CN105462974A

CN105462974A - Long-chain non-coding RNA participating in human cell ionization radiation stress responses and application thereof

Info

Publication number: CN105462974A
Application number: CN201510067990.5A
Authority: CN
Inventors: 周光明; 李朋飞; 胡文涛; 裴海龙; 李冰燕; 黑国庆
Original assignee: Suzhou University
Current assignee: Suzhou boaolong Technology Co., Ltd
Priority date: 2015-02-09
Filing date: 2015-02-09
Publication date: 2016-04-06
Anticipated expiration: 2035-02-09
Also published as: CN105462974B

Abstract

The invention relates to an ionization-radiation-sensitive long-chain non-coding RNA, in particular to a long-chain non-coding RNA participating in human cell ionization radiation stress responses and an application thereof. A transcript of the long-chain non-coding RNA has the nucleotide sequence shown as SEQ ID No.1. The new long-chain non-coding RNA participating in human cell ionization radiation stress responses has the advantages that a target RNA fragment is provided for further developing a detection biological marker used after the human body suffers from ionization radiation, a candidate lncRNA is also provided for researching a signal channel damaged after human cells are radiated, and certain application prospects are possibly achieved in the cancer therapy aspect.

Description

The long-chain non-coding RNA of participant's somatocyte ionizing rays stress reaction and application thereof

(1) technical field

The present invention relates to a kind of long-chain non-coding RNA of ionizing radiation sensitive, specifically a kind of long-chain non-coding RNA of participant's somatocyte ionizing rays stress reaction and application thereof.

(2) background technology

Along with the mankind are to the further investigation of DNA and the RNA function in own cells, " rubbish " RNA fragment being considered to not have proteins encoded function is in the past disclosed the critical function in human physiological functions gradually.Have a class not have the RNA fragment of coded protein ability, each fragment is greater than 200 bases, is defined as long-chain non-coding RNA, i.e. lncRNA.The expression of lncRNA is subject to developmental regulation, is tissue and cell-specific.Quite a few lncRNA is only positioned at nucleus.They comprise is permitted eurypalynous transcript, structurally similar mRNA, is sometimes also transcribed into the antisense transcript of encoding gene originally.LncRNA is considered to perform important adjusting function, also closely bound up with disease progression.LncRNA is by being combined with DNA or RNA or exercising its function with protein binding.Some lncRNA are actually the precursor of some rna regulation (as microRNA or piwiRNA).With miRNA unlike, lncRNA does not have a kind of general binding mode, it come in a number of different ways regulate gene expression and albumen synthesis.Research finds, some lncRNA take part in the primary process of gene regulating, comprises chromatin and modifies and structure and direct transcriptional control.Gene regulating may occur with cis (cis) or trans (trans).The rear function of transcribing of lncRNA comprises rna regulation processing event, as montage, editor, location, translation and degraded.In recent years, increasing lncRNA was found by scientist and studied.Now, bio-science field has confirmed that lncRNA take part in apoptosis, apparent legacy, x chromosome inactivation, the generation of cancer and transfer, multiple important biological procedureses such as mescenchymal stem cell conversion.In addition, research also finds lncRNA differential expression in kinds cancer, comprises leukemia, mammary cancer, liver cancer, colorectal carcinoma and prostate cancer.The other diseases of lncRNA imbalance also comprises cardiovascular disorder, nervous system disorders and immune-mediated disease.But, whether also have the participation of lncRNA in this important biological procedures of the stress reaction produced after human body cell is subject to ionizing rays, be not also proved to be.

(3) summary of the invention

The object of the invention is to provide a kind of long-chain non-coding RNA and application thereof of newfound participant's somatocyte ionizing rays stress reaction.

Technical scheme of the present invention is as follows:

A long-chain non-coding RNA for participant's somatocyte ionizing rays stress reaction, its transcript has the nucleotide sequence as shown in SEQIDNo.1.

SEQIDNo.1 sequence is as follows:

Due to the singularity of nucleotide sequence, the variant of polynucleotide shown in any SEQNO:1, as long as itself and this polynucleotide have more than 90% homology, and has identical function, then all belongs to the row of scope.The variant of described polynucleotide refers to a kind of polynucleotide sequence having one or more Nucleotide and change.The variant of these polynucleotide can make raw allelic variant or the varient of non-life, comprises and replaces varient, Deletion variants and insertion varient.As known in the art, allelic variant is the replacement form of polynucleotide, and it may be the replacement of a multiple Nucleotide, disappearance or insertion, but can not from changing in fact its function.

The invention still further relates to described long-chain non-coding RNA detects mark application as human body ionizing rays.Nucleotide fragments of the present invention ubiquity in human body cell, primer can be designed according to nucleotide sequence, PCR instrument is used to amplify this nucleotide fragments, after cell is subject to ionizing rays, extracts the total serum IgE of cell, carries out the expression level change that quantitative PCR experiment can detect this nucleotide fragments.

Concrete, described long-chain non-coding RNA is for the preparation of the biology detector differentiating different irradiation type.

The invention still further relates to described long-chain non-coding RNA and prepare the application in antitumor drug.Due to lncRNA differential expression in kinds cancer, comprise leukemia, mammary cancer, liver cancer, colorectal carcinoma and prostate cancer, therefore long-chain non-coding RNA of the present invention may.

Concrete, described long-chain non-coding RNA can be used for the medicine preparing inducing apoptosis of tumour cell or inhibition tumor cell propagation.

Beneficial effect of the present invention is mainly reflected in: the long-chain non-coding RNA that the invention provides a kind of new participant's somatocyte ionizing rays stress reaction, target RNA fragment is provided for developing the biomarker detected after human body is subject to ionizing rays further, also for researching human body cell be subject to radiation after occur damage signal path research provide candidate lncRNA, may also have certain application prospect in oncotherapy.

(4) accompanying drawing explanation

Fig. 1 is the expression level changes of 4 kinds of lncRNA after HeLa cell is subject to 2GyX x ray irradiation x; Fig. 2 is the expression level changes of 4 kinds of lncRNA after HeLa cell is subject to 2Gy Ion Irradiation on Multi-walled Carbon.

(5) embodiment

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

1, experimental technique

1) cell cultures

Select stable cervical cancer tumer line HeLa cell (purchased from American ATCC), use 1640 culture medium culturing, be placed in the cell culture incubator of 37 DEG C of constant temperature, 5% carbonic acid gas and cultivate; First wash twice with aseptic PBS when going down to posterity, then use 1% trysinization 1 minute, until cell after culture dish digests, resuspended with 1640 substratum; Be divided in new culture dish, add 1640 substratum and carry out enlarged culturing.

2) radiation treatment

The HeLa cell cultivated is divided into control group and experimental group, and often organizing about has 1 × 10 ⁸individual cell; Adopt the X ray excited irradiation instrument of biological experiment electric energy (model FaxitronRX-650) to carry out ionizing radiation experiment, voltage is 50KV, and dose rate is 0.675Gy/ minute, and experimental group accepts 2GyX x ray irradiation x, and control group is irradiation not; Again experimental group and control group are put into cell culture incubator after irradiation and cultivate 4 hr collections cells.

3) RNA is extracted

Two groups of cells are taken out from cell culture incubator, sucking-off substratum, wash twice with aseptic PBS, whole for PBS sucking-off; Trizol reagent is added, about every 5 × 10 in Tissue Culture Dish ⁶individual cell adds 1mL reagent, blows and beats several times, make the whole cracking of cell with pipettor; Proceed in the EP pipe without RNA enzyme with the cell of Trizol reagent cracking, tube wall performs mark.

4) RNA order-checking

The laboratory sample of experimental group and control group is put into be filled in the bubble chamber of dry ice, delivers to Shanghai branch office of Hua Da genome company and carries out the order-checking of lncRNA complete sequence, and add up the differential expression of lncRNA between two groups of laboratory samples.After obtaining raw data, we use transcript profile comparing software TopHat2 will filter ribosomal reading frame comparison on reference genome, and TopHat supports not rely on the segmentation comparison with reference to gene annotation, is more conducive to us and finds new transcript.TopHat utilizes BowtieBowtie as comparison " engine ", the reading frame in comparison not being broken into segment, and infers the position of reading frame segmentation and segmentation.For the reading frame in initial non-comparison, TopHat can build the reference set in a splice site under the prerequisite not relying on known annotation.The comparison of RNA-seq sequence not only contributes to finding variable sheer and new transcript, can also for the identification of the gene of expressing and quantitatively.Reading frame comparison is to postgenome, will assemble with Cufflinks, consider coverage to there is gap and cause transcript to assemble incomplete situation, we are more prone to the assembling mode (referenceannotationbasedtranscripts, BRAT) based on reference sequences.Cufflinks can construct manual read's frame according to reference to transcript, and make up the impact of low cover degree in order-checking, these reading frames will be used from assembling with the sequence one in comparison.The Transcriptional fragments that final step assembles out will compare with reference to gene, and the fragment roughly consistent with known transcript will be removed.Cuffdiff is the supporting a quantitative differences analysis software of Cufflinks, it with two samples in contrast and experimental group, calculate gene or the expression amount FPKM value of transcript in two samples, and whether detection there are differences expression.Cuffdiff first adds up the fragment number of all transcripts of each gene, is converted into the expression amount of transcript, then they is added on corresponding gene.As other instruments, cuffdiff also uses negative binomial distribution model to estimate the degree of gene or transcript differential expression.

5) sequencing result and checking is analyzed

According to the result of order-checking, analyze the lncRNA sequence between experimental group and control group with significant difference expression, obtain 26 kinds altogether; According to the primer of the sequences Design quantitative PCR of 26 kinds of lncRNA; Primer by Sangon Biotech (Shanghai) Co., Ltd. on behalf of synthesis; Repeat the method for culturing cell and lysing cell above, obtain 2GyX x ray irradiation x experimental group cell Trizol lysate and cellular control unit Trizol lysate.

6) realtime fluorescent quantitative PCR experiment

Every 1mLTrizol lysate adds 200uL chloroform, concuss 30 seconds, leaves standstill 3 minutes, 12000 revs/min, 4 DEG C of low-temperature centrifugations 15 minutes; Draw supernatant 400uL to proceed in new EP pipe, add the Virahol of equivalent, leave standstill 10 minutes on ice; 12000 revs/min, 4 DEG C of low-temperature centrifugations 15 minutes; Supernatant discarded, the precipitation obtained is RNA, washes precipitation with the DEPC alcohol 0.8mL of prepare 75%; 12000 revs/min, 4 DEG C of low-temperature centrifugations 10 minutes; Supernatant discarded, after natural air drying to be precipitated, adds 60 DEG C of DEPC water and dissolves; Measure the concentration of the total serum IgE extracted; Adopt the mRNA Reverse Transcription box of GeneCopoeia company to carry out total serum IgE reverse transcription, in every 25uL system, add 2ug total serum IgE; Reverse transcription adopt program be 45 DEG C one hour, 85 DEG C 5 minutes, 4 DEG C of preservations; The cDNA that reverse transcription obtains is diluted 5 times of templates as quantitative PCR; Primer dilution is the concentration of 2uM; Adopt GeneCopoeia company mRNA PCR kit for fluorescence quantitative and prepare system and setting program according to specification sheets; Calculate the expression change multiple of target lncRNA transcript between experimental group and control group according to the Ct value of fluorescent quantitative PCR experiment, and map according to the result of three independent experiments.

7) carbon ion radiation checking

Repeat the step of x-ray irradiation experiment, within 4 hours, extract RNA after HeLa cell accepts 2Gy Ion Irradiation on Multi-walled Carbon, again carry out quantitative PCR experiment, verify the expression change of 26 kinds of lncRNA after being subject to Ion Irradiation on Multi-walled Carbon; Carbon ion ray is provided by Lanzhou Heavy Ion Cyclotron National Laboratory experimental ring shallow-layer terminal.

2, experimental result:

Have found the lncRNA relevant to human body cell ionizing rays response expression, after being subject to ionizing rays, these 26 kinds of lncRNA have the change of significant expression level, wherein there are 4 kinds for newfound lncRNA transcript, called after XLOC_012077, XLOC_012764, XLOC_014016 and XLOC_023611 respectively, the 4 kinds of lncRNA expression level after HeLa cell is subject to 2GyX x ray irradiation x changes see Fig. 1; Wherein nucleotide sequence shown in XLOC_014016 and SEQIDNo.1, its primer is: 5 '-TGGAGACCGAGGCAAGAAT-3 ' and 5 '-ATAAACCCAGCCAGGAACCA-3 '.

The expression level of 4 kinds of lncRNA after HeLa cell is subject to 2Gy Ion Irradiation on Multi-walled Carbon changes see Fig. 2.From Fig. 1, Fig. 2, after being subject to ionizing rays, these 4 kinds of lncRNA have the change of significant expression level, point out 4 kinds of lncRNA all to can be used as the biomarker detected after human body is subject to ionizing rays.

Claims

1. a long-chain non-coding RNA for participant's somatocyte ionizing rays stress reaction, its transcript has the nucleotide sequence as shown in SEQIDNo.1.

2. long-chain non-coding RNA as claimed in claim 1 detects the application of mark as human body ionizing rays.

3. apply as claimed in claim 2, it is characterized in that described long-chain non-coding RNA is for the preparation of the biology detector differentiating different irradiation type.

4. long-chain non-coding RNA as claimed in claim 1 is preparing the application in antitumor drug.