CN108588222A - A kind of hepatocellular carcinoma auxiliary diagnosis or disease surveillance kit and application - Google Patents
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Abstract
The invention discloses a kind of hepatocellular carcinoma auxiliary diagnosis or disease surveillance kit, the oligonucleotide probe of in situ hybridization detection long-chain non-coding RNA HCCL5 expression is included in kit.The present invention provides strong biology tool for the auxiliary diagnosis and disease surveillance of hepatocellular carcinoma, has far-reaching clinical meaning and important popularizing application prospect.
Description
Technical field
The invention belongs to oncomolecularbiology fields, and in particular to a kind of hepatocellular carcinoma auxiliary diagnosis or disease surveillance
Kit and application.
Background technology
Hepatocellular carcinoma (hepatocellular carcinoma, HCC) is a kind of common malignant tumour, according to 2012 annual reports
Accuse estimation worldwide, new cancer cases 78.2 ten thousand in 1 year (only China just occupies 50%)；Hepatocellular carcinoma is simultaneously
It is also the third-largest cause of the death of global cancer mortality, there are within 2012 nearly 74.5 ten thousand deaths.Although the illness rate of liver cancer is continuous
Increase, but still lacks effective treatment means.Meanwhile liver cancer be one have destructive disease, developed country and development in
5 years overall survivals (overall survival, OS) of country are usual<20%；And the ratio of the death rate and incidence is
0.95, again show that the death of patients with hepatocellular carcinoma is high and survival rate is low.Hepatocellular carcinoma is that a kind of clinical prognosis is extremely bad
Fatal disease, consequently found that diagnosis of hepatoma and disease surveillance biomarker are necessary.
It is more than 200 that long-chain non-coding RNAs (long non-coding RNAs, lncRNAs), which are broadly described as length,
The RNA molecule of a nucleotide (nucleotides, nt), the structure feature with many mRNA, including contain poly (A) tail,
5 '-cap and promoter structures, without conservative open reading frame.It is now recognized that lncRNAs lacks protein coding energy
Power can be positioned in nucleus and cytoplasm.With the rise of high-flux sequence and other emerging investigative techniques, lncRNAs
Biological function gradually understood extensively.More and more evidences show that lncRNAs is acted as in extensive cell processes
With, including cell growth, cell survival, cell migration, cell invasion and cell differentiation etc..In short, lncRNAs can be tumour
Treatment provides new target spot, shows promise as diagnosing tumor and the new biomarker of disease surveillance.
The development of Oncogenome and its Relational database is provided a great convenience to cancer research person, a large amount of tumour
It is many that gene information and on-line analysis tool so that electronic cloning (in silico cloning) becomes screening cancer relative new gene
Highest one kind of cost performance in multi-method.We utilize the ends electronic cloning technology combination cDNA rapid amplifying technology recently
It is related new that (rapid-amplification of cDNA ends, RACE), successful clone and splicing obtain a hepatocellular carcinoma
Gene and its overall length, and find that the gene is a long-chain non-coding RNA (long non-using Bioinformatics Prediction
Coding RNA, lncRNA).
Invention content
The present invention is directed to overcome the deficiencies of the prior art and provide a kind of hepatocellular carcinoma auxiliary diagnosis or disease surveillance reagent
Box and application.
Inventor in hepatocellular carcinoma and cancer beside organism, has detected the long-chain non-coding RNA using the method for in situ hybridization
Expression, as a result show the lncRNA HCCL5 (sequence of long-chain non-coding RNA HCCL5 such as SEQ ID NO.4 institutes
Show, it is compared in ncbi database, it is found that it is 5 ' to extend 86 nucleosides on the basis of LOC105371083
Acid, 3 ' increase poly A tract.) the positive expression rate in Tissues of Hepatocellular Carcinoma is higher than cancer beside organism (p<0.05).Therefore it is directed to
The detection reagent of the lncRNA can be used for the auxiliary diagnosis of hepatocellular carcinoma.Subsequent further enlarged sample, is examined using in situ hybridization
The expression for surveying lncRNA HCCL5 in human liver cancer tissue and normal liver tissue, the results show that with Normal liver tissue groups
Compare, lncRNA HCCL5 in Bile duct adenocarcinoma, Hepatocellular carcinoma and
The positive expression rate in Metastatic adenocarcinoma dramatically increases, and the trend (p risen successively is presented<0.05).
Meanwhile compared with the Human Hepatocellular Carcinoma Tissues G1-2 phases, the positive expression rates of the lncRNA HCCL5 in the Human Hepatocellular Carcinoma Tissues G3 phases
Increase.In addition, compared with women Tissues of Hepatocellular Carcinoma, the positive expression rates of the lncRNA HCCL5 in male's Tissues of Hepatocellular Carcinoma
Increase (p<0.05), this is almost the same with incidence ratio of the hepatocellular carcinoma in masculinity femininity.Therefore for the lncRNA's
Detection reagent cannot be only used for the auxiliary diagnosis of hepatocellular carcinoma, and can be additionally used in hepatocellular carcinoma for the detection reagent of the lncRNA
Disease surveillance.
Therefore, in situ hybridization detection length is included in hepatocellular carcinoma auxiliary diagnosis or disease surveillance kit of the present invention
The oligonucleotide probe of chain non-coding RNA HCCL5 expression.
The oligonucleotide probe of the in situ hybridization detection long-chain non-coding RNA HCCL5 expression includes following few core
At least one of thuja acid probe：
5’-ACCTA CAGAG TATGA TTATT CATCT TTACA TTGCC-3’(SEQ ID NO.1)；
5’-TCTAG ACCTT TGCTT GTGTT GTTTG TTCTC TGCCT-3’(SEQ ID NO.2)；
5’-CTTCC AGACC AGTTT GGTGG TGCCC CTAAT CCACA-3’(SEQ ID NO.3)。
The present invention provides strong biology tool for the auxiliary diagnosis and disease surveillance of hepatocellular carcinoma, has
Far-reaching clinical meaning and important popularizing application prospect.
Description of the drawings
Figure 1A-B are that in situ hybridization detects hepatocellular carcinoma and corresponding cancer beside organism's (test set；N=30 the lncRNA in)
The expression representativeness colored graph of HCCL5；Figure 1A：Expression of the LncRNA HCCL5 in Adjacent liver tissue represents
Figure, wherein four pictures of upper row are 100 times, lower four pictures of row are 400 times；Figure 1B：LncRNA HCCL5 exist
Expression in Hepatocellular carcinoma, wherein four pictures of upper row are 100 times, lower four pictures of row are
400 times；
Fig. 2 is that in situ hybridization detects hepatocellular carcinoma and corresponding cancer beside organism's (test set；N=30 the lncRNA in)
The expression statistical chart of HCCL5；Using Chi-square Test analysis lncRNA HCCL5 in Adjacent liver tissue and
Expression variation in Hepatocellular carcinoma, as a result shows tables of the lncRNA HCCL5 in Tissues of Hepatocellular Carcinoma
It is higher than cancer beside organism (p up to positive rate<0.05)；
Fig. 3 A-E are the expression (verification that in situ hybridization detects lncRNA HCCL5 in human liver cancer tissue and normal liver tissue
Collection；N=208) representative colored graph；Fig. 3 A：Expression of the LncRNA HCCL5 in Normal liver tissue represents figure,
Wherein left side picture is 100 times, and the right picture is 400 times；Fig. 3 B：LncRNA HCCL5 are in Bile duct
Expression in adenocarcinoma represents figure, and wherein left side picture is 100 times, and the right picture is 400 times；Fig. 3 C：LncRNA
Negative expression of the HCCL5 in Hepatocellular carcinoma represents figure, and wherein left side picture is 100 times, the right figure
Piece is 400 times；Fig. 3 D：Positive expressions of the LncRNA HCCL5 in Hepatocellular carcinoma represents figure, wherein
Left side picture is 100 times, and the right picture is 400 times；Fig. 3 E：LncRNA HCCL5 are in Metastatic adenocarcinoma
In expression represent figure, wherein left side picture be 100 times, the right picture be 400 times；
Fig. 4 is expression (the verification collection that in situ hybridization detects lncRNA HCCL5 in human liver cancer tissue；N=208 it) unites
Meter figure.LncRNA HCCL5 are compared in different liver cancer patient Pathological Diagnosis (Normal using Chi-square Test
Liver tissue, Bile duct adenocarcinoma, Hepatocellular carcinoma and Metastatic
Adenocarcinoma the expression variation in), the results show that compared with Normal liver tissue groups, lncRNA
HCCL5 is in Bile duct adenocarcinoma, Hepatocellular carcinoma and Metastatic
The positive expression rate in adenocarcinoma dramatically increases, and the trend (p risen successively is presented<0.05)；
Fig. 5 is that in situ hybridization detects expression feelings of the lncRNA HCCL5 in patients with hepatocellular carcinoma difference classification G1-2 and G3
Condition (verification collection；N=208)；The results show that compared with the Human Hepatocellular Carcinoma Tissues G1-2 phases, lncRNA HCCL5 are in human liver cell
The positive expression rate of cancerous tissue G3 phases increases (p<0.05)；
Fig. 6 is that in situ hybridization detects expression of the lncRNA HCCL5 in patients with hepatocellular carcinoma different sexes male and female
Situation (verification collection；N=208).The results show that compared with women Tissues of Hepatocellular Carcinoma, lncRNA HCCL5 are in male liver cell
The positive expression rate in cancerous tissue increases (p<0.05), this and incidence ratio of the hepatocellular carcinoma in masculinity femininity basic one
It causes.
Specific implementation mode
Electronic cloning screens and PCR amplification new gene overall length
Electronic cloning technology obtains the est sequence of 5 Unknown Functions, the 5th article of progress subsequent experimental of initial option.According to normal
RACE methods are advised, Nest-PCR amplifications is carried out, the complete of HCCL5 genes is successfully obtained after 3 ' RACE and the splicing of 5 ' RACE amplifications
Long sequence；It is long-chain non-coding RNA to Bioinformatics Prediction, therefore preliminary designation is lncRNA HCCL5, sequence such as sequence
SEQ ID 4 in table.
The in situ hybridization oligonucleotide probe design of LncRNA HCCL5
In order to which using the expression of in-situ hybridization method detection long-chain non-coding RNA HCCL5, we devise needle
To the oligonucleotide probe 3 of in situ hybridization detection lncRNA HCCL5 expression.
Oligonucleotide probe such as table 1 for hybridization in situ technique detection lncRNA HCCL5 expression：
Table 1
| In situ hybridization probe sequence | SEQ ID in sequence table |
| LncRNA HCCL5 probes 1 | SEQ ID NO.1 |
| LncRNA HCCL5 probes 2 | SEQ ID NO.2 |
| LncRNA HCCL5 probes 3 | SEQ ID NO.3 |
Each gene specific oligonucleotides probe sequence of above-mentioned design is synthesized using chemical synthesis process.
Oligonucleotide probe labelling kit and in situ hybridization detection reagent
LncRNA HCCL5 oligonucleotide probes hybridization solution, the anti-digoxin of biotinylation mouse, SABC-POD, DAB dyeing examination
Agent；
Other main reagents and material
It is 3% citric acid, 2 × SSC, 0.5 × SSC, 0.2 × SSC, in situ hybridization PBS, pepsin (× 10), pre- miscellaneous
Hand over liquid, confining liquid, biotinylated peroxidase, the special coverslip of in situ hybridization, POLY-L-LYSINE, DEPC, 20% glycerine
Deng.
The operating process of LncRNA HCCL5 hybridization in situ detection kit
(1) main solution formula (table 2, table 3, table 4, table 5, table 6)
1. PBS buffer solution (1 ×；In situ hybridization use)
The preparation of 21 × PBS buffer solution of in situ hybridization of table
| Reagent | Quality (g) |
| NaCl | 30.00 |
| Na2HPO4·12H2O | 6.00 |
| NaH2PO4·2H2O | 0.40 |
Add distilled water to 1L, adjusts pH to 7.2-7.6 (room temperature)
②DEPC·water
The preparation of table 3DEPCwater
| Reagent | Volume (mL) |
| DEPC | 1.0 |
| Distilled water | 1000.0 |
After vortex oscillation is to mix well, 37 DEG C are placed for 24 hours, during which, are taken out vortex at regular intervals and are shaken up；High pressure is gone out
Bacterium 30min, it is cooling after 4 DEG C it is spare.
3. 3% citric acid
The preparation of 4 citric acid of table (3%)
| Reagent | Quality or volume |
| Citric acid (C6H8O7·H2O；g) | 3.0 |
| Distilled water (mL) | 100.0 |
| DEPC(mL) | 0.1 |
Incubator overnight, high pressure sterilization 30min are kept in dark place spare.
4. SSC (2 ×, 0.5 × and 0.2 ×)
The preparation of table 5SSC (2 ×)
| Reagent | Quality (g) |
| NaCl | 17.520 |
| Sodium citrate 2H2O | 8.820 |
HCl and NaOH is used to adjust so that pH=7.0 (room temperature), after being settled to 1000mL, is added 1 ‰ DEPC, shaking table mistake
Night, high pressure sterilization 30min are kept in dark place spare.
Points for attention：Experiment before using autoclaved DEPCwater 2 × SSC is diluted to respectively 0.5 × SSC and
0.2 × SSC is for use.
5. glycerine (20%)
The preparation of the glycerine of table 6 20%
Shaking table ambient temperature overnight, high pressure sterilization 30min, saves backup.
(2) fixed
1. fixer is 4% paraformaldehyde/0.1M PBS (pH=7.2-7.6), contain 1 ‰ DEPC.Room temperature fixes 20-
30min；
2. distilled water fully washs, dry -20 DEG C of the refrigerator that is placed on is preserved and (be can save 2 weeks or more).
(3) it is dehydrated
1. according to H2O2(30%)：Pure methanol=1：50 ratio prepares mixed liquor, uses mixed liquor to handle tissue at room temperature
Chip 30min；
2. distilled water washs 3min-5min/ times × 3 times.
(4) exposure mRNA nucleic acid fragments
1. the Fresh of pepsin：According to concentrated type pepsin：It drips 3% citric acid=2：The dilution proportion of 1mL
Pepsin；
2. pepsin organization chip：It is added dropwise on the pepsin to organization chip of diluted fresh, 37 DEG C or room temperature
5s-120s is digested, can not also be digested sometimes；
3. PBS is washed：In situ hybridization is washed 5min/ times × 3 times with PBS buffer solution；
4. washing：Distillation washing 3min-5min/ times × 1 time.
(5) it fixes afterwards
1. points for attention：It is just needed after pepsin digestion, if pepsin digestion is not used, this step is negligible；
2. fixing：Fixer is 1% paraformaldehyde/0.1M PBS (pH=7.2-7.6), contains 1 ‰ DEPC.Using solid
It is fixed that organization chip room temperature is also fixed into 10min；
3. washing：Distillation washing 3min-5min/ times × 3 times.
(6) prehybridization
1. preparing wet box：In order to keep humidity, first plus the glycerine of 20mL 20% is to clean and dry hybridizing box bottom
Portion；
2. prehybridization：Add appropriate prehybridization solution to organization chip, 38-42 DEG C of incubation 2h-4h of insulating box siphons away extra liquid
Body is not washed.
(7) hybridize
1. hybridizing：Add appropriate hybridization solution to organization chip, covered, 38-42 DEG C of hybridized overnight of insulating box
2. points for attention：Remember to use the special coverslip of in situ hybridization, and its protective film is opened into use；
Hybridization temperature and time are adjusted or optimized according to hybridisation events.
(8) post-hybridization washing
1. 2 × SSC is washed：Coverslip is opened, is embathed 5min/ times × 2 times using 37 DEG C of 2 × SSC；
2. 0.5 × SSC is washed：37 DEG C of 0.5 × SSC is washed 15min/ times × 1 time；
3. 0.2 × SSC is washed：37 DEG C of 0.2 × SSC is washed 15min/ times × 1 time；
4. points for attention：If there is unspecific staining, it is secondary to repeat 0.2 × SSC washings 15min/ times × (1-2).
(9) confining liquid is added dropwise
1. confining liquid is added dropwise to organization chip, 37 DEG C of incubation 30min；
2. getting rid of surplus liquid, do not wash.
(10) the anti-digoxin of biotinylation mouse is added dropwise
1. the anti-digoxin of biotinylation mouse is added dropwise to organization chip, 37 DEG C are incubated 60min or incubation at room temperature 120min；
2. in situ hybridization is washed 5min/ times × 4 times with PBS buffer solution；
3. points for attention：It is sure not to embathe using other buffer solutions or distilled water.
(11) SABC is added dropwise
1. SABC is added dropwise to organization chip, 37 DEG C are incubated 20min or incubation at room temperature 30min；
2. being washed 5min/ times × 3 times with PBS using in situ hybridization；
3. points for attention：It is sure not to embathe using other buffer solutions or distilled water.
(12) biotinylated peroxidase is added dropwise
1. being added dropwise on biotinylated peroxidase to slice, 37 DEG C are incubated 20min or incubation at room temperature 30min；
2. being washed 5min/ times × 4 times with PBS using in situ hybridization.
(13) DAB develops the color
1. the preparation of DAB color developing agents：Respectively plus color developing agent A, B, C each one is dropped in 1mL distilled water, fully blows and beats mixing
(also can autogamy DAB chromogenic reagents)；
2. being added on slice, thin piece using DAB color developing agents；
3. controlling colour developing under microscope, generally<In 30min
4. fully washing
5. points for attention：If can continue to develop the color without background appearance when controlling colour developing under mirror.
(14) it redyes
1. haematoxylin is redyed when necessary, it is not necessary to when this step can be ignored；
2. fully washing.
(15) mounting
1. dehydration of alcohol, dimethylbenzene is transparent, mounting.
In-situ hybridization method detects the expression (test set) of lncRNA HCCL5 in liver cancer patient tissue
Hepatocellular carcinoma and corresponding cancer beside organism's (test set are detected using in situ hybridization；N=30 the lncRNA in)
The subcellular localization of HCCL5 and expression, and lncRNA HCCL5 are analyzed in Adjacent liver tissue using Chi-square Test
With the expression variation in Hepatocellular carcinoma.The results show that lncRNA HCCL5 are primarily targeted for cytoplasm,
And the positive expression rate in Tissues of Hepatocellular Carcinoma is higher than cancer beside organism (Fig. 1,2).
In-situ hybridization method enlarged sample detects the expression (verification collection) of lncRNA HCCL5 in liver cancer patient tissue
Further enlarged sample detects lncRNA HCCL5 in human liver cancer tissue and normal liver tissue using in situ hybridization
Expression (verification collection；N=208), and the lncRNA HCCL5 positives are compared in the whole crowd being included in and negative group exists
Pathological Diagnosis(Normal liver tissue、Bile duct adenocarcinoma、
Hepatocellular carcinoma and Metastatic adenocarcinoma), in the patients with hepatocellular carcinoma being included in
The lncRNA HCCL5 positives and negative group whether there is difference in terms of Grade (G1-2 and G3) and gender (male and female).
The results show that compared with Normal liver tissue groups, lncRNA HCCL5 are in Bile duct
Expression sun in adenocarcinoma, Hepatocellular carcinoma and Metastatic adenocarcinoma
Property rate dramatically increase, and the trend (Fig. 3,4) that rises successively is presented.Meanwhile compared with the Human Hepatocellular Carcinoma Tissues G1-2 phases,
The positive expression rates of the lncRNA HCCL5 in the Human Hepatocellular Carcinoma Tissues G3 phases increases (Fig. 5).In addition, with women Tissues of Hepatocellular Carcinoma
It compares, the positive expression rates of the lncRNA HCCL5 in male's Tissues of Hepatocellular Carcinoma increases (Fig. 6), this is with hepatocellular carcinoma in male
Incidence ratio in women is almost the same.
As a result interpretation
It is observed under low power and high-power microscope respectively using fluorescence microscope, looks first at target gene in target
Intracellular subcellular localization：It is positioned at nucleus, cytoplasm or cell membrane.
Again respectively detection institute in a organized way in the expression of lncRNA HCCL5 be that positive or feminine gender is presented.Criterion is：
(1) cell dye-free is judged as feminine gender, is denoted as " 0 "；(2) it is the positive, including light brown, brown and dark brown that cell, which dyes brown,
Color is denoted as " 1 ".
In order to reduce the subjective factor of appraisal result as much as possible, above-mentioned standard is pressed respectively respectively by two pathologists
Diagosis and interpretation.
Statistical analysis
Mean ± standard deviation (mean ± SD) the form expression of all data, n >=8.Wherein, the comparison between group and group is equal
Using x2Test or Fisher exact test；Work as p<0.05, it is believed that difference is statistically significant.Use statistics software
SPSS17.0 carries out data analysis, and GraphPad Prism 5 draw statistical graph.
SEQUENCE LISTING
<110>Central South University
<120>A kind of hepatocellular carcinoma auxiliary diagnosis or disease surveillance kit and application
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 35
<212> DNA
<213>It is artificial synthesized
<400> 1
acctacagag tatgattatt catctttaca ttgcc 35
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<213>It is artificial synthesized
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tctagacctt tgcttgtgtt gtttgttctc tgcct 35
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<213>It is artificial synthesized
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cttccagacc agtttggtgg tgcccctaat ccaca 35
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<213>It is artificial synthesized
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catgcgtaga taaaagtaaa taaacgccct caagttagga atgaacggag gaaatgggtt 60
aaacgagaat aaataattga aggcagagag gtaaagaact tcctctttca acgcatctca 120
aaatttggcg tggatgcaga tcacctacag agtatgatta ttcatcttta cattgccttg 180
ttttgagatg atgaaactga ggttcaagcc aatgacaagc ctgaagccac atggctagtg 240
agtggcagag tcagaactca aacccaaatc cacctgaccc caaacttcat gtatcttgaa 300
ctacaggctc catctcactg ttcacactct cacccacctc cacccatcaa aggtgcctct 360
agacctttgc ttgtgttgtt tgttctctgc ctgacatgcc tccccctcag tcgctctcag 420
ttgaaacctg gcttagtcat caagtgcctg ctcagcagca agacctcttc tcttcacaga 480
gcctttgttg agccccacca ctggaattaa cctcttcctt ccagaccagt ttggtggtgc 540
ccctaatcca cagtttccca acctcagccc tactgacact ggggctggat aatctttgtc 600
gtgggggctg tcgtgtgcac tgcaggatat ggagcagtac ccctgctttc cacccacgag 660
atgccatggc accctcctca agttgcgaca accaaaatat gtcttcagac attgccactg 720
tcccctaggg tactaaatca cctctatgga gaaccactgt cctaagctac aatactcact 780
gccctctgtt ttgagcctaa aaaaaaaaaa aaaaaaaaaa aaa 823
Claims (3)
1. a kind of hepatocellular carcinoma auxiliary diagnosis or disease surveillance kit, which is characterized in that comprising in situ in the kit
The oligonucleotide probe of hybridization check long-chain non-coding RNA HCCL5 expression.
2. kit as described in claim 1, which is characterized in that the in situ hybridization detects long-chain non-coding RNA
The oligonucleotide probe of HCCL5 expression includes at least one of following oligonucleotide probe：
5’-ACCTA CAGAG TATGA TTATT CATCT TTACA TTGCC-3’；
5’-TCTAG ACCTT TGCTT GTGTT GTTTG TTCTC TGCCT-3’；
5’-CTTCC AGACC AGTTT GGTGG TGCCC CTAAT CCACA-3’。
3. long-chain non-coding RNA HCCL5 is in preparing hepatocellular carcinoma auxiliary diagnosis or disease surveillance biomarker
Using.
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| CN2017104668491 | 2017-06-20 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107022625A (en) * | 2017-05-09 | 2017-08-08 | 中南大学 | A kind of human liver cell carcinogenesis development related long-chain non-coding RNA, amplification detection method and application |
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| CN103547682A (en) * | 2011-01-14 | 2014-01-29 | 新加坡科技研究局 | Gene signatures for use with hepatocellular carcinoma |
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| CN107022625A (en) * | 2017-05-09 | 2017-08-08 | 中南大学 | A kind of human liver cell carcinogenesis development related long-chain non-coding RNA, amplification detection method and application |
| CN107022625B (en) * | 2017-05-09 | 2020-06-19 | 中南大学 | Long-chain non-coding RNA related to occurrence and development of human hepatocellular carcinoma, amplification detection method and application |
Also Published As
| Publication number | Publication date |
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| CN108588222B (en) | 2019-10-25 |
| CN107475362A (en) | 2017-12-15 |
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