CN103146693B - Long chain non-coding RNA (Ribonucleic Acid) gene and application method thereof - Google Patents

Abstract

The invention relates to a newly cloned full-length sequence of a long chain non-coding RNA (Ribonucleic Acid) gene and an application method thereof. The gene can be detected in a separation sample by methods including a hybrid method or an amplification method; and the gene can be used as a tumor marker, in particular a marker of liver cancer. According to the gene sequence, a real-time quantification PCR (Polymerase Chain Reaction) primer and an in situ hybridization probe are designed and synthesized; and by detecting the expression level of the long chain non-coding RNA in a liver cancer clinical case sample, expression of the long chain non-coding RNA in liver cancer is up-regulated remarkably, and prognosis of patients with liver caner with high expression of long chain non-coding RNA is poorer. According to the gene sequence, a RNA eukaryotic expression vector in a short hairpin structure for closing the expression of the long chain non-coding RNA by RNA interference is designed and synthesized; and the expression of the long chain non-coding RN in a liver cancer cell line A is inhibited successfully by using the vector.

Description

Long-chain non-coding RNA and application method thereof

Technical field

The invention belongs to oncomolecularbiology field, be specifically related to long-chain non-coding RNA sequence and the application method thereof of a new clone.

Background technology

Primary hepatocarcinoma is the Asia occurred frequently tumour regional with part Africa, and its case fatality rate occupies the second of China's malignant tumour, and five year survival rate only has 5% left and right, and the control of liver cancer and study of incident mechanism thereof are the important topics in China's medical scientific always.Hepatocellular carcinoma (hepatocellular carcinoma, HCC) be a kind of main Types of primary hepatocarcinoma, although had new progress aspect the current diagnosis in hepatocellular carcinoma and treatment, still there is the problem of a large amount of the unknowns in the precise mechanism that development and transfer occurs for it.

Long-chain non-coding RNA (long non-coding RNA, lncRNA) be the non-coding RNA that a class transcript length exceedes 200 Nucleotide, research finds that a lot of lncRNA have important regulative in the developing of tumour, and it has the tumor growth of inhibition and promotes the biological actions such as metastases.LncRNA can be divided into five types according to its gene location: lncRNA (intergenic lncRNA) between lncRNA (intronic lncRNA) and gene in just lncRNA (sense lncRNA), antisense lncRNA (antisense lncRNA), two-way lncRNA (bidirectional lncRNA), gene.Be considered to transcribe the lncRNA of " noise " before always, because of the critical function in generegulation, become at present the another study hotspot in non-coding RNA field after miRNA.In recent years research shows, the multiple important regulation processes such as lncRNA participates in X chromosome silence, genomic imprinting and chromatin modification, transcriptional activation, transcribes interferences, the interior transport of core.LncRNA is expected to become the new mark of diagnosing tumor, treatment and prognosis, simultaneously also for the treatment of tumour provides new target spot.

New-generation sequencing (Next Generation Sequencing, NGS) fast development of technology has been brought the research of genomic level into a new stage, manyly become possibility based on complete genomic research, RNA (the RNA-Sequencing that checks order, RNA-Seq) be exactly to utilize whole RNA that new-generation sequencing technology is transcribed biological sample to carry out high-flux sequence, transcribe the important new technology of one of group (transcriptome) data to obtain sample.

We utilize new-generation sequencing technology to carry out RNA-Seq to patients with hepatocellular carcinoma biopsy specimen and normal control hepatic tissue sample recently, in liver cancer sample, detect that Chromosome 11q13 .1 region exists adjacent several obvious RNA-Seq fignal center, and do not have in normal control tissue, and this chromosomal region there is no known login at present, and prompting may exist the new gene of one or more the unknowns.We confirm that these RNA-Seq peaks are from same new gene, and have cloned this full length gene sequence as clue.This full length gene sequence is 3526bp, the multiple transcripts of transcribed one-tenth, the sequence input ORF finder software of this full length gene is carried out to open reading frame prediction, do not find obvious open reading frame, long-chain non-coding RNA of this genes encoding (long non-coding RNA, lncRNA) is described.

We from liver cancer and normal control tissue after extracting RNA by reverse transcription, real-time quantitative PCR, has detected the expression of this lncRNA, result shows that this lncRNA raises (P<0.001) at Expression In Hepatocellular Carcinoma.We utilize again the method for in situ hybridization subsequently, further in large sample, have verified that this lncRNA expresses significantly rise (P<0.001) in liver cancer.Therefore can be used for the auxiliary diagnosis of liver cancer for the detection preparation of this lncRNA.Survival curve analysis finds that this lncRNA expresses its survival time of high patient and is shorter than this lncRNA and expresses low patient (P=0.024).Therefore also can be for the prognosis judgement of liver cancer for the detection preparation of this lncRNA.

We also for the sequences Design of this lncRNA gene the short hairpin RNA expression vector disturbing for RNA, utilize this carrier in hepatoma cell line, to suppress the expression of this lncRNA, and find to suppress this lncRNA and express the growth that can suppress hepatoma cell line.Therefore also there is the potential use of gene therapy of liver cancer for the inhibitory preparation of this lncRNA.

Summary of the invention

The object of this invention is to provide the sequence of a new clone lncRNA gene, and provide the application method of this lncRNA gene, and can adopt the method including hybrid method or TRAP in separating sample, to detect this gene, can be using this gene as tumor markers, the especially marker of liver cancer.

The lncRNA gene of new clone of the present invention, its transcript sequence is arbitrary in the nucleotide sequence shown in SEQ ID NO:1-12 in sequence table; Or with sequence table in the sequence shown in arbitrary of SEQ ID NO:1-12 there is the nucleotide sequence of 90% above homology; Or with sequence table in the sequence shown in arbitrary of SEQ ID NO:1-12 through thio-modification or/and methoxyl group is modified the nucleotide sequence obtaining.

Described long-chain non-coding RNA can be used as tumor markers.Described tumour is selected from liver cancer.

In separating sample, detect a method for above-mentioned sequence, comprise hybrid method or TRAP.

Described hybrid method comprises in situ hybridization, and described TRAP comprises fluorescence real-time quantitative PCR.

The present invention has found the long-chain non-coding RNA sequence of a new clone first, and prove specific expressed in liver cancer tissue of this gene by the method such as in situ hybridization, fluorescence real-time quantitative PCR, for auxiliary diagnosis and the prognosis prediction of liver cancer provide strong biology tool, a rna interference vector with the latent gene therapy potentiality of liver cancer and prospect is also provided, has there is profound significance and generalization.

Brief description of the drawings

Fig. 1 is hepatocellular carcinoma and normal control sample RNA-Seq result and the site plan on karyomit(e) thereof,

Show that we have found a new gene by RNA-seq;

The fignal center that wherein A:RNA-Seq finds is positioned at Chromosome 11q13 .1 section; The position relationship of B:RNA-Seq fignal center and contiguous gene; C: the contrast of RNA-Seq result in hepatocellular carcinoma (HCC) and normal control (Normal) sample, and this new full length gene sequence the primer design diagram of clonal expansion;

Fig. 2 is that RT-PCR confirms that the RNA-Seq peak of Chromosome 11q13 .1 section is transcribed from same gene and montage and becomes multiple transcripts;

A: carry out RT-PCR amplification, the electrophoresis result of PCR product with primer 1F and 2R and 1F and 3R taking liver cancer tissue cDNA as template; B: the different transcript montage modes that the PCR product of primer 1F and 2R and 1F and 3R amplification is found after TA-clone, order-checking; C: primer 3F and 4R and 4F and 5R carry out the result of RT-PCR amplification; D: the different transcript montage modes that the PCR product of primer 3F and 4R and 4F and 5R amplification is found through TA-clone, order-checking;

Fig. 3 is GeneRacer clone new gene 5 ' and 3 ' full length sequence;

A:GeneRacer experimental strategy sketch; B:3 ' GeneRacer PCR product electrophoresis result;

This gene 5 ' end and 3 ' that C:5 ' and 3 ' GeneRacer sequencing result obtain is held different splicing forms;

Fig. 4 is gene and 12 kinds of different transcripts of new clone, and open reading frame predicts the outcome and is shown as long-chain non-coding RNA;

A: the 12 kinds of representative transcript splicing forms of new clone gene that obtain by order-checking; B:ORF Finder predicts this new gene, does not find obvious open reading frame, shows this genes encoding long-chain non-coding RNA;

Fig. 5 is that real-time quantitative PCR detects the result of this lncRNA at 10 routine normal liver tissues and 36 routine Expression In Hepatocellular Carcinomas;

Wherein: N represents healthy tissues, T represents liver cancer tissue;

Fig. 6 is that in situ hybridization detects the expression (right side) of new clone lncRNA in normal liver tissue (left side) and liver cancer tissue;

Show that this lncRNA does not express in normal liver tissue, and in liver cancer tissue high expression level;

Fig. 7 is the lncRNA survival curve analytical results of new clone;

According to the expression level of new clone lncRNA, 51 routine liver cancer patients are divided into groups, lncRNA high expression level patient group (28 routine patients, fine dotted line) survival time is shorter than the low expression of lncRNA patient (23 routine patients, heavy line), shows that the lncRNA of new clone can be used for liver cancer patient prognosis judgement;

Fig. 8 designs and builds shRNA expression vector for the lncRNA of this new clone, disturbs the expression of this lncRNA in hepatoma cell line HepG2;

Show that 4 shRNA carriers of design all can disturb the expression of this lncRNA in HepG2 cell, four shRNA carrier mixing transfectional cells, effect is better;

Fig. 9 is the growth curve that shRNAs disturbs cell after the expression of new clone lncRNA in liver cancer cell HepG2,

Show that the expression of new clone lncRNA in interference cell can suppress the growth of HepG2 cell.

Embodiment

Further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.

One, new lncRNA gene clone and analysis

1. materials and methods:

1.1 reagent and test kit

The conventional biochemical reagents such as agarose (agrose), glue reclaim test kit purchased from Shanghai Hua Shun biotechnology company limited, and 1kbladder DNA molecular amount Marker is purchased from Invitrogen company; Utilize

the high-quality RNA of Mini Kit (Qiagen) extraction agent box extracting, SuperScript ^tMrNA reverse transcription is become cDNA by III (Invitrogen) test kit.

1.2 new-generation sequencing technology are carried out RNA order-checking

After people's liver cancer tissue sample and the total RNA of normal liver tissue sample extraction, cross column purification, retain RNA more than 200nt;

Then enrichment mRNA, and by the method for divalent cation heat by mRNA fragmentation; Taking mRNA short-movie section as template; With hexabasic base random primer reverse transcription synthetic double chain cDNA, purified, end reparation, add base A, add the step of sequence measuring joints, reclaim fragment through agarose gel electrophoresis, the increased preparation of sequencing library of the performing PCR of going forward side by side; Increasing through cBot in the library building, checks order with illumina solexa;

Illumina solexa being carried out to the result of RNA order-checking analyzes: comprise the sequence of removing inferior quality, pollution, joint, RNA sequencing result and human genome reference sequences are compared and shone upon, in liver cancer sample, detect that Chromosome 11q13 .1 region exists adjacent several obvious RNA-Seq fignal center, and do not have in normal control tissue, and this chromosomal region there is no known login at present, and prompting may exist the new gene of one or more the unknowns.

1.3 clone new gene sequences

In order whether to verify at No. 11 detected each RNA-Seq peaks of karyomit(e) from same RNA sequence, we have designed respectively forward (Forward for each RNA-Seq peak, F) primer and oppositely (Revise, R) primer, utilize the corresponding combination of primers of adjacent peak to carry out pcr amplification. approximate location and the direction at each primer place are shown in Fig. 1 C, and corresponding sequence is as follows:

1F：5’-CCCTTGTTTAGCCTCTGCTG-3’，2F：5’-CCTCTGTCACTGCCACAAAA-3’，

2R：5’-CATGTGGTGAGTGGCTGTG-3’，3F：5’-TCACTTACCCCTGCGACTCT-3’，

3R：5’-CATCACAGGGTGTGTTTTGC-3’，4F：5’-GCCCTGCTCTATCAGCAAAC-3’，

4R：5’-GCCTGCTGAGTTTGCTGATA-3’，5R：5’-CCCAAACCAAGCTGACAAAT-3’。

SEQ ID NO:13-20 in corresponding sequence table successively.

5 ' of unknown RNA holds and 3 ' end full length sequence to utilize GeneRacer test kit (Invitrogen) to increase, gene-specific primer (Gene Specific Primer for this project RNA sequence for GeneRacer reaction, GSP) approximate location and direction are shown in Fig. 1 C, and sequence is as follows:

Racer R1:5 '-GCAGAGGCTAAACAAGGGGGTCCTG-3 ', SEQ ID NO:21 in sequence table;

Racer F5:5 '-CACTGAATGCCCCACGTCAAAGAAA-3 '; SEQ ID NO:22 in sequence table;

The product of RT-PCR or GeneRacer PCR all uses TA clone test kit (Invitrogen) to connect, transform, and selects the order-checking of positive colony Sanger method, to obtain the sequence of Insert Fragment.

1.4 sequential analyses and Bioinformatics Prediction software used

New-generation sequencing technology is carried out the new gene of RNA-Seq discovery, we verify with traditional Sanger method order-checking, and cloned the full length sequence of this gene with Sanger method order-checking, the Sanger method sequence DNAStar software (DNASTAR Inc.) obtaining that checks order is assembled and is proofreaded; The potential open reading frame of new clone gene (Open Reading Frame, ORF) adopt the on-line analysis software ORF Finder (www.ncbi.nlm.nih.gov/gorf/gorf.html) of state-run bioinformation institute of the U.S. (National Center for Biotechnology Information, NCBI) exploitation to predict; Whether peptide sequence input Pfam (http://pfam.sanger.ac.uk/) database and the known protein structure domain of potential open reading frame coding have homology.

2 results

2.1RNA-Seq is at the multiple adjacent RNA fignal centers of Chromosome 11q13 .1 area discover

By new-generation sequencing technology to the RNA high-flux sequence (RNA-Seq) in hepatocellular carcinoma biopsy and normal control sample, and with human genome reference sequences (Build37.3, Hg19) for masterplate, RNA-Seq sequence is assembled, we find to have 6 adjacent obvious RNA fignal centers (Fig. 1 C) in Chromosome 11q13 .1 region (Figure 1A) apart from (Figure 1B) near No. 11 the short arm of a chromosome end physical distance 65.6Mb in liver cancer tissue, except only having about 50 order-checkings, read long (reads) support at the 5th peak, all the other each peaks have and exceed 100 reads and support, be up to nearly 400 reads and support (Fig. 1 C lower part), in same chromosomal region normal control sample, there is no or only has the reads (Fig. 1 C upper part) of trace, show that this chromosome segment has been transcribed out one or more RNA sequence in detected liver cancer tissue, and transcriptional level is higher, the chromosome region at these RNA fignal center places does not have known login (Figure 1B).

In order to verify that these RNA-Seq peaks are from several independently RNA sequences or from same RNA sequence on earth, we have designed respectively forward and reverse primer (Fig. 1 C) for each RNA-Seq peak, then taking liver cancer tissue cDNA as template, match and carry out pcr amplification with primer corresponding to adjacent peak.

Transcribe from same gene at 2.2RT-PCR confirmation multiple RNA-Seq peak, 11q13.1 region

Taking liver cancer tissue cDNA as masterplate, carry out pcr amplification with primer 1F and 2R, PCR product finds to amplify the band of many entries through agarose gel electrophoresis, primer 1F and 3R have obtained multi-ribbon (Fig. 2 A) too. by the object band purifying of tapping rubber respectively, TA clone, selecting positive colony checks order, find that the PCR product that primer 1F and 2R obtain has covered the 1st, 2 and 3 RNA-Seq peaks, and there are at least 2 kinds and transcribe rear splicing form, same, the PCR product of primer 1F and 3R amplification has covered 1st ~ 4 RNA-Seq peaks, exist at least 3 kinds to transcribe rear splicing form (Fig. 2 B).

Increase and also obtained similar result (Fig. 2 C and Fig. 2 D) with assembly PCRs such as primer 3F and 4R, 4F and 5R, in fact transcribe from same gene at these adjacent RNA-Seq peaks that confirmation Chromosome 11q13 .1 section records, the DNA fragmentation of their correspondences is the different exons of same gene, and has multiple montage mode by the RNA of PCR and known this genetic transcription of sequencing result. utilize our increased transcript of the more kinds of types of this gene of primer 1F and 5R.

What obtain due to pcr amplification is DNA double chain, only from sequencing result, we also cannot judge the actual direction of this RNA, yet do not obtain this RNA5 ' and 3 ' end full length sequence, therefore we further utilize GeneRacer test kit increase this RNA 5 ' and 3 ' hold total length.

2.3GeneRacer clone RNA full length sequence

GeneRacer the primer [GeneRacer test kit (Invitrogen) provides] is shown in Fig. 3 A, first use acid Pyrophosphate phosphohydrolase (the Tobacco Acid Pyrophosphatase of tobacco, TAP) remove RNA5 ' end cap (cap) structure, then connect one section of specific RNA joint sequence (left end heavy black line bar part in this joint sequence [being provided by GeneRacer test kit (Invitrogen)] Fig. 3 A) at RNA5 ' the end RNA ligase enzyme (RNA ligase) of removing cap sequence, next the oligo-dT that uses 5 ' end to increase another section of specificity joint is primer [this joint sequence is provided by GeneRacer test kit (Invitrogen)], RNA is carried out to reverse transcription, obtain the Article 1 chain of cDNA, now cDNA has just added respectively two sections of specific joint sequences at two.For the joint sequence at these two ends, GeneRacer test kit (Invitrogen) provides respectively the GeneRacer consensus primer of 5 ' and 3 ' end, in our interested goal gene sequence, design again gene-specific primer (gene specific primer, GSP, i.e. aforesaid Racer R1 and Racer F5) with public 5 ' or 3 ' GeneRacer combination of primers carry out pcr amplification and just can obtain our interested gene 5 ' and 3 ' and hold full length sequence.

Be template by liver cancer tissue RNA with the cDNA (two has added joint) of GeneRacer test kit reverse transcription, utilize the GeneRacer3 ' consensus primer in 2 gene specific GeneRacer primer Racer R1 and Racer F5 and the test kit designing in Fig. 1 C to carry out pcr amplification (Fig. 3 B), result Racer R1 and GeneRacer3 ' consensus primer fail to expand object band, Racer F5 and GeneRacer3 ' primer have expanded the object band being perfectly clear, show the 3 ' end of Racer F5 near goal gene, the 6th the 6th exon that RNA-Seq peak is exactly this new gene in its corresponding Fig. 1 C. it is template that the Hela cell cDNA providing in GeneRacer test kit is provided, Racer F5 and GeneRacer3 ' primer do not expand specific band yet, show that this gene do not express or express very weak in Hela cell.In Fig. 3 B, Racer F5 and GeneRacer3 ' consensus primer amplified production electrophoresis result are except having a very strong object band, above this band, also have two weak bands (in figure shown in arrow), show that this gene 3 ' end may exist alternative splicing equally.By PCR product TA clone, the order-checking of Sanger method, we have obtained the sequence (Fig. 3 C right-hand component) of three kinds of different transcripts of this new gene 3 ' end. and next we use Racer R1 and GeneRacer5 ' consensus primer to increase, and obtained the sequence (Fig. 3 C left-hand component) of 6 kinds of different transcripts of this new gene 5 ' end.

The gene of 2.4 new clones has multiple transcripts and there is no obvious open reading frame

We are further a large amount of PCR in other liver cancer tissue samples, and TA Cloning and sequencing is analyzed, and has obtained the more transcript sequence of this gene, and Fig. 4 A is the 12 kind transcript splice modes higher in Expression In Hepatocellular Carcinoma frequency ratio.The ORF finder software of transcript sequence input NCBI different this gene is carried out to open reading frame prediction, find that this gene does not have obvious open reading frame (all potential open reading frame are all less than 500bp).All potential ORF codings are translated as to albumen or structural domain known in peptide sequence and Pfam database compares, also find no homology, show this gene long-chain non-coding RNA (long non-coding RNA, lncRNA) of may encoding.

Two, fluorescence real-time quantitative PCR

1. materials and methods:

10 routine normal liver tissue and 36 routine liver cancer tissue extracted total RNA, 2 μ g RNA, after reverse transcription becomes cDNA, carry out fluorescence real-time quantitative PCR.New clone lncRNA primer is SEQ ID NO:23 in 5'-GTCCGTCAGTCCCTCACCT-3'(sequence table) and 5'-ATACAGCCCCAGACCCAAAC-3'(sequence table in SEQ ID NO:24).

Be 5'-TCTTAGCTGAGTGTCCCGCG-3'(sequence table SEQ ID NO:25 for the 18S primer that contrasts) and 5'-ATCATGGCCTCAGTTCCGAA-3'(sequence table in SEQ ID NO:26),

Fluorescence real-time quantitative PCR reaction system

Fluorescence real-time quantitative PCR reactions steps

194℃ 5min

295℃ 10sec

358℃ 30sec

472℃ 20sec

5Plate read

682℃ 30sec

7Plate read

8Go to step 2 for more 39 times

9Perform melting curve from 55.0℃to 95.0℃,read every 0.2℃,hold for 1 sec

Reaction finishes amplification curve and the melting curve of rear confirmation fluorescence real-time quantitative PCR, and the expression intensity of each gene, according to after CT value (threshold cycle values), reference gene (18S) markization, adopts group t-test inspection to calculate P value.

2. result

The lncRNA of new clone does not express or expresses very low in normal control tissue, and in liver cancer tissue high expression level P<0.001(Fig. 5)

Three, in situ hybridization

1. material method

1) design of oligonucleotide probe

Utilize DNA Club software that the lncRNA sequence of new clone is converted to its complementary sequence.Utilize the probe design function of Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3.cgi/), taking the complementary sequence of each gene as template, design oligonucleotides probe in its reading frame, probe parameter setting is basically identical, GC content is 40-60%, and Tm is 65-70 DEG C.Root Ju gene order length with the interval of 200 or 300 bases, designs the oligonucleotide probe of 30 base length of 5-6 bar under identical conditions.Use the online BLAST software (http://www.ncbi.nlm.nih.gov/BLAST/Blast.cgi) in the state-run information biology of U.S. research centre (NCBI) to carry out exact matching to designed probe, filter out best specific 3 oligonucleotide probes of Crossbreeding parameters.

2) oligonucleotide probe with the in situ hybridization of contrast healthy tissues for liver cancer

The lncRNA of new clone and with oligonucleotide probe length corresponding to the house-keeping gene GAPDH comparing be 30 bases, oligonucleotide probe 5 ' end is phosphate group, 3 ' end is hydroxyl, probe sequence is the complementary sequence of these gene cDNAs.The detailed sequence of gene oligonucleotide probe is as follows.Oligonucleotide probe adopts chemical synthesis process synthetic.

The lncRNA probe of new clone:

SEQ ID NO:27 in 5'-CATTGTTTTAATGCTTCCAGATGAGTTTGTATC-3'(sequence table)

SEQ ID NO:28 in 5'-CTTTGGAAATAGTGCAGACACAAAACTAGGG-3'(sequence table)

SEQ ID NO:29 in 5'-GTGTGTTTTGCAGTCTCCTTGTTTGTTTCTTC-3'(sequence table)

The corresponding probe of house-keeping gene GAPDH:

SEQ ID NO:30 in 5'-CCACTTTACCAGAGTTAAAAGCAGCCCTGG-3'(sequence table)

SEQ ID NO:31 in 5'-CAGTAGAGGCAGGGATGATGTTCTGGAGAG-3'(sequence table)

SEQ ID NO:32 in 5'-GTCAGAGGAGACCACCTGGTGCTCAGTGTA-3'(sequence table)

3) oligonucleotide probe labelling kit and in situ hybridization detection reagent

Dig Oligonucleitide Tailing Kit (2 ^ndgeneration) test kit (Roche company), anti-digoxin-horseradish peroxidase complex detection kit (Anti-Digoxigenin-POD, Fab fragments, Roche company).Strengthen the TSA signal amplifying system (TSA of expressed in situ detection signal ^tMbiotin System, NEL700 test kit, PerkinElmer company).DAB staining kit (Beijing Zhong Shan company).20 × SSC, T 500 (Dextran sulphate), deionized formamide (Deionized Formamide), polyadenylic acid (polyadenylic acid, Poly A), poly deoxyadenylic acid (polydeoxyadenylic acid, Poly dA), frog essence DNA (the denatured and sheared salmon spermDNA that sex change is sheared, ssDNA), yeast transfer RNA (yeast t-RNA, tRNA), DTT, 50 × Denhardts ' s solution, PBS buffer, stomach en-K, BSA (bovine serum albumin(BSA)), trolamine (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%Tween20) acetic anhydride, blocking-up reagent (Blocking reagent agent, Roche company).

4) other main agents and material

Dehydrated alcohol, 90% alcohol, 70% alcohol, 50% alcohol, turps, distilled water, PBS damping fluid (pH7.2～7.4, NaCl137mmol/L, KCl2.7mmol/L, Na ₂hPO ₄4.3mmol/L, KH ₂pO ₄1.4mmol/L); 3% methyl alcohol-hydrogen peroxide solution (80% methyl alcohol and the configuration of 30% hydrogen peroxide); 0.01mol/L citrate buffer (citrate buffer, CB, pH6.0 ± 0.1,9ml0.1M citric acid solution and 41ml0.1M sodium citrate solution add in 450ml distilled water after provisional configuration correction work liquid pH value again); 0.1% trypsinase; Hematorylin; 1% hydrochloride alcohol (configuration of 1ml concentrated hydrochloric acid+99ml70% alcohol); The special mounting glue of micro-array tissue (PTS Cure Mount II); Special cap slide (480 × 240mm ²) customize in Zhengzhou Glassware Factory.Leica low melting point (58 DEG C) paraffin, domestic beeswax, raw spirit, dimethylbenzene, 10% neutral paraformaldehyde (0.01mol/L, pH7.4, DEPC distilled water and the preparation of PBS damping fluid), Hematorylin, Yihong, neutral mounting natural gum, cover glass, slide glass.

5) mark of oligonucleotide probe

Utilize 3-tailing DIG Olignucleutide Kit to carry out oligonucleotide probe mark, reaction system is as follows.

100pmol oligonucleotide+ddH2O=9μl(control:control oligonucleutide5μl+ddH ₂O4μl)

Mix, slightly centrifugal.37 DEG C of water-bath 30min, add 2ul EDTA (0.2M, pH8.0) stopped reaction.

6) purifying after oligonucleotide probe mark

In order to increase the purity of label probe, need carry out purifying to the probe of mark, concrete operations are as follows:

A) probe reaction mixture (22 μ l)+2.5 cold ethanol of μ l4M LiCL+75 μ l100% (20 DEG C).

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

C) 13.000 × g4 DEG C of centrifugal 15min.

D) abandon supernatant, by ice-cold 70% (V/V) washing with alcohol of 50 μ l.

E) 13.000xg4 DEG C, centrifugal 5min.

F) abandon supernatant, 4 DEG C, vacuum is dry.

G) with the heavy molten probe of aseptic double-distilled water.

7) tissue slice hybridization pre-treatment

A) tissue slice of 4 DEG C of preservations is placed in 58 DEG C of roasting sheet 30min, melted surface paraffin.

B) the dimethylbenzene 3 × 5min that dewaxes successively.

C) step alcohol washing, 100% alcohol 2 × 2min → 95% alcohol 1 × 5min → 70% alcohol 1 × 5min → 50% alcohol 1 × 5min → DEPC water washing 2 × 3min → DEPC-PBS washs 2 × 5min.

D) drip 300 μ l stomach en-K (10 μ g/ml) in section above, 37 DEG C of digestion 20min.

E) cut into slices into PBS (0.1M PBS+2mg/ml L-glutamic acid) washing 1min, stopped reaction.

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

G) fixing 10min after 4% paraformaldehyde (0.1M PBS dissolving) for section, room temperature.

H) to organize positive intensity for hybridization in order increasing, section to be carried out to acetylize processing.Cut into slices into 0.25% diacetyl oxide Buffer I (0.1M trolamine), room temperature 10min.

I) 1M PBS washing 2 × 5min.

8) tissue prehybridization and hybridization

A) prehybridization: the prehybridization solution of-20 DEG C of preservations, be first placed in 37 DEG C and hatch 60min, the consumption of prehybridization solution is every square centimeter of section area 12.5 μ l, the Parafilm of corresponding size covers section, prehybridization 2 hours in 37 DEG C of wet boxes.(prehybridization solution composition comprises: 2XSSC, 10%Dextran sulphate, 1X Denhardt ' s solution, 50mM Phosphate Buffer (PH7.0), 50mM DTT, 250 μ l, 100 μ g/ml poly A, 5 μ g/ml poly dA, 250 μ g/ml yeast t-RNA, 500 μ g/ml ssDNA, 47%Deionized formamide).

B) remove Parafilm, get rid of prehybridization solution, section is placed in 2 × SSC 5min.

C) hybridization: 37 DEG C of hybridization spend the night (18-20h).Each section adds 250 μ l hybridization solutions and covers with Parafilm.In prehybridization solution, add corresponding probe just to become hybridization solution.Hybridization solution is prepared in the time of prehybridization, places 37 DEG C and hatches, and probe is fully dissolved in hybridization solution, and this experiment mixes with many oligonucleotide probes, is mixed with probe hybridization liquid by each probe 500ng/ml concentration.Digoxin tailing labelling kit label probe concentration basis: the concentration of each probe by it during with positive quantitatively probe when detection reaction colour developing compare and the naked probe mark reaction theory probe output of 100pmol30 base is the concentration that two kinds of standards of 900ng are carried out COMPREHENSIVE CALCULATING and go out label probe.

D) post-hybridization washing, 2 × SSC is immersed in section, and 10min, throws off Parafilm.Shake washing on shaking table successively, 2 × SSC (0.5%SDS), 2 × 15min → 0.25 × SSC (0.5%SDS), 2 × 15min.

9) color developing detection reaction after hybridization

A) adopt Anti-Digoxigenin-POD to detect digoxigenin-probe and be combined mixture with object RNA; TSA amplification system strengthens the positive signal of in situ hybridization reaction solution reaction, DAB colour developing.

B) section goes in TNT damping fluid, 3 × 5min.

C) drip TNB blocking-up damping fluid, 300 μ l/TMAs, room temperature, 30min.

D) do not wash, suck unnecessary blocker, the Anti-Digoxigenin-POD (TBS+0.1%Triton X-100+1% blocker) of 1:100 dilution, room temperature 4 hours.

E) TNT Buffer (0.1M Tris-HCl, PH7.5,0.15M NaCL, 0.05%Tween20) washes 3 × 5min.

F) the upper signal that drips of section amplifies reagent Biotinyl Tyamid, 300 μ l/TMAs, (Biotinyl Tyramid stock solution: Biotinyl Tyramid is dissolved in 0.2ml DMSO, Biotinyl Tyramid working fluid: 1 × diluent, 1:50 dilution Biotinyl Tyramid stock solution), room temperature 10 minutes.

G) TNT washes, 3 × 5min.

H) section drips SA-HRP (strepto-avidin-horseradish peroxidase), 300 μ l/TMAs, room temperature 30min.

I) TNT washes, 3 × 5min.

J) aquae destillata washing, 1 × 1min.

K) DAB colour developing, controls color reaction under microscope.

L) Hematorylin is redyed,

M) alcohol step dehydration, chip drying.

N) drip the special mounting glue of tissue slice, the cover glass cover plate of dimension, crosslinked section 1min under the ultraviolet lamp that section adhesive tape transfer system is equipped with.

10) result judgement and standard

Application Nikon E600 binocular optical microscope is observed respectively under low power and high power lens, and first the positive expression signal of object observing RNA is in the intracellular location of object observing: be positioned at nucleus, cytoplasm or cytolemma.

Carry out comprehensive grading with the intensity of this detection rna expression position positive signal and two kinds of standards of cell count of positive expression respectively again, judging criterion is: (1) judges according to positive cell dyeing intensity: a. cell dye-free, remember 0 point; B. to dye light brown be the weak positive to cell, remembers 1 point; C. cell dye brown and painted without background, or cell to dye dark-brown and have light brown background be the medium positive, remember 2 points; D. cell is dyed dark-brown and is colored as strong positive without background, remembers 3 points.(2) express number score according to positive cell: a. expresses without positive cell, remembers 0 point; B. positive expression cell count≤25%, remembers 1 point; C.25% < positive cell number < 50%, remembers 2 points; D. positive expression cell count >=50%, remembers 3 points (because each interlacing point diameter is only 0.9mm, the target complete cell of each interlacing point that counts under 10 × low power lens when statistics positive cell number).

In order to reduce the subjective factor of appraisal result as far as possible,, in the situation that not knowing each interlacing point character, judge separately and mark by one of above-mentioned standard respectively by two pathology experts, then both scorings are multiplied each other, result is: 1. 0 point of person finally counts 0 point, thinks negative and expresses; 2. 1 point and 2 points of persons finally count 1 point, think weak positive expression; 3. 3 points and 4 points of persons finally count 2 points, think medium positive expression; 4. 6 points and 9 points of persons finally count 3 points, think that strong positive expresses.

11) analysis and statistical software

Application SPSS13.0 statistical software carries out statistical analysis to experimental result, relatively uses between two χ ²test or Fisher exact test, correlation analysis adopts Spearmen correlation method; P < 0.05 is that difference has statistical significance.Survival curve analysis adopts Kaplan-Meier method and log-rank test; Multivariate analysis adopts Cox ' s proportional hazards model; P < 0.05 is that difference has statistical significance.

2 results

1) expression of the lncRNA of new clone in liver cancer and normal control tissue

The lncRNA of new clone all has expression in 51 routine liver cancer, it is expressed all (Fig. 6 right side) in cytoplasm and karyon, and 46 examples in 92%(50 example healthy tissues sample) normal liver tissue in the expression that is negative, all the other 8% are weak expression (Fig. 6 left side), have obvious significant difference (P<0.001) between the two.

2) Kaplan-Meier survival analysis

We have carried out Effect of follow-up visit by telephone to all 51 routine liver cancer patients, inquire in detail their start time, treatment situation, have or not recurrence, have or not and suffer from again other diseases, recurrence and death time etc., and survival time and state are registered, and the survival analysis that the expression of new clone lncRNA in liver cancer tissue and patient's survival time and state are carried out, lncRNA high expression level patient's mean survival time of finding new clone is 16.09 months, 4 years (48 months) interior 82.1%(23/28) patient dead, and the patient of the low expression of new clone lncRNA, mean survival time has exceeded 24.53 months, 34.8%(8/23 only in 4 years (48 months)) dead (Fig. 7) of patient.The lncRNA that new clone is described is a molecule marker relevant to prognosis in hcc, and this lncRNA expresses high, patient's poor prognosis.

Four, build the expression of shRNA carrier interference new clone l ncRNA

1. material method

1) reagent and test kit

Restriction enzyme HindIII, BglII, EcoR I and Cla I, T4DNA ligase enzymes etc. are purchased from TakaRa company;

TRIZOLTM Reagent（Invitrogen）；

Plasmid extraction test kit (#D6943-01, OMEGA);

Glue reclaims test kit (#M5212, OMEGA);

Reverse transcription test kit (#A3500, Promega);

Microbiotic G418(Ameresc).

2) design of shRNA

First by the Block-It RNAi designer software of the lncRNA sequence input Invitrogen company of new clone, find the shRNA best target of this lncRNA, select 4 best corresponding target sequences as follows:

ShRNA-1:5 '-GGACAAGGTCCAAGCTCTTAC-3 ' (SEQ ID NO:33 in sequence table)

ShRNA-2:5 '-GCAGTGCAGCAGTATCATTGC-3 ' (SEQ ID NO:34 in sequence table)

ShRNA-3:5 '-GCAGCAGTATCATTGCTTAGC-3 ' (SEQ ID NO:35 in sequence table)

ShRNA-4:5 '-GCAGCTCTGTGTGAGATTTGT-3 ' (SEQ ID NO:36 in sequence table)

Using widely used in human genome without any the Scramble sequence of target spot as negative control, its sequence is as follows:

Scramble:5 '-GACACGCGACTTGTACCAC-3 ' (SEQ ID NO:37 in sequence table)

For these 4 lncRNA target sequences, and Scramble sequence, according to the pSUPER of OligoEngine company carrier specification sheets, design can form oligonucleotide strand and the reverse complementary sequence thereof of hairpin structure, after their annealing, can form the two ends DNA double chain with restriction enzyme site BglII and HindIII sticky end respectively.The concrete DNA double chain that needs each synthetic oligonucleotide sequence and their pairings to return rear formation is as follows:

shRNA-1：

5’-GATCCCC GGACAAGGTCCAAGCTCTTAC TTCAAGAGA GTAAGAGCTTGGACCTTGTCC TTTTTA-3’

3’-GGG CCTGTTCCAGGTTCGAGAATG AAGTTCTCT CATTCTCGAACCTGGAACAGG AAAAATTCGA-5’shRNA-2：

5’-GATCCCC GCAGTGCAGCAGTATCATTGC TTCAAGAGA GCAATGATACTGCTGCACTGC TTTTTA-3’

3’-GGG CGTCACGTCGTCATAGTAACG AAGTTCTCT CGTTACTATGACGACGTGACG AAAAATTCGA-5’shRNA-3：

5’-GATCCCC GCAGCAGTATCATTGCTTAGC TTCAAGAGA GCTAAGCAATGATACTGCTGC TTTTTA-3’

3’-GGG CGTCGTCATAGTAACGAATCG AAGTTCTCT CGATTCGTTACTATGACGACG AAAAATTCGA-5’shRNA-4：

5’-GATCCCC GCAGCTCTGTGTGAGATTTGT TTCAAGAGA ACAAATCTCACACAGAGCTGC TTTTTA-3’

3’-GGG CGTCGAGACACACTCTAAACA AAGTTCTCT TGTTTAGAGTGTGTCTCGACG AAAAATTCGA-5’Scramble

5′-GATCCCC GACACGCGACTTGTACCAC TTCAAGAGA GTGGTACAAGTCGCGTGTC TTTTTA-3′

3 '-GGG CTGTGCGCTGAACATGGTG AAGTTCTCT CACCATGTTCAGCGCACAG AAAAATTCGA-5 ' is SEQ ID NO:38-47 in corresponding sequence table successively.

The part of underscore is the lncRNA sequence of shRNA target.Article two, after the DNA of complementary pairing annealing, the left side is the sticky end of restriction enzyme BglII, and the right is the sticky end of HindIII.

3) shRNA vector construction

8 strand oligo sequences that above-mentioned 4 shRNA of chemosynthesis are corresponding, are dissolved into 20 μ M by synthetic oligo with oligo annealing buffer, and complementary strand is respectively got 10 μ l and mixed.Then by oligo mixture in PCR instrument 95 DEG C heating 5 minutes, then naturally cool to room temperature, form double-stranded oligo fragment.

With BglII and Hind III double digestion pSUPER plasmid, reclaim the carrier segments of 3.1kb, the DNA of the sticky end after annealing and carrier that enzyme cuts back to close are mixed according to the ratio of the amount of 3:1, use T4 ligase enzyme, 16 DEG C of connections are spent the night.Transformed E .coil competence, select transformant, bacterium colony PCR and order-checking qualification, cut with Cla I and EcoR I enzyme the pSUPER plasmid building again, 2% agarose DNA gel electrophoresis, taking blank pSUPER as blank, the positive colony of object fragment has been inserted in judgement, after positive colony sequence verification for the expression of new clone lncRNA in interference cell.

4) cell cultures and transfection

Hepatoma cell line HepG2 is purchased from Chinese Academy of Sciences Shanghai biochemical cell institute, and cell cultures RPMI1640 used trains base and foetal calf serum, and peptic cell trypsinase used is Gibco company of U.S. product.

Liver cancer cell HepG2 good growth conditions is pressed to 2 × 10 ⁵individual cells/well is inoculated in 6 orifice plates, 6 orifice plates is placed in to 37 DEG C, 5%CO ₂in incubator, treat that culturing cell grows to 50-70% density and can start the transfection of shRNA expression vector; Transfection process is as follows:

In aseptic EP pipe, add the lipofectamine2000 of 3 μ l in 100 μ l serum free mediums, to mix standing 5min;

The shRNA expression vector of structure is added in 100 μ l serum free mediums; Then mix with the 100 μ l serum free medium gentlenesses of the above-mentioned lipofectamine of comprising, room temperature leaves standstill 30 minutes, makes DNA and liposome form complex body;

With D-Hank's liquid washed cell 3 times;

To in said mixture, add 800 μ l serum free mediums (antibiotic-free), after gentleness mixes, add 1 hole in 6 orifice plates;

6 orifice plates are placed in to CO ₂in incubator, cultivate 6 hours for 37 DEG C, then abandon supernatant, add perfect medium to continue to cultivate 48 hours.

5) real-time quantitative PCR detects the effect that shRNA disturbs lncRNA to express:

By the HepG2 cell extracted total RNA after the transfection of various shRNA carrier, 2 μ g RNA, after reverse transcription becomes cDNA, carry out fluorescence real-time quantitative PCR.New clone lncRNA primer is SEQ ID NO:23 in 5'-GTCCGTCAGTCCCTCACCT-3'(sequence table) and 5'-ATACAGCCCCAGACCCAAAC-3'(sequence table in SEQ ID NO:24)

Be 5'-TCTTAGCTGAGTGTCCCGCG-3'(sequence table SEQ ID NO:25 for the 18S primer that contrasts) and 5'-ATCATGGCCTCAGTTCCGAA-3'(sequence table in SEQ ID NO:26),

Fluorescence real-time quantitative PCR reaction system

Fluorescence real-time quantitative PCR reactions steps

194℃ 5min

295℃ 10sec

358℃ 30sec

472℃ 20sec

5Plate read

682℃ 30sec

7Plate read

8Go to step2for more39times

9Perform melting curve from55.0℃to95.0℃,read every0.2℃,hold for1sec

Reaction finishes amplification curve and the melting curve of rear confirmation fluorescence real-time quantitative PCR, and the expression intensity of each gene, according to after CT value (threshold cycle values), reference gene (18S) markization, adopts group t-test inspection to calculate P value.

5) impact on liver cancer cell growth after cell counting observation shRNA interference lncRNA:

By the shRNA expression vector balanced mix of 4 kinds of target new clone lncRNA, the HepG2 cell of transfection digested after 24 hours from 6 orifice plates, again with 1 × 10 ⁴the density in/hole is inoculated in 24 orifice plates, taking the cell of transfection Scramble shRNA expression vector as control group.24 holes of every group of each inoculation.

When cell after every kind of shRNA transfection is same in every day subsequently, get 3 holes, digestion HepG2 cell wherein, under the microscope counting.And draw cell growth curve.

2. result

1) shRNA disturbs the effect that lncRNA expresses:

After these four shRNA carrier transfection HepG 2 cells, all can significantly lower the expression level of the lncRNA of new clone in HepG2 cell, four shRNA carriers are mixed and merge transfection HepG 2 cell, this lncRNA down-regulated expression more remarkable (Fig. 8).

2) impact on liver cancer cell growth after shRNA interference lncRNA:

Because four kinds of shRNA carriers mix, disturb the lncRNA expression effect of new clone best, we mix this four kinds of shRNA expression vector transfection HepG 2 cells, taking Scramble as contrast, carry out cell counting every day, discovery utilizes shRNA technology to disturb after the expression of this lncRNA, liver cancer cell growth slack-off (Fig. 9).The expression that prompting suppresses this new clone lncRNA has obvious tumor-inhibiting action in liver cancer.These four kinds of shRNA expression vectors that we build and derivative reagent thereof have the effect of potential gene therapy.

Claims (1)

1. a long-chain non-coding RNA, is characterized in that, its transcript sequence is arbitrary in the nucleotide sequence shown in SEQ ID NO:1-12 in sequence table.

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