CN107937545A - Applications of the circEMILIN2 in pituitary adenoma biomarker - Google Patents
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Abstract
The invention discloses applications of the circEMILIN2 (circRNA EMILIN2) in pituitary adenoma biomarker, the specially purposes technical field of natural ribonucleotide, circRNA EMILIN2 long 1925bp, 2892486 to 2890561 on the 18th article of chromosome of the mankind, EMILIN2 is the covering gene of the circular rna, detect the amount of circRNA EMILIN2 in the pituitary adenoma tissues sample from the object, the wherein circRNA EMILIN2 of relatively low amount are strong for the object Invasiveness of Pituitary Adenomas, it is and related with the object prognosis mala possibility increase.Circular rna has the features such as mechanism stable, abundance and tissue specific expression.Therefore circRNA EMILIN2 have the application prospect as pituitary adenoma biomarker.
Description
Technical field
The present invention relates to applications of the circEMILIN2 (circRNA-EMILIN2) in pituitary adenoma biomarker, specifically
For the purposes technical field of natural ribonucleotide.
Background technology
Pituitary adenoma (abbreviation hypophysoma) is one of most common intracranial tumors of the mankind, accounts for the 10%- of intracranial tumors
15%.Hypophysoma histologically belongs to benign tumour, but some hypophysomas are in invasive growth, wrapping or infringement surrounding tissue
One of the main reason for structure, Partial tumors operation is difficult to thoroughly cut off, be tumor recurrence.According to the biological scholarship and moral conduct of hypophysoma
For Non-Invasive hypophysoma, invasive pituitary adenomas and hypophysis cancer can be divided into.Invasive pituitary adenomas between Non-Invasive hypophysoma and
Between hypophysis cancer, its Histological Study belongs to benign, and biological property is but like pernicious.Invasion and Non-invasive pituitary adenoma
Clinical manifestation, prognosis are significantly different.The necrosis of invasive pituitary adenoma, palsy, capsule become incidence apparently higher than Non-Invasive
Pituitary adenoma.Invasive growth cause surgical operation be difficult to it is complete cut tumour, and common drug bromine it is hidden stop, growth hormone release inhibiting hormone is to this
The effect of class tumour, is poor；Invasive growth equally limits its sensitiveness to radiotherapy, and the radiotherapy of saddle area is easy to cause normally
The damage of the important features such as hypophysis, hypothalamus and optic nerve.These factors cause invasive pituitary adenoma Postoperative recurrent rate high, swell
Knurl remnant tissue increases fast.It is clear that the treatment to invasive pituitary adenomas is field of neurosurgery and therapeutic field of tumor face
The huge challenge faced.Therefore, find with the relevant molecular labeling of Invasiveness of Pituitary Adenomas for treating in time and improving prognosis extremely
Close important.
Circular rna is a kind of long-chain non-coding RNA that can close cyclization, is had been found that in virus, plant, archeobacteria
The presence of a large amount of circular rnas.In recent years, the seminar such as Norman E Sharpless passes through high-throughput techniques and bioinformatics
Analysis is speculated in mammalian cell there are a large amount of endogenous circular rna s, and passes through Northern blot, two-dimentional gel
The biochemical test means such as electrophoresis and reverse primer PCR are confirmed.Part achievement in research, which discloses circular rna s, to be passed through
Consumption shears corpusculum, with reference to the mode such as rna plymerase ii and absorption miRNA, participates in core base in regulating cell biobehavioral
The expression of cause.Research shows that circular rna s abnormal expressions in kinds of tumor cells, prompt it may be with the occurrence and development of tumour
It is closely related.
The content of the invention
It is an object of the invention to provide circEMILIN2 (circRNA-EMILIN2) in pituitary adenoma biomarker
Application.
To achieve the above object, the present invention provides following technical solution：CircEMILIN2 (circRNA-EMILIN2) exists
Application in pituitary adenoma biomarker, circEMILIN2 long 1925bp, 2892486 on the 18th article of chromosome of the mankind
It is the covering gene of the circular rna to 2890561, EMILIN2.
The described method includes detection the pituitary adenoma tissues sample from the object in circRNA-EMILIN2 amount,
The wherein circRNA-EMILIN2 of relatively low amount is strong for the object Invasiveness of Pituitary Adenomas, and can with the object prognosis mala
Energy property increase is related.
Expressed the present invention also provides circRNA-EMILIN2 in pituitary adenoma biomarker, the expression bag
Containing following steps：
Step 1: invasion and Non-invasive pituitary adenoma tissue RNA extractions；
Step 2: total serum IgE reverse transcription cDNA；
Step 3: cDNA is timed quantitative PCR detection, circRNA-EMILIN2 in sample is detected after reaction
PCR identification sequence, then contrasted with PCR product sequence results knowable to circRNA-EMILIN2 in pituitary adenoma tissues
In expressed.
Further preferably, invasion and Non-invasive pituitary adenoma tissue RNA extraction method are phases in the step one
With, specific extracting method is：2 grams of organization materials are weighed, 250 microlitres of RNAisoPlus is added, after the grinding of hand electric device, turns
Move in the centrifuge tube of 1.5 milliliters of RNase-free, 12000 turns 4 DEG C centrifuge 10 minutes, go 200 microlitres of supernatant to add 1.5 milliliters
In the centrifuge tube of RNase-free, add after 1 milliliter of RNAiso Plus is newly mixed and be stored at room temperature 5 minutes.Add 200 microlitres
Chloroform, after fully mixing, is stored at room temperature 3 minutes, 12000 turns 4 DEG C centrifuge 15 minutes, take supernatant 250ul, and 500 microlitres of addition is different
Propyl alcohol is stored at room temperature 10 minutes after mixing, and 12000 turns 4 DEG C centrifuge 10 minutes, discard supernatant, add 75% ethanol, overturn and mix
Afterwards, centrifuge 5 minutes for 7500 turns 4 DEG C, discard supernatant, be stored at room temperature 5 minutes, add 15-20 microlitres of Nuclease-Free water
Dissolving.The OD values and concentration of Nanodrop measuring and calculating gained liquid.
Further preferably, the method for total serum IgE reverse transcription cDNA is in the step two：According to reverse transcription reagent box
PrimeScriptTMRT reagent Kit with gDNA Eraser illustrate, add 1 microgram RNA and carry out genomic DNA
Remove, specifically add 10 microlitres of DNA enzymatic, reaction buffer and Nuclease-Free water polishing reaction systems, 42 DEG C of PCR instrument is incubated
Educate 2 minutes.Then carry out reverse transcription, the specific random primer added needed for reverse transcription, buffer solution, reverse transcriptase and
20 microlitres of reaction systems of Nuclease-Free water polishing, 37 DEG C of PCR instrument are incubated 15 minutes, and 85 DEG C are incubated 30 seconds, and 4 DEG C of insulations will
Reverse transcription product 1: 10 dilutes, and qPCR mixtures are prepared on ice.
Preferably, it is as follows that eDNA is timed quantitative PCR detection reaction condition in the step three：95 DEG C first
Lower pre-degeneration 30 seconds, is then denatured 5 seconds for 95 DEG C, and 56 DEG C are annealed 30 seconds, and 72 DEG C extend 30 seconds, and 40 circulations, heat melts the stage 95
Heat is melted 30 seconds at being denatured 60 seconds, 56 DEG C at DEG C, and 95 DEG C of heat are melted 30 seconds.
The beneficial effects of the invention are as follows：(1) purposes of circRNA-EMILIN2 is proposed.(2) research of the invention shows
The circRNA-EMILIN2 of abnormal amount for the object Invasiveness of Pituitary Adenomas it is strong, and with the object prognosis mala possibility
Increase related.(3) circular rna has the features such as mechanism stable, abundance and tissue specific expression.And circRNA-EMILIN2
Had differences in invasive pituitary adenoma and expression quantity in Non-invasive pituitary adenoma tissue of patient, therefore circRNA-
EMILIN2 has the application prospect as pituitary adenoma biomarker.
Brief description of the drawings
The PCR that Fig. 1 is circRNA-EMILIN2 identifies sequence and PCR product sequencing result comparison diagram；
Fig. 2 is circRNA-EMILIN2 solubility curve figures；
Fig. 3 is to use circRNA-EMILIN2 examination primer pair pituitary adenoma patients tissue samples expression quantity the selection results
Figure；
Fig. 4 is the suggestion structure chart and primer location figure of circRNA-EMILIN2.
Embodiment
Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete
Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work
Embodiment, belongs to the scope of protection of the invention.With reference to embodiment, the present invention is described further
1st, experiment material, clinical sample：19 pituitary adenomas (8 invasive pituitary adenomas, 9 Non-Invasive pituitary glands
Knurl) tissue samples have Subsidiary Second Hospital, Dalian Medical Univ. to be provided.
Reagent：CircRNA-EMILIN2 examinations primer is synthesized by Invitrogen companies；
RNAiso Plus (article No. 9109) are purchased from Takara companies；
Chloroform is purchased from big chemical reagent (Tianjin) Co., Ltd forever；
Isopropanol is purchased from big chemical reagent (Tianjin) Co., Ltd forever；
Ethanol is purchased from Sinopharm Chemical Reagent Co., Ltd.；
Reverse transcription reagent box PrimeScriptTMRT reagent Kit with gDNA Eraser (article No.s：RR047Q)
Purchased from Takara companies；
SuperReal PreMix Plus (SYBR Green) (article No.：FP205-02) purchased from Tiangeng biochemical technology (north
Capital) Co., Ltd；
Nuclease-Free water (article No. RT121) are purchased from root biochemical technology (Beijing) Co., Ltd.
2nd, experimental method, invasion or Non-invasive pituitary adenoma tissue RNA extraction method are：2 grams of organization materials are weighed,
250 microlitres of RNAiso Plus are added, after the grinding of hand electric device, are transferred in the centrifuge tube of 1.5 milliliters of RNase-free,
12000 turns 4 DEG C centrifuge 10 minutes, go in the centrifuge tube of 200 microlitres of additions of supernatant, 1.5 milliliters of RNase-free, add 1 milliliter
RNAiso Plus are stored at room temperature 5 minutes after newly mixing.200 microlitres of chloroform is added, after fully mixing, is stored at room temperature 3 minutes,
12000 turns 4 DEG C centrifuge 15 minutes, take supernatant 250ul, add after 500 microlitres of isopropanols mix and are stored at room temperature 10 minutes, and 12000
Turn 4 DEG C to centrifuge 10 minutes.Supernatant is discarded, adds 75% ethanol, is overturned after mixing, 7500 turns 4 DEG C centrifuge 5 minutes, discard supernatant,
It is stored at room temperature 5 minutes, adds 15-20 microlitres of Nuclease-Free water dissolving, the OD values of Nanodrop measuring and calculating gained liquid
And concentration.
3rd, expression quantity examination, according to reverse transcription reagent box PrimeScriptTM RT reagent Kit with gDNA
Eraser illustrate, add 1 microgram RNA carry out genomic DNA removal, specifically add DNA enzymatic, reaction buffer and
10 microlitres of reaction systems of Nuclease-Free water polishing, 42 DEG C of PCR instrument are incubated 2 minutes.Reverse transcription is then carried out, it is specific to add
Random primer needed for reverse transcription, 20 microlitres of buffer solution, reverse transcriptase and Nuclease-Free water polishing reaction systems, PCR instrument
37 DEG C are incubated 15 minutes, and 85 DEG C are incubated 30 seconds, 4 DEG C of insulations.
Configure reaction mixture
B.Real-Time PCR (SYRB Green methods)
Reverse transcription product 1: 10 is diluted, qPCR mixtures are prepared on ice：
Sample is put into qPCR instruments and is run, program is as follows
4th, referring to Fig. 1 and Fig. 2, Fig. 1 is that the PCR of circRNA-EMILIN2 identifies pair of sequence and PCR product sequence results
Than figure, Fig. 2 is circRNA-EMILIN2 solubility curve figures.Test result indicates that the identification sequence of circRNA-EMILIN2 with
PCR product sequencing result contrasts successfully, and circRNA-EMILIN2 is expressed in pituitary adenoma tissues.
Fig. 3 uses the result figure of circRNA-EMILIN2 examination primer pair pituitary adenoma patients sample expression quantity examinations.It is real
Test the result shows that expression of the circRNA-EMILIN2 in invasive pituitary adenoma significantly reduces, average expression amount (Ct values) is poor
3, the expression quantity equivalent to circRNA-EMILIN2 in invasive pituitary adenoma is 0.125 times in Non-invasive pituitary adenoma.
So big differential expression has given application prospects of the circRNA-EMILIN2 as invasive pituitary adenoma biomarker.
Although an embodiment of the present invention has been shown and described, for the ordinary skill in the art, can be with
Understanding without departing from the principles and spirit of the present invention can carry out these embodiments a variety of changes, modification, replace
And modification, the scope of the present invention is defined by the appended.
Sequence table
<110>Subsidiary Second Hospital, Dalian Medical Univ.
<120>Applications of the circEMILIN2 in pituitary adenoma biomarker
<141> 2018-01-04
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1926
<212> DNA
<213>The mankind (homo sapiens)
<400> 1
ataatgaacc cagccaattc tcagagccca ggaagacttt gtccccaact ggtacagcac 60
aaccaagctg gggggtagat ccaaaagagg ggcctcagga acttcaggaa aagaagatac 120
aggtgctaga ggagaaggtt cttcgactca caaggacggt tcttgacctc cagtcttccc 180
ttgctggagt gagtgaaaat ctcaaacatg ccactcagga tgatgccagt agaacacggg 240
caccagggct cagcagccag caccccaagc ctgacaccac tgttagtgga gacacagaaa 300
cgggccagag tcctggtgtc ttcaacacta aggaatctgg catgaaggac atcaagtctg 360
aattggctga agtcaaagat actctaaaga acaaaagtga caagctggaa gagctggatg 420
gaaaagtgaa gggctacgaa gggcagctca gacagctcca ggaagcagct cagggcccga 480
cggtgaccat gacaaccaac gaactctacc aagcctatgt ggacagtaag atcgacgccc 540
tgagagagga gctcatggag ggcatggaca gaaagctggc tgacctgaaa aactcatgtg 600
agtacaagct cactggcctc cagcagcagt gtgatgacta tgggagcagc tacctgggag 660
tgatagagct cataggggag aaggaaacaa gcctgagaaa agaaataaat aacctccgag 720
cccggctaca ggagccttca gcccaggcaa attgctgcga cagtgaaaag aatggtgaca 780
ttggtcaaca gatcaagaca ttggaccaga aaatcgagag agttgctgaa gccaccagaa 840
tgctgaatgg aagactggac aatgagtttg accgccttat agttccagag ccagatgtgg 900
attttgatgc aaaatggaat gaactcgatg caaggatcaa tgtgacggag aagaacgctg 960
aagaacattg cttttacatt gaggaaaccc ttcggggcgc cattaatgga gaggtgggtg 1020
acttgaagca gcttgttgat cagaaaatac agtctctgga agaccgtctg gggagcgttc 1080
tcctacagat gaccaataac actggtgcag agctcagtcc cccaggggca gcagccctgc 1140
caggagtgtc agggtcagga gatgaacggg tcatgatgga attaaaccac ctgaaggaca 1200
aagttcaagt tgttgaagac atttgcctgc tgaacatcca gggaaagcct catgggatgg 1260
aaggtgcctt gccaaacagg gaagaccgcg cagtacgcga cagcctgcac cttttgaaat 1320
ctctcaacga cacgatgcac aggaagtttc aagaaaccga acaaaccatc cagaaacttc 1380
aacaggattt tagttttctt tattctcaat taaaccacac agaaaatgat gtgactcatc 1440
ttcaaaagga aatgagcaat tgtagagcag gtgaaaacgc tggcatgggt aggttcacta 1500
aggtgggtga gcaagaaagg acagtggaca ccctgccgtc cccccagcac cccgtggctc 1560
attgctgcag tcagctggag gagaggtggc agaggttgca gagccaggtc atctcggagc 1620
tggatgcttg taaggaatgc acgcaggggg tccagaggga ggtctccatg gtggagggca 1680
gggtgtctca tatggagaaa acttgcagca agctggactc tatctcagga aatcttcaga 1740
ggatcaagga ggggctcaac aagcatgtca gcagcctgtg gaactgtgtc aggcagatga 1800
acggaacgct caggtcgcat tccagagaca tttctggcct gaagaattca gtccagcagt 1860
tctacagcca cgtcttccag atttctactg atttgcaaga tctggtcaaa tttcagccat 1920
cagcaa 1926
<210> 2
<211> 151
<212> DNA
<213>The mankind (homo sapiens)
<400> 2
ccatcagcaa ataatgaacc cagccaattc tcagagccca ggaagacttt gtccccaact 60
ggtacagcac aaccaagctg gggggtagat ccaaaagagg ggcctcagga acttcaggaa 120
aagaagatac aggtgctaga ggagaaggtt c 151
Claims (6)
- Applications of the 1.circEMILIN2 in pituitary adenoma biomarker, it is characterised in that：CircRNA-EMILIN2 long 1925bp, 2892486 to 2890561, EMILIN2 is the covering gene of the circular rna on the 18th article of chromosome of the mankind, The base sequence of the circRNA-EMILIN2 such as SEQIDNO：Shown in 1.
- 2. circRNA- in application as claimed in claim 1, including pituitary adenoma tissues sample of the detection from the object The circRNA-EMILIN2 of the amount of EMILIN2, wherein relatively low amount for the object Invasiveness of Pituitary Adenomas it is strong, and with it is described right As the increase of prognosis mala possibility is related.
- Expression of the 3.circEMILIN2 in pituitary adenoma biomarker, it is characterised in that：Specific expression includes following Step：Step 1: invasion and Non-invasive pituitary adenoma tissue RNA extractions；Step 2: total serum IgE reverse transcription cDNA；Step 3: cDNA is timed quantitative PCR detection, the PCR of circRNA-EMILIN2 in sample is detected after reaction Identify sequence, then contrasted with PCR product sequence results knowable to circRNA-EMILIN2 carried out in pituitary adenoma tissues Expression.
- 4. expression according to claim 3, it is characterised in that：Invasion and Non-Invasive pituitary gland in the step one Tumor tissue RNA extraction method is identical, and specific extracting method is：2 grams of organization materials are weighed, add 250 microlitres of RNAiso Plus, after the grinding of hand electric device, is transferred in the centrifuge tube of 1.5 milliliters of RNase-free, and 12000 turns 4 DEG C centrifuge 10 minutes, Go in the centrifuge tube of 200 microlitres of additions of supernatant, 1.5 milliliters of RNase-free, add room temperature after 1 milliliter of RNAiso Plus is newly mixed 5 minutes are stood, adds 200 microlitres of chloroform, after fully mixing, is stored at room temperature 3 minutes, 12000 turns 4 DEG C centrifuge 15 minutes, take Supernatant 250ul, adds after 500 microlitres of isopropanols mix and is stored at room temperature 10 minutes, 12000 turns 4 DEG C centrifuge 10 minutes, discard Clearly, 75% ethanol is added, is overturned after mixing, 7500 turns 4 DEG C centrifuge 5 minutes, discard supernatant, are stored at room temperature 5 minutes, add 15- 20 microlitres of Nuclease-Free warer dissolvings, the OD values and concentration of Nanodrop measuring and calculating gained liquid.
- 5. expression according to claim 3, it is characterised in that：Total serum IgE is anti-in the step twoTranscription cDNA method be：According to reverse transcription reagent box PrimeScriptTM RT reagent Kit with gDNA Eraser illustrate, add 1 microgram RNA carry out genomic DNA removal, specifically add DNA enzymatic, reaction buffer and 10 microlitres of reaction systems of Nuclease-Free water polishing, 42 DEG C of PCR instrument are incubated 2 minutes, then carry out reverse transcription, specific to add Random primer needed for reverse transcription, 20 microlitres of buffer solution, reverse transcriptase and Nuclease-Free water polishing reaction systems, PCR instrument 37 DEG C are incubated 15 minutes, and 85 DEG C are incubated 30 seconds, and 4 DEG C of insulations, reverse transcription product 1: 10 is diluted, and qPCR mixing is prepared on ice Thing.
- 6. expression according to claim 3, it is characterised in that cDNA is timed quantitative PCR inspection in the step three It is as follows to survey reaction condition：Pre-degeneration 30 seconds at 95 DEG C first, is then denatured 5 seconds for 95 DEG C, 56 DEG C are annealed 30 seconds, 72 DEG C of extensions 30 Second, 40 circulation, heat is melted be denatured 60 seconds, 56 DEG C at 95 DEG C of stage at heat melt 30 seconds, 95 DEG C of heat are melted 30 seconds.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980955A (en) * | 2021-03-05 | 2021-06-18 | 南昌大学第二附属医院 | Application of EMILIN2 as drug-resistant detection, treatment and prognosis molecular target of glioma temozolomide |
Citations (2)
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| CN103937886A (en) * | 2014-04-02 | 2014-07-23 | 南京大学 | Real-time fluorescence quantitative PCR detection kit for quantitative detection of GGPPS1 genes, detection method and application |
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2018
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|---|---|---|---|---|
| CN103937886A (en) * | 2014-04-02 | 2014-07-23 | 南京大学 | Real-time fluorescence quantitative PCR detection kit for quantitative detection of GGPPS1 genes, detection method and application |
| CN105950768A (en) * | 2016-07-01 | 2016-09-21 | 江苏医诺万细胞诊疗有限公司 | Kit for auxiliary diagnosis of multiple tumors by taking micro ribonucleic acid (RNA) combination as tumor marker, and detection method of kit |
Non-Patent Citations (1)
| Title |
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| WU SILIN等: "Novel Biomarkers for Non-functioning Invasive Pituitary Adenomas were Identified by Using Analysis of microRNAs Expression Profile", 《BIOCHEM GENET》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112980955A (en) * | 2021-03-05 | 2021-06-18 | 南昌大学第二附属医院 | Application of EMILIN2 as drug-resistant detection, treatment and prognosis molecular target of glioma temozolomide |
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Application publication date: 20180420 |