CN107586781A - Liver cancer marker lncRNA ENST00000620463.1 and its application - Google Patents
Liver cancer marker lncRNA ENST00000620463.1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of liver cancer marker lncRNAENST00000620463.1 and its application.The invention provides a kind of tumor markers, is a kind of LncRNA, is named as ENST00000620463.1, is the RNA shown in the sequence 1 of sequence table.The corresponding DNA of ENST00000620463.1 are the DNA shown in the sequence 2 of sequence table.Present invention firstly discovers that ENST00000620463.1 conspicuousnesses raise in liver cancer.It is demonstrated experimentally that shRNA disturbs silence ENST00000620463.1, it can effectively suppress propagation, migration and the invasion and attack of liver cancer cells, new way is provided for the personalized treatment of liver cancer.
Description
Technical field
The present invention relates to biomedicine field, and in particular to a kind of liver cancer marker lncRNA ENST00000620463.1
And its application.
Background technology
Primary hepatoma (hepatocellular carcinoma, HCC) (hereinafter referred to as liver cancer) is that China is common
Malignant tumour, case fatality rate occupies second cancer, annual about 350,000 people that fall ill.Onset of liver cancer rate and the death rate occupy always
It is high not under, because its grade of malignancy is high, disease progression is fast, Most patients be in middle and advanced stage when clinically finding, the death rate and again
Hair rate is high, poor prognosis.Current conventional therapy means mainly include surgery excision, liver transfer operation, local ablation therapy, trans-hepatic artery
Chemoembolization, radiotherapy, molecular targeted therapy, complex treatment etc., but therapeutic effect is not often very good, moderate or advanced liver cancer
Treatment is even more unsatisfactory.
The occurrence and development of liver cancer are a complicated biological processes.There are some researches show the occurrence and development of liver cancer with
HBV virus proteins (such as HBx) raise the transcription factors such as c-myc, c-jun, NF- κ B, AP-2, β-catenin in liver cell
Expression it is relevant.In addition, numerous studies show that the occurrence and development of human liver cancer and IGF (IGF), liver are thin
The intracellular growth factor (HGF), transforminggrowthfactor-α (TGF- α), EGF (EGF), transforming growth factor-β (TGF-
β), the expression of a variety of growth factors such as VEGF (VEGF) and abnormal signal are closely related.However, liver cancer is sent out
The Molecular Biology Mechanism research of raw, development and transfer is not yet clear and definite.
Long-chain non-coding RNA (long non-coding RNA, lncRNA) is a kind of not encoding proteins, transcript length
Non-coding RNA molecule more than 200nt, it is the accessory substance in transcription to be initially believed to, without any biological function.By it
Position relationship between mRNA is roughly divided into five types：(1) just long-chain non-coding RNA；(2) antisense long-chain non-coding
RNA；(3) two-way long-chain non-coding RNA；(4) subtype long-chain non-coding RNA is included；(5) long-chain non-coding RNA is (i.e. between gene
Large intergenic noncoding RNA, llncRNA).In whole gene group transcription product, the ratio shared by lncRNA
Ratio of the example considerably beyond mRNA.Increasing studies have shown that lncRNA is in genetic transcription and translation, epigenetic, cell point
Important regulating and controlling effect has been played in the vital movements such as change, and unconventionality expression in kinds of tumors be present, take part in tumour
The processes such as occurrence and development, infiltration and transfer, by studying LncRNA biological function and its regulatory mechanism in tumour, energy
Enough full appreciations and the generation fierceness for understanding disease, are expected to turn into new potential tumor mark and drug targets, are tumour
Diagnosis and prognosis provide new Research Thinking.
The content of the invention
It is an object of the invention to provide a kind of mark, especially early diagnosis or targeted therapy for tumour, liver cancer
Early diagnosis or targeted therapy.
The invention provides a kind of tumor markers, is a kind of LncRNA, is named as ENST00000620463.1, is sequence
RNA shown in the sequence 1 of list.The corresponding DNA of ENST00000620463.1 are the DNA shown in the sequence 2 of sequence table.
There is at least more than 75% homology, at least more than 85% homologous from people and with the RNA or described DNA
Property, at least more than 90% homology, at least more than 95% homology, the polynucleotides of at least more than 98% homology fall within this
The protection domain of invention.
The present invention also protects product (product first) the answering in reagent preparation box for detecting the RNA or described DNA
With；The purposes of the kit is auxiliary diagnosing tumour.
The present invention also protects a kind of kit, including for detecting the RNA or described DNA product (product first)；Institute
The purposes for stating kit is auxiliary diagnosing tumour.
Product described in any of the above (product first) can be the product of DNA expressions described in detection sample to be tested.It is described
Product concretely detects primer, probe, chip, nucleic acid film bar, preparation or the reagent of DNA expressions described in sample to be tested
Box.The product concretely special primer pair, is made up of primers F and primer R；
The primers F is following (e1) or (e2)：
(e1) single strand dna shown in the sequence 3 of sequence table；
(e2) sequence 3 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3
The DNA molecular of identical function；
The primer R is following (e3) or (e4)：
(e3) single strand dna shown in the sequence 4 of sequence table；
(e4) sequence 4 is had by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4
The DNA molecular of identical function.
Sample to be tested described in any of the above can be people source sample.
Sample to be tested described in any of the above can be cell, tissue, internal organs, body fluid (blood, lymph), digestive juice, expectoration,
Alveole bronchus cleaning fluid, urine or excrement.
The system that the present invention is used for auxiliary diagnosis tumour, is system A or system B.
The system A includes the material and data processing equipment for being used to detect the expression of the DNA；At the data
Module 1 and module 2 are set in reason device, the module 1 has function shown in following (c1), and the module 2 has following (c2) institute
Show function：
(c1) it is respectively compared the expression for being derived from DNA described in the in vitro sample of person under test and normal healthy controls person；
(c2) according to the comparative result of (c1) according to being identified below whether the person under test is tumor patient：If it is good for described
Health collator compares, the up-regulation of the expression of DNA described in the in vitro sample of the person under test, then the person under test is or candidate
For tumor patient；Conversely, then the person under test is not or candidate is not tumor patient.
The system B includes the material and data processing equipment for being used to detect the expression of the DNA；At the data
Module 3 and module 4 are set in reason device, the module 3 has function shown in following (d1), and the module 2 has following (d2) institute
Show function：
(d1) it is respectively compared the in vitro sample for other normal structures being derived from beyond person under test's suspected tumor tissue and the tissue
DNA expression described in this；
(d2) according to the comparative result of (d1) according to being identified below whether the person under test is tumor patient：If with it is described just
Often tissue is compared, and the expression up-regulation of DNA described in the suspected tumor tissue of the person under test, then the person under test is or waited
Elect tumor patient as；Conversely, then the person under test is not or candidate is not tumor patient.
In vitro sample described in any of the above can be cell, tissue, internal organs, body fluid (blood, lymph), digestive juice, expectoration,
Alveole bronchus cleaning fluid, urine or excrement.
The special primer is to falling within protection scope of the present invention.
The present invention also protects the RNA or described DNA as target spot in auxiliary diagnosis tumour and/or neoplasm targeted therapy
And/or the application in screening tumour medicine.
The tumour medicine is prevention and/or the medicine for the treatment of tumour.
The method of the screening tumour medicine comprises the following steps：(1) using candidate substances processing expression and/or containing
State DNA system；The parallel control for not using candidate substances to handle is set；(2) after completing step (1), described in detection architecture
DNA expression；Compared with parallel control, if using candidate substances handle system described in DNA expression quantity it is notable
Reduction (reducing by more than 20%, relatively adding reduces by more than 50, more reduction by more than 80%), the candidate substances can be as the swollen of candidate
Tumor medicine.
The system can be cell system, subcellular fraction system, solution system, organizational framework, organ systems or animal system.
The present invention also protect for suppress the RNA or described DNA expression material in product (product second) is prepared
Using；The purposes of the product is at least one of following (f1)-(f7)：
(f1) cancer cell multiplication is suppressed；
(f2) cancer cell-apoptosis is promoted；
(f3) cancer metastasis is suppressed；
(f4) cancer cell diffusion is suppressed；
(f5) metastases are suppressed；
(f6) tumour diffusion is suppressed；
(f7) tumour is treated.
The present invention also protects a kind of product (product second), the thing that its active component is expressed for the suppression RNA or described DNA
Matter；The purposes of the product is at least one of following (f1)-(f7)：
(f1) cancer cell multiplication is suppressed；
(f2) cancer cell-apoptosis is promoted；
(f3) cancer metastasis is suppressed；
(f4) cancer cell diffusion is suppressed；
(f5) metastases are suppressed；
(f6) tumour diffusion is suppressed；
(f7) tumour is treated.
The material of expression " suppress the RNA or described DNA " described in any of the above can be using the DNA or its transcript as
Target sequence and the disturbing molecule that can suppress the DNA expression or transcription, specifically may include shRNA (children purpura nephritis), small interference
RNA (siRNA), dsRNA, Microrna, antisensenucleic acids, or, can express or be formed the shRNA, siRNA are dsRNA, micro-
The construction of tiny RNA, antisensenucleic acids.
" material for suppressing the RNA or described DNA expression " can be compound or chemotherapeutics.The chemotherapeutics can wrap
Include micro-pipe activator, alkylating agent, anti-superfluous raw antimetabolite, platinum-like compounds, the alkylating agents of DNA mono-, antitumor antibiotics agent, anti-generation
Thank agent, microtubule stabilizing agent, tubulin destabilizing agent, hormone antagonist, topoisomerase enzyme inhibitor, protein kinase suppression
Agent, HMG-COA inhibitor, CDK inhibitor, cyclin inhibitors, flesh day protease inhibitors, metalloprotein enzyme level
Agent, antisensenucleic acids, triple helix DNA, virus, bacterium and the exotoxin reagent of aptamer or molecular modification.
Described the material of expression " suppress the RNA or described DNA " concretely shRNA, be shRNA1 or shRNA2 or
shRNA3。
The shRNA1 is made up of positive-sense strand 1 and antisense strand 1；The positive-sense strand 1 is as shown in the sequence 5 of sequence table；It is described
Antisense strand 1 is as shown in the sequence 6 of sequence table.
The shRNA2 is made up of positive-sense strand 2 and antisense strand 2；The positive-sense strand 2 is as shown in the sequence 7 of sequence table；It is described
Antisense strand 2 is as shown in the sequence 8 of sequence table.
The shRNA3 is made up of positive-sense strand 3 and antisense strand 3；The positive-sense strand 3 is as shown in the sequence 9 of sequence table；It is described
Antisense strand 3 is as shown in the sequence 10 of sequence table.
Product described in any of the above (product second) can be pharmaceutical composition.
Described pharmaceutical composition includes " material for suppressing the RNA or described DNA expression " described in any of the above.
Described pharmaceutical composition may also include pharmaceutically acceptable carrier.The pharmaceutically acceptable carrier can be slow
Electuary, emulsifying agent, suspending agent, stabilizer, preservative, physiological saline, excipient, filler, coagulating agent and blender, interface are lived
Property agent, diffusant or defoamer.
Described pharmaceutical composition may also include pharmaceutical acceptable carrier.Described pharmaceutical acceptable carrier can be virus, micro-capsule, liposome,
Nano particle or polymer and its any combination.The delivering supporting agent of described pharmaceutical acceptable carrier can gather for liposome, biocompatibility
Compound (including natural polymer and synthetic polymer), lipoprotein, more skins, polysaccharide, lipopolysaccharides, artificial viral envelope, inorganic (bag
Include metal) particle and bacterium or virus (such as baculoviral, adenovirus and retrovirus), bacteriophage, sticking grain or plasmid
Carrier.
Described pharmaceutical composition can also with the drug combination of other treatment tumour, other therapeutic compound can with it is main
Active component be administered simultaneously, or even be administered simultaneously in same composition.
Described pharmaceutical composition can also be with single composition or the dosage form list different from main active component
Solely give other therapeutic compounds.The Fractional of main component can be administered simultaneously with other therapeutic compounds, and its
Its dosage can be administered alone.Over the course for the treatment of, can be according to the order of severity of symptom, the frequency of recurrence and therapeutic scheme
Physiologic response, adjust the dosage of pharmaceutical composition of the present invention.
The shRNA1 or shRNA2 or shRNA3 fall within protection scope of the present invention.
Tumour described in any of the above can be entity tumor or neoplastic hematologic disorder.The tumour concretely liver cancer, lung cancer, colon
Cancer, colorectal cancer, breast cancer, oophoroma, cervical carcinoma, stomach cancer, kidney, cancer of pancreas, prostate cancer, lymthoma, glioma or
Melanoma.
Cancer cell described in any of the above can be liver cancer cells.The liver cancer cells concretely HepG2 cell lines,
Huh7 or SMMC7721.
The present inventor's in-depth study by general, by high throughput method, using high-flux sequence, detect liver
LncRNA expression in cancerous tissue and cancer beside organism, find that wherein there is the lncRNA fragments of obvious differential expression, inquire into
Its relation between the generation of liver cancer, so as to which the early detection for liver cancer and targeted therapy find more preferable approaches and methods.
By screening, present invention firstly discovers that ENST00000620463.1 conspicuousnesses raise in liver cancer.It is demonstrated experimentally that shRNA is disturbed
It silence ENST00000620463.1, can effectively suppress propagation, migration and the invasion and attack of liver cancer cells, be the personalization of liver cancer
Treatment provides new way.
Brief description of the drawings
Fig. 1 is to detect expression figures of the ENST00000620463.1 in liver cancer patient using QPCR.
Fig. 2 is ROC curve figures of the ENST00000620463.1 in liver cancer patient.
Fig. 3 is to detect expression figures of the ENST00000620463.1 in liver cancer cells using QPCR.
Fig. 4 is that expression of the detection transfection siRNA on ENST00000620463.1 in liver cancer cells influences figure.
Fig. 5 is the influence figure that ENST00000620463.1 cell proliferations are detected using CCK8.
Fig. 6 is influence figures of the detection ENST00000620463.1 to the Clone formation colony of cell.
Fig. 7 is influence figures of the detection ENST00000620463.1 to hepatoma cell apoptosis.
Fig. 8 is that Transwell cells detect ENST00000620463.1 to fucosylation and the influence figure of invasion and attack；
Wherein, it is influence figures of the ENST00000620463.1 to fucosylation to scheme A, and figure B is ENST00000620463.1 to liver
The influence figure of cancer cell invasion.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, it is conventional method unless otherwise specified.Test material used in following embodiments, it is certainly unless otherwise specified
What routine biochemistry reagent shop was commercially available.In a particular embodiment of the present invention, experiment is all according to being at least repeated 3 times
Into, result data is represented in a manner of mean+SD, is counted using the statistical softwares of SPSS 20.0
Analysis, difference between the two is examined using t, it is believed that has statistical significance as P ＜ 0.05.
The screening of embodiment 1, liver cancer marker
Cancerous tissue and pairing cancer beside organism to 20 liver cancer patients carry out two generation sequencing analysis (the equal informed consent of patient,
The acquirement of above-mentioned all samples is by the agreement of the committee of organizational ethics), a liver cancer marker is found, for one kind
LncRNA, ENST00000620463.1 is named as, as shown in the sequence 1 of sequence table.The corresponding DNA of ENST00000620463.1
As shown in the sequence 2 of sequence table.Compared with cancer beside organism, expressions of the ENST00000620463.1 in liver cancer tissue shows
Work is higher than cancer beside organism.
The differential expression of embodiment 2, qPCR sequence verifications ENST00000620463.1
1st, the cancerous tissue of 102 liver cancer patients of collection and cancer beside organism, the equal informed consent of patient, above-mentioned all samples take
Obtain and pass through the agreement of the committee of organizational ethics.
2nd, the tissue RNA of 1 each sample of extraction step, and reverse transcription is cDNA.
3rd, each sample cDNA obtained using step 2 carries out qPCR detections ENST00000620463.1 expression feelings as template
Condition (using GAPDH as reference gene).Using primers F 1 and R1 detections ENST00000620463.1 expression, using primer
The expression of F2 and R2 detection GAPDH genes.
F1：5 '-GGTGTAAGCGGTGGTTA-3 ' (sequence 3 of sequence table)；
R1：5 '-CCAGTTCTGGGCTGGGATGT-3 ' (sequence 4 of sequence table)；
F2：5’-GGTGGTCTCCTCTGACTTCAACA-3’；
R2：5’-GTTGCTGTAGCCAAATTCGTTGT-3’.
As a result it is as shown in Figure 1.As a result show, compared with cancer beside organism, ENST00000620463.1 tables in liver cancer tissue
Raised up to level, difference has statistical significance (P ＜ 0.001).
Embodiment 3, ENST00000620463.1 analyze in patient's cancerous tissue and the ROC curve of cancer beside organism
Using the Receiver Operating Characteristics of the pROC bags analysis ENST00000620463.1 in R language, it is accurate to calculate binomial
Confidence space, draw ROC curve.
As a result it is as shown in Figure 2.ENST00000620463.1 AUC is 0.85, and with preferably specificity and sensitivity
Property, illustrate that the diagnosis that ENST00000620463.1 is applied to liver cancer has higher accuracy.
The differential expression of embodiment 4, ENST00000620463.1 in liver cancer cell lines
HepG2 cell lines (Japanese JCRB cell banks, article No. are extracted respectively：JCRB1054), Huh7 (Japanese JCRB
Cell bank, article No.：JCRB0403), SMMC7721 (Beijing consonance cell bank, resource number：3111C0001CCC000087) and
Normal liver cell system LO2 (Beijing consonance cell bank, resource number：Total serum IgE 3131C0001000200006), and reverse transcription is
cDNA.The cDNA of each cell is used as template, the expression for carrying out qPCR detections ENST00000620463.1 (uses GAPDH
As reference gene).Using primers F 1 and R1 detections ENST00000620463.1 expression, detected using primers F 2 and R2
The expression of GAPDH genes.
F1：5’-GGTGTAAGCGGTGGTTA-3’；
R1：5’-CCAGTTCTGGGCTGGGATGT-3’；
F2：5’-GGTGGTCTCCTCTGACTTCAACA-3’；
R2：5’-GTTGCTGTAGCCAAATTCGTTGT-3’.
As a result it is as shown in Figure 3.As a result show, compared with normal liver cell system LO2, ENST00000620463.1 is in liver cancer
Express in cell HepG2, Huh7, SMMC7721 and raise, difference has statistical significance (P ＜ 0.001).
Embodiment 5, ENST00000620463.1 silence
1st, four groups of shRNA are designed for ENST00000620463.1, it is specific as follows shown：
(1) negative control shRNA sequences (shRNANC)：
Positive-sense strand：5′-UUCUCCGAACGUGUCACGU-3′；
Antisense strand：5’-ACGUGACACGUUCGGAGAA-3′；
(2)shRNA1：
Positive-sense strand：5 '-AACUGGUCCUUCUCGAGGUAUAGCUU-3 ' (sequence 5 of sequence table)；
Antisense strand：5 '-AAGCUAUACCUCGAGAAGGACCAGUU-3 ' (sequence 6 of sequence table)；
(3)shRNA2：
Positive-sense strand：5 '-ACAGGGCUCUCUCGAGGUAUAGCUU-3 ' (sequence 7 of sequence table)；
Antisense strand：5 '-AAGCUAUACCUCGAGAGAGCCCUGU-3 ' (sequence 8 of sequence table)；
(4)shRNA3：
Positive-sense strand is 5 ,-AAGACAAUCAUUCUCGAGGUAUAGC-3 ' (sequence 9 of sequence table)；
Antisense strand is 5 ,-AAGCUAUACCUCGAGAGAGCCCUGU-3 ' (sequence 10 of sequence table).
2nd, human hepatoma cell strain Huh7 is pressed 2 × 105Individual cells/well is inoculated into six porocyte culture plates, at 37 DEG C,
5%CO2Cell culture 24h in incubator；Four groups of shRNA (concentration is 20nM/ holes) of step 1 are used into liposome transfection respectively
Reagent 2000 (Invitrogen companies) transfection Huh7 cells (transfection method is with reference to body transfection reagent reagent specification), after 48h
Cell is collected, extracts total serum IgE, and reverse transcription is cDNA.CDNA is used as template, carries out qPCR detections
ENST00000620463.1 expression (using GAPDH as reference gene).Detected using primers F 1 and R1
ENST00000620463.1 expression, the expression of GAPDH genes is detected using primers F 2 and R2.
F1：5’-GGTGTAAGCGGTGGTTA-3’；
R1：5’-CCAGTTCTGGGCTGGGATGT-3’；
F2：5’-GGTGGTCTCCTCTGACTTCAACA-3’；
R2：5’-GTTGCTGTAGCCAAATTCGTTGT-3’.
As a result it is as shown in Figure 4.As a result show, compared to negative control shRNA groups, shRNA1 groups, shRNA2 and shRNA3 groups
ENST00000620463.1 expression can be significantly reduced, difference has statistical significance (P ＜ 0.01, P ＜ 0.05, P ＜
0.05)。
Embodiment 6, CCK8 detection cell proliferation experiments
1st, the Huh7 cells of logarithmic proliferation phase are inoculated in 96 orifice plates, per hole 2 × 103Individual cell, at 37 DEG C, 5%CO2Training
Support cell culture 24h in case.
2nd, 96 orifice plates of step 1 are taken, shRNANC the and shRNA1 transfection HepG 2 cells for respectively designing embodiment 5 (turn
Dyeing method is with reference to body transfection reagent reagent specification), 10 μ l/ hole CCK8 examinations are added after 24h, 48h, 72h and 96h is transfected respectively
Agent；ELIASA detection A450nm light absorption value is used after addition CCK8 after 2h.
As a result it is as shown in Figure 5.As a result show, transfect shRNA1 groups vitro growth rates substantially low control group cell give birth to
Long speed, difference have statistical significance (P ＜ 0.01), and the above results show that ENST00000620463.1 expression can promote
Enter the growth of liver cancer cells.
Embodiment 7, soft-agar cloning form experiment
1st, the Huh7 cells of logarithmic proliferation phase are inoculated in 6 orifice plates, per hole 1.5 × 105Individual cell, at 37 DEG C, 5%CO2Training
Support in case and cultivate 24h.
2nd, (transfection method is with reference to body transfection examination for shRNANC and shRNA1 transfection the Huh7 cells respectively designed embodiment 5
Agent reagent specification), cell is collected after 24h is transfected, cell concentration is adjusted using the DMEM culture mediums containing 20% hyclone
For 5 × 103Individual cell/ml, obtains cell suspension.
3rd, the low melting point that two concentration are respectively 1.2% (weight/mass percentage composition) and 0.7% (weight/mass percentage composition) is prepared
Agar liquid glucose, after autoclaving, maintain in 40 DEG C of water-baths.
4th, take 1.2% (weight/mass percentage composition) low melting point agar liquid glucose in 1 parts by volume step 3 and 1 parts by volume 2 ×
After the mixing of DMEM culture mediums, 2 × gentamicin (100 μ g/mL) and the calf serum of 20% (percent by volume) are added, is mixed
Close liquid.Take in 3ml mixed liquors injection diameter 6cm plates and place 5min cooled and solidifieds, be placed in as bottom-layer agar standby in incubator.
5th, take 0.7% (weight/mass percentage composition) low melting point agar liquid glucose in 1 parts by volume step 3 and 1 parts by volume 2 ×
After the mixing of DMEM culture mediums, the cell suspension that 0.2ml steps 1 obtain, the diameter that after fully mixing prepared by implantation step 3 are added
In 6cm plates, double agar layers are formed, each experimental group repeats 4 samples.After top-layer agar solidification, plate is inserted 37 DEG C,
Cultivate, every 3 days plus DMEM culture medium 1.5ml, co-culture 14 days in 5%CO2 incubators.
6th, the plate of step 4 is taken out, 90min is dyed with the gentian violet that 1ml concentration is 0.005%.Plate is placed on down
Micro- Microscopic observation is put, every group of cell randomly selects 10 low-power fields, and the number of cell clones of formation is calculated under mirror.
As a result it is as shown in Figure 6.As a result show, compared with control group, transfect the thin of shRNA1-ENST00000620463.1
Born of the same parents organize single cell clone Colony forming number and significantly reduced (P ＜ 0.001).
The influence of embodiment 8, ENST00000620463.1 to hepatoma cell apoptosis
1st, the Huh7 cells of logarithmic proliferation phase are inoculated in 12 orifice plates, per hole 1.0 × 105Individual cell, at 37 DEG C, 5%CO2
24h is cultivated in incubator.
2nd, (transfection method is with reference to body transfection examination for shRNANC and shRNA1 transfection the Huh7 cells respectively designed embodiment 5
Agent reagent specification), cell, Annexin/FITC dyeing detection Apoptosis are collected after 24h is transfected.
As a result it is as shown in Figure 7.As a result show, compared with control group, transfect the thin of shRNA1-ENST00000620463.1
Born of the same parents organize apoptosis rate rise (P ＜ 0.001), and the result illustrates, ENST00000620463.1 expression inhibiting liver cancer cells
Apoptosis.
Embodiment 9, cell migration and Matrigel
1st, Matrigel is ice bath melted under aseptic condition, carries out 20 times of dilutions with PBS, is layered on the volume in 50 μ l/ holes
On the poly- carbonic acid vinegar film of Transwell cells.37 DEG C of placement 4h, take out after Matrigel glue aggregates into gel, gently suction out
Upper strata separates out liquid.The serum-free medium containing BSA that 50 μ l are added in per hole carries out hydration process, 37 DEG C of placements to basilar memebrane
30min。
2nd, the Huh7 cells of logarithmic proliferation phase are inoculated in 24 orifice plates, per hole 5 × 104Individual cell, at 37 DEG C, 5%CO2Training
Support in case and cultivate 24h.
3rd, (transfection method is with reference to body transfection examination for shRNANC and shRNA1 transfection the Huh7 cells respectively designed embodiment 5
Agent reagent specification), after 24h is transfected, plasma-free DMEM medium culture 12h is changed, cell is collected, using serum-free DMEM
Culture medium adjustment cell concentration is 5 × 105Individual cell/ml, obtains cell suspension.
4th, the μ l of cell suspension 200 (migration experiment is 100 μ l, and Matrigel is 200 μ l) for taking step 3 to prepare are added to
In Transwell cells.Room adds 1640 culture mediums of the 500 μ l containing FBS under 24 orifice plates.Cell is put into cell culture incubator
Cultivate 24h.
5th, after completing step 4, small ventricular cell is first rinsed 2 times with PBS, room temperature in DAPI working solutions is put into and dyes 5-
20min.Rinsed 2 times with PBS, be put into fluorescence microscopy Microscopic observation and count.
As a result it is as shown in Figure 8.As a result show, compared with control group, transfect the thin of shRNA1-ENST00000620463.1
Born of the same parents organize cell migration and invasive ability is decreased obviously (P ＜ 0.001), as a result illustrate that ENST00000620463.1 can promote
Enter the migration and invasion and attack of liver cancer cells.
Claims (10)
1.RNA, for following (a1) or (a2)；
(a1) RNA shown in the sequence 1 of sequence table；
(a2) from people and with (a1) have at least more than 75% homology, at least more than 85% homology, at least 90% with
The polynucleotides of upper homology, at least more than 95% homology, at least more than 98% homology.
2.DNA, for following (b1) or (b2)；
(b1) RNA shown in the sequence 2 of sequence table；
(b2) from people and with (b1) have at least more than 75% homology, at least more than 85% homology, at least 90% with
The polynucleotides of upper homology, at least more than 95% homology, at least more than 98% homology.
3. require application of the product of DNA described in 1 RNA or claim 2 in reagent preparation box for test right；Institute
The purposes for stating kit is auxiliary diagnosing tumour.
4. a kind of kit, including the product for DNA described in test right 1 RNA of requirement or claim 2；The examination
The purposes of agent box is auxiliary diagnosing tumour.
It is system A or system B 5. for the system of auxiliary diagnosis tumour；
The system A includes the material and data processing equipment for being used for the expression that test right requires 2 DNA；It is described
Module 1 and module 2 are set in data processing equipment, the module 1 has function shown in following (c1), and the module 2 has as follows
(c2) function shown in：
(c1) it is respectively compared the expression for being derived from DNA described in the in vitro sample of person under test and normal healthy controls person；
(c2) according to the comparative result of (c1) according to being identified below whether the person under test is tumor patient：It is if right with the health
Compared according to person, the expression up-regulation of DNA described in the in vitro sample of the person under test, then the person under test is or candidate is swollen
Knurl patient；Conversely, then the person under test is not or candidate is not tumor patient；
The system B includes the material and data processing equipment for being used for the expression that test right requires 2 DNA；It is described
Module 3 and module 4 are set in data processing equipment, the module 3 has function shown in following (d1), and the module 2 has as follows
(d2) function shown in：
(d1) it is respectively compared in the in vitro sample for other normal structures being derived from beyond person under test's suspected tumor tissue and the tissue
The expression of the DNA；
(d2) according to the comparative result of (d1) according to being identified below whether the person under test is tumor patient：If with the normal group
Knit and compare, the expression up-regulation of DNA described in the suspected tumor tissue of the person under test, then the person under test is or candidate is
Tumor patient；Conversely, then the person under test is not or candidate is not tumor patient.
6. a species-specific primers pair, it is made up of primers F and primer R；
The primers F is following (e1) or (e2)：
(e1) single strand dna shown in the sequence 3 of sequence table；
(e2) have by sequence 3 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 3 identical
The DNA molecular of function；
The primer R is following (e3) or (e4)：
(e3) single strand dna shown in the sequence 4 of sequence table；
(e4) have by sequence 4 by the substitution of one or several nucleotides and/or missing and/or addition and with sequence 4 identical
The DNA molecular of function.
7. DNA described in RNA described in claim 1 or claim 2 controls as target spot in auxiliary diagnosis tumour and/or cancer target
Treat and/or screen the application in tumour medicine.
8. for suppressing application of the material of DNA expression described in RNA described in claim 1 or claim 2 in product is prepared；
The purposes of the product is at least one of following (f1)-(f7)：
(f1) cancer cell multiplication is suppressed；
(f2) cancer cell-apoptosis is promoted；
(f3) cancer metastasis is suppressed；
(f4) cancer cell diffusion is suppressed；
(f5) metastases are suppressed；
(f6) tumour diffusion is suppressed；
(f7) tumour is treated.
9. a kind of product, the material that its active component is expressed for DNA described in RNA described in suppression claim 1 or claim 2；
The purposes of the product is at least one of following (f1)-(f7)：
(f1) cancer cell multiplication is suppressed；
(f2) cancer cell-apoptosis is promoted；
(f3) cancer metastasis is suppressed；
(f4) cancer cell diffusion is suppressed；
(f5) metastases are suppressed；
(f6) tumour diffusion is suppressed；
(f7) tumour is treated.
10.shRNA, it is shRNA1 or shRNA2 or shRNA3；
The shRNA1 is made up of positive-sense strand 1 and antisense strand 1；The positive-sense strand 1 is as shown in the sequence 5 of sequence table；The antisense
Chain 1 is as shown in the sequence 6 of sequence table；
The shRNA2 is made up of positive-sense strand 2 and antisense strand 2；The positive-sense strand 2 is as shown in the sequence 7 of sequence table；The antisense
Chain 2 is as shown in the sequence 8 of sequence table；
The shRNA3 is made up of positive-sense strand 3 and antisense strand 3；The positive-sense strand 3 is as shown in the sequence 9 of sequence table；The antisense
Chain 3 is as shown in the sequence 10 of sequence table.
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| CN108796064A (en) * | 2018-06-22 | 2018-11-13 | 北京艾瑟尔生物医学研究有限公司 | The marker of auxiliary detection Acute Exposed Altitude hypobaric hypoxia environment related myocardium damage |
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| CN109517894A (en) * | 2018-08-31 | 2019-03-26 | 清华大学 | One kind non-coding RNA biomarker relevant to liver cancer and its application |
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| CN108796064A (en) * | 2018-06-22 | 2018-11-13 | 北京艾瑟尔生物医学研究有限公司 | The marker of auxiliary detection Acute Exposed Altitude hypobaric hypoxia environment related myocardium damage |
| CN108841961A (en) * | 2018-07-26 | 2018-11-20 | 泰山医学院 | Application of the LINC01702 in the diagnostic kit of preparation hepatocellular carcinoma |
| CN108841961B (en) * | 2018-07-26 | 2019-07-05 | 泰山医学院 | Application of the LINC01702 in the diagnostic kit of preparation hepatocellular carcinoma |
| CN109517894A (en) * | 2018-08-31 | 2019-03-26 | 清华大学 | One kind non-coding RNA biomarker relevant to liver cancer and its application |
| CN109628594A (en) * | 2018-12-27 | 2019-04-16 | 河北医科大学第二医院 | One kind long-chain non-coding RNA relevant to liver cancer and its application |
| CN110804613A (en) * | 2019-12-09 | 2020-02-18 | 南京苏粤再生医学研究院有限公司 | Application of siRNA for targeted inhibition of lncRNA-00861 gene expression in liver cancer treatment |
| CN110804613B (en) * | 2019-12-09 | 2023-08-15 | 南京苏粤再生医学研究院有限公司 | Application of siRNA for targeted inhibition of lncRNA-00861 gene expression in liver cancer treatment |
| CN111518906A (en) * | 2020-05-12 | 2020-08-11 | 广州医科大学附属第五医院 | Application of lncRNA01622 in recurrence prediction and treatment of hepatocellular carcinoma |
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