CN109182530A - hepatocellular carcinoma RNA biomarker - Google Patents
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Abstract
The present invention provides hepatocellular carcinoma biomarker, and the hepatocellular carcinoma biomarker is selected from mRNA marker: ZIC5, C12orf75, C1QL1, TMEM74, GNAZ, FAM65C and PZP；LncRNA marker: LNC-TOB2P1, LOC100499489, LNC-NMRAL2p, LNC-NBPF22P, LINC01093, LOC100130899, LOC200772 and LNC-FENDRR；MicroRNA marker: miR-200a-3p, miR-200b-3p, miR-139-5p and miR-10-3p and circRNA marker: circAKR1B10, circAKR1C3, circHMGCS1 and circC3P1.
Description
Technical field
The present invention relates to biology and medical domain, are demonstrated in hepatocellular carcinoma by high-flux sequence screening a series of
Different types of RNA biomarker.
Background technique
Hepatocellular carcinoma (HCC) is one of most common malignant tumour in world wide, and Asia and Africa are to fall ill in the world
The highest country of rate, only China just accounts for the half in Asia and African case.Due to lacking discovery early and treatment in time, life in 5 years
Rate is deposited lower than 20% (McGlynn KA, et al.2011；Lee S S,et al.2012).
The early diagnosis and therapy for being found to be cancer of biomarker for cancer provides possible solution (Cho W
C S.2014).It is mostly in the cancer of late stage stage when most of cancer patients are detected, this is mainly due to clinical diagnosises to lack
Few sensitive and special biomarker.Therefore, it early diagnoses and the Accurate classification of disease is the key that treatment of cancer project
Factor.And this needs will have the application of more effective biomarker.For decades, the discovery of various types of biomarkers and
Using being made that very big contribution for the treatment of many kinds of Disease Clinicals and diagnosis, compare other kinds of biomarker, RNA is raw
Substance markers have many advantages, such as with high sensitivity and specificity, relative to normal as a kind of emerging biomarker
The protein marker seen, RNA biomarker cost is lower, compared to DNA biomarker, can dynamically observe that RNA enters
The state and adjustment process (Xi X, et al.2017) of cell.In addition, some novel RNA, such as annular RNA marker tool
There is stability height in plasma or serum, is not easy the features such as being degraded.The present invention is by high-flux sequence to more in hepatocellular carcinoma
The rna transcription of seed type originally carries out screening and being verified in HCC clinical sample, demonstrates 23 kinds of RNA biomarkers altogether.
Much all screens or be verified in HCC for the first time.These biomarkers can be used as the clinic of hepatocellular carcinoma
The research and development target spot of diagnosing and treating drug.There is stronger reference and application value to the future therapeutic of HCC.
Summary of the invention
The present invention provides effective RNA biomarkers special in hepatocellular carcinoma.A variety of RNA that inventor filters out are raw
Object marker has important application value and meaning for the clinical diagnosis and treatment of hepatocellular carcinoma patient and medicament research and development.
Specifically, the present invention provides the following terms:
It is mRNA:ZIC5, C12orf75 selected from the group being made of the following terms 1. hepatocellular carcinoma biomarker,
C1QL1, TMEM74, GNAZ, FAM65C and PZP, sequence is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID
Shown in NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.
2. hepatocellular carcinoma biomarker is the lncRNA:LNC-TOB2P1 selected from the group being made of the following terms,
LOC100499489, LNC-NMRAL2p, LNC-NBPF22P, LINC01093, LOC100130899, LOC200772 and LNC-
FENDRR, sequence is respectively such as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID
Shown in NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15.
3. hepatocellular carcinoma biomarker is the microRNA:miR-200a- selected from the group being made of the following terms
3p, miR-200b-3p, miR-139-5p and miR-10-3p, sequence respectively such as SEQ ID NO.16, SEQ ID NO.17,
Shown in SEQ ID NO.18 and SEQ ID NO.19.
4. hepatocellular carcinoma biomarker is the circRNA:circAKR1B10 selected from the group being made of the following terms,
CircAKR1C3, circHMGCS1 and circC3P1, sequence is respectively such as SEQ ID NO.20, SEQ ID NO.21, SEQ ID
Shown in NO.22 and SEQ ID NO.23.
5. the reagent for detecting the hepatocellular carcinoma biomarker according to any one of 1-4 is in preparation for diagnosing
Purposes in the diagnosticum of hepatocellular carcinoma.
6. the purposes according to 5, wherein described for detecting the hepatocellular carcinoma biology mark according to any one of 1-4
The reagent of will object is the primer for detecting the hepatocellular carcinoma biomarker.
Detailed description of the invention
7 kinds of mRNA biomarkers that Fig. 1 present invention filters out, wherein N: normal liver tissue by cancer, T: liver cancer tissue.
8 kinds of lncRNA biomarkers that Fig. 2 present invention filters out, wherein N: normal liver tissue by cancer, T: liver cancer group
It knits.
4 kinds of microRNA biomarkers that Fig. 3 present invention filters out, wherein N: normal liver tissue by cancer, T: liver cancer group
It knits.
4 kinds of circRNA biomarkers that Fig. 4 present invention filters out, wherein N: normal liver tissue by cancer, T: liver cancer group
It knits.
Specific embodiment
Following embodiment is provided to more fully understand the present invention, is not intended to limit the present invention.Experiment in following embodiment
Method is conventional method unless otherwise specified.The experimental materials used in the following example is routine unless otherwise specified
Biochemical reagents shop purchase gained.
Tissue sample source: the cancerous tissue of all hepatocarcinoma patients used and cancer beside organism are all from Chinese science in embodiment
The first affiliated hospital of technology university.
By extracting the total serum IgE of liver cancer tissue and cancer beside organism respectively, high-flux sequence screening is carried out and in 21 HCC diseases
It is verified in the clinical sample of people.
Embodiment
The collection of 1. hepatocarcinoma patient tissue of embodiment:
The cancerous tissue of all fresh hepatocarcinoma patients and cancer beside organism both are from attached in China Science & Technology University first
Hospital.All operations, which have all been signed by the audit of Ethics Committee of China Science & Technology University and patient and family members, knows
Letter of consent.Ethics examination & approval number are as follows: USTCEC201700007.Concrete operations are as follows: patient's cancerous tissue and cancer beside organism cut in operation
Within waiting except the half an hour after in vitro, the tissue of soya bean size is removed from these tissues respectively, was impregnated with DEPC water
The clamping of operation surgical forceps gently, which is put into rinse in DEPC water, removes extra remained blood.Then RNAhold buffer is put into
(TransGen) it is temporarily saved in.
Embodiment 2.RNA extracting
1) tissue is fully ground with electric homogenizer, about 200ul is added in the 1.5mL EP pipe of no RNA enzyme at the beginning
Trizol reagent, it is to be ground sufficiently after add 800ul trizol.Then slight concussion mixes.Operation on ice into
Row.
2) 200 μ l chloroforms are added, gently shakes, is stored at room temperature 5-10 minutes.
3) in 4 DEG C of centrifuges, 12000g is centrifuged 15min, is layered solution.
4) after being centrifuged, it is seen that solution is divided into three layers, and upper layer is clarification water phase, and middle layer and lower layer are organic phase.Wherein
RNA is distributed in upper strata aqueous phase.It is albumen and DNA in organic phase.
5) upper strata aqueous phase is carefully sucked out into the 1.5ml EP pipe of new no RNA enzyme, be careful not to touch middle layer with
Exempt to lead to DNA pollution, isometric isopropanol is added, mixes, be placed in -80 DEG C of refrigerators and stand 20 minutes.The step can be with
RNA precipitate is helped, and keeps the integrality of RNA.
6) 12000g is centrifuged 15 minutes in 4 DEG C of centrifuges, comes out RNA precipitate.
7) 1ml 75%-80% ice ethyl alcohol is added, 7500g is centrifuged 5 minutes and cleans one time in 4 DEG C of centrifuges.
8) ethyl alcohol is outwelled, is dried 10 minutes, appropriate DEPC water dissolution RNA is added.
Embodiment 3.RNA purifies (DNase I digestion)
5 μ l 10X Reaction Buffer, 5 μ l RQ1 RNase- are added in the RNA for taking 39.5 μ l DEPC water to dissolve
Free DNase I, 0.5 μ l RNase Inhibitor digest 30 minutes in 37 degree of water-baths.
5 μ l Stop Solution are added, 72 degree water-bath water-bath 5 minutes, make DNase I inactivate.
3M sodium acetate (PH 5.2) (50:1) and 300 μ l dehydrated alcohols are added, is put into -80 DEG C of refrigerators and precipitates 120 minutes
More than.
12000g is centrifuged 15 minutes in 4 DEG C of centrifuges, abandons supernatant.
1ml 75%-80% ice ethyl alcohol is added, 7500g is centrifuged 5 minutes and cleans one time in 4 DEG C of centrifuges.Ethyl alcohol is outwelled,
Room temperature is dried 10 minutes, and appropriate DEPC water dissolution RNA is added
4. reverse transcription of embodiment (Promega reverse transcription system)
1) RNA concentration is measured, 300ng-500ng RNA is taken to be inverted.1 μ l random primer/Oligo is added
DT/gene specific primer, with DEPC water polishing to 10 μ l.
2) it is put on 72 degree of metal baths and is denaturalized 5 minutes, be then placed within 2 minutes on ice
It is added in above-mentioned 10 μ l system:
This system is put into PCR instrument, carries out reverse transcription by following reaction:
25 DEG C 5 minutes；42 DEG C 60 minutes；70 DEG C 15 minutes.
3) cDNA obtained can suitably dilute, in subsequent PCR experiment.
5. real-time quantitative PCR of embodiment (each RNA primer sequence used is shown in Table 1 to table 4)
1) real-time PCR system are as follows:
2) above-mentioned system is the 50ul system of a sample, this system is added in 96 orifice plate apertures and is gone, Mei Gefu
Hole 15ul, each sample do 3 multiple holes.
3) 96 orifice plates are sealed with heat-sealing film, is centrifuged, upper machine.
4) real-time PCR is run by following procedure:
5) data are analyzed: being used 2.2 software analysis data of pikoreal software, used house-keeping gene ACTB (base
Because of registration number: NM_001101.4) it is used as internal reference, the CT value for subtracting ACTB with collected CT value is organized by tumor group and cancer respectively,
The difference DELTA 1 and Δ 2 obtained, then calculates separately 2-Δ1With 2-Δ2Magnitude i.e. be respectively tumor tissues and cancer beside organism it is opposite
In the expression quantity of ACTB.(primer sequence for ACTB gene is F:CCAACACAGTGCTGTCTGG, R:
GAGTACTTGCGCTCAGGAG)。
List of primers (table 1- table 4)
(wherein F: forward primer, R: reverse primer, SL:Stem-Loop sequence)
Table 1
Table 2
Table 3
Table 4
As a result:
1. screening and verifying 7 kinds of mRNA biomarkers.
The result being sequenced according to high-throughput RNA-seq.We filter out respectively ZIC5, C12orf75, C1QL1, TMEM74,
This four genes of GNAZ, FAM65C and PZP have the variation of conspicuousness.Wherein compared to normal liver tissue ZIC5 by cancer,
The expression quantity of C12orf75, C1QL1, TMEM74, GNAZ obviously rise in liver cancer tissue, and opposite FAM65C and PZP are in liver cancer
Decline clearly (Fig. 1) in tissue.
2. screening and verifying 8 kinds of lncRNA biomarkers.
The discovery of our screening verifications LNC-TOB2P1, LOC100499489, LNC-NMRAL2p and LNC-NBPF22P this four
The expression of kind lncRNA significantly increases in liver cancer tissue.And other four kinds of lncRNA, LINC01093, LOC100130899,
LOC200772 and LNC-FENDRR expression quantity in normal liver tissue is high, and declines inside hepatocellular carcinoma tumor tissue very bright
Aobvious (Fig. 2).
3. screening and verifying 4 kinds of microRNA biomarkers.
Our screening verifications go out miR-200a-3p, these three microRNA of miR-200b-3p, miR-139-5p are thin in liver
It is obviously lowered in born of the same parents' cancerous swelling tumor tissue.And miR-10-3p significantly rises (Fig. 3) in Tissues of Hepatocellular Carcinoma.
4. screening and verifying 4 kinds of circRNA biomarkers.
We have screened four kinds of annulars RNA:circAKR1B10, circAKR1C3, circHMGCS1 and circC3P1 altogether.
Wherein significantly risen with expression of the normal liver tissue ratio circAKR1B10 and circAKR1C3 by cancer inside tumor tissues, and
Expression decline of the circHMGCS1 and circC3P1 in Tissues of Hepatocellular Carcinoma is extremely obvious, and circC3P1 is also to be sent out for the first time
It is existing.Furthermore the PCR product of circRNA has passed through further sequence verification with typical circRNA feature (Fig. 4).
Claims (6)
- It is mRNA:ZIC5, C12orf75 selected from the group being made of the following terms 1. hepatocellular carcinoma biomarker, C1QL1, TMEM74, GNAZ, FAM65C and PZP, sequence is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID Shown in NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7.
- 2. hepatocellular carcinoma biomarker is the lncRNA:LNC-TOB2P1 selected from the group being made of the following terms, LOC100499489, LNC-NMRAL2p, LNC-NBPF22P, LINC01093, LOC100130899, LOC200772 and LNC- FENDRR, sequence is respectively such as SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID Shown in NO.12, SEQ ID NO.13, SEQ ID NO.14 and SEQ ID NO.15.
- 3. hepatocellular carcinoma biomarker is the microRNA:miR-200a-3p selected from the group being made of the following terms, MiR-200b-3p, miR-139-5p and miR-10-3p, sequence is respectively such as SEQ ID NO.16, SEQ ID NO.17, SEQ Shown in ID NO.18 and SEQ ID NO.19.
- 4. hepatocellular carcinoma biomarker is the circRNA:circAKR1B10 selected from the group being made of the following terms, CircAKR1C3, circHMGCS1 and circC3P1, sequence is respectively such as SEQ ID NO.20, SEQ ID NO.21, SEQ ID Shown in NO.22 and SEQ ID NO.23.
- 5. the reagent for detecting hepatocellular carcinoma biomarker described in any one of -4 according to claim 1 is used in preparation Purposes in the diagnosticum of diagnosing hepatocellular carcinoma.
- 6. purposes according to claim 5, wherein described for detecting liver described in any one of -4 according to claim 1 The reagent of cell cancer biomarker is the primer for detecting the hepatocellular carcinoma biomarker.
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| CN113136427A (en) * | 2020-01-17 | 2021-07-20 | 中国科学技术大学 | Application of mitochondrial circular RNA as hepatocellular carcinoma biomarker |
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