CN104059887A - Application of long non-coding RNA (ribonucleic acid) Ovo12-AS - Google Patents

Abstract

The invention relates to the field of physiological and biological medicines, and particularly relates to long non-coding RNA (lncRNA) for regulating islet beta cell proliferation and functions and application thereof. The novel lncRNA Ovo12-AS for regulating islet beta cell proliferation and functions is screened and discovered by establishing a pregnant mouse model, separating islet of a pregnant mouse and a non-pregnant mouse to detect the lncRNA differential expression levels of the mice. Over expression of Ovo12-AS in mouse islet primary broken cells and mouse islet beta cell line Min6 discovers that the Ovo12-AS can remarkably promote proliferation of beta cells and plays a role in inhibiting cell apoptosis. The lncRNA Ovo12-AS can be used for inhibiting islet cell apoptosis and promoting proliferation of islet cells, and can be used for preparing and screening medicaments for treating diabetes mellitus.

Description

The application of long-chain non-coding RNA Ovol2-AS

Technical field

The present invention relates to physiology and biomedical sector, be specially long-chain non-coding RNA (lncRNA) and the application thereof of regulation and control beta Cell of islet proliferation and function.

Background technology

Diabetes are metabolic diseases of a kind of multi-pathogenesis, and feature is chronic hyperglycemia, follow because of insulin secretion and (or) act on sugar, fat and the protein metabolism disorder that defect causes.In diabetes generation evolution, beta Cell of islet quantity and function all play an important role to its Clinical course.In recent years diabetes in the whole world particularly China sickness rate rise rapidly, within 2010, China shows in the result of nearly 100,000 people having been carried out to long term follow-up investigation, China 18 years old and be grown up above in sample, the diabetes estimation morbidity of diagnosing according to the up-to-date clinical criteria in the world is 11.6%, approximately 1.139 hundred million people, make the research in this field also even more important and urgent, disclosing unknown regulatory mechanism and treating target spot is global investigator's common objective.

The quantity of beta Cell of islet and function play an important role in diabetes generation evolution, and wherein the propagation of β cell has become the key link of diabetes study.More and more research trends are in thinking that islet beta cell function defect is the core link that development occurs diabetes, and β cell function defect main manifestations is two aspects: the minimizing of β cell quantity and insulopathic.No matter be 1 type or diabetes B, all have beta Cell of islet quantity defect, therefore inquire into and affect the focus that the cause and mechanism of β cell quantity and function becomes global diabetes study field.Beta Cell of islet quantity is mainly by four kinds of mechanism regulatings: β cellular replication (the β cell having broken up carries out mitotic division), β cell newborn (reaching maturity of precursor cell), β apoptosis (apoptosis) and the expansion of β cell, the variation of any regulatory mechanism all can directly affect the quantity of β cell, and these four kinds of mechanism can regulate and control according to the changes in demand of the different steps of growing or internal metabolism the quantity of β cell.Research is found, mouse is under insulin resistant or Obesity, can there is the increase of nearly 30 times in β cell quantity, and people's p-Isletβ cell quantity under identical physiological and pathological state only can increase 30-40%, the Beta cell proliferation rate of adult's pancreas islet is very low, necrotomy mankind pancreas discovery β cellular replication (the positive β cell of Ki67) is just down to less than 0.2% about 5 years old, and in adult pancreas, almost can not find the β cell of the Ki67 positive, this explanation becomes the propagation of beta Cell of islet in human body very limited under normal physiological situation, therefore how to allow the functional beta Cell of islet quantity of diabetic recover to become the emphasis of diabetes study.

Many important research results disclose in recent years, although mouse β cell can be transformed by a small amount of precursor cell under specific environment, but most of β cells are still bred by self-replication, be replicated in embryonic stage and the neonatal period of beta Cell of islet, can increase fast, and after wean until to fully-developed in the time its quantity can change hardly, still all very slow in its reproduction speed of mankind's pancreas islet in ripe rodent, but β cell has still retained the ability that its acceleration copies, in some special physiological and pathological situations, such as gestation, hyperglycemia, injury of pancreas] and around in the situation of insulin resistant (fat state etc.) copying of beta Cell of islet can obviously increase.

Long-chain non-coding RNA (lncRNA) is the non-coding RNA that length is greater than 200 Nucleotide.Within 2002, researchist has found first the new transcript-lncRNA of this class in to the large-scale order-checking process in mouse total length complementary DNA (cDNA) library, according to lncRNA position with respect to protein coding gene on genome, lncRNA can be fallen into 5 types: in justice (sense), antisense (antisense), two-way (bidirectional), gene, between (intronic) and gene (intergenic), this position relationship has very great help for the function of inferring lncRNA.Current research shows that the possible source of lncRNA comprises following aspect: the gene structure of (1) coded protein interruption occurs and is transformed into lncRNAs, such as Xist lncRNA, (2) result of chromatin restructuring, two non transcribed genes and another independently gene recombinate side by side and produce the lncRNAs containing a plurality of exons, (3) the backward shift position product in Noncoding gene reproduction process, (4) local contiguous generation of inline copy, (5) in genome, insert a swivel base composition and produce the non-coding lncRNA of function, as BC1 and BC200.Studies show that, in mammalian genes group sequence, 1% transcript producing is codified albumen, and the transcript that has 4%-9% to produce is lncRNA, originally lncRNA is considered to " noise " that genome is transcribed, be the by product that rna plymerase ii is transcribed, do not have biological function, the lncRNA in Mammals compares with protein coding gene, the length of lncRNA is generally shorter, and the poor and expression level of conservative property is starkly lower than protein coding gene.Yet large quantity research shows in recent years, lncRNA plays a significant role in many bioprocesss such as epigenetic regulation, cell cycle regulating, dosage compensation effect and cytodifferentiation regulation and control.LncRNA does not have a kind of general binding mode, it can come regulate gene expression and albumen to synthesize in a different manner, can on many levels, to genetic expression, regulate and control, comprise that the regulation and control of epigenetics level, transcriptional level control and post-transcriptional level regulate and control several aspects.

In the mechanism of regulate gene expression, there is general character in lncRNA, mainly from epigenetics, transcriptional level and 3 aspects of post-transcriptional level, realize the regulation and control to genetic expression, lncRNA mainly may have the function of the following aspects: (1), by transcribing in protein coding gene upstream promoter district, disturbs the expression of downstream gene; (2) by suppressing rna plymerase ii or mediation chromatin reconstruct and histone modification, affect downstream gene expression; (3) by the transcript with protein coding gene, form complementary two strands, and then disturb the shearing of mRNA, thereby produce different shear-forms; (4) by the transcript with protein coding gene, form complementary two strands, further under the effect of Dicer enzyme, produce endogenic siRNA, the expression level of regulatory gene; (5) by being attached on specified protein, lncRNA transcript can regulate the activity of corresponding protein; (6) as structural constituent and Protein formation nucleic acid protein complex body; (7), by being attached on specific protein, change the kytoplasm location of this albumen; (8) as microRNA, as the precursor molecule of miRNA, piRNA is transcribed.And just because of lncRNA can be from transcribing the regulation and control that participate in protein coding gene with post-transcriptional level, these genes comprise disease generation genes involved and development related gene etc., by participating in regulation protein encoding gene, lncRNA can affect intracellular signal transduction approach, also can affect the signal transduction pathway in organism growth course.At present reported that lncRNA can exert an influence to the generation of various diseases and development, comprises neural disease, the diseases such as cardiovascular disorder and kinds of tumors cancer.

In recent years the research of long-chain non-coding RNA (lncRNA) is attracted wide attention, and the relevant lncRNA of beta Cell of islet studies still very limited.Exploring the new lncRNA playing an important role in β cell, not only can disclose the important mechanisms of regulation and control β cell quantity and function, also will provide new treatment target spot, is the important scientific issues of urgently opening up in diabetes study.

Summary of the invention

The present invention aims to provide the lncRNA of regulation and control beta Cell of islet proliferation and function, and above-mentioned lncRNA is used for regulating and controlling beta Cell of islet propagation and suppresses Intra-islet Apoptosis.

Set up the Gestation period this ripe Beta cell proliferation model of mouse, the pancreas islet of the separated Gestation period and non-pregnancy mouse detects its lncRNA differential expression level.Method with microarray analysis lncRNA express spectra, the lncRNA of differential expression in utilized lncRNA chip detection gestation and non-pregnant mouse pancreas islet, GO (Geneontology) functional analysis and pathway path analysis have been carried out, screened and found the lncRNA of new regulation and control beta Cell of islet proliferation and function: result shows, according to the analysis to lncRNA chip, found that the expression of approximately 834 kinds of lncRNA in the Gestation period and the non-pregnancy mouse islets exists significant difference, protein coding gene in conjunction with differential expression has carried out GO analysis, pathway analyzes, by analyzing the dependency of lncRNA-mRNA on expression level, built lncRNA-mRNA coexpression regulated and control network.According to result, gene expression abundance is higher and change multiple and have significantly AK078687, AK080649, AK154298, AK007144, AK149154 and AK162104 etc., the result of the realtime PCR of these lncRNA also with chip on result basically identical.Wherein AK007144 gene expression abundance in mouse islets is abundanter, variation differs greatly, and because this lncRNA is positioned on the antisense strand of protein coding gene ovol2, belongs to the lncRNA of antisense overlap, according to the principle of lncRNA name, by its called after Ovol2-AS.

Cross expression Ovol2-AS in breaing up cell and mouse islets β clone Min6 mouse islets is primary,, observe the impact of Ovol2-AS on beta Cell of islet proliferation and apoptosis; And further detect the effect of Ovol2-AS to Beta cell proliferation related pathways.There is significant difference in the expression level of Ovol2-AS, cross beta Cell of islet growth quickening, the increase of S phase cell proportion of expressing Ovol2-AS group in pregnant and non-pregnancy mouse islets, and the ratio of viable apoptotic cell reduces; And further prove, Ovol2-AS can make Akt phosphorylation level in Min6 cell increase, and also increases GSK-3 β phosphorylation level simultaneously, and then affects the level of the propagation genes involveds such as c-myc.Result shows, crosses expression Ovol2-AS, can significantly promote the propagation of β cell, and play the effect of inhibited apoptosis.

Ovol2-AS can promote the propagation of beta Cell of islet and suppress islet beta-cell apoptosis, and this effect may realize by activating AKT/GSK-3 β path.

Therefore, long-chain non-coding RNA Ovol2-AS can be used for suppressing the apoptosis of islet cells, for promoting the propagation of islet cells; Can also for the preparation of or the medicine of screening preparation treatment diabetes.

At islet cells, cross expression Ovol2-AS and can promote insulinoma cell proliferation, suppress Intra-islet Apoptosis.Described islet cells is beta Cell of islet Min6 or mouse primary islet cells.In beta Cell of islet Min6, cross expression Ovol2-AS and can also activate Akt/GSK-3 β path in beta Cell of islet Min6.

The present invention utilizes the model of pregnant mouse, and the pancreas islet by the separated Gestation period and non-pregnancy mouse detects its lncRNA differential expression level.By the method for microarray analysis lncRNA express spectra, by analyzing the expression level of the Gestation period and non-pregnancy pancreas islet lncRNA, for continuing function and the mechanism of the relevant lncRNA of Study Mouse beta Cell of islet propagation, provide rich in natural resources.Meanwhile, we have also built the expression plasmid of crossing of relevant lncRNA, and have packed slow virus, realize crossing on beta Cell of islet Min6 clone and mouse primary pancreas islet and express, and have confirmed really to exist in gravidic Beta cell proliferation process the participation of lncRNA.Further function and the relevant regulatory mechanism thereof of screening and exploration lncRNA, may provide for the treatment of diabetes new thinking.

Accompanying drawing explanation

Fig. 1 is in embodiment 1, the box map analysis of the lncRNA chip of mouse control group and 6 samples of gestation group

Fig. 2 is in embodiment 1, and the GO that gestation group mouse islets significance is expressed rising gene analyzes

Fig. 3 is in embodiment 1, and gestation group mouse islets significance is expressed the path (Pathway) of rising gene and analyzed

Fig. 4 is in embodiment 1, and 14.5 days groups (pregnancy) of gestation and control group (control) mouse islets lncRNA expression level difference are verified

Fig. 5 is in embodiment 2, the expression level of Ovol2-AS in Min6 cell after transfection pcDNA3.1-Ovol2-AS

Fig. 6 is in embodiment 2, the growth curve of Min6 cell after transfection pcDNA3.1-Ovol2-AS

Fig. 7 is in embodiment 2, and Min6 clone transfection pcDNA3.1-Ovol2-AS crossed expression plasmid after 48 hours, and immunofluorescence detects the positive expression of Ki67

Fig. 8 is in embodiment 2, and clone Min6 transfection Ovol2-AS crossed after expression plasmid and control plasmid after 48 hours, by the result of the positive rate of flow cytometer detection E du

Fig. 9 is in embodiment 2, and flow detection and analysis Min6 clone transfection Ovol2-AS crosses after expression plasmid and control plasmid apoptotic impact

Figure 10 is in embodiment 2, and flow detection and analysis Min6 clone transfection Ovol2-AS crosses after expression plasmid and control plasmid apoptotic difference

Figure 11 is in embodiment 2, and mouse primary is broken up islet cells transfection transfection Ovol2-AS and crossed fluorescence photo after expression plasmid and control plasmid

Figure 12 is in embodiment 2, and apoptosis in situ detection is crossed expression Ovol2-AS to the apoptotic impact of mouse primary β

Figure 13 is in embodiment 2, P-AKT, and T-AKT, P-GSK3 β, the protein diversity of T-GSK3 β in the Min6 of transfection Ovol2-AS and control plasmid clone expressed

Figure 14 is in embodiment 2, in Min6 clone, excessively after expression Ovol2-AS, breeds genes involved change level

Embodiment

Embodiment 1

One, main experiment material: the Gestation period mouse and the primary islet cells of control group

Main agents: HBSS (10 *), (1 *) (Invitrogen company); Collagenase XI (Sigma company); Trizol lysate (Invitrogen company).

Two, experimental technique

(1) set up pregnant mouse model

Buy SPF level C57BL/6 female mice and male mice, in SPF level Animal House, raise: sub-cage rearing (4 every cages of mouse), mouse can freely ingest and drink water.Envrionment temperature 21 ± 1 degree of Animal House, humidity 55 ± 10%, diel rhythm 12-12 hour.Part female mice is control group, and another part female mice and male mice mate according to the ratio of 2:1, gets the gestation mouse of 14.5 days, and the mouse of gestation is again confirmed pregnant time by embryo's Phenotypic Observation when separated pancreas islet.

(2) separated pancreas islet from control group and the gestation C57BL/6 female mice of 14.5 days:

1, anesthetized animal: adopt 1% vetanarcol intraperitoneal injection anesthetized mice (dosage is 50mg/kg).

2, by mouse in being fixed on animal operator's console, belly, fixes four limbs upward; With 75% cotton ball soaked in alcohol wiping belly, sterilization.Get median abdominal incision, with eye scissors, open belly and be a U-shaped otch; Fully expose abdominal organs, will on its liver, turn over, duodenum is turned over to the left, small intestine and caecum turn over left, and the passivity common bile duct that dissociates, closes and clamp common bile duct and duodenum intersection with Wen's blood vessel clamp, be major duodenal papilla place, to avoid collagenase to enter from here enteron aisle.

3, with 5ml syringe, be filled collagenase XI working fluid (10 * XI Collagenase Type is 1 * XI Collagenase Type with 1xHBSS dilution, and consumption is 4-5ml/ pancreas), disposable venous infusion needle be fixed on to injector head and connect, the bubble of emptying inside.With disposable venous infusion needle, the common bile duct place below gall-bladder punctures (common hepatic duct bifurcation below position) under the microscope.

4, fix and insert after choledoch-venous needle, push gently a little collagenase XI working fluid, examining under a microscope pancreas has a little full, and common bile duct does not have sepage around, with eye scissors, break heart rapidly, stop blood flow, in order to avoid sneak into too much hemocyte in pancreas.Continue slowly to inject about 2-3ml collagenase XI working fluid, during injection, can feel that syringe resistance increases, pancreas is filled to pressure increases gradually, until pancreas stops after completely full.

5, by pancreas and soft the separating of tweezers for surrounding tissue, find spleen, the spleen full pancreas of ining succession is taken off, be put in 15ml centrifuge tube, in centrifuge tube, added 2-3ml collagenase XI working fluid in advance, note spleen not being sneaked in centrifuge tube.

6, then 15ml centrifuge tube is placed in to 37 ℃ of water-baths and digests, general digestion time is about 18 minutes, during, when digesting by 15 minutes, 16 minutes, 17 minutes, use respectively hand concuss centrifuge tube 1 time, to accelerate the digestion of pancreatic tissue.During to about 18 minutes, the visible digestion of visual inspection is organized and is fine sand shape completely.

7, after digestion, to 15ml centrifuge tube, add 1 * HBSS of 10ml precooling on ice, stop digestion, in order to avoid excessively digestion, 4 degree, 1000rpm (accelerate 9, slow down 9), centrifugal 1 minute.Supernatant is discarded, 1 * the HBSS that adds 10ml left and right, connects the resuspended precipitation of the soft piping and druming of Glass tubing with rubber suction bulb, and precipitation and liquid are drawn on 40 object screen clothes, through screen filtration, fall the tissue block that cannot digest (as fatty tissue etc.), collect filtrate in new 15ml centrifuge tube.With 1 * HBSS, the filtered liquid of collection is filled to 10ml to 4 degree, centrifugal 1 minute of 1000rpm.

8, supernatant discarded liquid, gradient centrifugation, 4 degree, centrifugal 20 minutes of 1500rpm (accelerate 5, slow down 0).The supernatant of collecting after centrifugal is added to 10ml1 * HBSS liquid, recentrifuge, 4 degree, centrifugal 1 minute of 1000rpm (accelerate 9, slow down 9).Supernatant is discarded, in centrifuge tube, add 10ml1 * HBSS liquid, make islet cells resuspended, wash one time, 4 degree, 1000rpm (accelerate 9, slow down 9), centrifugal 1 minute.Supernatant is discarded, and the 1 * HBSS that adds 10ml to contain 0.5%BSA, blows and beats resuspended precipitation with plastic suction tube, and mixing liquid is sucked to 10cm culture dish, picking pancreas islet under stereoscopic microscope.

9,, for extracting the pancreas islet of RNA, choose to 1.5ml EP pipe 4 degree, 1000rpm (accelerate 9, slow down 9), centrifugal 1 minute, sop up supernatant, every pipe adds 1ml Trizol lysate, is stored in-80 degree, or the pancreas islet of choosing is put into cryopreservation tube, 4 degree, 1000rpm, after centrifugal 1 minute, supernatant is abandoned in suction, directly deposits in liquid nitrogen container.

(3) mouse primary pancreas islet RNA extracts

With aforesaid method, get 3 groups of 14.5 days female mice pancreas islet of gestation, every group is comprised of 7 mouse islets, is labeled as preg1, preg2, preg3; 3 groups of the female mice pancreas islet of control group age-matched, every group is comprised of 7-8 mouse islets, is labeled as control1, control2, control3.Adopt Trizol reagent method, extract total RNA respectively, then use reverse transcriptase polymerase chain test kit (A3500, Promega) to make cRNA from the primary pancreas islet of mouse separation, reverse transcription reaction operation is undertaken by Promega specification sheets.And carry out RNA quality control detection, cRNA is carried out also having carried out mark quality control after mark.RNA concentration is on average between 200-500ng/ μ l, and 28SRNA approaches 1:1 than the brightness of 18SRNA band, and the A260/A280 ratio of RNA is between 1.8-2.1, and A260/A230 ratio is greater than 1.8.Quality is good, meets Mouse4x44K lncRNA expression array microarray gene expression requirement of experiment.The expression profiling that becomes biological company limited to adopt mouse lncRNA Array (4x44K, ArrayStar) to complete by upper Haikang.

(4) mouse lncRNA chip analysis

1, mouse lncRNA Array (4x44K, ArrayStar) microarray hybridization analysis

Application Agilent gene expression hybridization kit (Agilent p/n5188-5242), Hybridization Chamber, stainless (Agilent p/n G2534A), Hybridization Chamber gasketslides (Agilent p/n G2534-60003), Hybridization oven (Agilent p/n G2545A), Hybridization oven rotator for Agilent Microarray, Hybridization Chambers (Agilent p/nG2530-60029) detects.

2, lncRNA chip data analysis

Total RNA of each sample carries out Mouse4x44K lncRNA expression array microarray gene expression spectrum analysis.On this mouse long-chain non-coding RNA chip, containing having an appointment 40000 kinds of lncRNA, its database is from NCBI RefSeq, UCSC, RNAdb2.0, NRED, Fantom3.0, UCRs.Tumor-necrosis factor glycoproteins and to be less than the non-coding RNA of 200bp disallowable, the probe of the corresponding coupling of each transcript, the positive contrast of some house-keeping genes.First data analysis is applied Agilent GeneSpring GX v11.5.1 software and is proofreaied and correct raw data, after raw data having been carried out to fractile correction, to having at least 3 above markd lncRNA of sample band to carry out the lncRNAs differential expression screening of further control group and preg group, the ratio of control group and preg being organized to each pattern detection signal carries out log2 conversion, balance, carries out t-test analysis and calculates p-values; In the t-test of certain lncRNA between two groups analyzes, obtain p-value≤0.05, and otherness multiple >=1.5 o'clock, just think the lncRNA of control group and preg group there were significant differences property expression.

Result shows, the Gestation period mouse islets and non-pregnant control group mice pancreas islet between have about more than 800 kinds of lncRNA to have significance differential expression, the expression level of approximately 380 kinds of lncRNA raises in Gestation period mouse islets, the expression level of approximately 454 kinds of lncRNA is lowered in Gestation period mouse islets, by visible 6 samples of box figure (Box-Plot), respectively organize data level interior and that respectively organize between data and roughly approach, between group, homogeneity is better.The lncRNA level that can compare differential expression between control group (control) and gestation group (preg) by scatter diagram (Scatter-Plot), between visible group, lncRNA difference is more obvious.Fig. 1 is the box map analysis of chip, and the coordinate axis that Y-axis is logarithmetics represents the ratio of the numerical value after red green two fluorescent signal stdn.

To having the gene of remarkable expression level difference to carry out GO analysis in the Gestation period and non-pregnancy control mice pancreas islet, carrying out biological procedures, molecular function and subcellular components classification annotation analyzes, by central tendency from high to low (the less central tendency of p-value is higher) listed the concentrated classification of significant difference expressing gene, result shows, the gene that in Gestation period mouse islets, significance expression level raises is mainly relevant with cell cycle, cell fission, DNA replication dna.The biology path that can find out the effect gene that differential expression occurs is analyzed in Pathway enrichment, to there being the gene of significance differential expression to carry out KEGG Pathway annotation, analyze, by central tendency from high to low (the less central tendency of p-value is higher) list the concentrated biology path list of significant difference expressing gene, result shows that in Gestation period mouse islets, expressing the gene raising mainly participates in the processes such as DNA replication dna, nitrogen metabolism, p53 signal path and oocyte maturation.Illustrate corresponding Gestation period lncRNA of significantly raising of expression level may be by regulating and controlling corresponding cyclin, participate in the breeding that corresponding biological pathway etc. regulates beta Cell of islet.Fig. 2 has shown the histogram that GO analyzes, and Fig. 3 has shown the histogram that Pathway analyzes.

3, Gestation period mouse islets proliferation period pancreas islet lncRNA differential expression is analyzed and statistics

Select arbitrarily 112 lncRNA (otherness multiple >=2 of differential expression in Gestation period mouse islets, p value < 0.05), carry out cluster analysis, result shows, compare with not pregnant control group mice pancreas islet, the Gestation period mouse islets have part lncRNA up-regulated, part lncRNA down-regulated expression, in group, homogeneity is better, and group difference is more obvious.

Analyze express spectra data, occur to express in the lncRNA changing, gene expression abundance is higher and change multiple and have significantly AK078687, AK080649, AK154298, AK007144, AK149154 and AK162104 etc.Compare with not pregnant control group, the expression level of these several lncRNA in mouse islets obviously raises (table 1) in the Gestation period.

14.5 days mouse islets lncRNA differential expressions of table 1 contrast and gestation are analyzed

4. Gestation period mouse islets proliferation period pancreas islet lncRNA differential expression checking

The gestation mouse islets of 14.5 days and the part lncRNA expression level of control group mice pancreas islet are carried out to the checking of realtime PCR.Result is visible, and AK078687, AK080649, AK154298, AK007144, AK149154 and AK162104 expression level in 14.5 days mouse islets of gestation are compared and had significant difference with control group, have increased corresponding multiple.This result and lncRNA expression chip result are basically identical.As Fig. 4.

Wherein AK007144 gene expression abundance in mouse islets is abundanter, variation differs greatly, and because this lncRNA is positioned on the antisense strand of protein coding gene ovol2, belongs to the lncRNA of antisense overlap, according to the principle of lncRNA name, by its called after Ovol2-AS.

Take ABI7300Real-time PCR as example, the differential expression lncRNA level that real-time quantitative PCR detects is consistent with express spectra, wherein, Ovol2-AS gene expression abundance in mouse islets is higher and variation multiple is remarkable, and prompting Ovol2-AS may breed relevant with Gestation period pancreas islet.

Embodiment 2Ovol2-AS (AK007144) regulation and control beta Cell of islet proliferation and apoptosis

Further by experiment in vitro, study the impact of Ovol2-AS on beta Cell of islet proliferation and apoptosis.In the primary pancreas islet of mouse Min6 clone and separation and Culture, cross respectively the Ovol2-AS that expresses Ovol2-AS plasmid and slow virus packing, by multiple means such as CCK-8, Edu, ki67, detect the propagation situation of cell, and apply the situation of AnnexinV/7-AAD test kit and TUNEL kit detection cell apoptosis, by original position and flow cytometer detection, jointly analyze transfection Ovol2-AS respectively and whether can exert an influence to mouse islets Beta cell proliferation and apoptosis.

(1) build Ovol2-AS expression vector

(1) obtain Ovol2-AS full length fragment according to the full length fragment sequence of the upper Ovol2-AS of NCBI, design primer, selected restriction enzyme site; 5 ' end restriction enzyme site is Hind III, and 3 ' end restriction enzyme site is EcoR I, and corresponding protection base is added at two ends; primer sequence, as table 2, is delivered to company synthetic.

Table 2.Ovol2-AS synthetic primer sequence

Take mouse DNA after template pcr amplification, get amplified production and be connected to pcDNA3.1 plasmid, form pcDNA3.1-Ovol2-AS plasmid, transfection competence bacillus coli DH 5 alpha is also cultivated, and chooses cloning and sequencing.Extracting plasmid, NheI and EcoRV enzyme cut back to close object fragment purifying.

(2) slow virus of Ovol2-AS expression vector packing

1, get skeleton lentiviral vectors PDS087-V2_Pl-TO-V5NheI and EcoRV enzyme and cut product recovery purifying, be connected with the object fragment of above-mentioned recovery, connect product transformed competence colibacillus bacterium, coated plate incubated overnight, random choose single bacterium colony that falls completely from flat board, by bacterium inspection PCR method, detect picking positive colony sequence verification.

The bacterium liquid amplification culture that checks order correct, utilizes Axygen plasmid extraction test kit and method extracting plasmid to carry out OD and measures quality inspection.

2, the packing of slow virus and virus titer are measured

Above-mentioned plasmid solution and liposome are mixed into plasmid liposome complex, transfection logarithmic phase 293T cell again, collecting cell culture supernatant after 48 disappearances, centrifugal removal cell and fragment, and filter with 0.45um filter, ultracentrifugation under virus stock solution used 50000g 2 is removed to supernatant, be resuspended in 1ml DMEM nutrient solution, packing tubule, is positioned over-80 ℃ and saves backup.And by the titre of fluorescence spectrometry slow virus liquid.

(3) mouse primary pancreas islet and beta Cell of islet are the virus transfection of Min6 cell

Cultivating respectively mouse primary islet cells (8-10 week age, SPF level C57BL/6 female mice) and beta Cell of islet is Min6 cell.Adopt liposome to carry out transient transfection, step is with reference to Lipofectamine2000 test kit.After transfection 48 hours, add that Edu or Thapsigargin breed or apoptosis experiment.

(4) result

1, Ovol2-AS crosses to express and promotes that beta Cell of islet is min6 propagation

In Min6 clone, transient transfection pcDNA3.1-Ovol2-AS crosses expression plasmid and contrast pcDNA3.1 plasmid respectively, after 48h, receive RNA, realtime PCR detects its expression level, result the has shown transfection expression level obviously raise (Fig. 5) of crossing Ovol2-AS in the clone of expression plasmid.

In Min6 clone, transient transfection pcDNA3.1-Ovol2-AS crosses expression plasmid, detects respectively cell viability (cell counting kit-8 in transfection after 24 hours, 48 hours, 72 hours, 96 hours; Cck8), and draw the growth curve (Fig. 6) of cell after transfection, result shows, the clone Min6 that experimental group expresses Ovol2-AS excessively compares with control group, the viable cell number of its each time point all has increase in various degree, especially more obvious with the cell number difference of 72 hours and 96 hours, experimental group viable cell number increases 25%-30% left and right than contrast group viable cell number, after statistical analysis, show, two groups of viable count object differences have significant difference (P is less than 0.01).

The creep plate cell transient transfection pcDNA3.1-Ovol2-AS of 8 orifice plates crosses expression plasmid and contrast pcDNA3.1 plasmid, after transfection 48h, and fixed cell, creep plate cell is done ki67 dyeing.The demonstration of immunofluorescence result, compares with control group, and the ratio that Ovol2-AS crosses expression group ki67 positive cell obviously increases (Fig. 7).

24 orifice plate cell transient transfection pcDNA3.1-Ovol2-AS cross expression plasmid and contrast pcDNA3.1 plasmid, after transfection 48h, change general fresh training liquid, according to the ratio of 1:1000, add 10mM Edu simultaneously, mix cell DNA, after 4 hours with 4% paraformaldehyde fixed cell, carry out flow cytometer showed, the ratio that flow cytometer showed result was presented at the Edu stained positive cell after expression Ovol2-AS is increased to 25% left and right by 21%, compared to control group, cross in the Min6 clone express Ovol2-AS Edu positive cell ratio 21% left and right (Figure 14) that raise.

2, to cross expression inhibiting beta Cell of islet be Min6 apoptosis to Ovol2-AS

In clone Min6, transient transfection control plasmid and pcDNA3.1-Ovol2-AS cross expression plasmid, transfection adds the Thapsigargin of short apoptosis agent 1uM concentration after 48 hours, act on and after 24 hours, receive cell and carry out Annexin-FITC/7-AAD dyeing, then flow detection and analysis, method is with reference to FITC Annexin V apoptosisDetection Kit (BD).Result shows, compare with control group, the ratio that Ovol2-AS crosses expression group early apoptosis of cells cell (the Annexin-FITC positive/7-AAD negative cells) reduces, the ratio of survivaling cell (the equal negative cells of Annexin-FITC/7-AAD) increases, and the ratio of late period apoptosis or dead cell (the equal positive cell of Annexin-FITC/7-AAD) is without obviously changing (Fig. 9).

3, Ovol2-AS crosses the apoptosis of expression inhibiting mouse primary beta Cell of islet

Mouse primary pancreas islet overnight incubation, in good condition after pancreas islet reparation, with being broken up as unicellular after 0.05% trysinization pancreas islet, be seeded in 8 orifice plates, transient transfection control plasmid and pcDNA3.1-Ovol2-AS cross expression plasmid simultaneously, transfection adds 1uM Thapsigargin after 48 hours, process after 6 hours, receive fixing saturatingization of cell sample and carry out TUNEL dyeing, apoptosis in situ detection, step is with reference to In Situ Cell Death Detection Kit, TMR red (roche), Olympus BX51 fluorescence microscope, the ratio of counting tunel staining cell number and insulin positive cell number is also carried out statistical study.

Through fluorescence microscopy Microscopic observation, take pictures (Figure 11), the ratio of statistical study TUNEL positive cell after counting, result demonstration, compares with control group, and the TUNEL positive cell ratio of experimental group drops to 2.9% left and right (Figure 11) by 6.6%.

4, can to activate beta Cell of islet be Akt/GSK-3 β path in Min6 to Ovol2-AS

In clone Min6, transient transfection control plasmid and pcDNA3.1-Ovol2-AS cross expression plasmid, transfection was received cell protein after 72 hours, because Akt/GSK-3 β path is for regulating the important path of Beta cell proliferation, so we have detected the level of p-Akt, result shows, in crossing the Min6 cell of expressing Ovol2-AS, Akt phosphorylation level obviously increases, and detect the level of GSK-3 β and find that equally its phosphorylation level raises, and total Akt and total GSK-3 β level constant (Figure 13).These results suggest that expression Ovol2-AS can activate Akt/GSK-3 β path.

Equally, we transient transfection control plasmid and pcDNA3.1-Ovol2-AS in clone Min6 cross expression plasmid, transfection was received RNA after 48 hours, realtime PCR detects propagation genes involved level (primer sequence is as following table), compare with control group as seen, cross cyclin D2 in the Min6 clone of expressing Ovol2-AS, c-myc, the expression level of cpb1 obviously raises, difference between two groups has statistical significance (p < 0.05), the expression level of cyclin B2 raises, p27, the expression level of unc5c has decline (Figure 14), this explanation Ovol2-AS can cause the variation of propagation genes involved level, thereby promote beta Cell of islet propagation.

Real-time PCR primer sequence

Claims (8)

1. long-chain non-coding RNA Ovol2-AS is for suppressing the apoptosis of islet cells.

2. long-chain non-coding RNA Ovol2-AS is for promoting the propagation of islet cells.

Long-chain non-coding RNA Ovol2-AS for the preparation of or the medicine of screening preparation treatment diabetes.

4. a method that promotes insulinoma cell proliferation, is characterized in that, crosses expression Ovol2-AS in islet cells.

5. described in claim 4, promote the method for insulinoma cell proliferation, it is characterized in that, described islet cells is beta Cell of islet Min6 or mouse primary islet cells.

6. a method that suppresses Intra-islet Apoptosis, is characterized in that, crosses expression Ovol2-AS in islet cells.

7. described in claim 6, suppress the method for Intra-islet Apoptosis, it is characterized in that, described islet cells is beta Cell of islet Min6 or mouse primary islet cells.

8. a method that activates Akt/GSK-3 β path in beta Cell of islet Min6, is characterized in that, crosses expression Ovol2-AS in beta Cell of islet Min6.