CN107760685A - Long-chain non-coding RNA ROR application - Google Patents
Long-chain non-coding RNA ROR application Download PDFInfo
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Abstract
The invention discloses a kind of application of long-chain non-coding RNA ROR (linc ROR).Material and the material of suppression linc ROR expression and/or function respectively application in product that preparation be used for auxiliary diagnosis and treatment esophageal squamous cell carcinoma of the application particularly for detection linc ROR.Experiment shows that esophageal squamous cell cancer cell multiplication, invasion and attack and migration, resistance ability and tumor stem cell sample characteristic can be suppressed by suppressing linc ROR expression, be played a significant role in the occurrence and development of esophageal squamous cell carcinoma.A variety of microRNA include simultaneously：Has miR 15b 5p, has miR 33a 5p, has miR 129 5p, has miR 145 5p, has miR 206 can target linc ROR.The invention provides the novel targets of the diagnosis for esophageal squamous cell carcinoma and treatment, linc ROR genes can be used for the new product for preparing diagnosis and/or treatment esophageal squamous cell carcinoma as novel targets mark.The present invention has important clinical significance to the treatment level for improving esophageal squamous cell carcinoma.
Description
Technical field
The invention belongs to biomedical sector, is related to a kind of application of long-chain non-coding RNA-ROR (linc-ROR), specifically
It is related to the purposes that linc-ROR is used in esophageal squamous cell carcinoma (abbreviation esophageal squamous cell carcinoma) in preparing target spot mark.
Background technology
The cancer of the esophagus is one of most common malignant tumor of digestive tract in the world, and squamous cell carcinoma (abbreviation squamous carcinoma) is that its is main
One of histological type.China is Esophageal Cancer area, and the morbidity and death of the cancer of the esophagus of the whole world more than 50% occur in China,
And wherein there are about 90% is esophageal squamous cell carcinoma.The continuous rise of the incidence of disease of the cancer of the esophagus and the death rate in recent years is to threaten human health
Major issue, its postoperative five year survival rate only has 15%-30%.Therefore, the molecular mechanism pair of cancer of the esophagus occurrence and development is disclosed
In the cancer of the esophagus early diagnosis and to seek effective therapy target be very necessary.
Recent studies indicate that the gene for only having fraction (1%-2%) in full-length genome and transcript profile has coding egg
White function, and most of is non-coding RNA, the nearly or completely function without encoding proteins.This kind of RNA and chromatin
The various biological processes such as restructuring, open gene expression, post-transcriptional control are relevant.In the non-coding RNA with adjustment effect,
Long-chain non-coding RNA and the major class of small non-coding RNA two can be divided into (using 200nt as boundary) according to its transcript length.Grind
Study carefully and find that long-chain non-coding RNA is relevant with generation, the development of some tumours, can be as new tumor marker and specific
Therapy target.
Long-chain non-coding RNA-ROR (linc-ROR) between gene, belongs to long-chain non-coding RNA between gene, positioned at chromosome
18q21.31 total length 2.6kb.Loewer in 2010 et al. has found that linc-ROR can promote cell to reprogram, and makes differentiated
Cell dedifferentes to induced pluripotent stem cells, while is able to maintain that the characteristic of embryonic stem cell and induced pluripotent stem cells,
Therefore the lincRNA is also named.In recent years multiple studies have shown that linc-ROR unconventionality expression in kinds cancer be present, such as
Carcinoma of endometrium, breast cancer, hepatocellular carcinoma, cancer of pancreas etc., but its specific mechanism of action not yet illustrates completely.
The content of the invention
It is an object of the invention to provide a kind of long-chain non-coding RNA-ROR new purposes.
First, present invention protection suppress the material of long-chain non-coding RNA-ROR expression and/or function it is following it is any in
Using：
(A1) product for treating esophageal squamous cell carcinoma is prepared；
(A2) esophageal squamous cell carcinoma is treated.
Second, present invention protection suppress the material of long-chain non-coding RNA-ROR expression and/or function it is following it is any in
Using：
(a1) product for suppressing tumour growth is prepared, or suppresses tumour growth；The tumour is esophageal squamous cell carcinoma；
(a2) product for suppressing esophageal squamous cell cancer cell multiplication is prepared, or suppresses esophageal squamous cell cancer cell multiplication；
(a3) product for suppressing esophageal squamous cell carcinoma cell invasion is prepared, or suppresses esophageal squamous cell carcinoma cell invasion；
(a4) product for suppressing esophageal squamous cell carcinoma cell migration is prepared, or suppresses esophageal squamous cell carcinoma cell migration；
(a5) product for reducing esophageal squamous cell carcinoma cellular drug resistance is prepared, or reduces esophageal squamous cell carcinoma cellular drug resistance；
(a6) product of the tumor stem cell sample characteristic for reducing esophageal squamous cell carcinoma cell is prepared, or to reduce esophageal squamous cell carcinoma thin
The tumor stem cell sample characteristic of born of the same parents.
Wherein, the medicine in (a5) is the conventional radiotherapy and chemotherapy medicine for being clinically used to treat esophageal squamous cell carcinoma.In the present invention
One embodiment in, the medicine is specially chemotherapeutic drugs Cisplatin.
Wherein, the tumor stem cell sample characteristic is this area technical term, refer to a small set of cell in tumour have it is similar
In the characteristic of stem cell (such as there is infinite multiplication, self-renewing, resistance ability).
The tumor stem cell sample characteristic for reducing esophageal squamous cell carcinoma cell can specifically be presented as following any one or more：
(a) expression of stem cell labeling thing in esophageal squamous cell carcinoma cell is reduced；(b) the balling-up ability of esophageal squamous cell carcinoma cell is reduced；(c)
Reduce the expression of epithelial-mesenchymal conversion (EMT) mark of correlation thing in esophageal squamous cell carcinoma cell.Wherein, the stem cell labeling
Thing is selected from：KLF4、NANOG、CD44、OCT4、SOX2；Epithelial-mesenchymal conversion (EMT) the mark of correlation thing is vimentin.
In above two application, the material of the suppression long-chain non-coding RNA-ROR expression and/or function can be any
The material of long-chain non-coding RNA-ROR expression and/or function can be suppressed.
In one embodiment of the invention, the material tool for suppressing long-chain non-coding RNA-ROR expression and/or function
Body is targeting long-chain non-coding RNA-ROR siRNA (siRNA).The siRNA of the targeting long-chain non-coding RNA-ROR
It is made up of positive-sense strand and antisense strand, the positive-sense strand is complementarily shaped to double-strand, the sequence such as SEQ of the positive-sense strand with the antisense strand
Shown in ID No.2, the sequence of the antisense strand is as shown in SEQ ID No.3.
Further, the material of the suppression long-chain non-coding RNA-ROR expression and/or function is alternatively through chemical modification
The siRNA of targeting long-chain non-coding RNA-ROR afterwards.The siRNA of the targeting long-chain non-coding RNA-ROR is by positive-sense strand and instead
Adopted chain composition, the positive-sense strand are complementarily shaped to double-strand, the sequence such as SEQ ID No.2 institutes of the positive-sense strand with the antisense strand
Show, the sequence of the antisense strand is as shown in SEQ ID No.3.
In one embodiment of the invention, the chemical modification is specially cholesterol modification.Specifically, the cholesterol
Modify in the siRNA of the targeting long-chain non-coding RNA-ROR 5 ' ends.The specially product of commercialization, reference can be made to Shang Haiji
Agate company's site http://www.genepharma.com/show.phpCtype=0＆coupid=567＆cateid=
111.The purpose of cholesterol modification is to improve the efficiency (similar to the lipid bilayer of cell membrane to mix) that siRNA enters cell.
In another embodiment of the present invention, the material for suppressing long-chain non-coding RNA-ROR expression and/or function
Specially target long-chain non-coding RNA-ROR microRNA.The microRNA of the targeting long-chain non-coding RNA-ROR is selected from
It is as follows：hsa-miR-15b-5p、hsa-miR-33a-5p、hsa-miR-129-5p、hsa-miR-145-5p、hsa-miR-206.
Further, the sequence of the hsa-miR-15b-5p is specific as shown in SEQ ID No.4；The hsa-miR-
33a-5p sequence is specific as shown in SEQ ID No.5；The sequence of the hsa-miR-129-5p is specific such as SEQ ID No.6 institutes
Show；The sequence of the hsa-miR-145-5p is specific as shown in SEQ ID No.7；The sequence of the hsa-miR-206 is specific such as
Shown in SEQ ID No.8.
In one embodiment of the invention, the pipe squamous cell carcinoma is specially esophageal squamous cell carcinoma cell line EC9706 or Eca-
109。
3rd, the present invention protect for detect long-chain non-coding RNA-ROR material it is following it is any in application：
(B1) product for auxiliary diagnosis esophageal squamous cell carcinoma is prepared；
(B2) auxiliary diagnosis esophageal squamous cell carcinoma.
In one embodiment of the invention, it is described be used to detecting long-chain non-coding RNA-ROR material particularly for
Detect long-chain non-coding RNA-ROR primer pair.The primer pair is as two shown in SEQ ID No.9 and SEQ ID No.10
The primer pair of bar single stranded DNA composition.
Further, the material for being used to detect long-chain non-coding RNA-ROR is alternatively for detecting long-chain non-coding
RNA-ROR real time fluorescent quantitative detection preparation.The real time fluorescent quantitative detection for being used to detect long-chain non-coding RNA-ROR
Contain the primer pair being made up of two single stranded DNAs shown in SEQ ID No.9 and SEQ ID No.10 in preparation.
The present invention suppresses linc-ROR expressions, extracting in esophageal squamous cell carcinoma cell line, with the siRNA of design synthesis
Reverse transcription after RNA, the expression that real time fluorescent quantitative method demonstrates linc-ROR decline；Detect the propagation of cell, invasion and attack migrate, be resistance to
The change of medicine ability and stem cell-like properties, as a result show cell propagation, invasion and attack migration, resistance after the expression for suppressing linc-ROR
Ability and stem cell-like properties are suppressed.With bioinformatic analysis targeting linc-ROR microRNA, and pass through reality
When fluorescent quantitation and RNA co-immunoprecipitations a variety of microRNA of method validation include hsa-miR-15b-5p, hsa-miR-
33a-5pa, hsa-miR-129-5p, hsa-miR-145-5p and hsa-miR-206 can target linc-ROR.Pass through nude mice
Knurl internal injection is carried out with the targeting linc-ROR of cholesterol modification siRNA after plantation knurl success into knurl experiment, as a result
Display experimental group knurl weight and volume are substantially less than control group.Prompt linc-ROR can be as the new of esophageal squamous cell carcinoma targeted therapy
Target spot.The present invention for the selection of esophageal squamous cell carcinoma targeted therapy novel targets provides theoretical foundation, have far-reaching clinical meaning with
Important popularizing application prospect.
Brief description of the drawings
Fig. 1 is that its table after linc-ROR is disturbed in the method detection esophageal squamous cell carcinoma cell line using real-time fluorescence quantitative PCR
Up to level.
Fig. 2 is the influence after silence linc-ROR to esophageal squamous cell carcinoma cell lines Tumor characteristic.A：CCK8 methods detect EC9706
Cell growth curve；B：Plate clone testing inspection EC9706 Cell clonalities；C：Detect transwell cells
EC9706 cell migration abilities；D：Detect EC9706 cell invasion abilities in transwell cells；E：CCK8 methods detect cisplatin treated
EC9706 cytoactives afterwards；F：Real time fluorescent quantitative method detects EC9706 stem cell labeling thing mRNA level in-sites；G：Ball test is examined
Survey Eca-109 cell self-renewal abilities.
Fig. 3 is Targeted-control linc-ROR microRNA.A：Transfect microRNA analogies and water is expressed to linc-ROR
Flat influence；B：With influence of the different microRNA inhibitor to linc-ROR expressions.
Fig. 4 is that silence linc-ROR can suppress tumour growth in experiment in vivo.A：Tumor volume；B：Knurl weight；C：
Immunohistochemical staining detection CD44, vimentin expression.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc., unless otherwise specified, are commercially obtained in following embodiments.
Embodiment 1, linc-ROR can be as the auxiliary diagnosis of esophageal squamous cell carcinoma and the novel targets of targeted therapy
Linc-ROR sequence is as shown in SEQ ID No.1.
First, materials and methods
(1) main agents and consumptive material
1st, cell culture and transfection main agents and consumptive material
EC9706 cell lines (Shanghai Fu Xiang bio tech ltd)；Eca-109 cell lines (the multiple auspicious biotechnology in Shanghai
Co., Ltd), product link is respectively http://www.xiangbio.com/enstyle/product_9034853.html
http://www.xiangbio.com/enstyle/product_8389053.html；(the U.S. of nutrient solution RPMI 1640
Gibco companies)；Opti-MEM I (Gibco companies of the U.S.)；Dimethyl sulfoxide (DMSO) DMSO (Beijing Solarbio companies)；Tryptose
Enzyme EDTA digestive juices (Beijing Solarbio companies)；Mycillin mixed liquor (Beijing Solarbio companies)；20×PBS
Solution (Shanghai Sheng Gong bio-engineering corporations)；Hyclone (Biological Industries companies of Israel)；T25
Tissue Culture Flask (Corning companies of the U.S.)；15mL, 50mL centrifuge tube (Corning companies of the U.S.)；6 orifice plates, 12 orifice plates, 24
Orifice plate, 96 orifice plates (Corning companies of the U.S.)；Lipofectamine 2000Regent (Invitrogen companies of the U.S.)；
SiRNA and control RNA (the design synthesis of Shanghai GenePharma companies)；MicroRNA analogies and inhibitor (miRNA analogies
It is to simulate the endogenous miRNAs of organism, is synthesized with the method for chemical synthesis, endogenous miRNA function can be strengthened.And
MiRNA inhibitor is the inhibitor specifically for target miRNA special in cell of chemical modification, please respectively referring to company web page
http://www.genepharma.com/show.phpCtype=0＆coupid=557＆cateid=113 and http://
www.genepharma.com/show.phpCtype=0＆coupid=556＆cateid=113；Shanghai GenePharma is public
Department's design synthesis).
Linc-ROR siRNA sequences：
Linc-ROR disturbs positive sequence：5’-GGAGAGGAAGCCUGAGAGUdTdT-3’(SEQ ID No.2)；
Linc-ROR disturbs reverse sequence：5’-ACUCUCAGGCUUCCUCUCCdTdT-3’(SEQ ID No.3).
The Linc-ROR siRNA modified through cholesterol：Cholesterol is modified in the 5 ' ends of the Linc-ROR siRNA.For
The product of commercialization, referring to Shanghai Ji Ma company's sites http://www.genepharma.com/show.phpCtype=0＆
Coupid=567＆cateid=111.
MicroRNA is related to following 5 kinds：
hsa-miR-15b-5p：5’-UAGCAGCACAUCAUGGUUUACA-3’(SEQ ID No.4)；
hsa-miR-33a-5p：5’-GUGCAUUGUAGUUGCAUUGCA-3’(SEQ ID No.5)；
hsa-miR-129-5p：5’-CUUUUUGCGGUCUGGGCUUGC-3’(SEQ ID No.6)；
hsa-miR-145-5p：5’-GUCCAGUUUUCCCAGGAAUCCCU-3’(SEQ ID No.7)；
hsa-miR-206：5’-UGGAAUGUAAGGAAGUGUGUGG-3’(SEQ ID No.8)；
As above 5 kinds of microRNA analogies are respectively hsa-miR-15b-5p mimics, hsa-miR-33a-5p
Mimics, hsa-miR-129-5p mimics, hsa-miR-145-5p mimics, hsa-miR-206mimics；Inhibitor point
Wei not hsa-miR-15b-5p inhibitor, hsa-miR-33a-5p inhibitor, hsa-miR-129-5p
Inhibitor, hsa-miR-145-5p inhibitor, hsa-miR-206inhibitor (sequence such as Tables 1 and 2).
Table 1miRNA mimics sequences
Table 2miRNA inhibitor sequences
2nd, cell function experiment main agents and consumptive material
CCK8 (Japanese colleague company)；Transwell cells (Costar companies of the U.S.)；Crystal violet (Beijing Solarbio
Company)；4% paraformaldehyde (Beijing Solarbio companies)；Matrigel (BD Biosciences companies of the U.S.)；Neoplatin
(Qilu Pharmaceutical Co., Ltd.).
3rd, ball test
DMEM/F12 fluid nutrient mediums (Hyclone companies of the U.S.)；B27 additives (GBICO companies of the U.S.)；Recombined human table
Skin cell growth factor (EGF, Peprotech companies of the U.S.)；Recombination human basic fibroblast growth factor (bFGF, the U.S.
Peprotech companies)；Phosphate buffer (containing 5% trehalose) (connection section biology)；It is low to stick orifice plate (U.S. Corning public affairs
Department).
4th, RNA extracts main agents and consumptive material
E.N.Z.A.Total RNA Kit I (OMEGA, R6834-01)；(Tianjin richness space fine chemistry industry is limited for dimethylbenzene
Company)；Absolute ethyl alcohol (Tianjin Fu Yu Fine Chemical Co., Ltd)；(given birth in Shanghai without enzyme pipe (1.5mL, 2mL) and without enzyme pipette tips
Thing engineering services Co., Ltd)；Beta -mercaptoethanol (Tianjin Fu Yu Fine Chemical Co., Ltd)；Pyrocarbonic acid diethyl ester
DEPC (Beijing Tiangeng biotechnology company).
5th, RNA reverse transcriptions main agents and consumptive material
SuperQuick RT MasterMix (for Real-Time PCR) (Beijing health be ShiJi Co., Ltd, CW2391M).
6th, RNA Fluorescent quantitative PCRs detection main agents and consumptive material
UltraSYBR Mixture (Low ROX) (Beijing health be ShiJi Co., Ltd, CW2601M)；Eight connecting leg (the U.S.
Applied Biosystems companies)；96 orifice plates (Applied Biosystems companies of the U.S.).
7th, RNA co-immunoprecipitation reactions (RIP)
EZ-Magna RIP kits (Millipore companies of the U.S.)；Ago2 antibody (Abcam companies of Britain)；IgG antibody
(Millipore companies of the U.S.).
8th, immunohistochemistry (immunohistochemistry, IHC) dyeing main agents and consumptive material
Adhere to slide (Shi Tai experiment equipments Co., Ltd, Jiangsu)；Deparaffinization reagents (29490, Leica)；Antigen retrieval
Liquid (ER20134, Leica)；Antibody elution liquid (W0080, Leica)；Antibody diluent (Dako companies, Germany)；Dimethylbenzene (my god
Fu Yu Fine Chemical Co., Ltd of Jinshi City)；Absolute ethyl alcohol (Tianjin Fu Yu Fine Chemical Co., Ltd)；Primary antibody：CD44、
Vimentin (company of Zhong Shan Golden Bridge, Beijing)；General secondary antibody (company of Zhong Shan Golden Bridge, Beijing).
(2) method
1st, cell culture and transfection
Esophageal cancer cell cultivates cell with 1640 culture mediums containing 10% hyclone and 1% mycillin mixed liquor
In 37 DEG C, 5%CO2Cultivated in incubator.SiRNA and microRNA analogies or suppression are transfected using Lipofectamin2000
Agent, concrete operation step is referring to product description.
2nd, cell function is tested
2.1CCK8 methods detect ability of cell proliferation
The different disposal group cell of identical quantity is seeded in 96 orifice plates, all processing group orifice plates are placed in incubator
It is middle to cultivate 0 day, 1 day, 2 days, 3 days respectively；After taking out orifice plate according to time point, detected not under the conditions of 450nm using ELIASA
With the OD values for the treatment of group；Detected value is recorded, draws cell growth curve figure.
2.2 plate clones experiment detection Cell clonality
The different disposal group cell of identical quantity is seeded in 6 orifice plates and cultivated two weeks, with PBS, 4% paraformaldehyde
With 1% violet staining 20min after fixed cell 30min；dH2O is cleaned to after colourless, and IMAQ simultaneously calculates cell clone group
Block number mesh.
2.3Tanswell cells experiment detection cell invasion and transfer ability
2.3.1 cell invasion ability detects
Cell transfecting is inoculated in 24 orifice plates for 24 hours, carries out cell invasion ability detection：By the culture of the bases of RPMI 1640
Liquid:Matrigel matrigel=1:8 (volume ratios) dilution is mixed, and the matrigel covering that 45 μ L have been configured is added in every hole
The upper chamber face of transwell cells bottom film, it is incubated 3 hours in incubator；After the completion of incubation, under transwell cells
600 μ L and the basic culture solutions of 100 μ L RPMI 1640 are separately added into room and upper chamber, 30min is incubated in incubator；After transfecting
Cell dissociation after be prepared into single cell suspension, with the basic culture solutions of RPMI 1640 be resuspended cell；Taken out after completion aquation small
Room, 200 μ L cell suspensions are added after absorbing small indoor culture medium, add RPMI 1640 culture mediums of the 600 μ L containing 20%FBS in
The cell for filling cell suspension is placed in one after in 24 orifice plates, culture plate is placed in incubator after cultivating 48 hours with 4%
Paraformaldehyde fix 20min under the conditions of 4 DEG C；1% crystal violet dye liquor dyes 15min, and IMAQ is simultaneously analyzed.
2.3.2 cell migration ability detects
Need not be 24 hours with Matrigel matrigels coating upper chamber, incubation time during the detection of cell migration ability, its
Remaining step is consistent with the detection of cell invasion ability.
2.4 cells resistance abilities detect
5,000 EC9706 cell is inoculated with into 96 orifice plates, after handling cell by cell transfecting method according to experimental design
Culture be replaced by after 1 day after the nutrient solution of the neoplatin containing various concentrations (kinds of tumor chemotherapeutics) cultivate 0 day, 1 day,
2 days, 3 days；Cytoactive is detected with CCK8.
3rd, cell balling-up ability detects
By 1 × 104Individual Eca-109 cells are inoculated in six orifice plates of ultralow adhesion, with containing 20ng/mL EGF, 20ng/mL
BFGF and 2%B27 DMEM/F12 cultures, are placed in 37 DEG C, 5%CO2Cultivated in incubator.
4th, real time fluorescent quantitative
Cell RNA is extracted using E.N.Z.A TOTAL RNA KIT II kits, applies SuperQuick RT afterwards
MasterMix kits synthesize cDNA, are examined using UltraSYBR Mixture (Low ROX) real-time fluorescence quantitative PCR kit
Linc-ROR expressions are surveyed, using GAPDH as reference gene, response procedures are shown in Table 3.Wherein, detected for real time fluorescent quantitative
The primer sequence of linc-ROR expression：
Linc-ROR forward primers：5’-TTCAGTTCCCTAAAGTCACCC-3’(SEQ ID No.9)；
Linc-ROR reverse primers：5’-GTCCTTCTAAGCCTCTGTTGC-3’(SEQ ID No.10).
Table 3qRT-PCR response procedures
5th, RNA co-immunoprecipitation reactions (RIP)
RNA co-immunoprecipitation reactions, 10 μ g Ago2 antibody and IgG antibody are detected with EZ-Magna RIP reagents, specifically
Step reference reagent box specification.
6th, experiment in vivo
6.1 nude mices are into knurl
Nude mice is raised in the zoopery for reaching SPF (specific pathogen free condition) rank
Room.Animal feeding and experiment process are according to the guideline operation of ethics comittees of Xinjiang Medicine University, animal during experiment
Freely absorb feed and water.Precuring starts to build Transplanted tumor model for 5 days, and 1.5 × 10 are inoculated with nude mice bilateral axillary regions6/ 0.2mL's
EC9706 cells, every group 6, inject in left side after macroscopic subcutaneous nodule occurs in most of nude mice and modified through cholesterol
Targeting linc-ROR siRNA, right side injection PBS as a control group, every three days injection medicine, each 5nmol/
0.2mL, mouse is put to death after three weeks, takes tumor tissue to fix, embedded.
6.2IHC
Tissue is fixed in 10% formalin fixer, is embedded after fixed 24h, is prepared wax stone, cuts white tiles, IHC dyeing.
Antibody concentration SOX9 (1:600)；CD44(1:50)；vimentin(1：600).
7th, statistical method
Statistical analysis is carried out to related data in experiment using SPSS17.0 softwares.Cell function experiment is carried out three times
Independent repeated trials, its result is represented with mean ± standard deviation (X ± S), using bilateral paired-sample t test, p<0.05 has
Statistical significance, if p<0.05, marked with " * "；If p<0.01, with " * * " are marked；If p<0.001, with " * * * " are marked.Chart
Using the software development of Graghpad Prism 5.0.
2nd, result and analysis
1st, linc-ROR RNA interferings Efficiency testing
Selectively targeted linc-ROR siRNA is detected (by SEQ ID No.2 with quantitative real-time PCR
The double-stranded RNA formed with two single stranded RNA complementations shown in SEQ ID No.3) the horizontal changes of linc-ROR after processing, as a result show
Show that experimental group significantly reduces (Fig. 1) compared with control group linc-ROR levels.
2nd, influences of the silence linc-ROR to esophageal squamous cell carcinoma cell tumour characteristic
With the growth curve of EC9706 cell lines after CCK8 methods detection silence linc-ROR, its result, which is shown in, have been transfected
Multiplication capacity into rear 48 and 72 hours tumour cells reduces compared with control group, and difference has statistical significance (A in Fig. 2).In addition,
Experimental result, which is formed, using plate clone shows that the Clone formation number of tumour cell after silence linc-ROR is few and agglomerate is more right
It is small according to organizing, show that the individual cells multiplication capacity after processing reduces (B in Fig. 2).It is above-mentioned test result indicates that, test in vitro
Middle linc-ROR can promote the multiplication capacity of esophageal squamous cell carcinoma cell line.
In order to further verify linc-ROR in the developing effect of esophageal squamous cell carcinoma, the silence in EC9706 cell lines
After linc-ROR, the migration of cell and invasive ability significantly reduce (C and D in Fig. 2) compared with control group.Neoplatin
(cisplatin, Cis) is common tumor chemotherapeutic drug, the EC9706 after the Cis processing silences linc-ROR of various concentrations
Cell line simultaneously detects cytoactive, and its result shows the resistance ability reduction of cell after silence linc-ROR, has with control group
(when Cis concentration is 1.5625,3.125,6.25 μ g/mL, p value is equal for significant difference<0.05) (E in Fig. 2).
It is horizontal that we have detected in interference EC9706 cells tumor stem cell marker mRNA after linc-ROR expression
Change, it is found that the stem cell labeling thing expressions such as KLF4 and NANOG are remarkably decreased (F in Fig. 2).Oesophagus after interference linc-ROR
Cancerous cell line Eca-109 balling-up ability significantly reduces compared with control group；With at 3 μ g/mL Cis while silence linc-ROR
Reason, the balling-up ability of cell is minimum (G in Fig. 2), and these results show that silence linc-ROR can significantly inhibit cancer of the esophagus balling-up
Cell is acted on the chemoresistance of cis-platinum.
Summary result shows that silence linc-ROR can suppress the propagation of esophageal squamous cell carcinoma cell line, Clone formation, invade
Attack and migrate, resistance and tumor stem cell characteristic.
3rd, predict and verify targeting linc-ROR microRNA
Competitive endogenous RNA hypothesis thinks that long-chain non-coding RNA can trap what is dissociated as " miRNA sponges "
MicroRNA, make there are downstream elements the microRNA abundance of regulating and controlling effect to reduce, with antagonism microRNA effect so as to shadow
Ring the protein expression level of downstream elements.It can be targetted simultaneously with bioinformatics software RNA22 software predictions in the present invention
Linc-ROR and downstream elements SOX9 microRNA.
We use microRNA analogies transfectional cells, change (A in Fig. 3) horizontal detection linc-ROR, filter out
Effect 5 microRNA the most obvious.In order to further verify influence that selected microRNA is expressed linc-ROR, subsequently
Using microRNA inhibitor transfectional cells, the horizontal changes of detection linc-ROR, as a result as shown in B in Fig. 3,5 kinds are being transfected
The horizontal changes of linc-ROR are the most notable after the mixture of microRNA inhibitor, and foundation is provided for follow-up test.
4th, silence linc-ROR can suppress tumour growth in vivo
Based on preliminary in vitro result of study, follow-up we further verify linc-ROR to esophageal squamous cell carcinoma by experiment in vivo
The facilitation of occurrence and development.After EC9706 cells establish nude mice cancer of the esophagus plantation knurl model, the targeting of cholesterol modification is utilized
Linc-ROR siRNA carries out knurl internal injection treatment.Volume (volume=length × wide is carried out to the knurl body removed2/ 2) and quality
Measurement, the volume and quality for as a result finding the knurl body after silence linc-ROR be substantially less than control group (A and B in Fig. 4), table
The growth of nude mice plantation knurl can be suppressed after bright silence linc-ROR.IHC detects stem cell labeling thing CD44 and EMT mark of correlation thing
Vimentin expression, as a result show that expression of the experimental group compared with control group CD44 and vimentin reduces (C in Fig. 4), it is and external
Experimental result is consistent.
Result of this example indicate that：Linc-ROR can promote esophageal squamous cell carcinoma cell propagation, invasion and attack migration, resistance and
Self-renewal capacity；Hsa-miR-15b-5p, hsa-miR-33a-5p, hsa-miR-129-5p, hsa-miR-145-5p, hsa-
MiR-206 can simultaneously targeting in lincROR and downstream elements SOX9；Linc-ROR can turn into potential esophageal squamous cell carcinoma
Auxiliary diagnosis and therapy target.Tumour progression can be prevented or postpone by the expression or activity that suppress linc-ROR, so as to reach
To the purpose for improving patient's prognosis.
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Claims (10)
1. suppress the material of long-chain non-coding RNA-ROR expression and/or function it is following it is any in application：
(A1) product for treating esophageal squamous cell carcinoma is prepared；
(A2) esophageal squamous cell carcinoma is treated.
2. suppress the material of long-chain non-coding RNA-ROR expression and/or function it is following it is any in application：
(a1) product for suppressing tumour growth is prepared, or suppresses tumour growth；The tumour is esophageal squamous cell carcinoma；
(a2) product for suppressing esophageal squamous cell cancer cell multiplication is prepared, or suppresses esophageal squamous cell cancer cell multiplication；
(a3) product for suppressing esophageal squamous cell carcinoma cell invasion is prepared, or suppresses esophageal squamous cell carcinoma cell invasion；
(a4) product for suppressing esophageal squamous cell carcinoma cell migration is prepared, or suppresses esophageal squamous cell carcinoma cell migration；
(a5) product for reducing esophageal squamous cell carcinoma cellular drug resistance is prepared, or reduces esophageal squamous cell carcinoma cellular drug resistance；
(a6) product of the tumor stem cell sample characteristic for reducing esophageal squamous cell carcinoma cell is prepared, or reduces esophageal squamous cell carcinoma cell
Tumor stem cell sample characteristic.
3. application according to claim 1 or 2, it is characterised in that：The suppression long-chain non-coding RNA-ROR expression and/
Or the material of function is targeting long-chain non-coding RNA-ROR siRNA；
The siRNA of the targeting long-chain non-coding RNA-ROR is made up of positive-sense strand and antisense strand, the positive-sense strand and the antisense
Chain is complementarily shaped to double-strand, and the sequence of the positive-sense strand is as shown in SEQ ID No.2, the sequence such as SEQ ID of the antisense strand
Shown in No.3.
4. application according to claim 1 or 2, it is characterised in that：The suppression long-chain non-coding RNA-ROR expression and/
Or the material of function is the siRNA of the targeting long-chain non-coding RNA-ROR after chemical modification；
The siRNA of the targeting long-chain non-coding RNA-ROR is made up of positive-sense strand and antisense strand, the positive-sense strand and the antisense
Chain is complementarily shaped to double-strand, and the sequence of the positive-sense strand is as shown in SEQ ID No.2, the sequence such as SEQ ID of the antisense strand
Shown in No.3.
5. application according to claim 4, it is characterised in that：The chemical modification is modified for cholesterol；
Specifically, the cholesterol is modified in the siRNA of the targeting long-chain non-coding RNA-ROR 5 ' ends.
6. application according to claim 1 or 2, it is characterised in that：The suppression long-chain non-coding RNA-ROR expression and/
Or the material of function is targeting long-chain non-coding RNA-ROR microRNA；
The microRNA of the targeting long-chain non-coding RNA-ROR is selected from as follows：has-miR-15b-5p、has-miR-33a-
5p、has-miR-129-5p、has-miR-145、has-miR-206。
7. application according to claim 6, it is characterised in that：The sequence of the has-miR-15b-5p such as SEQ ID
Shown in No.4；The sequence of the has-miR-33a-5p is as shown in SEQ ID No.5；The sequence of the has-miR-129-5p is such as
Shown in SEQ ID No.6；The sequence of the has-miR-145-5p is as shown in SEQ ID No.7；The sequence of the has-miR-206
Row are as shown in SEQ ID No.8.
8. for detect long-chain non-coding RNA-ROR material it is following it is any in application：
(B1) product for auxiliary diagnosis esophageal squamous cell carcinoma is prepared；
(B2) auxiliary diagnosis esophageal squamous cell carcinoma.
9. application according to claim 8, it is characterised in that：The material for being used to detect long-chain non-coding RNA-ROR
For the primer pair for detecting long-chain non-coding RNA-ROR；
The primer pair is the primer pair being made up of two single stranded DNAs shown in SEQ ID No.9 and SEQ ID No.10.
10. application according to claim 8, it is characterised in that：The material for being used to detect long-chain non-coding RNA-ROR
To detect preparation for the real time fluorescent quantitative for detecting long-chain non-coding RNA-ROR；
Contain in the real time fluorescent quantitative detection preparation for being used to detecting long-chain non-coding RNA-ROR by SEQ ID No.9 and
The primer pair of two single stranded DNAs composition shown in SEQ ID No.10.
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| CN108588220A (en) * | 2018-04-26 | 2018-09-28 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma long-chain non-coding RNA LINC01419 molecular markers and its application |
| CN109536502A (en) * | 2018-12-19 | 2019-03-29 | 浙江大学医学院附属妇产科医院 | A kind of PCR internal reference suitable for Trophoblastic Tumor blood plasma excretion body miRNA |
| CN110205386A (en) * | 2019-07-11 | 2019-09-06 | 中国医科大学附属盛京医院 | One kind miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its application |
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| IT201900004571A1 (en) * | 2019-03-27 | 2020-09-27 | Univ Degli Studi Di Milano Bicocca | MiR-129 inhibitor and its uses |
| CN111920961A (en) * | 2020-08-14 | 2020-11-13 | 福建医科大学附属协和医院 | Medicine for treating cancer |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108588220A (en) * | 2018-04-26 | 2018-09-28 | 汕头大学医学院附属肿瘤医院 | Esophageal squamous cell carcinoma long-chain non-coding RNA LINC01419 molecular markers and its application |
| CN109536502A (en) * | 2018-12-19 | 2019-03-29 | 浙江大学医学院附属妇产科医院 | A kind of PCR internal reference suitable for Trophoblastic Tumor blood plasma excretion body miRNA |
| CN109536502B (en) * | 2018-12-19 | 2020-02-18 | 浙江大学医学院附属妇产科医院 | PCR (polymerase chain reaction) internal reference applicable to plasma exosome miRNA of patient with gestational trophoblastic tumor |
| IT201900004571A1 (en) * | 2019-03-27 | 2020-09-27 | Univ Degli Studi Di Milano Bicocca | MiR-129 inhibitor and its uses |
| CN110205386A (en) * | 2019-07-11 | 2019-09-06 | 中国医科大学附属盛京医院 | One kind miRNA molecule miR-33a-5p relevant to carcinoma of endometrium and its application |
| CN110205386B (en) * | 2019-07-11 | 2022-12-02 | 中国医科大学附属盛京医院 | miRNA molecule miR-33a-5p related to endometrial cancer and application thereof |
| CN111118154A (en) * | 2020-01-16 | 2020-05-08 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
| CN111118154B (en) * | 2020-01-16 | 2022-10-18 | 华南协同创新研究院 | Application of LINC01272 in preparation of tumor detection reagent and/or treatment drug |
| CN111920961A (en) * | 2020-08-14 | 2020-11-13 | 福建医科大学附属协和医院 | Medicine for treating cancer |
| CN111920961B (en) * | 2020-08-14 | 2023-09-08 | 福建医科大学附属协和医院 | Medicine for treating cancer |
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