CN106884016A

CN106884016A - The expression vector of long-chain non-coding RNA LINC00472, tumor suppression reagent and its application

Info

Publication number: CN106884016A
Application number: CN201710076769.5A
Authority: CN
Inventors: 曾朝阳; 李桂源; 李小玲; 熊炜; 龚朝建; 张姗姗; 郭灿; 熊芳; 廖前进; 周钰娟; 周鸣; 王贺冉
Original assignee: Central South University
Current assignee: Central South University
Priority date: 2017-02-13
Filing date: 2017-02-13
Publication date: 2017-06-23
Anticipated expiration: 2037-02-13
Also published as: CN106884016B

Abstract

The invention discloses a kind of expression vector of long-chain non-coding RNA LINC00472, tumor suppression reagent and its application, it is used to prepare the preparation for suppressing growth of tumour cell and propagation, according to the lncRNA sequences, the carrier for expression of eukaryon for overexpression LINC00472 is designed and synthesized, and nanosphere will be made on the carrier loaded nano silicon particles to polylysine modification, LINC00472 is successfully given expression in Nasopharyngeal Carcinoma Cell Line, it is suppressed that the growth of nasopharyngeal carcinoma cell.The nano silicon particles of polylysine modification of the invention, can protect LINC00472 carriers to be degraded from nuclease, extend action time, there is transfection efficiency higher, be conducive to further development and application.

Description

The expression vector of long-chain non-coding RNA LINC00472, tumor suppression reagent and its application

Technical field

The invention belongs to oncomolecularbiology field, and in particular to the expression of long-chain non-coding RNA LINC00472 is carried Body, tumor suppression reagent and its application.

Background technology

Nasopharyngeal carcinoma is common head-neck malignant tumor occurred frequently, is susceptible to metastasic cervical lymph nodes, and prognosis is poor.Research Show, the generation development of this tumour is polygenes participation, multi-step, multistage complex process, the wherein mistake of tumor suppressor gene Activation with oncogene living has played the effect of key.

It has been always oncomolecularbiology research field since TP53 genes (coding p53 albumen) were cloned from 1979 One of most noticeable important CDKN2.Substantial amounts of evidence shows that TP53 genes are logical by regulating and controlling a series of signal transduction Wide participation Several Kinds of Malignancy in road develops.When cell is stimulated by various carcinogenic factors, p53 protein activations And by the expression of transcriptional control downstream key target gene, arresting cell cycle, Cell differentiation inducing activity, active cell aging and wither Die, stabilization, regulation energetic supersession and suppression Tumor Angiongesis that DNA damage reparation maintains genome are participated in, so as to prevent to swell Knurl develops.Existing research finds that TP53 genes are including most common in the Several Kinds of Malignancy including nasopharyngeal carcinoma There is TP53 gene unconventionalities in one of mutator, 33%~76% patient.Therefore, recover nasopharyngeal carcinoma in TP53 gene functions or Signal path downstream is reversed, the treatment to nasopharyngeal carcinoma has great importance.

Long-chain non-coding RNAs (long non-coding RNAs, lncRNAs) be a class length more than 200nt, lack The RNAs molecules of special complete ORFs, nothing or few encoding histone functions.It has recently been demonstrated that lncRNAs By epigenetic, transcription and the extensive regulatory gene of post-transcriptional level expression, they take part in biology growing, development, The regulation and control of the important vital movement such as aging and death.Increasing research shows the unconventionality expression or afunction of lncRNAs Generation development with tumour is closely related.Some lncRNAs molecules have great importance in terms of the diagnosis and treatment of tumour, And new molecular labeling can be judged as tumor prognosis.At present, the mankind lncRNAs genes cloned are more than 40,000 It is individual, but overwhelming majority lncRNA Unknown Function.

In order to find the downstream lncRNAs and its application value in nasopharyngeal carcinoma of TP53 gene regulations in nasopharyngeal carcinoma, I TP53 genes are transfected in Nasopharyngeal Carcinoma Cell Line, collect cell a new generation lncRNAs chip detections of different time points The expression of known lncRNA.The result of chip detection shows that the differential expression of lncRNA LINC00472 is most notable, its table Up to substantially rise, LINC00472 is pointed out to have the effect of CDKN2.

We construct the carrier for expression of eukaryon of expression LINC00472, are transfected into human nasopharyngeal epithelioma 1, cultivate in vitro System and nude mice shifting confirm that lncRNA LINC00472 can substantially suppress the propagation of nasopharyngeal carcinoma cell in growing knurl In vivo model, press down The growth of tumour cell processed, therefore LINC00472 is a new tumor suppression lncRNA.By LINC00472 carrier for expression of eukaryon Load to and Nanoparticulate Carriers for Gene Delivery is made on the nano silicon particles of polylysine modification, the silicon nanometer of polylysine modification Grain, can protect LINC00472 carriers to be degraded from nuclease, extend action time, and have transfection efficiency higher.

The content of the invention

Expression vector, tumor suppression reagent and its application it is an object of the invention to provide long-chain non-coding RNA LINC00472. The expression vector obtained using LINC00472 sequences can prepare the preparation for suppressing nasopharyngeal carcinoma cell.

The expression vector of long-chain non-coding RNA LINC00472, the sequence of long-chain non-coding RNA LINC00472 is shown in SEQ NO:1。

The expression vector of described long-chain non-coding RNA LINC00472, selection Nhe I and EcoR V restriction enzyme sites are used for Digestion pcDNA3.1 carriers, and LINC00472 sequences are inserted into the site, obtain the table of long-chain non-coding RNA LINC00472 Up to carrier.

The expression vector step for building long-chain non-coding RNA LINC00472 is as follows：

1) it is template to transfect the cDNA of the HNE2 cells of TP53 gene plasmids, using TaKaRa LAEnzyme is carried out PCR expands total length LINC00472 sequences；

LINC00472 full length sequence amplimers are as follows：

Sense primer：5’-atgcgaggctggggccggtt-3’

Anti-sense primer：5’-tttagactccaaatggctgttttatt-3’

In upstream and downstream, 5 ' ends of primer are respectively plus restriction enzyme Nhe I and EcoR V recognition sites and protection alkali After base, primer sequence is as follows：

Sense primer：5’-AGGAGCTAGC atgcgaggctggggccggtt-3’

Anti-sense primer：5’-ATGCGATATCtttagactccaaatggctgttttatt-3’；

2) PCR reaction conditions are as follows：

PCR reactions steps：

3) by through Nhe I and EcoR V double digestion rear electrophoresis, glue is returned again after PCR primer electrophoresis, glue reclaim purpose fragment Receive；

4) pcDNA3.1 plasmids are through Nhe I and EcoR V double digestion rear electrophoresis glue reclaim purpose fragments；

4) and 5) 5) with T4DNA ligases connection step glue reclaim product, you can obtain for eukaryotic expression lncRNA The vector plasmid of LINC00472；

6) by the pcDNA3.1 eukaryon expression plasmid transformed competence colibacillus comprising LINC00472 full length sequences that 5) step is obtained Escherichia coli, to expand plasmid.

Described expression vector, the preparation of nasopharyngeal carcinoma cell is suppressed for preparing,

The preparation for suppressing nasopharyngeal carcinoma cell is by the nano silicon particles of polylysine modification and long-chain non-coding RNA The expression vector mixing of LINC00472 stands, and obtains the nano silicon particles of polylysine modification.

The nano silicon particles of polylysine modification are entered with the microemulsion self-assembling technique of OP-10, hexamethylene, ammoniacal liquor The synthesis of row nano silicon particles, and using nano silicon particles surface can and ion electrostatic interaction carry out poly-D-lysine surface and repair Decorations, prepare.

The nano silicon particles process for preparing polylysine modification is as follows：

1) OP-10, hexamethylene and ammoniacal liquor are mixed, be stirred at room temperature it is uniform after add the positive different ester of silicic acid, continue to stir to poly- Close and complete, add equal-volume acetone, ultrasonic disperse, centrifugation, distilled water washs three times, is collected by centrifugation and is deposited in 80 DEG C of dryings, grinds The thin nano silicon particles for obtaining particle size range 10-50nm；Wherein H₂O and OP-10 and H₂The mol ratio of O and the different ester of positive silicic acid for 2~ 10th, ammonia concn is that 1.6~28%, molar concentration of the different ester of positive silicic acid in hexamethylene is 0.1~3mol/L；

2) nano silicon particles are resuspended in 0.6M NaCO by 0.1~10mg/ml₃In solution, ultrasonic disperse, centrifugation is abandoned Clearly, then by sediment it is resuspended in the PBS of pH 7.4 by 0.1~10mg/ml, ultrasonic disperse adds polylysine, final concentration of 4~15nmol/mL, fully mixes, and room temperature is mixed to shake；Centrifugation, abandons supernatant, and precipitation is resuspended in distilled water by 0.1~10mg/ml, Obtain the nano silicon particles of polylysine modification.

The preparation of described suppression nasopharyngeal carcinoma cell, by the nano silicon particles ultrasonic disperse of polylysine modification, per milli The expression vector that nano particle suspension adds 10~50ug long-chain non-coding RNAs LINC00472 is risen, mixing, being stored at room temperature makes it With reference to.

The present invention constructs the carrier for expression of eukaryon of expression LINC00472, is transfected into human nasopharyngeal epithelioma 1, trains in vitro The system of supporting and nude mice shifting confirm that lncRNA LINC00472 can substantially suppress the propagation of nasopharyngeal carcinoma cell in growing knurl In vivo model, Suppress the growth of tumour cell, therefore LINC00472 is a new tumor suppression lncRNA.LINC00472 eukaryotic expressions are carried Body loads to and Nanoparticulate Carriers for Gene Delivery is made on the nano silicon particles of polylysine modification, the silicon nanometer of polylysine modification Particle, can protect LINC00472 carriers to be degraded from nuclease, extend action time, and have transfection efficiency higher.

Brief description of the drawings

Fig. 1 is the inducible lncRNA LINC00472 expressions of TP53 genes；

A：Real time fluorescent quantitative is used after transfecting TP53 expression vectors 0,12,24 and 48 hours in Nasopharyngeal Carcinoma Cell Line PCR detects the expression of TP53 genes, and TP53 mrna expressions are significantly raised；

B：Western is used after transfecting TP53 expression vectors 0,12,24 and 48 hours in Nasopharyngeal Carcinoma Cell Line Blotting methods detect the expression of TP53 genes, and TP53 expression of gene protein levels are significantly raised；

C：Luciferase reporting is used after transfecting TP53 expression vectors 0,12,24 and 48 hours in Nasopharyngeal Carcinoma Cell Line Gene activated methods detect the p53 albumen of TP53 gene expressions as the transcriptional activity of nuclear factor, and p53 transcriptional activities are notable Raise；

D：TP53 expression vectors are transfected in Nasopharyngeal Carcinoma Cell Line to be examined using lncRNA chips after 12,24 and 48 hours The change of intracellular lncRNA expressions is surveyed, this figure shows up-regulated most significant 30 after transfection TP53 genes in cell Individual lncRNA genes, LINC00472 is outlined in figure；

E：Real-Time Fluorescent Quantitative PCR Technique verifies the result of lncRNA chips, lncRNA in cell after transfection TP53 genes The expression of LINC00472 is significantly raised；

Fig. 2 is that the growing state that lncRNA LINC00472 suppress nasopharyngeal carcinoma cell is imported in nasopharyngeal carcinoma cell；

A：Vitro growth rates subtract after growth curve experiment shows to transfect LINC00472 expression vectors in nasopharyngeal carcinoma cell Slowly；

B：After stream type cell analyzer detection transfection LINC00472 expression vectors, cell cycle of human nasopharyngeal carcinoma blocks in G2/M Phase；

C：After stream type cell analyzer detection transfection LINC00472 expression vectors, the nasopharyngeal carcinoma cell showed increased of apoptosis；

Fig. 3 is that zoopery confirms that lncRNA LINC00472 suppress growth and the proliferative conditions of nasopharyngeal carcinoma cell；

A：After LINC00472 expression vectors are transfected in the nasopharyngeal carcinoma cell, it is subcutaneous to be expelled to nude mice armpit, measures within every 5 days Knurl size is grown in shifting, and LINC00472 can significantly inhibit the propagation of nasopharyngeal carcinoma cell；

B：Put to death nude mice after 30 days, gross examination of skeletal muscle shows to express again LINC00472 group shiftings, and to grow knurl smaller；

C：To put to death take out shifting and grow knurl and compare and measure shifting after nude mice and grow knurl size, again expression LINC00472 group shiftings grow knurl compared with It is small；

Fig. 4 is the expression feelings that LINC00472 in the in vitro sample of nasopharyngeal carcinoma is detected using the method for real-time fluorescence quantitative PCR Condition；

Expression of the LINC00472 in nasopharyngeal carcinoma (T) in normal control (N) sample than significantly lowering；

Fig. 5 is expressions of the in situ hybridization detection LINC00472 in mucous membrane by nasopharyngeal carcinoma and cancer；Left figure is cancer side group Knit, right figure is tissues of nasopharyngeal carcinoma (multiplication factor：200 ×), LINC00472 is relatively low in nasopharyngeal carcinoma level；

Fig. 6 is the prognosis correlation analysis of the expression with patient of LINC00472 in nasopharyngeal carcinoma；LINC00472 expression it is low or The patient not expressed is dead faster.

Specific embodiment

The present invention is further illustrated below in conjunction with specific embodiment, is not intended to limit the present invention.

Embodiment 1, imports TP53 genes in nasopharyngeal carcinoma cell, and lncRNA LINC00472 expression is significantly raised

1. materials and methods：

1.1 reagents and kit

The conventional biochemical reagents such as agarose (agrose), glue reclaim kit are purchased from Shanghai China Shun's limited public affairs of bioengineering Department, utilizesMini Kit (Qiagen) extraction agents box extracts high-quality RNA, SuperScript^TMIII (Invitrogen) kit is by RNA reverse transcriptions into cDNA.Luciferase reporter gene detection kit Dual-Luciferase Reporter Assay System are purchased from Promega companies.Nasopharyngeal carcinoma cell HNE2 used by the present invention is Central South University's tumour Research institute preserves.RPMI1640 trainings base and hyclone used by cell culture, and trypsase used by vitellophag is U.S. State's Gibco Products.LncRNA chips are Agilent Products, and the Chip scale is 4*180K, wherein lncRNAs probes 46506.

TP53 eukaryon expression plasmids are purchased from Clontech companies of the U.S., the pp53- for detecting p53 protein transcriptions activity TA-luc luciferase reporting plasmids are purchased from green skies company.

Real time fluorescent quantitative detection primer sequence has：

TP53 upstream region of gene primers：5’-CCCTTCCCAGAAAACCTACC-3’

TP53 downstream of gene primers：5’-CTCCGTCATGTGCTGTGACT-3’

Internal reference house-keeping gene GAPDH sense primers：5’-ACCACAGTCCATGCCATCAC-3’

Internal reference house-keeping gene GAPDH anti-sense primers：5’-TCCACCACCCTGTTGCTGTA-3’

LINC00472 sense primers：5’-GCATCTGTCAACGCTCCTCT-3’

LINC00472 anti-sense primers：5’-CCGCGTTCTTTGTGTGTCTC-3’

Western blotting detect that p53 albumen and the antibody of reference gene GAPDH expression are purchased from Cell Signaling Technology companies.

1.2 cell culture and transfection

The good nasopharyngeal carcinoma cell HNE2 of growth conditions is pressed 2 × 10⁵Individual cells/well is inoculated in 6 orifice plates, by 6 orifice plates It is placed in 37 DEG C, 5%CO₂In incubator, treat that cultured cells starts TP53 carrier for expression of eukaryon by growing to 50-70% density Transfection；Transfection process is as follows：

Add the lipofectamine 2000 of 3 μ l that standing is mixed in 100 μ l serum free mediums in aseptic EP pipes 5min；By in TP53 expression vectors 100 μ l serum free mediums of addition；Then with the above-mentioned 100 μ l comprising lipofectamine Serum free medium is gently mixed, and is stored at room temperature 30 minutes, DNA is formed complex with liposome；Washed with D-Hank's liquid Cell 3 times；800 μ l serum free mediums (antibiotic-free) will be added in said mixture, it is gentle mix after add 6 orifice plates 1 hole；6 orifice plates are placed in CO₂In incubator, 37 DEG C are cultivated 6 hours, then abandon supernatant, add complete medium to continue to cultivate 12nd, 24 and 48 hours.

1.3 real time fluorescent quantitative methods detect the expression of TP53 genes and LINC00472

Nasopharyngeal carcinoma cell HNE2 transfects TP53 carrier for expression of eukaryon and cell was collected after 0,12,24 and 48 hours, and extracting is thin Total serum IgE in born of the same parents, 2 μ g RNA, into after cDNA, carry out real-time fluorescence quantitative PCR through reverse transcription.

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescence quantitative PCR reactions steps

Reaction confirms the amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene after terminating After according to CT values (threshold cycle values), reference gene (GAPDH) markization, checked using group t-test and calculated P values.1.4Western blotting detect the expression of p53 albumen

Nasopharyngeal carcinoma cell HNE2 transfects TP53 carrier for expression of eukaryon and cell was collected after 0,12,24 and 48 hours, and extracting is thin Total protein in born of the same parents, conventional Western blotting methods detect the expression of p53 albumen, and GAPDH is used as reference gene.

1.5 luciferase reporter genes detect the transcriptional activity of p53

Nasopharyngeal carcinoma cell HNE2 transfects TP53 carrier for expression of eukaryon, while transfecting pp53-TA-luc luciferase reporting matter Grain, collected cell, according to Promega companies luciferase reporter gene detection kit specification in 0,12,24 and 48 hours Detection p53 transcription factor activities.

LncRNA expression changes in 1.6lncRNA chip detections transfection TP53 gene posterior nasopharynx cancer cells

Illustrated by Agilent Products, extracting cell total rna, purifying, random primer synthesis cDNA, and in cDNA subscripts Note fluorescence Cy3, with lncRNA chip hybridizations, scans after washing after chip hybridization on Agilent special chip scanner, obtains The fluorescence intensity of each probe points on chip is obtained, special-purpose software analysis is converted into corresponding lncRNA expressions.Compare nasopharynx After cancer cell HNE2 transfection TP53 carrier for expression of eukaryon in 0,12,24 and 48 hour cells lncRNA expression, it is poor Different expressing gene collection of illustrative plates.

2. result

Expression effect after 2.1TP53 eukaryotic expression plasmids nasopharyngeal carcinoma cells HNE2：

After TP53 eukaryotic expression vector transfection HNE2 cells, TP53mRNA (figures are can detect within 12,24 and 48 hours 1A) the expression with p53 albumen (Figure 1B) is significantly raised, and the p53 albumen for giving expression to has stronger transcription factor activity (figure 1C)。

The expression of part lncRNA is significantly changed in 2.2 transfection TP53 carrier for expression of eukaryon posterior nasopharynx cancer cells：

Using lncRNA chips, we have detected transfection TP53 0,12,24 and 48 hours posterior nasopharynx cancer cells of expression vector The expression status of lncRNA in HNE2, part lncRNA expressions there occurs that (Fig. 1 D list up-regulated to significant change Most significant 30 lncRNA).Then, our expressions to LINC00472 in HNE2 cells after transfection TP53 carriers are used Real time fluorescent quantitative method is verified, it was demonstrated that the expression of LINC00472 significantly raises (Fig. 1 E) really, shows that TP53 is thin in HNE2 The expression of LINC00472 is regulated and controled in born of the same parents, in view of TP53 genes are one of most important CDKN2s, prompting LINC00472 can Can also have CDKN2 function.

Embodiment 2, lncRNA LINC00472 suppress cell cycle of human nasopharyngeal carcinoma retardance, inducing cell apoptosis

1. materials and methods

1.1 reagents and kit

Restriction enzyme Nhe I and EcoR V and T4DNA ligases etc. are purchased from TakaRa companies；TRIZOL^TMReagent (Invitrogen)；Plasmid extraction kit, glue reclaim kit (OMEGA)；Reverse Transcriptase kit (Promega)；Protease K, DNase I, RNAsin, RNase A (GBICOL companies)；Methyl thiazoly tetrazolium assay (MTT, Sigma)；Antibiotic G418 (Ameresc)。

The structure of 1.2pcDNA3.1-LINC00472 carrier for expression of eukaryon

We select the overexpression that pcDNA3.1 empty vectors (from Invitrogen companies) build LINC00472 to carry Body.We select Nhe I and EcoR V restriction enzyme sites for digestion pcDNA3.1 carriers and LINC00472 sequences are inserted into the position Point.

Build pcDNA3.1-LINC00472 eukaryotic vector steps as follows：

1) it is template to transfect the HNE2 cell cDNAs of TP53 plasmids, using TaKaRa LAEnzyme enters performing PCR amplification Total length LINC00472 sequences.LINC00472 full length sequence amplimers are as follows：

Sense primer：5’-atgcgaggctggggccggtt-3’

Anti-sense primer：5’-tttagactccaaatggctgttttatt-3’

Upstream：5’-AGGAGCTAGCAtgcgaggctggggccggtt-3 ' (underscore part is Nhe I recognition sites)

Downstream：5’-ATGCGATATCTttagactccaaatggctgttttatt-3 ' (knows underscore part for EcoR V Other site)

2) PCR amplifications, LINC00472 full length sequences, PCR reaction conditions are as follows：

PCR reactions steps

3) by through Nhe I and EcoR V double digestion rear electrophoresis, glue is returned again after PCR primer electrophoresis, glue reclaim purpose fragment Receive.

4) pcDNA3.1 plasmids are through Nhe I and EcoR V double digestion rear electrophoresis glue reclaim purpose fragments.

4) and 5) 5) with T4DNA ligases connection step glue reclaim product, you can obtain can be used for eukaryotic expression lncRNA The vector plasmid of LINC00472.

1.3 prepare the coated nano silicon particles of poly-D-lysine

The coated nano silicon particles of poly-D-lysine are carried out with OP-10/ hexamethylenes/ammonia microemulsion self-assembling technique The synthesis of nano silicon particles (silica nanoparticle, SiNP), and using nano silicon particles surface can and by ion Electrostatic interaction, prepares the nano silicon particles of polylysine modification；Described nano particle can be prepared by following methods：

1) OP-10 (NPE), hexamethylene and ammoniacal liquor are mixed, the uniform rear positive silicic acid of addition is stirred at room temperature Different ester (TEOS), continues to stir to polymerization completion, adds equal-volume acetone, and ultrasonic disperse, centrifugation, distilled water is washed three times, from The heart is collected and is deposited in 80 DEG C of dryings, finely ground to obtain nano silicon particles (SiNP, particle size range 10-50nm).Wherein H₂O and OP-10 and H₂The mol ratio of O and TEOS be 2~10, ammonia concn be molar concentrations of 1.6~28%, TEOS in hexamethylene for 0.1~ 3mol/L。

2) SiNP is resuspended in 0.6M NaCO by 0.1~10mg/ml₃In solution, supernatant is abandoned in ultrasonic disperse, centrifugation, then Sediment is resuspended in PBS (pH 7.4) by 0.1~10mg/ml, ultrasonic disperse, add polylysine (final concentration of 4~ 15nmol/mL), fully mix, room temperature is mixed to shake；Centrifugation, abandons supernatant, and precipitation is resuspended in distilled water by 0.1~10mg/ml, obtained To the nano silicon particles of polylysine modification.

3) by modified nano silicon particles ultrasonic disperse, every milliliter of nano particle suspension adds 10~50ug LINC00472 tables Up to carrier, mixing, being stored at room temperature combines it.

1.4 cell culture and transfection

The good nasopharyngeal carcinoma cell HNE2 of growth conditions is pressed 2 × 10⁵Individual cells/well is inoculated in 6 orifice plates, by 6 orifice plates It is placed in 37 DEG C, 5%CO₂In incubator, treat that cultured cells starts LINC00472 eukaryotic expressions by growing to 50-70% density The transfection of carrier；Transfection process is as follows：

The poly-D-lysine of the carrying LINC00472 eukaryon expression plasmids that 100 μ l prepare is added to repair in aseptic EP pipes The nano silicon particles suspension of decorations, gently mixes with 100 μ l serum free mediums；With D-Hank's liquid washed cell 3 times；Will be above-mentioned 800 μ l serum free mediums (antibiotic-free) are added in mixture, 1 hole in 6 orifice plates is added after gentle mixing；By 6 orifice plates It is placed in CO₂In incubator, 37 DEG C are cultivated 6 hours, then abandon supernatant, add complete medium to continue overnight incubation.With carrying The nano silicon particles of the polylysine modification of pcDNA3.1 empty carriers are used as experiment contrast.

1.5MTT cell proliferation experiments

1) digest cell obtained in the previous step, cell is counted with cell counter, will transfection LINC00472 and The cell of pcDNA3.1 empty carriers is inoculated in 96 orifice plates, and 1000 cells are inoculated with per hole, and every kind of cell is inoculated with 5 holes, as a result takes Its average.

2) 37 DEG C, 5%CO2 incubators culture 6 hours, after after cell attachment, the μ l of MTT liquid (5mg/ml) 20 is added per hole.After It is continuous to be incubated 4 hours, terminate culture, discard nutrient solution.150 μ l DMSO are added per hole, is vibrated 10 minutes, dissolve crystal.

3) 490nm wavelength is selected, zeroing hole is concurrently set, on enzyme-linked immunosorbent assay instrument, each hole absorbance is determined simultaneously Record result.

4) every 24 hours repeat steps ibid, detect 6 days altogether.With absorbance as ordinate, interval time is horizontal seat Mark and draw MTT curves processed.

5) test in triplicate.With each time point as abscissa, absorbance is ordinate, draws cell growth curve.

1.6 flow cytometry analysis cell cycles

1) trypsin digestion cell when cultured cells reaches 85% fusion, 1200rpm/min centrifugation 5min, collects cell and sinks Form sediment.

2) 1xPBS re-suspended cells, 1200rpm/min is centrifuged 5min, collects cell.Repeat this step 2 time.

3) 1xPBS re-suspended cells, add 70% ethanol of precooling to fix cell pellet overnight.

4) 1000rcf/min, is centrifuged 5min, collects cell precipitation.

5) PH7.4PBS washed cells 1 time, adds PBS re-suspended cells, and adjust cell concentration to Ix 10 after centrifugation⁶/ ml。

6) propidium iodide (PI) dyeing liquor (PI containing 50mg/L, 1g/L Triton X-100,100g/L RNase) is added Mix, 4 DEG C of lucifuges are incubated 30min.

7) flow cytometer FACStar (U.S. company BD) detections.The signal of reception is right through Cellquest software processings Detect that the fluorescence intensity of cell is analyzed.Experiment is repeated 3 times.

1.7 flow cytometry analysis natural death of cerebral cells rates

1) when cultured cells reaches 85% fusion, digest each group cell with pancreatin and be collected in centrifuge tube, while collecting Each group supernatant suspension cell.Note gently blowing and beating cell, it is to avoid pancreatin excessively digests.

2) merge each group cell respectively and be transferred in centrifuge tube, 1000rpm centrifugation 5min abandon supernatant and collect cell, PBS After gently resuspended, cell count.

3) 5~100,000 re-suspended cells are taken, 1000rpm centrifugation 5min abandon supernatant, add 195ul Annexin V-FITC knots Close liquid gently re-suspended cell.

4) 5ul Annexin V are added, is gently mixed.Lucifuge is incubated at room temperature 10min.

5) 1000rpm centrifugations 5min, abandons supernatant, adds 190ul Annexin V-FITC combinations liquid gently re-suspended cell.

6) 10ul PI dyeing liquors are added, is gently mixed, ice bath lucifuge.

7) flow cytomery is carried out immediately, and Annexin V-FITC are green fluorescence, and PI is red fluorescence.

2. result

2.1LINC00472 suppresses the growth of nasopharyngeal carcinoma cell

After transfection LINC00472 carrier for expression of eukaryon, compared with the cell of transfection empty carrier, the life of nasopharyngeal carcinoma cell HNE2 Speed long significantly slows down (Fig. 2A).

2.2LINC00472 suppresses the growth of nasopharyngeal carcinoma cell by arresting cell cycle, inducing cell apoptosis

Flow cytometry analysis show that after transfecting LINC00472 in nasopharyngeal carcinoma cell HNE2, G2/M phases cell proportion shows Writing increases, and S phases and G0/G1 phases cell proportion are reduced, and show that LINC00472 can make it by HNE2 cell blocks in the G2/M phases Cell division slow speed (Fig. 2 B).Meanwhile, apoptotic cell ratio is obvious after transfecting LINC00472 in nasopharyngeal carcinoma cell HNE2 Increase (Fig. 2 C), this is also one of the reason for LINC00472 suppresses Growth of Nasopharyngeal Carcinoma.

Embodiment 3, nude mice moves LINC00472 in growing knurl model and suppresses Growth of Nasopharyngeal Carcinoma

1. materials and methods

8 male BALB/C nude mices, 4 week old, weight is 19 ± 2g, is bought in the Shanghai limited public affairs of Si Laike experimental animals Department.All equal quality inspections of nude mice are qualified, in experimental animal portion of Central South University, raised under the conditions of no-special pathogen (SPF).

The preparation of the nano silicon particles of LINC00472 construction of eukaryotic expression vector and polylysine modification, cell culture With transfection equivalent integers 2.

Transfect the HNE2 cells of LINC00472 or pcDNA3.1 empty vectors, and the HNE2 cells for not making any treatment (Mock) 2 × 10 are respectively taken⁶The subcutaneous of nude mice oxter is injected separately into, the growing state of comparison of tumor is observed, from the 5th day, vernier is used Slide calliper rule are moved across skin measurement grows knurl size, and nude mice is put to death at the 30th day, takes out shifting and grows knurl measurement, compares each group shifting and grows the big of knurl It is small.

2. result

Nude mice shifting is grown knurl model and is further characterized by, and lncRNA LINC00472 can suppress the growth (Fig. 3) of nasopharyngeal carcinoma cell

Embodiment 4, real time fluorescent quantitative method detection confirms that LINC00472 is lowered in nasopharyngeal carcinoma

1. materials and methods：

9 normal nasopharyngeal mucous epitheliums and 9 tissues of nasopharyngeal carcinoma extracted total RNAs, 2 μ g RNA through reverse transcription into after cDNA, Carry out real-time fluorescence quantitative PCR.LINC00472 sense primers are 5'-GCATCTGTCAACGCTCCTCT-3' and anti-sense primer 5'-CCGCGTTCTTTGTGTGTCTC-3'。

GAPDH sense primers for compareing are 5'-ACCACAGTCCATGCCATCAC-3' and anti-sense primer 5'- TCCACCACCCTGTTGCTGTA-3',

Real-time fluorescence quantitative PCR reaction system

Real-time fluorescence quantitative PCR reactions steps

Reaction confirms the amplification curve and melting curve of real-time fluorescence quantitative PCR, the expression intensity root of each gene after terminating After according to CT values (threshold cycle values), reference gene (GAPDH) markization, checked using group t-test and calculated P values.

2. result

LncRNA LINC00472 express relatively low in normal control tissue, and the low expression or not in tissues of nasopharyngeal carcinoma Expression P<0.001 (Fig. 4)

Embodiment 5, in situ hybridization detection finds that expression of the LINC00472 in nasopharyngeal carcinoma is related to patient's prognosis

1. materials and methods：

1.1 design and synthesize hybridization probe

In order to detect the expression of LINC00472 using in-situ hybridization method, we are devised in situ hybridization inspection Survey the oligonucleotide probe and each 3 of two groups of in situ hybridization oligonucleotide probes of positive control of LINC00472 expression.

For the oligonucleotide probe of in situ hybridization detection LINC00472 expression：

LINC00472 probes 1：5’-CAAAACTAGGATTCTGTCTTTGAGGCAGAC-3’

LINC00472 probes 2：5’-AAGAATGGAAGTGGGAAGAGAGAAAGACTT-3’；

LINC00472 probes 3：5’-TAATTCAGGGGTATCCGTCTTAGTTTAGGC-3’.

Positive control probe (detection house-keeping gene GAPDH)：

GAPDH probes 1：5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'

GAPDH probes 2：5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'

GAPDH probes 3：5’-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3’.

The synthesis of sequence oligonucleotide probe synthesizes the few core of above-mentioned each gene specific of design using chemical synthesis process Thuja acid probe sequence.

1.2 oligonucleotide probe labelling kits and in situ hybridization detection reagent

Digoxin oligonucleotides tailing reagent (Dig Oligonucleitide Tailing Kit 2^ndGeneration, Roche companies), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab Fragments, Roche company), the TSA signal amplifying systems (TSA of enhancing detection of expression signal in situ^TM Biotin System, NEL700 kit, PerkinElmer companies), DAB staining kits (Beijing Zhong Shan companies), 20x sodium citrates Cushioning liquid (saline sodium citrate, SSC), dextran sulfate (Dextran sulphate), deionized formamide (Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA), is denatured frog essence DNA (the denatured and sheared of shearing Salmon sperm DNA, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), dithiothreitol (DTT) (DTT), 50x Deng Han Family name's buffer solution (Denhardts ' s solution), phosphate buffer (PBS buffer), pepsin K, bovine serum albumin(BSA) (BSA), triethanolamine (TEA), TNB Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.5%Blocking Reagent), TNT Buffer (0.1M Tris-HCl, pH7.5,0.15M NaCL, 0.05%Tween 20), acetic anhydride, resistance Disconnected reagent (Blocking reagent agent, Roche companies).

1.3 other main agents and material

Absolute ethyl alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turpentine oil, distilled water, PBS (pH7.2~ 7.4, NaCl 137mmol/L, KCl 2.7mmol/L, Na₂HPO₄4.3mmol/L, KH₂PO₄1.4mmol/L)；3% methyl alcohol- Hydrogen peroxide solution (80% methyl alcohol and 30% hydrogen peroxide are configured)；0.01mol/L citrate buffers (citrate buffer, It is interim in CB, pH6.0 ± 0.1,9ml 0.1M citric acid solutions and 41ml 0.1M sodium citrate solutions addition 450ml distilled water With postponing correction work liquid pH value again)；0.1% trypsase；Haematoxylin；1% hydrochloride alcohol (1ml concentrated hydrochloric acids+99ml 70% Alcohol is configured)；Mounting glue (PTS Cure Mount II)；Special cover glass (480 × 240mm²) customize in Zhengzhou glass apparatus Factory.Leica low melting points (58 DEG C) paraffin, domestic beeswax, absolute alcohol, dimethylbenzene, 10% neutrality paraformaldehyde (0.01mol/L, PH7.4, DEPC distilled water and PBS are prepared), haematoxylin, Yihong, neutral mounting natural gum, cover glass, slide.

1.4 label probes

Oligonucleotide probe mark is carried out using 3-tailing DIG Olignucleutide Kit, reaction system is such as Under.

100pmol oligonucleotide+ddH₂O=9 μ l (control:control oligonucleutide 5μ l+ddH₂O 4μl)

Mix, be slightly centrifuged.37 DEG C of water-bath 30min, plus 2 μ l EDTA (0.2M, PH 8.0) stopped reactions.

Purified after 1.5 oligonucleotide probes mark

In order to increase the purity of label probe, marked probe need to be purified, concrete operations are as follows：

1) cold ethanol (- 20 DEG C) of+2.5 μ l 4M LiCL+75 μ l of probe reaction mixture (22 μ l) 100%

2) -70 DEG C of precipitations 60min, or-20 DEG C of 2h.

3) 13.000 × g, 4 DEG C of centrifugation 15min.

4) supernatant is abandoned, is washed with 70% ice-cold (V/V) ethanol of 50 μ l.

5) 4 DEG C of 13.000xg, are centrifuged 5min.

6) supernatant, 4 DEG C of dryings of vacuum are abandoned.

7) with the aseptic double-distilled water molten probe of weight.

1.6 in situ hybridizations detection achieves the expression of LINC00472 in paraffin section

Paraffin section hybridizes pre-treatment

1) paraffin section of 4 DEG C of preservations is placed in 58 DEG C of roasting piece 30min, melted surface paraffin.

2) dimethylbenzene dewaxes 3 × 5min successively.

3) step ethanol wash, 100% 1 × 5min of alcohol 2 × 2min → 95% alcohol 1 × 5min → 70% alcohol → 50% 1 × 5min of alcohol → 2 × 3min of DEPC water washings → DEPC-PBS washs 2 × 5min.

4) 300 μ l pepsins K (10 μ g/ml) are added dropwise in section, 37 DEG C digest 20min.

5) to cut into slices and wash 1min, stopped reaction into PBS (0.1M PBS+2mg/ml glutamic acid).

6) cut into slices into 0.2N HCL, 20-30min is reacted in 37 DEG C, increase the permeability of tissue.

7) section 4% paraformaldehyde (0.1M PBS dissolvings) fixes 10min, room temperature afterwards.

8) in order to increase tissue positive intensity for hybridization, acetyl treatment is carried out to section.Cut into slices into 0.25% acetic anhydride Buffer I (0.1M triethanolamines), room temperature 10min.

9) 1M PBS wash 2 × 5min.

Prehybridization and hybridization

Prehybridization：- 20 DEG C of prehybridization solutions of preservation, are first placed in 37 DEG C of incubation 60min, and the consumption of prehybridization solution is 50 μ l, Parafilm carries out lid section, prehybridization 2 hours in 37 DEG C of wet box.(prehybridization solution composition includes：2XSSC, 10%Dextran Sulphate, 1X Denhardt ' s solution, 50mM Phosphate Buffer (PH 7.0), 50mM DTT, 250 μ l, The μ g/ml yeast of 100 μ g/ml poly A, 5 μ g/ml poly dA, 250 t-RNA, 500 μ g/ml ssDNA, 47% Deionized formamide)。

1) Parafilm is removed, prehybridization solution is got rid of, section is placed in 5min in 2 × SSC.

2) hybridization reaction：37 DEG C of hybridized overnights (18-20h).Each section adds 250 μ l hybridization solutions and is carried out with Parafilm Lid.Corresponding probe is added in prehybridization solution just turns into hybridization solution.Hybridization solution is prepared in prehybridization, places 37 DEG C of incubations, is made Probe is completely dissolved in hybridization solution, and this experiment is mixed with a plurality of oligonucleotide probe, is matched somebody with somebody by each probe 500ng/ml concentration It is made probe hybridization solution.Digoxin tailing labelling kit label probe concentration basis：The concentration of each probe by its with Develop the color the naked probe mark reaction theory spy compared with 30 bases of 100pmol when the positive quantifies probe during detection reaction Pin yield carries out the concentration that COMPREHENSIVE CALCULATING goes out label probe for two kinds of standards of 900ng.

3) post-hybridization washing, section immersion 2 × SSC, 10min, throws off Parafilm.Successively in shake washing on shaking table, 2 × SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.

Color developing detection reaction after hybridization

1) digoxigenin-probe and mRNA combination compounds are detected using Anti-Digoxigenin-POD；TSA amplification systems The positive signal of enhancing in situ hybridization reaction solution reaction, DAB colour developings.

2) during section goes to TNT buffer solutions, 3 × 5min.

3) TNB blocking buffer solutions, 300 μ l/TMAs, room temperature, 30min is added dropwise.

4) unnecessary blocking agent, 1 are sucked:Anti-Digoxigenin-POD (the TBS+0.1%Triton X- of 100 dilutions 100+1% blocking agents), room temperature 4 hours.

5) TNT Buffer (0.1M Tris-CL, pH7.5,0.15M NaCL, 0.05%Tween 20) washings, 3x5min。

6) signal is added dropwise in section and amplifies reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid Storage liquid：Biotinyl Tyramid are dissolved in 0.2ml DMSO, Biotinyl Tyramid working solutions：1 × dilution, 1:50 Dilution Biotinyl Tyramid storages liquid), room temperature 10 minutes.

7) TNT is washed, 3 × 5min.

8) section is added dropwise SA-HRP (strepto- avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min.

9) TNT is washed, 3 × 5min.

10) distilled water washing, 1 × 1min.

11) DAB colour developings, chromogenic reaction is controlled under microscope.

12) haematoxylin is redyed,

13) dehydration of alcohol step, chip drying.

14) mounting glue is added dropwise, the cover glass cover plate of dimension is crosslinked section 1min under uviol lamp.

1.7 results judge and standard

Application Optics microscope is observed under low power and high power lens respectively, looks first at the positive expression of target RNA Signal is in the intracellular positioning of object observing：Positioned at nucleus, cytoplasm or cell membrane.

Carried out with two kinds of standards of cell number of the intensity of the detection rna expression position positive signal and positive expression respectively again Comprehensive grading, criterion is：(1) judge according to positive cell dyeing intensity：A. cell dye-free, remembers 0 point；B. cell is dyed Light brown is weakly positive, remembers 1 point；C. cell dyes brown and dyes dark-brown and have light brown to carry on the back without background coloration, or cell Scape is moderate positive, remembers 2 points；D. cell dyes dark-brown and is strong positive without background coloration, remembers 3 points.(2) according to positive cell Expression number score：A. no positive cell expression, remembers 0 point；B. positive expression cell number≤25%, remembers 1 point；C.25% ＜ is positive thin Born of the same parents' number ＜ 50%, remembers 2 points；D. positive expression cell number >=50%, remembers 3 points.

It is respective by one of above-mentioned standard respectively by two pathology experts in order to reduce the subjective factor of appraisal result as far as possible Judged and scored, then both are scored multiplication, as a result for：1. 0 point of person is finally calculated as 0 point, it is believed that radiolucent table reaches；2. 1 point Finally 1 point is calculated as with 2 points of persons, it is believed that weakly positive is expressed；3. 3 points and 4 points of persons are finally calculated as 2 points, it is believed that moderate positive is expressed；④ 6 assign to 9 points of persons is finally calculated as 3 points, it is believed that strong positive is expressed.

1.8 analyses and statistical software

Statistical analysis are carried out to experimental result using SPSS13.0 statistical softwares, is compared use χ two-by-two²Test or Fisher Exact test, correlation analysis use Spearmen correlation methods；P ＜ 0.05 are that difference is statistically significant. Survivorship curve analysis is using Kaplan-Meier method and log-rank test；Multi-variables analysis uses Cox ' s proportional hazards model；P ＜ 0.05 are that difference is statistically significant.

2 results

Expression in 2.1lncRNA LINC00472 nasopharyngeal carcinoma

LncRNA LINC00472 have expression (Fig. 5 is left) in normal nasopharyngeal epithelium, and low in the tissues of nasopharyngeal carcinoma of part Expression or not (Fig. 5 is right).

The relation of expression Yu the Nasopharyngeal Carcinoma Patients prognosis of 2.2LINC00472

We have carried out Effect of follow-up visit by telephone to 250 Nasopharyngeal Carcinoma Patients, and their start time, treatment feelings have been inquired in detail Condition, whether there is recurrence, whether there is suffer from again other diseases, recurrence and death time etc., and registering life span and state, and to nasopharynx The survival analysis that the expression of lncRNA LINC00472 is carried out with the life span of patient and state in cancerous tissue, finds It is (Fig. 6) long that LINC00472 expression patient's mean survival times high express patient that is low or not expressing compared with LINC00472.Explanation LINC00472 is a molecular labeling related to nasopharyngeal carcinoma prognosis, and the lncRNA expresses low, patient's poor prognosis.

SEQUENCE LISTING

<110>Central South University

<120>The expression vector of long-chain non-coding RNA LINC00472, tumor suppression reagent and its application

<130>Nothing

<160> 17

<170> PatentIn version 3.3

<210> 1

<211> 9515

<212> RNA

<213>The sequence of long-chain non-coding RNA LINC00472

<400> 1

augcgaggcu ggggccgguu gccuaccggc cgcuucucgc cgaggcaguc cagacuuuuc 60

ccccggcggu gcccgcucca agacagcauc ugucaacgcu ccucuucucc ccuccuccuc 120

cugccgggcc gggcuccgcc ggcugcggcc gagaggacgc gggacccggc gcggugagcc 180

caucagcugu caggcgagcg gcgaagcggc uggagggcgg cgagagacac acaaagaacg 240

cggugggcgg cggcggcgaa aggggacggc aacuccuccc cgcgcccgcc ggugccaccg 300

ccggccgugc uuguuccgag gccgcgcaga caaugcggcc gggcucgucc ccgcgugccc 360

cagagugcgg agcgcccgcg cucccccgac cccaacuuga ccgucucccg gcucgcccag 420

cccccucccg ggguaggggc gcccccuccc uccgguggcc ggcgaaggaa gucgguccgc 480

ggccgcagau cccggcaacu ugcgaaccgg gaaaaguuug cggcgccucc gcggggcggc 540

gcgacgcggc ccgccccucg cguccgcggu caucgcgggu gacuuucucg acucgucguc 600

agccggggcc gcagcgcggc cgguggggac ugcggggcgg gccggagucc guccgagggc 660

ucccgcaccu cgggcugcgg auuucaggua cuccacuggg cauuuucucu ucauggagcu 720

uauagcaaca aaaguguuuu acaaaacacc agacauguug gcaucaucuc uuaguugggc 780

cauguggcca gggaacccuu caugaaacca ccucugcagu ccuggagagu ugcuguagaa 840

gaaggaaaag agaggaagac aacacaacac aggaggggau aaguaccucu ggacacaaug 900

gaaguaguag uuguugagca gagggaaagg gauaggagaa ggauguggac ugcagugcug 960

cagugccagg uaauaacuuu gaucauguac cuccuuggcu caggugcugu cucuucuacu 1020

cuccuccuua cuuguacuac cuuauuuagc acuuaauuau auacuuucuu gacucacuuu 1080

gagacacacu gauuaugugg ucugccucaa agacagaauc cuaguuuugg auuuuagcau 1140

uacugaucua uagccuugca ggggcuuaag gauggcagcu gucucucucc cuugggaucu 1200

agcuuuugca aauauugaag ggaagagauu ucuuugaucu uauaggaaua gauccacagu 1260

uccucuccag auuugcuuuu auagagaagu gugugucacc aaaacaaugg agauacgguc 1320

agagaggccc cuacccccag aauuuuaaua uaauuuuacu uucuggggca uuuauucuug 1380

acaaauacuu ucucuccuau cagcucacuu guuccucaca aaaacccuau aagauauuaa 1440

uauuauuaaa cagaccccua ugaagcaccu caagucuuuc ucucuuccca cuuccauucu 1500

ugcacaccuu uugcuagacu agucucugcg uucaucacau cagguuucua aaauaaaagu 1560

ccccuaauaa cuuucuauuu guucucucau cauauugaac cuuuuuugcc uggcuuauaa 1620

gguggccuua aaaccccauu uuccucuaaa cauugucucu guaguucagg aauauggcag 1680

gcucaacacu augcuugagc ccauauugcu ucccucauua gugaugccuc cucucagacc 1740

acccagauuc uacucauucu ucaauuucca gcuucuccuu ggagccuucc cagaucauua 1800

gcuucuuaaa aaucccagcu uucucugaau ucugauggua gcccccguua uuuggacaau 1860

uauguccugg ucuuaaaguc cuaaugggaa aaaaaaguac augauggacu ucauguagag 1920

aagaaagcac uggacugagu uuuaagacag cugcuaagga gcugugugag ccaaaccagg 1980

uuacuuuacc ucucuggauc uauauuugcu gaucuguuaa guggagacaa uugggucaga 2040

uaaucuuggu ggucuuugcu uauucugagg uucuuggaca acuuggccua guucacgugg 2100

gaugggauug aagaucuuac ugaguucuug gauggaaaug ucaguaaugg gcacuuagug 2160

ccuucaggac uuguucuaua cuaucccaga aauuucugau gcuaaacuug gcaauuuuua 2220

aaugucauau uccuuuucuc caaacucuuu aaccacaaga auucagggaa gguuaugggg 2280

ggugacuuag uuaucuggau aauacauauu guggacaaua uagcauuauc cuugggaaug 2340

auuucauauu guugaauaaa uaggaucaac uucuugauaa accuuagucc caagcauuuc 2400

aucuucauug gauaguguua cauaguaaua uauuuauguu uucuuuuaau cauuucauaa 2460

cuuggaaaau acuaacauag ucaaaacucu aggguaggug auacaugagu uucuguagua 2520

aucugguugg agacauguug uaauucugua uauauaugua cauuuauccc augcauguua 2580

ugccuaaacu aagacggaua ccccugaauu aagaggugcu guuauacauu gaccaggcuu 2640

aagaauaucu cuuuaaagug ugucgacauu uaauugaccu uuggaaguuc auucuguuaa 2700

ucauacucaa agugcuaaag cuaugguuga cugcucuggu guuuuuauau ucauucgugc 2760

uuuagcauau aaauucuuca gcauaauugc uacuuauuua gcaagaguuu ccuuuauuug 2820

aaaaugugag uugugcuugu auuuuugugu cuuucuuucu uucuuucuuu uuuuaaacuu 2880

ugcuucaggc uggguagugg uagagguuug aauuaaaaug uuuuccuguc aguaguugua 2940

ugaggugugg cuucuuaauc uagaaguaau ggaaauuucu ugcuuaaaug aagugcacuu 3000

uucuuccugc agaaguuaga acucuuuuag acaagcuuaa cacuucaaaa cuguauaucu 3060

uuaacagaaa cuuuuggaaa uuuuuaaauu ccauuugugu agauuaaaaa uaucaaugcu 3120

aauacuuucu auuaaauaag gauuuaucua uuuucauaaa accucuaauu auauuccuug 3180

aauugcuaug aguagaauuu uguuguaaac acaauauuuu uaaaaaguca aauauauuuu 3240

aaaaaucuua acauuauauu acuauaacuu agagaacagg uguuuacaug uuuaaaauuu 3300

ucuaggccuu aaauuuguga aaucagucuu ucagaaguua uuuuuaauau aguuaucugu 3360

guuacagguu uauaaguuga cauuucaauu uaaaauuuuu ucaauuuaua uuucaaugac 3420

caggaaaaaa uaguuaaacu uauuccuaaa uuuuaauaga aaaauaaaaa accacauagu 3480

uguuaauuuc ugguaauuua ucuugagacu ccaggcaugu uuuuagaaca uuuuuaccuu 3540

aaaagcaauu uugaaauagg gaaauuaaag uuuaaagcag aguuuucacu uuaaugaggu 3600

aauuuuauac uucccagaua gguaggccca uauucaagaa cacucugauc ucuguuuaua 3660

guaaguuuug augccauuau aucauuuacu uauucacgca uuauaagcau aguagauagc 3720

cccauuguca cgaagcuggc auuauuuucu ugucugucau ucaauauugu ccccuuuuuu 3780

uuuucuguuu ucaucauagc cguccuuuuc auuuuauugu agcccugacu cgaaaugaaa 3840

guaaguuuga acucuuaaaa aagaguuccu caaacucuuu uuuaagaaua uacagugaaa 3900

ugucuuaaau aauauuaucu aaauaauacc uaaagaaugc aaaugacuug caaugcugaa 3960

uagucagaca caggauguuc caugcuaagg guagacucau auuuccacuu uauaaaaugu 4020

gguuucagaa aacaaauaga uguguuucuu ccuuaauaau uaguguuuca ggaccaaagu 4080

ugacuaaaua uggaaguuua agauaaaacc cgguuucuua gggaaucuuu cucaguccuu 4140

aguaauucug uagaauugau gauagagugg caacuucaau acacucagua uucuuuucug 4200

uuugaagucu guuuugccau uggaaaaaac uuauuguugu uugaauuugg guauauguau 4260

ucacagaugg uuuacuuuau uuuuauuuug uuuaaacaaa uacuuucaug ucccaaacac 4320

ugagacaugu uucauguccu aaacacuuua uaaauauuag aauauuuaau ccccuuaacu 4380

cuguaguuau aauuaucuuc auuuuacaau ugagaaaacu aagauacgac guuuuauuua 4440

augguaacgc cuuaaguaag gggagaguga uaaguguaag cagaguggaa aacuuaguag 4500

ugcuaaggag caacagaagu augugcaaga guuugggaag augugucugc uuggaaagga 4560

ucagggacuc agcugggauc agucuuaaag agcaacaugu ccucauuccu caauuuuaag 4620

ccuuugggcu cuagaaguaa agggcaaacu uagaggaggc acuccucagg uuucagcugc 4680

cuguuccggc cuggugaggu ggcuguacug gcucaugugu uucucauagg gcuacaugag 4740

ccuuuauguc uuaccuccuc cucauuucuu ggcugacucc ugcuuuuuug gaggguuuaa 4800

cuucucucuc agcuagaagg uuaggcauug caaauaccag uggauaauuu uuuucuuagc 4860

uuuaacccca gcccauuuca acccccucuu ugcccuuugu auauucuuuu gaaaauauga 4920

uccaguagug uuuaugaaug uguguugugu aaaauuuaga gauugauguu aaacaacaga 4980

auuaaaggac aaagcugucu uuuuuguugg aauuggggau gggagagcag cucaaagugg 5040

gaaauaugga gaaaggagag acauugugga aaagaagaga uaaauugagg ggaaaaaaag 5100

aggaugaaaa aaugaagaga gagaauuacu gaugugcuuc acacccacac cuccucucca 5160

acuucacagu aacagaggca augucacuau cuuuuaccug acaagugcuu uaccagggag 5220

uugugccugg caucguggga guggaaaauu uuucccuuuu gcauuuugag acuugggugu 5280

gcugcuuuaa gaugccacug guaucuaagu aauguuuguu uauacuggau auuuuucucu 5340

guagccuuug uggauuggac uuauaauaua aacaauauug uuagcagaac ugcagguacu 5400

cuauugcuau guuuauuuua uuaguuaaaa aacuaccaga gcccuaggac uucugagcac 5460

auuuagaaaa uaccagaggc aauuuuaaga cuagcauuag aaagaaagaa aagaauaaga 5520

auaaaacuaa gacuugcaua ccuuagaauc cuaaccaaug uggugauuuu uaaaaauuaa 5580

uguuucagag acucuaaauu aggguaguug guuucaucaa uuacauuuuu uaaguuaaua 5640

uaaagugcuu uuguauguaa auucucuuaa uagauaauuu agguaaaaug aaaguguggc 5700

auuuuuuguu ccauagaaug uaaaacuaac gucagaauaa gaauucuuuu gggaauuuag 5760

gauuuuuuug augcuucuca aaucaucugu aaugacuuuu auuuauagaa aagguagauu 5820

auucauuagu uuuuaugaaa aacacucagu uuaaauagcc ggguauggug gcgcauggcu 5880

guagucccag cuacucagga ggcugggcac gagaauugcu ugaaccugug aggcagaggu 5940

ugcagugagc caagaucgca ccacugcacu ccagccuggu ccagagugag auuccgucuc 6000

gaacaaaaca aaacaaaaau auugagcuaa ucuaaauggu aaaaaucuag auuugaagaa 6060

auuuauuuua aaauaguugu ucauuuguca aauagaaaag uaugaucuua gcagguuugg 6120

uuagaucccu aucugaaaca aaguaaacua aagauguuuu ucuugaucaa ugcauagcaa 6180

uaacugcugu ucaaacaugu gaggaaucac caguaaauug ggggauagaa agcaggaaaa 6240

ggagcuuuuu ucucuuauuu uuuuucauuu aucaguuuuu cccauuuguc cauccuuucc 6300

uauaaucaug gaaauuuuua uuuuuuacac uccucucccc auuuuuuuau ccuagauugc 6360

caccacacca agguuuucua gaguuaggca gaaagagaac cugggccuag augcuaaugg 6420

aaacuauccc augcuuucuc agacucucuu uccuccccag agacaagagg agcaaaacag 6480

gaguugaugg cucauugcug uugcuucugu ggcugcccug uuugguugac cugagcuuuu 6540

uuuaaaaacc uccacucuug acgcuuaacu cuaugaauaa aauuagggcc agucacacug 6600

uauuuucauc aucaguuauc ccuagugauu caaauuuagc acaaaccugc ugugaguaca 6660

uauuaauuag gugaauacag uagaaugcaa ucauaacaug agucuuuuaa uaacuauucu 6720

uuguuugauu uuuauuuuag aaguuugaag uuauuuuguu gaaaaucuag aaaaucuuua 6780

ugugagcaac ugagguucgg guagcccaca acaguuuaaa ucaaacauua acuugcagua 6840

aaacacuacu gaaagugucu uugauuuuga ggugcagaga gcuguuucaa guuaaguugc 6900

uauauuaguc uguucucaug cugcuaauaa agacauaccu gcaaccgggc aauuuauuaa 6960

aaaaaaaaag agguuuaauu gacugacagu uccacauggc uggggaggcc ucaccgucau 7020

ggugaaagag caagggaugu cuuacauggu ggcaggcaag aaguuugugc agaggaacuc 7080

cuguuuauaa aaccaccaga ucucaugaga cuuacucacu accaggagaa caggaugcgg 7140

gaaaccaccu cggugauuca guuaucugca ccuggcccca cccuugacac auguugauua 7200

uuacaauuca aggugagauu uggguaggua cacagccaaa ccauaucagc ugcccauucu 7260

ccugugcagu gacauaaagg ucugauaacc ugauuuaaag cuuggacucu aagauuuuuu 7320

uuuuucuuuu uuugagucag guuaucucug ucgcucaagc uggaguucag uggcaugaac 7380

agggcucauu gcagccucaa ccuuccaggu ucaagugauu cucuugccuc aaccuccuga 7440

guaucugggg cuacaggcau gugccaccgu gccaggcuaa uauuuuuauu uuuauuuuug 7500

uaaagauggg gucucacuuc ugggcucaag caaucuuucc ucagccuccc aaagugcugg 7560

gauuauaggu gcgagccacu gcacccugcu guaaagaauu ucauaucccu guuuucggug 7620

ugcuguguca cuguuugcca uggguaaagg uauaugaauc ucuuuuauuu ugcugcuugc 7680

aacccaauuc ccaugccaaa cccaauuugu auccaaacaa acaauauugg agacaaguuc 7740

uucuauagaa agcuucauaa gccaguuacu gacuacugug ucuguaaaua uacucaaacu 7800

auuuuguagu cuugcuauuu uaaaaaaugu gugggugugg gaagugaugu ugaaugaauu 7860

caggguaauu ggugguucuu uguauuagga aaacucacua guuaagggaa gucuguuuuc 7920

augauucaug uauguuuaac uuaaaaaaaa auucuuccau ccuucucucc aguuuccacu 7980

cuaccucaua aauucaagua augaaggaau uuaaauaaua gaauuuuaua acugaaagga 8040

uuuuggagag aaucuauuaa cccuccuacu acaguugagg aaauagaugu ccagugaggu 8100

caaaugagau cauguagcua guuuaaaaac ccaaauaaca uuuuaguagu uuguuuccau 8160

uaguaaacac uguucuguuu gauuugcccu aacauguuca uaaucagagu cauaauaagg 8220

aaaaaucacu aauaugugug ugguacuuuc uugugcauac cucagucacu ucucaaaaga 8280

ggucuguaag gugucgguau ccugguuucc auucagggag agaacagucu augagccuaa 8340

gagaccuguc ugugccaguc acuaucagaa cagucuaaug cuaugugucu uccuaccuug 8400

gcaaaagcau uggcuggucu cuggacaguu cucauagacc acaggauugg gcagggggga 8460

gccacaucuu cauguuaggg cucuaaccug uaccaauuau uaacuguugu cucuauuuca 8520

ucauuucuua aagggaaaaa aaagcacuuu uuaaagagca ugagauuuua aauuuuucaa 8580

aagaaggaug cuggaucauu auaaguaagg cauguuuuaa agaucuucuu aucaacagcu 8640

uaaaaauuag ccaguauaua cugaacaucc acuguauaaa aagcacauua uggggugcuc 8700

caguagguac agcagugauc gagacagaau cauagccuuc agggaagagg uauaugcaau 8760

guucucuccu uccaaagauu accuagauag agaacuguuc auaagaaaua ucuugguacu 8820

gucuacauua cucuugagau auaaaauaua uauuauuucu uacgucugaa augagucagu 8880

uguuucuaaa acaauuugua uuaaaguauu cacauuaaaa auuuguaaaa uacagaaaag 8940

uagagagaaa aaaucaugaa uuuuuaacau gguaaugauu auaucaguuu cguucuuggc 9000

guuucuuuaa cuugauggga uagcuaaugu uuuuugucau ugcauagcuu uguaaacuug 9060

uccuaaggau uauauaauuu cagcaugagu uaguuucuaa guaugucaga aaucuagcuu 9120

uuuuguggua auuguuguuu uacacaaaau gauuaauaaa agacucuaaa cuaaccuugg 9180

cuuuagaaau gcuuuuuaaa uuuuaaaaua gugaaugagu agcagauuac uuucuaguau 9240

uucuuuaagg ucaccaaaau cacuuaacuu gcuuaaaaua uugauggaaa uaucaacuac 9300

guacaguuga ccuaaauuag aaaugggugu uuuuuaaaua gugaaauuua uaaauauaau 9360

auguauauuu uaaaauacag uuuauauuau agaguuaugu uccucuguaa uaauuucucu 9420

uuacacugaa ucauguucag uuuuuauuau gcagauaauu uaaucuuaca gcuuugaucu 9480

uuuuuaaaaa auaaaacagc cauuuggagu cuaaa 9515

<210> 2

<211> 20

<212> DNA

<213>TP53 upstream region of gene primers

<400> 2

cccttcccag aaaacctacc 20

<210> 3

<211> 20

<212> DNA

<213>TP53 downstream of gene primers

<400> 3

ctccgtcatg tgctgtgact 20

<210> 4

<211> 20

<212> DNA

<213>Reference gene GAPDH specific PCR forward primers

<400> 4

accacagtcc atgccatcac 20

<210> 5

<211> 20

<212> DNA

<213>Reference gene GAPDH specific PCR reverse primers

<400> 5

tccaccaccc tgttgctgta 20

<210> 6

<211> 20

<212> DNA

<213>LINC00472 forward primers

<400> 6

gcatctgtca acgctcctct 20

<210> 7

<211> 20

<212> DNA

<213>LINC00472 reverse primers

<400> 7

ccgcgttctt tgtgtgtctc 20

<210> 8

<211> 20

<212> DNA

<213>LINC00472 full length sequences expand sense primer

<400> 8

atgcgaggct ggggccggtt 20

<210> 9

<211> 26

<212> DNA

<213>LINC00472 full length sequences expand anti-sense primer

<400> 9

tttagactcc aaatggctgt tttatt 26

<210> 10

<211> 30

<212> DNA

<213>LINC00472 full length sequences amplification sense primer containing restriction enzyme site and protectiveness base

<400> 10

aggagctagc atgcgaggct ggggccggtt 30

<210> 11

<211> 36

<212> DNA

<213>LINC00472 full length sequences amplification anti-sense primer containing restriction enzyme site and protectiveness base

<400> 11

atgcgatatc tttagactcc aaatggctgt tttatt 36

<210> 12

<211> 30

<212> DNA

<213>In situ hybridization detection LINC00472 probes 1

<400> 12

caaaactagg attctgtctt tgaggcagac 30

<210> 13

<211> 30

<212> DNA

<213>In situ hybridization detection LINC00472 probes 2

<400> 13

aagaatggaa gtgggaagag agaaagactt 30

<210> 14

<211> 30

<212> DNA

<213>In situ hybridization detection LINC00472 probes 3

<400> 14

taattcaggg gtatccgtct tagtttaggc 30

<210> 15

<211> 30

<212> DNA

<213>GAPDH probes 1

<400> 15

ccactttacc agagttaaaa gcagccctgg 30

<210> 16

<211> 30

<212> DNA

<213>GAPDH probes 2

<400> 16

gtcagaggag accacctggt gctcagtgta 30

<210> 17

<211> 30

<212> DNA

<213>GAPDH probes 3

<400> 17

cagtagaggc agggatgatg ttctggagag 30

Claims

1. the expression vector of long-chain non-coding RNA LINC00472, the sequence of long-chain non-coding RNA LINC00472 is shown in SEQ NO:1。

2. the expression vector of long-chain non-coding RNA LINC00472 according to claim 1, it is characterised in that selection Nhe I and EcoR V restriction enzyme sites are used for digestion pcDNA3.1 carriers, and LINC00472 sequences are inserted into the site, obtain long-chain non- The expression vector of coding RNA LINC00472.

3. the expression vector of long-chain non-coding RNA LINC00472 according to claim 1, it is characterised in that

1) it is template to transfect the cDNA of the HNE2 cells of TP53 gene plasmids, using TaKaRa LAEnzyme enters performing PCR expansion Increase total length LINC00472 sequences；

LINC00472 full length sequence amplimers are as follows：

Sense primer：5’-atgcgaggctggggccggtt-3’

Anti-sense primer：5’-tttagactccaaatggctgttttatt-3’

After 5 ' ends of upstream and downstream primer add restriction enzyme Nhe I and EcoR V recognition sites and protection base respectively, Primer sequence is as follows：

Sense primer：5’-AGGAGCTAGCatgcgaggctggggccggtt-3’

Anti-sense primer：5’-ATGCGATATCtttagactccaaatggctgttttatt-3’；

2) PCR reaction conditions are as follows：

PCR reactions steps：

3) by after PCR primer electrophoresis, glue reclaim purpose fragment through Nhe I and EcoR V double digestion rear electrophoresis, glue reclaim again；

4) and 5) 5) with T4DNA ligases connection step glue reclaim product, you can obtain for eukaryotic expression lncRNALINC00472 Vector plasmid；

6) by the pcDNA3.1 eukaryon expression plasmid transformed competence colibacillus large intestines comprising LINC00472 full length sequences that 5) step is obtained Bacillus, to expand plasmid.

4. the expression vector described in claim 1 or 2 or 3, it is characterised in that suppress the preparation of nasopharyngeal carcinoma cell for preparing.

5. the preparation of nasopharyngeal carcinoma cell is suppressed, it is characterised in that by the nano silicon particles of polylysine modification and the non-volume of long-chain The expression vector mixing of code RNA LINC00472 stands, and obtains the nano silicon particles of polylysine modification.

6. it is according to claim 5 suppress nasopharyngeal carcinoma cell preparation, it is characterised in that

The nano silicon particles of polylysine modification are to carry out silicon with the microemulsion self-assembling technique of OP-10, hexamethylene, ammoniacal liquor The synthesis of nano particle, and using nano silicon particles surface can and ion electrostatic interaction carry out poly-D-lysine surface modification, Prepare.

7. it is according to claim 6 suppress nasopharyngeal carcinoma cell preparation, it is characterised in that prepare polylysine modification Nano silicon particles：

1) OP-10, hexamethylene and ammoniacal liquor are mixed, be stirred at room temperature it is uniform after add the positive different ester of silicic acid, continue to stir to being polymerized Into, equal-volume acetone is added, ultrasonic disperse, centrifugation, distilled water washs three times, is collected by centrifugation and is deposited in 80 DEG C of dryings, finely ground The nano silicon particles of particle size range 10-50nm；Wherein H₂O and OP-10 and H₂O and the mol ratio of the different ester of positive silicic acid are 2~10, ammonia Water concentration is that 1.6~28%, molar concentration of the different ester of positive silicic acid in hexamethylene is 0.1~3mol/L；

2) nano silicon particles are resuspended in 0.6M NaCO by 0.1~10mg/ml₃In solution, supernatant is abandoned in ultrasonic disperse, centrifugation, then Sediment is resuspended in the PBS of pH 7.4 by 0.1~10mg/ml, ultrasonic disperse, adds polylysine, final concentration of 4~ 15nmol/mL, fully mixes, and room temperature is mixed to shake；Centrifugation, abandons supernatant, and precipitation is resuspended in distilled water by 0.1~10mg/ml, obtained The nano silicon particles of polylysine modification.

8. according to claim any one of 5-7 suppression nasopharyngeal carcinoma cell preparation, it is characterised in that by poly-D-lysine The nano silicon particles ultrasonic disperse of modification, every milliliter of nano particle suspension adds 10~50ug long-chain non-coding RNAs LINC00472 Expression vector, mixing, being stored at room temperature combines it.