAC ATTTTTATGC TTCCACCAGC CAGTCATGGG ACCAGGCATT
GGGATACCAG GGCTTAAGGT GTCTTTCGGG TTCCTGTTTT GAACGTCCTA GCATGCATGT ATATATATAT
ACATACATAC ATATATATAT ATACACATTT AAGGCATGCA AACGAGCAAC TGCAGAGGCG TCTACAGTAC
TTAGCGTGTA GCAGGAACAT CCAGGCTCTC CAGGCAGTTC ACGGGTGCTT ACACAGCCTG ACGAGGTGCT
CGCCGTGCCG CCATCCTTTC CTTGAGCGAA CTGTACCCTG GGTTATGAAC CTTGTTTGCC TGATAGTTCA
GGGCATCAGT GCAGTAACAG GGCTGATGAG AGAGAGCTCC ACCAAAGCAG CAGCGCCATT GAGAAGGTAG
AAGACAGCTA TTTTAACGGA AGCTTCAGAA GCTCATGAGT CTATTCCCAG GAGTATGGGT AAAGTTTGCT
CATGCAAATG CTGCATTTCA TGAGTCCATG GGTTAAAGCA GTTACGGACC CTGGAAGATG GGAGATACTT
ACAAAAAGCA AAGCCCAGAT CTCCACACCT TCAAAGAGAA GATGCTCAAA TCTCCAATTT TCAGACCAGT
GCTTTTTTCA GTTATGCACT GGTATATGGC TGAAACTGTA GGTGGCATGG GAGACTTTGG TAAGTGTAGG
CCCTAGGATA TCTGTCCCAC CATTTTCCCT GTAACAAAAG TAAGGAATCT GTAA
The invention further relates to the markers of the identification long non-coding RNA to judge T2DM morbidity and treatment condition in preparation
Diagnostic products in application, the marker includes but is not limited to:
(1) in conjunction with described in the combination of primer/primer sets of the long non-coding RNA or fluorescent marker long non-coding RNA
Primer/primer sets；
(2) in conjunction with the small molecule compound of the long non-coding RNA；
(3) in conjunction with the large biological molecule of the long non-coding RNA, the large biological molecule includes but is not limited to: antibody
Or the antibody or antibody functional fragment of antibody functional fragment, fluorescent marker, rna binding protein or its function fragment, fluorescent marker
Rna binding protein or its function fragment.
Preferably, the nucleotide sequence of the primer sets or the primer sets of fluorescent marker such as SEQ ID NO.2 and SEQ
Shown in ID NO.3,
Lincpint Primer F (SEQ ID NO:2): 5'ACGCTCTCGGAATCTACGTG3',
Primer R (SEQ ID NO:3): 5 '-CTCCCGAAAGTGAGGCAAGG-3 '.
Beta-actin Primer F (SEQ ID NO:4): 5'AGGTCATCACTATTGGCAACGA 3',
Primer R (SEQ ID NO:5): 5'CACTTCATGATGGAATTGAATGTAGTT 3'.
The invention further relates to the markers comprising long non-coding RNA described in the identification for judging that T2DM falls ill
The reagent or kit of situation.
Reagent the invention further relates to the marker of long non-coding RNA described in the identification or comprising the marker
Or application of the kit in the incidence for judging T2DM.
The invention further relates to application of the long non-coding RNA in the drug of preparation treatment T2DM；
The invention further relates to the long non-coding RNAs in the diagnostic reagent for judging T2DM incidence as screening
Using.
The invention further relates to application of the long non-coding RNA in the drug as screening treatment T2DM.
Technical solution
Experimental animal
Each 40 using male B6.BKS-Leprdb (db/db) mouse of 8 week old and control mice, weight is on the left side 30g
The right side is bought from model animal research institute of Nanjing University (MARC).The male Balb/c mouse of 6-8 weeks size, each 50, weight
25g or so is bought from Nanjing Medical University's animal center.All experiments are entrusted by Nanjing Medical University's animal protection and ethics
Member's meeting (IACUC) is ratified.
Cell culture
15%FBS, 2.5mM β-sulfydryl is added in pancreas islet beta cell line (MIN6 cell) DMEM in high glucose culture medium culture
Ethyl alcohol, the streptomysin of 100U/ milliliters of penicillin and 100 mg/mls are cultivated in 37 DEG C, constant incubator containing 5%CO2.
The 1640 culture medium culture of RPMI of NIT-1 cell, 10%FBS, addition is dual anti-, at 37 DEG C, in the constant incubator containing 5%CO2
Culture.Culture medium is changed daily, is passed on when cell density reaches 80%-90%.Be from Shanghai Cell Bank of the Chinese Academy of Sciences purchase (on
Sea, China).NCIH1299, NCI-H1975, SK-MES-1 and 16HBE cell are cultivated in 1640 culture medium of RPMI；SPC-A1
It is cultivated in DMEM culture medium with PC9 cell.In complete medium containing 10% fetal calf serum, at 37 DEG C, 5%CO2's
It is cultivated in incubator.
RNA is extracted and quantitative PCR analysis
According to the operation instruction of reagent, total serum IgE is separated with Trizol reagent.Reverse transcription reaction application TaKaRa Prime
Script kit (TaKaRa, Dalian, China).Reverse Transcriptase kit carries out reverse transcription to 1 μ g total serum IgE, and final volume is 20 μ
l.Interpretation of result: the specificity and amplification efficiency of primer are analyzed, the atopic of primer is judged according to solubility curve.According to expansion
Increase curve and obtain Ct value, the analysis of target gene relative expression quantity is carried out using relative measurement and internal reference beta-actin.It calculates
Formula are as follows: 2^ (- △ Ct), △ Ct=Ct gene-Ctcontrol.
Cell transfecting
According to operating instruction with 2000 transfection reagent of liposome Lipofectamine transfect Lincpint-siRNA and
control-siRNA.MIN6 and NIT-1 cell fusion, which has expired, to be just inoculated into six orifice plates, then according to illustrating to operate.Transfection 48
After hour, cell is collected, is used for qRT-PCR or immunoblotting assay.
Cell Proliferation and cell apoptosis assay
Mtt assay detects cell Proliferation vigor: transfection cell first is cultivated by 8000, every hole cell kind in 96 holes afterwards for 24 hours
Plate, every 200 μ l of hole, is arranged 5 multiple holes.After 12h, culture medium is discarded, the MTT reaction solution of 20 μ l is added every hole, and 37 DEG C are protected from light and incubate
Educate 4h.Supernatant is abandoned, the dimethyl sulfoxide (DMSO) of 150 μ l is added every hole, shakes 10min, measures the light absorption value at 490nm wavelength.
The flow cytomery cell cycle: cell kind is transfected after cultivating 12h in 6 well culture plates.After 48h, with being free of
The pancreatin of EDTA gets off cell dissociation, and 4 DEG C, supernatant is abandoned in 1000g centrifugation, and PBS is washed 2 times.75% ethyl alcohol of pre-cooling is blown even thin
It is centrifuged after born of the same parents, overnight at -20 DEG C.Centrifugation discards supernatant, and PBS is washed 2 times.Each sample takes 450 μ l PBS to blow even centrifugation, 50 μ l's
Propidium iodide is added thereto, and 37 DEG C of water bath 30min are placed on after mixing.Centrifugation discards supernatant, and PBS blows even centrifugation, thin with streaming
Born of the same parents' instrument (BD company) is measured and is analyzed.Flow cytomery Apoptosis: by cell kind in 6 well culture plates, after cultivating 12h,
It is transfected.After 48h, cell dissociation is got off with the pancreatin without EDTA, 4 DEG C, supernatant is abandoned in 1000g centrifugation, and PBS is washed 2 times.
The remaining PBS of cell precipitation is exhausted, 30s is vibrated, cell is made to suspend.The combination buffer of 200 μ l calcium ions is added, then will
10 μ l Annexin V-FITC fluorescence probes are added, and gently oscillation mixes, avoid light place 30min.The propidium iodide of 5 μ l is added
In cell, oscillation is mixed, avoid light place 5min.400 μ l combination buffers are added thereto, and after oscillation mixes, use flow cytometer
Detection.
(GSIS) is tested in the insulin secretion of glucose stimulation
By MIN6 cell kind in 48 well culture plates, 60-70% is reached to cell density, culture medium is discarded, is transfected.48h
Afterwards, GSIS experiment is carried out.Preparation glucose-free Krebs-Ringer bicarbonate (Krb) (115mM NaCl,
4.7mM KCl,1.2mM MgSO4.7H2O,1.2mM KH2PO4,20mM NaHCO3,16mM HEPES,2.56mM CaCl2,
0.2%BSA) solution prepares low sugar liquid (2mM), high liquid glucose (20mM) according to experiment hole count.Low sugar liquid 200ul is added, discards.
Add the low sugar liquid of 200ul, discarded after 37 DEG C of culture 1h, 200ul low sugar liquid is added in every hole, and 37 DEG C of culture 1h collect supernatant to EP
Guan Zhong.Add 200ul high liquid glucose, 37 DEG C of culture 1h collect supernatant into EP pipe.Add the sour ethyl alcohol of 200ul, 4 DEG C overnight, take supernatant
Into EP pipe, it is placed on -80 DEG C.Insulin content is detected with radioimmunoassay (RIA).
The separation and culture and transfection of Mice Islet Cells
The extraction and purifying of mouse islets: match HBSS solution (KCl 0.4g, KH2PO 40.06g, MgCl2.6H2O
0.1g, MgSO4.7H2O 0.1g, CaCl2 0.1g, NaCl 8g, NaHCO3 0.35g, NaHPO4.7H2O 0.09g deionization
Water prepares clostridiopetidase A (Collagenase V HBSS solution is prepared, 1mg/ml) to IL, PH7.4), and mouse fasting 12h or more prepares penta
Barbital sodium, anesthetized mice.It cuts open the belly out chest, finds pancreas general pipeline, pricked with knot.Heart bloodletting is cut, bloodstain is dried, it is total to find pancreas
Pipe, is intubated.It is first slow rear fast to inject emitter.Pancreas is full generally to need 2ml.Pancreas is stripped down, the 50ml of sterilizing is placed on
In centrifuge tube, it is subsequently put on ice.Add Collagenase V to 5ml into 50ml centrifuge tube.37 degree of water-bath 25min.Centrifuge tube is taken
Out, it places on ice.Tissue is vortexed into silt shape by vortex centrifugal pipe.2 times of volumes of HBSS solution with serum are added, mix eventually
Only digest.Indigested tissue block is removed to new centrifuge tube with sieve.1000g (acceleration 9, deceleration 9), 2 minutes, 4 degree.
Supernatant is abandoned, the HBSS for being free of serum is added, centrifugation is washed one time.Abandon supernatant.Histopaque 5ml is added, piping and druming is mixed, is put into
In 10ml glass tube, HBSS, 5ml are slowly added to pasteur pipet.2000g (adds 9, subtract 1), and 20 minutes, 4 degree.After centrifugation, will in
Between white particle layer be sucked into big ware wait choose.It slightly chooses under the microscope.The culture of primary islet cells: primary islet cells
With RPMI-1640 culture medium, 10%FBS, dual anti-culture is cultivated in incubator, tall and slender overnight.Primary islet cells transfection:
After cultivating 12h, by the pancreas islet distribution of 200, every hole in 6 orifice plates.Take 10 μ l liposomes, 100pmol si-Lincpint, si-NC
It is added in 200 μ l of OPTI-MEMI, piping and druming mixes.It places 5 minutes at room temperature.Liposome is mixed and rouge with si-Lincpint
Plastid and si-NC mixing, piping and druming mix.It places 20 minutes at room temperature.1.6ml OPTI-MEMI is added in 6 orifice plates, will mix
Liquid uniformly instills, and pancreas islet is chosen to every hole.Culture medium is replaced after 6h, and pancreas islet is chosen to every hole.
Lincpint is interfered in animal body
The highest si-Lincpint#1 of jamming effectiveness is selected, Invitrogen company synthesizes the small interference sequence of living body.It will
The si-Lincpint of 50nmol be dissolved in 200ul PBS with syringe needle by tail vein (hydrodynamic intravenous
(IV) tail vein injections) it is injected into adult mice body, as si group, the random ordering of same volume is separately taken to be injected into
Year mouse, as NC group.After 48h, tested.
The glucose tolerance test (IPGTT) of intraperitoneal injection
By si-NC and si-Lincpint mouse fasting 12h.2mg glucose is needed according to every g weight, grape is injected intraperitoneally
Sugar.Volume (ml)=weight (g)/100.With 75% ethanol disinfection animal abdomen, and it is dry with cotton balls.Clench animal subject
Simultaneously glucose is injected intraperitoneally with syringe in back.Respectively before the injection 0,15,30,60,90,120 when, cut tail to survey blood glucose.
Immunoblotting assay and antibody
Cell protein pyrolysis product is separated by 10%SDS-PAGE, is transferred to 0.22 μm of NC film, with specific antibody
It is incubated for.Autoradiograph densitometry is to be quantized.Beta-actin antibody is used as compareing.EZH2, beta-
Actin antibody (1:1000) is bought from CST company.
ELISA method surveys mice serum insulin
Intravital mouse, orbital vein blood sampling.The blood for taking for 0,15,30,60,90,120 time points, is placed at room temperature for 30min,
It is centrifuged 3000g, 15min.Upper serum is collected, can be reserved in -20 DEG C.Seminal plasma fructose detection kit is placed into room temperature.With ddH2O handle
10X wash buffer is diluted to 1X, and every hole adds 300 microlitres of wash buffer, washes 3 times.Control and sample add 10 microlitres
Assay buffer, control and gauge orifice (0.2,0.5,1,2,5,10ng/ml) plus 10 microlitres of matrix solution；Standard
The titer of 10 microlitres of each concentration is added in hole, 10 microlitres of blood serum sample is added in sample well, all holes add 80 microlitres
Detection Ab is incubated at room temperature on shaking table 2 hours (all operations before this step are completed within 1 hour as far as possible).wash
Buffer is cleaned 3 times, 300 microlitres of every hole；100 microlitres of every hole enzyme solution, shaking table are incubated at room temperature 30min.It is sucked out,
Wash buffer is cleaned 6 times, 300 microlitres of every hole；It is sucked out, 100 microlitres of every hole substrate；Shaking table is incubated at room temperature 15min, meeting
There is blue.It is sucked out, 100 microlitres of every hole stop solution, blue flavescence color is read in microplate reader under 450nm wavelength.
Data processing
Data are analyzed with SPSS software, with double tail Student’S T examines group difference, correlation analysis linear regression
Model analysis.P < 0.05, P < 0.01 or P < 0.001 are significant difference.
Detailed description of the invention
Fig. 1 is in normal Balb/c mouse, the expression of Lincpint
1A: relative to the heart, liver, spleen, flesh, kidney, Lincpint relatively high expression in pancreatic tissue；
1B:Lincpint is 7 times of exocrine portion in mouse islets
Fig. 2 in db/db mouse islets, lower by Lincpint expression
2A has detected random blood sugar variation using 8 week old and 12 week old control mices and db/db mouse: with compare
Mouse is compared, and the random blood sugar of db/db mouse has raising (P < 0.05)；
2B-2C extracts the pancreas islet of control mice and db/db mouse, has detected Ins1, Ins2 by qPCR technology,
The expression of Lincpint: compared with 8 week old and 12 week old control mices, db/db mouse of the Lincpint in identical week old
37 ± 3% and 49 ± 5% (P < 0.05) are lowered in expression in pancreas islet,
2D: it after giving palmitate (palmitate 0.5mM) processing 48h in MIN6 cell, is detected by qPCR
The expression of Lincpint, as the result is shown: compared with the control group, the expression of Lincpint in palmitate processing group
Lower 37 ± 2% (P < 0.01)
Regulation of the expression of Fig. 3 .Lincpint by glucose
3A-B: compared with normal glucose concentration 25mM, Ins2, MafA are lowered in 5.5,11.1,33.3mM expression
3C: compared with normal glucose concentration 25mM, Lincpint lowers 28% ± 2% in 5.5,11.1,33.3mM expression,
32% ± 7%, 32% ± 3%, it is consistent with the expression trend of Ins2 and MafA
Fig. 4 lowers the secretion that Lincpint inhibits insulin in mouse cell lines
4A, 4B: compared with the control, the jamming effectiveness of Lincpint siRNA#1 is up to 80 ± 3% and 68 ± 3%；
4C, 4D: in interference group, under low sugar concn 2mM stimulation, the secretion no significant difference of insulin, but high glucose concentration
Under 20mM stimulation, the insulin secretion of interference group lowers 42%, while after lowering Lincpint, compared with the control group, into the cell
Insulin content reduce 22%.
Fig. 5 lowers the expression inhibiting insulin synthesis associated retroviral of Lincpint in mouse cell lines and primary pancreas islet
The expression of the factor
5A, 5C, 5E: Ins1 and Ins2 expression is lowered in interference group；
5B, 5D, 5F: after interference Lincpint, transcription factor MafA, Pdx1, Glut2mRNA horizontal expression is lowered；
The protein expression of 5G-H: interference group transcription factor MafA, Pdx1, Glut2 are reduced, consistent with mRNA expression
For Fig. 6 in MIN6 cell, the expression for lowering Lincpint can inhibit cell Proliferation to promote apoptosis
6A: after interference Lincpint, cell viability is suppressed；
6B-C: in interference group, the cell cycle is unaffected；
6D-E: in interference group, Apoptosis increases 8.5%；
6F-G: after interference Lincpint, pro apoptotic protein Bax, shearing-type caspase3, caspase9 expression increases, and resists
Apoptotic proteins Bcl-2 expression is reduced.
For Fig. 7 in normal adult Balb/c mouse, the expression for lowering Lincpint can weaken insulin secreting ability
7A: compared with the control group, the jamming effectiveness in interference group pancreas is 55 ± 7.9%；
7B-C: compared with the control group, 15min, the blood glucose of 30min interference group has raising, 0min, 15min, and 60min is done
The insulin level for disturbing group is declined；
7D: compared with the control group, the expression of Ins1, Ins2, MafA, Pdx1, Glut2 are lowered, the result with cellular level
Unanimously.
Specific embodiment
The present invention will be further explained by examples below, but does not limit the present invention.
Generality explanation:
The experimental method of the dated actual conditions in end in embodiment is substantially all and writes according to Sambrook, J et al.
" Molecular Cloning:A Laboratory guide (the 3rd edition) " (MolecularCloning:ALaboratoryManual, 3rded. Huang Peitang etc.
Translate, Science Press .2002.8) described in condition and method or according to condition proposed by material supplier and method into
Row, it is well known standard method that other technologies being not described in, which correspond to for those skilled in the art,.
Material of the invention: cell strain, siRNA interference sequence and the culture medium referred in the application has supply of commodities
Or with other approach can for obtained by the public, they are only for example, to the present invention be not uniquely, can be respectively with other suitable work
Have with biomaterial and replaces.
Embodiment 1 detects expression of the Lincpint in normal Balb/c mouse
The extraction of primary islet cells, with HBSS solution (KCl 0.4g, KH2PO 40.06g, MgCl2.6H2O 0.1g,
MgSO4.7H2O 0.1g, CaCl2 0.1g, NaCl 8g, NaHCO3 0.35g, NaHPO4.7H2O 0.09g deionized water is extremely
IL, PH7.4；Prepare clostridiopetidase A (Collagenase V is prepared with HBSS solution, 1mg/ml)；Mouse fasting 12h or more prepares penta bar of ratio
Appropriate sodium, anesthetized mice.；It cuts open the belly out chest, finds pancreas general pipeline, pricked with knot；Heart bloodletting is cut, bloodstain is dried, finds pancreas general pipeline, into
Row intubation；It is first slow rear fast to inject emitter.Pancreas is full generally to need 2ml.Pancreas is stripped down, the 50ml centrifuge tube of sterilizing is placed on
In, it is subsequently put on ice；Add Collagenase V to 5ml into 50ml centrifuge tube.37 degree of water-bath 25min；Centrifuge tube is taken out, is placed
On ice；Tissue is vortexed into silt shape by vortex centrifugal pipe.2 times of volumes of HBSS solution with serum are added, mixes and terminates digestion；
Indigested tissue block is removed to new centrifuge tube with sieve.1000g (acceleration 9, deceleration 9), 2 minutes, 4 degree；Supernatant is abandoned,
The HBSS for being free of serum is added, centrifugation is washed one time；Supernatant is abandoned, Histopaque 5ml is added, piping and druming mixes, and is put into 10ml glass
Guan Zhong is slowly added to HBSS, 5ml with pasteur pipet；2000g (adds 9, subtract 1), and 20 minutes, 4 degree；After centrifugation, by intermediate white
Stratum granulosum is sucked into big ware wait choose.It slightly chooses under the microscope.Primary islet cells RPMI-1640 culture medium, 10%FBS,
Dual anti-culture, is cultivated in incubator, tall and slender overnight.
0.1g is taken to organize, the islet cells that liquid nitrogen grinding sufficiently (at powdered) or hand are chosen, the PBS rinse of pre-cooling 2 times.Add
Enter the Trizol lysate of 1ml, blown and beaten and mixed with no enzyme pipette tips, stand 5min, lysate immigration has been marked in advance sterile
In centrifuge tube without enzyme 1.5ml.4 DEG C of 7500g are centrifuged 5 minutes, are taken supernatant that the chloroform of 1/5 volume is added, are mixed by inversion 30s, quiet
Set 2-4min.4 DEG C, 12000g centrifugation, 15min.Three layers (water phase-white precipitate-red organic matter) of solution point shifts aqueous layer
Into new 1.5ml centrifuge tube, try not to be drawn onto white precipitate.Isometric isopropanol is added, is gently mixed by inversion, places 5-
10min.4 DEG C, 12000g centrifugation, 10min.It inhales and abandons supernatant, the ethyl alcohol (now matching) of 1ml 75% is added, wash RNA precipitate.4 DEG C,
7500g centrifugation, 5min abandon supernatant.The alcohol of removal 75% as far as possible, dries, about 15min in room temperature.With no RNA enzyme water (20-
25 μ l) dissolution RNA precipitate.
The concentration of determination of uv absorption method measurement RNA.RNA concentration and purity, measurement are measured using ultraviolet specrophotometer
It is preceding first to be returned to zero with the DEPC water of dissolution RNA.Readings 1 is to indicate 40ng/ μ l at 260nm, the A260/A280's of RNA solution
Ratio is used for the detection of RNA purity, and ratio range shows to meet the requirements 1.8 to 2.1.Agarose gel electrophoresis identifies RNA's
Integrality.Prepare 1% agarose gel.Agarose is dissolved by heating, it is cooling, 1 μ l ethidium bromide (EB, 10mg/ml) is added.It shakes up
The glue that falls afterwards is placed in electrophoresis tank after glue condensation, is dipped in 1 × TAE buffer and balances 10min, for use.Point sample.By 1:4 (v/
V) 5 × nucleic acid electrophoresis sample-loading buffer is mixed with sample, accurately the RNA that each sample contains 1 μ g is added in gel pore.80V
Constant pressure electrophoresis 50min.After electrophoresis, result is observed in gel imager.
Tris- acetic acid (TAE) buffer formulation (1L) 50 ×:
Real-time quantitative PCR
The each tissue of Balb/c mouse, the total serum IgE of primary islet cells and exocrine cell, reverse transcription reaction application
TaKaRa PrimeScript kit (Dalian treasured bioengineering Co., Ltd).Reverse transcription reaction system is as follows:
Reverse transcription reaction condition is as follows: 37 DEG C of 15min (reverse transcription reaction)；(inactivation of reverse transcriptase is anti-by 85 DEG C of 5sec
It answers).According to Genebank provide gene order, design primer sequence,
QPCR application 7300PCR system (Applied Biosystems, Warrington, UK).CDNA sample uses three
Portion's method PCR amplification standardization program.Reaction system:
Reaction condition:
Interpretation of result: the specificity and amplification efficiency of primer are analyzed, the atopic of primer is judged according to solubility curve.
Ct value is obtained according to amplification curve, point of target gene relative expression quantity is carried out using relative measurement and internal reference beta-actin
Analysis.Calculation formula are as follows: 2^(-△Ct), △ Ct=Ct gene-Ct control.
The primer of Lincpint is as follows:
Primer F (SEQ ID NO:2):
5 '-ACGCTCTCGGAATCTACGTG-3 ',
Primer R (SEQ ID NO:3):
5’-CTCCCGAAAGTGAGGCAAGG-3’。
The result shows that relative to the heart, liver, spleen, flesh, kidney, Lincpint relatively high expression (P < 0.05) in pancreatic tissue
(Figure 1A).Expression of the Lincpint in mice pancreatic endo-exocrine portion is had detected using real-time quantitative PCR.As the result is shown
Lincpint is 7 times (P < 0.05) (Figure 1B) of exocrine portion in mouse islets.
Embodiment 2 in db/db mouse islets, lower by Lincpint expression
Db/db (diabetes) mouse is T2DM mouse model, is occurred in mouse No. 4 autosomal db genes recessive prominent
Become, be the spontaneous type-II diabetes mouse as caused by leptin receptor defect, is the research preferable animal model of T2DM.
Db/db mouse started hyperglycemia, hyperinsulinemia, insulin resistance occur at 2 weeks or so, fertilizer then occurred
It is fat, generally obviously increase to blood glucose after 4 weeks, pancreas islet beta cell is significantly destroyed, and random blood sugar reaches after eight weeks
To 20mmol/L or more, there is typical diabetic symptom.8 week old and 12 week old control mices and db/db mouse, detect simultaneously
Random blood sugar variation (Fig2.A): compared with control mice, the random blood sugar of db/db mouse has raising (P < 0.05).It mentions
The pancreas islet for taking control mice and db/db mouse has detected Ins1, the expression of Ins2, Lincpint by qPCR technology
(Fig2.B-C): compared with 8 week old and 12 week old control mices, Lincpint table in the db/db mouse islets of identical week old
Up to 37 ± 3% and 49 ± 5% (P < 0.05) are lowered, consistent with the mRNA level in-site variation of Ins1 and Ins2, prompt Lincpint can
The occurrence and development of T2DM can be taken part in.
Studies have reported that the generation of T2DM is related with the aggregation of fatty acid, the raising of long-term fatty acid levels in vivo
It can cause the damage of beta cell function and the death of beta cell, so as to cause the generation of diabetes.In vitro in cell line
Fatty acid treatment is given, GSIS function and the transcription [38] of insulin can be inhibited.We give palmitate in MIN6 cell
After (palmitate 0.5mM) handles 48h, the expression of Lincpint is detected by qPCR, as the result is shown (Fig.2D): with
Control group is compared, and the expression of Lincpint lowers 37 ± 2% (P < 0.01) in palmitate processing group, is prompted
Lincpint may be related with pancreas islet beta cell function damage in T2DM.
Embodiment 3 lowers the secretion that Lincpint inhibits insulin in mouse cell lines
In order to detect influence of the Lincpint to islet cell function, Invitrogen company design and synthesis mouse
Three interference sequences of Lincpint, and synthesized corresponding random ordering and compareed.We transfect in MIN6 and NIT-1 cell
3 interference sequences of Lincpint, real-time quantitative PCR detect their jamming effectiveness, as the result is shown (Fig.4A-B): with compare
It compares, the jamming effectiveness of Lincpint siRNA#1 is up to 80 ± 3% and 68 ± 3% (P < 0.01).
By MIN6 cell kind in 48 well culture plates, 60-70% is reached to cell density, culture medium is discarded, is transfected.48h
Afterwards, GSIS experiment is carried out.Preparation glucose-free Krebs-Ringer bicarbonate (Krb) (115mM NaCl,
4.7mM KCl,1.2mM MgSO4.7H2O,1.2mM KH2PO4,20mM NaHCO3,16mM HEPES,2.56mM CaCl2,
0.2%BSA) solution prepares low sugar liquid (2mM), high liquid glucose (20mM) according to experiment hole count.Low sugar liquid 200ul is added, discards.
Add the low sugar liquid of 200ul, discarded after 37 DEG C of culture 1h, 200ul low sugar liquid is added in every hole, and 37 DEG C of culture 1h collect supernatant to EP
Guan Zhong.Add 200ul high liquid glucose, 37 DEG C of culture 1h collect supernatant into EP pipe.Add the sour ethyl alcohol of 200ul, 4 DEG C overnight, take supernatant
Into EP pipe, it is placed on -80 DEG C.Insulin content is detected with radioimmunoassay (RIA).
Influence of the Lincpint to insulin synthesis and secretion is had detected first, and we used insulin secretion functions most
Strong MIN6 cell.After interfering Lincpint 48h in MIN6 cell, the change of amount of insulin secretion and insulin total amount is detected
Change as the result is shown (Fig.4C-D): in interference group, under low sugar concn 2mM stimulation, the secretion no significant difference (P=of insulin
0.66), but under high glucose concentration 20mM stimulation, 42% (P < 0.05) is lowered in the insulin secretion of interference group, is lowered simultaneously
After Lincpint, compared with the control group, intracellular insulin content reduces by 22% (P < 0.05).Result above prompt
The downward of Lincpint can impaired isle element secreting function.
For embodiment 4 in mouse cell lines and primary pancreas islet, the expression inhibiting insulin synthesis for lowering Lincpint is related
The expression protein immunoblotting (Western blot) of transcription factor
The denaturation of protein: 5 × lauryl sodium sulfate (SDS) protein electrophorese is added by the volume ratio of 1:4 in sample
Sample-loading buffer mixes, 99 DEG C of heating 10min, -20 DEG C of preservations.
Protein electrophoresis: the protein sample that denaturation is completed presses 40 holes μ g/, and it is solidifying that pre-fabricated modacrylic acyl ammonia is added
In the well of glue (SDS-PAGE)；The concentrated glue laminated contracting of albumen sample is first made with 80V voltage constant pressure, enters separation gel to sample
Afterwards, it switches to 120V voltage constant pressure and continues electrophoresis 45-50min, make the Protein Separation in sample.
SDS-PAGE glue formula:
4% concentration glue (total 4ml):
10% separation gel (total 6ml):
12.5% separation gel (8ml):
5 × Tris- glycine running buffer (1L):
Transferring film: the adhesive tape of specific position is switched to by conjunction according to the size of destination protein by albumen marker after electrophoresis
Suitable size (corner cut label) impregnates 10min with transferring film buffer.Film process: in advance cut out with an equal amount of filter paper of adhesive tape (on
Under it is each two layers) and cellulose acetate (NC) film (shear angle label), impregnate 10min in transferring film buffer.Half-dried film of walking around: transferring film dress
It sets and is successively put well from bottom to up according to the sequence of carbon anode plate, filter paper, NC film, gel, filter paper, cathode stainless steel plate, filter paper coagulates
Glue, NC film Accurate align, each step are both needed to bubble removing, cover cathode stainless steel plate.Power on, constant current 1mA/cm2, according to
The size of different molecular weight of albumen selects the suitable transferring film time.
1 × half-dried walk around film buffer formulation (1L):
Closing: after to transferring film, the NC film that displaced albumen being taken out, primary with 1 × TBST flushing membrane.Then with 1
5% skim milk that × TBST buffer (20mM Tris-HCl, 150mM NaCl, 0.05%Tween-20, pH7.6) is prepared
2h is closed, in room temperature with the binding site of nonspecific proteins in close membrane.
Antibody incubation: diluting primary antibody (EZH2, GAPDH, 1:1000) with 5% skim milk of 1 × TBST buffer,
4 DEG C, jog is overnight.1 × TBST buffer washes film, 5min × 3 time.It is separately added into 5% degreasing ox of 1 × TBST buffer
The horseradish peroxidase of the diluted anti-rabbit of milk or mouse is coupled IgG, and shaking is incubated for 2h in room temperature.1 × TBST buffer washes film,
10min × 3 time.
ECL detection: being protected from light and prepare ECL-plus developing solution, is added dropwise on placing smooth plastic packaging film after mixing, wait react
1min, with sandwiching NC film in plastic packaging film.Photographic film is covered in darkroom, according to the intensity of fluorescence determine exposure when
Between.
In order to inquire into whether influence of the Lincpint to pancreas beta cell insulin synthesis and secretion is since beta is thin
In born of the same parents caused by associated transcription factor variation, after we interfere Lincpint first in MIN6, NIT-1 cell and primary pancreas islet
The variation of two the transcripts Ins1 and Ins2 of insulin in beta cell are detected, as the result is shown (Fig.5A, C, E): interference group
Middle Ins1 and Ins2 expression lowers (P < 0.05).Then have detected the expression feelings that relevant transcription factor is synthesized to insulin
Condition, as the result is shown (Fig.5B, D, F): after interference Lincpint, under transcription factor MafA, Pdx1, Glut2mRNA horizontal expression
It adjusts (P < 0.05).In the protein expression situation (Fig.5G-H) of MIN6 cell detection transcription factor: interference group transcription factor
The protein expression of MafA, Pdx1, Glut2 reduce (P < 0.05), consistent with mRNA expression.Result above prompt is lowered
The reduction that Lincpint can cause insulin to synthesize.
For embodiment 5 in normal adult Balb/c mouse, the expression for lowering Lincpint can weaken insulin secreting ability
The highest si-Lincpint#1 of jamming effectiveness is selected, Invitrogen company synthesizes the small interference sequence of living body.It will
The si-Lincpint of 50nmol be dissolved in 200ul PBS with syringe needle by tail vein (hydrodynamic intravenous
(IV) tail vein injections) it is injected into adult mice body, as si group, the random ordering of same volume is separately taken to be injected into
Year mouse, as NC group.After 48h, tested.
The glucose tolerance test (IPGTT) of intraperitoneal injection: by si-NC and si-Lincpint mouse fasting 12h.According to
Every g weight needs 2mg glucose, and glucose is injected intraperitoneally.Volume (ml)=weight (g)/100.With 75% ethanol disinfection animal abdomen
Portion, and it is dry with cotton balls.It clenches animal subject back and glucose is injected intraperitoneally with syringe.Respectively before the injection 0,
When 15,30,60,90,120, tail is cut to survey blood glucose.
ELISA method surveys mice serum insulin: intravital mouse, orbital vein blood sampling.Took for 0,15,30,60,90,120 times
The blood of point is placed at room temperature for 30min, is centrifuged 3000g, 15min.Upper serum is collected, can be reserved in -20 DEG C.It will be in kit
Reagent places room temperature, and 10X wash buffer is diluted to 1X with ddH2O.Every hole adds 300 microlitres of wash buffer, washes 3 times；
Control and sample add 10 microlitres of assay buffer.Control and gauge orifice (0.2,0.5,1,2,5,10ng/ml) add 10 microlitres
The titer of 10 microlitres of each concentration is added in gauge orifice by matrix solution, in sample well plus 10 microlitres of serum sample
Product.All holes add 80 microlitres of detection Ab, and 2 hours are incubated at room temperature on shaking table, and (all operations before this step are as far as possible at 1
Completed in hour), wash buffer is cleaned 3 times, 300 microlitres of every hole；100 microlitres of every hole enzyme solution.Shaking table room
Temperature is incubated for 30min, is sucked out, and wash buffer is cleaned 6 times, 300 microlitres of every hole；It is sucked out, 100 microlitres of every hole substrate.It shakes
Bed incubation at room temperature 15min, it may appear that blue is sucked out, 100 microlitres of every hole stop solution, blue flavescence color, in 450nm wave
It is read in long lower microplate reader.
50nmol siRNA is dissolved in 0.2ml PBS, is rapidly injected in Mice Body by tail vein, in adult mice
The expression of exogenous closing Lincpint.It simultaneously will be in the out-of-order injection control group mice body of same volume.After 48h, it is detected
Jamming effectiveness in pancreas, as the result is shown (Fig.7A): compared with the control group, jamming effectiveness in interference group pancreas is 55 ±
7.9% (P < 0.05).
Interfere 48h after, carry out glucose tolerance test (IPGTT), detect injectable dextrose monohydrate before 0min and injection after
The blood glucose and insulin level of 15min, 30min, 60min, 90min, 120min, as the result is shown (Fig.7B-C): with control group
It compares, 15min, the blood glucose of 30min interference group has raising (P < 0.05), 0min, 15min, the insulin water of 60min interference group
It is flat to be declined (P < 0.05).
In living body level, the variation of associated transcription factor is detected by qPCR, as the result is shown (Fig.7D): with control group
It compares, Ins1, Ins2, MafA, Pdx1, (P < 0.05) is lowered in the expression of Glut2, consistent with the result of cellular level.
Primary Study of 6 Lincpint of embodiment in terms of mechanism
Caryoplasm separating experiment: the cell in big ware is washed 2 times with PBS；Pancreatin gets off cell dissociation, and 2mL culture medium terminates
Digestion, the sucking sterile no enzyme pipe of 2mL, 4 DEG C, 500g, 5 minutes；1mL PBS is added, abandons supernatant, 500 μ L Cell are added
Fractionation Buffer, gently blows and beats, put 5 on ice~after ten minutes, 4 DEG C, 500g, 5 minutes；Supernatant moves into new 2mL
Sterile no enzyme pipe, 4 DEG C, 500g, 5 minutes；500 μ L Cell fractionation Buffer are added in precipitating, after mixing, add
Enter 500 μ L 2 × Lysis/binding solution, piping and druming mixes, and discards supernatant after standing, 500 μ L of pre-cooling are added
Cell Disruption Buffer is vortexed and mixes；500 μ L dehydrated alcohols are added, piping and druming mixes, and adsorption column is put into collecting pipe
In, 700 μ L reaction solutions are added, 12,000g, 30s discard collection liquid in pipe, and follow the steps below: 700 μ L are added
Wash solution I, 12,000g, 30s, discard collecting pipe, and 500 μ L Wash solution 2/3,12 are then added,
000g, 30s, abandon collecting pipe, and maximum (top) speed is centrifuged void column 1min, discards collecting pipe；Adsorption column is put into new collecting pipe, is added 40
μ L Elution solution (water-bath is to 95 DEG C) 12,000g, 30s elution, adds 10 μ L and is eluted；It is dense to measure RNA
Degree, removes DNA reverse transcription, qPCR.
In MIN6 cell, the expression of Lincpint is detected by caryoplasm separating experiment, as the result is shown (Fig.8A):
Lincpint is mainly expressed in nucleus, prompts its mainly expression in transcriptional level control gene.It is done in MIN6 cell
Ezh2 is disturbed, the expression of transcription factor is detected, as the result is shown (Fig.8C-D): interference group Ins1, Ins2, transcription factor MafA,
The mRNA and protein level of Pdx1, Glut2 are lowered, consistent with the interference result of Lincpint group.Prompt Lincpint may lead to
Binding Ezh2 is crossed, silencing downstream target gene causes transcription factor expression to change, to influence beta cell function.
Claims (1)
1. the primer sets of one group of identification long non-coding RNA Lincpint are in the kit of preparation diagnosis T2DM incidence
Purposes, which is characterized in that the nucleotide sequence of the primer sets is as shown in SEQ ID NO:2 and SEQ ID NO:3.
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| Diverse Phenotypes and Specific Transcription Patterns in Twenty Mouse Lines with Ablated LincRNAs;Ka-Man Venus Lai等;《PLOS ONE》;20150314;第10卷(第4期);e0125522 |
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