CN106978418A - The shRNA interference sequences of people's PYCR1 genes and its application - Google Patents

The shRNA interference sequences of people's PYCR1 genes and its application Download PDF

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CN106978418A
CN106978418A CN201710067075.5A CN201710067075A CN106978418A CN 106978418 A CN106978418 A CN 106978418A CN 201710067075 A CN201710067075 A CN 201710067075A CN 106978418 A CN106978418 A CN 106978418A
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pycr1
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夏伟
叶颖
王国玉
庄菊花
倪晶
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Shanghai Seventh Peoples Hospital
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Abstract

The invention discloses the purposes of people's PYCR1 genes and its related related drugs.The invention discloses purposes of the people PYCR1 genes in oncotherapy, diagnosing tumor and medicine preparation.The present invention also further constructs people's PYCR1 genes small molecules interference RNA, people's PYCR1 gene RNAs construct, people PYCR1 genes interference slow virus, and discloses their purposes.The siRNA that the present invention is provided or the nucleic acid construct comprising the siRNA sequence, slow virus are capable of the expression of specificity suppression people's PYCR1 genes, especially slow virus, target cell can efficiently be infected, high efficiency suppresses the expression of PYCR1 genes in target cell, and then suppress the growth of tumour cell, promote apoptosis of tumor cells, it is significant in oncotherapy.

Description

The shRNA interference sequences of people's PYCR1 genes and its application
Technical field
The invention belongs to field of biological medicine, and in particular to a kind of shRNA interference sequences of people PYCR1 genes and its should With.
Background technology
With human lives be accustomed to and behavior pattern change, and the environmental pollution that brings of agro-industry evolution and people The reasons such as mouth aging, malignant tumour turns into the lethal principal disease of the mankind.IARC of the World Health Organization (IARC/WHO) latest data shows that the incidence of disease of the malignant tumour in the whole world is in rising trend, and 7,600,000 people die from evil within 2008 Property tumour, wherein 64% occur in developing country.In China, the situation that treatment and prevention of tumour faces is also extremely severe, newest report It has been shown that, between Past 30 Years, the morbidity and mortality of China's malignant tumour are in obvious ascendant trend；It was predicted that in future Between 20-30, the trend that China's Cancer Mortality and the death rate constantly rise will be continued to.Therefore, tumour is ground It is one of lasting study hotspot of life science and medical domain to study carefully.
The limitation of malignant tumour traditional remedies such as radiotherapy, chemotherapy and operative treatment promotes people to find new antitumor side Method.With going deep into tumor development molecular mechanism understanding, people have appreciated that tumour is a kind of genopathy, correct and occur The gene of defect can provide new hope for the treatment of tumour.Many gene therapy methods for being directed to tumour are developed in recent years Research, but the clinical efficacy so far into several genomic medicines of clinical practice is limited, and with potential, long-term , the side effect of delay, therefore, seek the novel targets for the treatment of and develop new gene therapy method to turn into domestic and international tumour base Because of the research direction for the treatment of.
RNA interference (RNA interference, RNAi) is a Gene silence, is a kind of double-stranded RNA (double-stranded RNA, dsRNA) molecule blocks the expression of specific gene in mRNA level in-site or makes the mistake of its silence The PTGS (Post-transcriptional gene silencing, PTGS) of journey, i.e. sequence-specific.Closely Nian Lai, RNAi turn into the available strategy of therapy of tumor.It can suppress proto-oncogene, the suppression being mutated using RNAi technology Oncogene, Cell cycle-related genes, anti-apoptotic related gene etc. express to suppress the occurrence and development of tumour.
PYCR1 is located at No. 17 chromosomes, and coding pyrrolin -5 carboxylate reductase 1 belongs to the carboxylate reductase man of pyrrolin -5 Race, its major function is that the carboxylic acid of pyrrolin -5 is reduced into proline, and PYCR1 is widely present in eucaryote, adjusts cell Interior signal transduction, and then a variety of extracellular stimulations are responded.
Multiple members of PYCR families are related to cancer generation development, and PYCR1 genes are considered as and age and/or aging Relevant gene, such as its in treatment as by genetic modification or modified it in animal in cutis laxa, wrinkly skin syndrome disease Internal expression carried out research, but only showed the tendency of experimental level.
The content of the invention
The invention discloses the purposes of people's PYCR1 genes and its related related drugs.The invention discloses people's PYCR1 bases Because of the purposes in oncotherapy, diagnosing tumor and medicine preparation.The present invention also further constructs people's PYCR1 gene small molecules RNA interfering, people's PYCR1 gene RNAs construct, people PYCR1 genes interference slow virus, and disclose their purposes. The siRNA that the present invention is provided or the nucleic acid construct comprising the siRNA sequence, slow virus being capable of specificity suppression people's PYCR1 bases The expression of cause, especially slow virus, can efficiently infect target cell, and high efficiency suppresses the expression of PYCR1 genes in target cell, entered And suppress the growth of tumour cell, and promote apoptosis of tumor cells, it is significant in oncotherapy.
Therefore, the first object of the present invention is to provide a kind of shRNA interference sequences of people PYCR1 genes, including DNAoligo positive strand sequences and anti-chain sequence, wherein, the positive strand sequence is：
5’-CCGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAG AAACTGTGTTTTTG-3’(SEQ ID NO1)；
The anti-chain sequence is：
5’-AATTCAAAAACACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGA GAGCAGAAACTGTG-3’(SEQ ID NO2)。
Preferably, in the shRNA interference sequences of people PYCR1 genes of the present invention, the DNA oligo normal chain sequences Row include siRNA target sequences with anti-chain sequence：
5’-CAGTTTCTGCTCTCAGGAA-3’(SEQ ID NO3)。
Present invention also offers a kind of shRNA slow virus carriers of people PYCR1 genes, the people PYCR1 genes ShRNA slow virus carriers are for the siRNA target sequences of people's PYCR1 genes：
5’-CAGTTTCTGCTCTCAGGAA-3’。
Another object of the present invention also resides in a kind of people PYCR1 gene siRNAs of offer, wherein, encode the people PYCR1 bases Because the siRNA siRNA target sequences for including being directed to people's PYCR1 genes are：
5’-CAGTTTCTGCTCTCAGGAA-3’。
A further object of the present invention also resides in offer people's PYCR1 gene siRNA nucleic acid carriers, wherein the people PYCR1 bases Because the siRNA target sequences that siRNA nucleic acid carriers include being directed to people's PYCR1 genes are：
5’-CAGTTTCTGCTCTCAGGAA-3’。
Preferably, in people PYCR1 gene siRNA nucleic acid carriers of the present invention, the people PYCR1 gene siRNAs Nucleic acid carrier is SEQ ID NO4：
5’-GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTG AAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACA CCGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAACT GTGTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCATCCACGAA TTCGGATCCATTAGGCGGCCGCGTGGATAACCGTATTACCGCCATGCATT AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATG GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTA ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTA AACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCC CTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCA-3’。
It is including following another aspect of the invention is the construction method that slow virus is disturbed there is provided a kind of people PYCR1 genes Step：
1) synthesis is for the DNAoligo normal chains and anti-chain of the RNA interfered target sequences of people's PYCR1 genes, the normal chain sequence It is classified as：
5’-CCGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAG AAACTGTGTTTTTG-3’；
The anti-chain sequence is：
5’-AATTCAAAAACACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGA GAGCAGAAACTGTG-3’；
2) double-stranded DNA of positive strand sequence and anti-chain the sequence anneals formation with cohesive end；
3) vector linearization being made by digestion, makes linearized vector and step 1) obtained double-stranded DNA is connected；
4) connection product is converted into competent escherichia coli cell, performing PCR identification is entered to obtained positive colony；
5) plasmid extraction, virus packaging.
Preferably, the present inventor PYCR1 genes interference slow virus construction method in, the step 3) in endonuclease reaction To add AgeI and EcoRI double digestions.
Preferably, in the construction method of the present inventor PYCR1 genes interference slow virus, the step 4) connection product turn Change competent escherichia coli cell specific method be：
Connection product is added in competent escherichia coli cell, ice bath；Heat shock, ice bath；Add the LB of antibiotic-free Fluid nutrient medium, shaking table concussion and cultivate；Bacterium solution is taken uniformly to be applied on the LB solid mediums containing Amp, in 37 DEG C of incubators Middle incubated overnight；Positive colony enters in performing PCR qualification process
What is used identifies primer for identification primer-F：5’-CCTATTTCCCATGATTCCTTCATA -3’(SEQ ID ) and identification primer-R NO5:5 '-GTAATACGGTTATCCACGCG-3 ' (SEQ ID NO6), detailed process is：Picking single bacterium colony Enter performing PCR amplification, electrophoresis, sequencing.
Built present invention also offers the shRNA interference sequences of above-mentioned people PYCR1 genes, people's PYCR1 genes RNA Body, people PYCR1 genes interference slow virus are preparing the growth of suppression tumour cell, promote apoptosis of tumor cells, prevent or treat Application in the medicine of tumour.
From subsequent embodiment upper and of the present invention, method of the invention and the sequence of offer, carrier and slow virus are extremely Rare advantages below：
PYCR1 genes are typically considered the gene relevant with age and/or aging, such as its treatment as cutis laxa, By genetic modification or modify its expression in animal body in the diseases such as wrinkly skin syndrome and carried out research, and only show The tendency of experimental level, does not embody the effect for suppressing related gene expression in cellular level.Secondly, PYCR1 genes RNAi suppressing methods are influenceed in cellular level by multilevel factor, in particular how select for people's PYCR1 genes SiRNA target sequences, how application lentivirus mediated RNAi methods realize suppress related gene expression all the time for puzzlement The Major Difficulties of related researcher.
And present inventors discovered unexpectedly that, using specific siRNA target sequences for people's PYCR1 genes, And the RNAi methods of suitable carrier and suitably lentivirus mediated, in expression of the reduction PYCR1 genes in tumour cell Afterwards, the propagation of tumour cell can effectively be suppressed.The study show that, PYCR1 genes are a proto-oncogenes, can promote tumour Cell breed, in tumour occurrence and development have important biological function, PYCR1 genes can as oncotherapy target Mark, the PYCR1 gene specifics silence of lentivirus mediated can as oncotherapy a kind of new tool.
Brief description of the drawings
Fig. 1 is one embodiment of the invention linear carrier restriction enzyme digestion and electrophoresis figure；
Fig. 2 is rna interference vector structure and positive clone identification schematic diagram in one embodiment of the invention；
Fig. 3 is one embodiment of the invention positive clone identification restriction enzyme digestion and electrophoresis figure；
Fig. 4 is linear carrier GV115 collection of illustrative plates figures in one embodiment of the invention；
Fig. 5 infects human liver cancer BEL-7404 cells after 3 days for PYCR1-RNAi slow virus in one embodiment of the invention, PYCR1mRNA expression block diagrams；
Fig. 6 infects human liver cancer SMMC-7721 cells after 3 days for PYCR1-RNAi slow virus in one embodiment of the invention, PYCR1mRNA expression block diagrams；
Fig. 7 infects human liver cancer BEL-7404 cells after 5 days for PYCR1-RNAi slow virus in one embodiment of the invention, Cause cell inhibitory effect figure；
Fig. 8 infects human liver cancer SMMC-7721 cells after 5 days for PYCR1-RNAi slow virus in one embodiment of the invention, Cause cell inhibitory effect figure；
Fig. 9 is infected after human liver cancer BEL-7404 cells for PYCR1-RNAi slow virus in one embodiment of the invention, cell Clonality declines signal block diagram；
Figure 10 is infected after human hepatocarcinoma BEL-7402 for PYCR1-RNAi slow virus in one embodiment of the invention, carefully Born of the same parents' clonality declines signal block diagram；
Figure 11 is infected after human liver cancer BEL-7404 cells for PYCR1-RNAi slow virus in one embodiment of the invention, is promoted Enter Apoptosis signal block diagram；
Figure 12 is infected after human liver cancer BEL-7404 cells for PYCR1-RNAi slow virus in one embodiment of the invention, is promoted Enter Apoptosis signal block diagram.
Embodiment
Further technical scheme is illustrated below by way of specific embodiment, it should be understood that be only this hair below Bright exemplary illustration, is not intended to limit the invention scope of the claims.
Embodiment 1：For the preparation of people PYCR1 gene RNAi slow virus
1RNA disturbance target points are designed and prepared by double-stranded DNA oligo
(1) effective siRNA target spot of the design synthesis for people's PYCR1 genes
PYCR1 (NM_153824) gene information is transferred from Genbank；Effective siRNA of the design for PYCR1 genes Target spot.Table 1 is effective siRNA target sequences for PYCR1 genes.
Table 1 targets effective siRNA target sequences of people's PYCR1 genes
(2)) DNA oligo sequent synthesis
According to having selected target sequence to design shRNA interference sequences, and suitable digestion with restriction enzyme is added at two ends Site is to complete vector construction.In addition, in the end of normal chain 3 ' addition TTTTT termination signals, and the addition of the end of anti-chain 5 ' terminates letter Number complementary series.Synthesizing single-stranded DNA oligo.
Single DNA Oligo of two ends I containing Age and EcoR the I restriction enzyme site cohesive ends of table 2
* CCGG：AgeI restriction enzyme sites；AATTC：EcoRI restriction enzyme sites；G：EcoRI restriction enzyme site complementary series.
(3) prepared by double-stranded DNA oligo
The single stranded DNA oligo dry powder of synthesis is dissolved in annealing buffer (20 μM of final concentration), 90 DEG C of water-baths 15min.Naturally cool to after room temperature, form the double-strand with cohesive end.
(4) prepared by linearized vector
50 μ l reaction systems are prepared according to NEB specifications, using AgeI and EcoRI double digestion GV115 carriers (such as Fig. 4 institutes Show) linearize it.
Reaction system
37 DEG C (optimum temperature) reacts 1h, afterwards gel extraction purpose fragment.
As shown in figure 1, swimming lane 1：1kb Marker：10kb, 8kb, 6kb, 5kb, 4kb, 3.5kb are followed successively by from top to bottom, 3kb, 2.5kb, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp；
Swimming lane 2：Vector plasmid after the linearisation of Age I and EcoR I double digestions；
Swimming lane 3：There is no the vector plasmid of digestion, electrophoresis result illustrates that linearized vector is successfully constructed.
2RNA interference slow virus carriers are built
(1) connect
20 μ l reaction systems are prepared according to Fermentas T4DNA Ligase specifications, by double-stranded DNA oligo and line Property carrier be connected.
1h-3h is reacted in 16 DEG C, connection product is named as psc45562, transformation experiment is carried out afterwards.
(2) convert
Connection product is converted into competent escherichia coli cell, Detailed operating procedures are as follows：
1) 10 μ l connection products psc45562 are added in 100 μ l competent escherichia coli cells, ice bath 30min.
2) 42 DEG C of heat shock 90sec, ice bath 2min.
3) the LB fluid nutrient mediums of 500 μ L antibiotic-frees are added, 200rpm is in 37 DEG C of shaking table concussion and cultivate 1hr.
4) take 150 μ l bacterium solutions to be uniformly applied on the LB solid mediums containing Amp, trained overnight in 37 DEG C of incubators Support.
(3) the PCR identifications of positive colony
1. rna interference vector is built and positive clone identification
As shown in Fig. 2 multiple cloning sites carrier (MCS) is cut to linear carrier by double enzymes (AgeI and EcoRI), pass through It is connected by T4DNAligase with dsDNA, and connection product is converted into competent escherichia coli cell, cultivates laggard performing PCR amplification With identification positive colony.
2. primer
3. PCR is expanded
According to the form below prepares 20 μ l PCR reaction systems, is template with sterile pipette tips picking single bacterium colony, enters performing PCR amplification, Reaction condition is：94℃3min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, 22 circulations；72℃5min.After PCR terminates, 5 μ l are taken Product, 1% agarose gel electrophoresis test strip.
4. electrophoresis loading explanation
As shown in figure 3, swimming lane 1：Negative control (ddH2O), external source pollution of nucleic acid causes false positive results in removal system； Swimming lane 2：From even control (empty carrier connects control group certainly)；Swimming lane 3：250bp Marker：5kb, 3kb are followed successively by from top to bottom, 2kb, 1.5kb, 1kb, 750bp, 500bp, 250bp, 100bp；Swimming lane 4-8：Monoclonal psc45562-1,2,3,4,5, as a result say It is bright：The nonvisualization of swimming lane 1, illustrates system pollution of nucleic acid；The empty carrier clone PCR clip size not connected into shRNA fragments is： 307bp (swimming lane 2).The positive colony PCR fragment size connected into shRNA fragments is：380bp (swimming lane 4-8), thus judges Psc45562-1,2,3,4,5 be positive colony, preserves qualification result and correctly clones, and is sequenced.
(4) positive colony sequencing interpretation of result
To identify that primer-F carries out positive colony sequencing, selecting the sequencing result clone completely the same with target sequence is used for Next step is tested.
Psc45562 sequencing results：
GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGA AAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACAC CGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAACT GTGTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCATCCACGAA TTCGGATCCATTAGGCGGCCGCGTGGATAACCGTATTACCGCCATGCATT AGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATG GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGC CCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTA ACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTA AACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCC CTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTAC ATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATC GCTATTACCA
* shRNA interference sequences Insert Fragment is marked with red font, and wherein AgeI restriction enzyme sites are destroyed.
3 plasmid extractions
Correct bacterium solution will be sequenced to transfer in the LB fluid nutrient mediums of 150ml antibiotic containing Amp, 37 DEG C of shaking table concussion trainings Support overnight.Plasmid is extracted according to EndoFree Maxi PlasmidKit specifications, the qualified plasmid of quality inspection enters down stream train.
Detailed operating procedures are as follows：
1.8000rpm centrifuging 4min collects thalline.
2. adding 7ml P1, concussion is mixed；
3. adding 7ml P3, overturn and mix 6~8 times, stand 5min；
4. adding 7ml P4, overturn and mix 6~8 times, ice bath 10min；
5.9000rpm centrifuges 10min, and supernatant is transferred in filter CS, and 10ml isopropanols are added after filtering and are mixed；
6. adding 2.5ml equilibrium liquid BL, 8000rpm centrifugation 2min into adsorption column, the waste liquid in collecting pipe is outwelled, will Pillar is put back to standby；
7. supernatant is poured into adsorption column in two times, 8000rpm centrifugation 2min abandon waste liquid；
8. adding 10ml rinsing liquids PW (having added absolute ethyl alcohol) into adsorption column, same rotational speed centrifugation 2min abandons useless Liquid, repeats the step once；
10. adding 3ml absolute ethyl alcohols into adsorption column, 8000rpm centrifugation 2min abandon waste liquid；
11.9500rpm skies get rid of 5min, remove the rinsing liquid of residual；
Adsorption column is transferred to a new Bai Guanzhong, 800 μ l elution buffers TB (first preheating), room temperature are added dropwise at post center 5min is placed, then 9500rpm centrifuges 2min；
12. the eluent in pipe is transferred in a clean 1.5mlEP pipes, -20 DEG C of preservations；
13. sampling electrophoresis, plasmid concentration, quality inspection are determined using spectrophotometer (Thermo_Nanodrop 2000).
14. the qualified plasmid of quality inspection is subjected to viral packaging.
4 slow virus are packed
Human embryonic kidney cells 293T, the incasing cells of slow virus, be anchorage dependence type into epithelioid cell, growth medium is DMEM (contains 10%FBS), and attached cell is through cultivating growing multiplication formation cell monolayer.Coli strain DH5 α, for expanding Slow virus carrier and auxiliary package carrier plasmid.
Three kinds of DNAs in RNAi plasmids, slow virus packaging system are extracted with the plasmid extraction kit of Qiagen companies (GV vector plasmids, the vector plasmids of pHelper 1.0, pHelper2.0 vector plasmids), is configured to 100ng/ul storing liquids.
24h before transfection, with the 293T cells of Trypsin Induced exponential phase, to be adjusted containing the culture medium of 10% serum Cell density about 5x 106Cell/15ml, is re-seeded into 10cm Tissue Culture Dish, 37 DEG C, the interior culture of 5%CO2 incubators. 24h can be used to transfection when cell density is up to 70%~80%；2h is replaced by serum free medium before transfection；To one sterilize from Prepared each DNA solution (the μ g of GV vector plasmids 20, pHelper 1.0 vector plasmid 15 μ g, pHelper is added in heart pipe μ g of 2.0 vector plasmid 10), the transfection reagent of respective volume is not well mixed, and adjustment cumulative volume is 1ml, is incubated at room temperature 15min；Mixed liquor is slowly added dropwise into 293T cell culture fluids, is mixed, and is cultivated in 37 DEG C, 5%CO2 cell culture incubators；Training Support and the culture medium containing transfection mixture is discarded after 6h, the PBS liquid cleaning for adding 10ml once, softly rocks culture dish to wash Abandoned after the transfection mixture for washing remnants；The cell culture medium 20ml containing 10% serum is slowly added to, is trained in 37 DEG C, 5%CO2 Support and continue to cultivate 48-72h in case.
Slow virus concentrates and purifying：According to cell state, the 293T for collecting 48h after transfection (transfection can be calculated as 0h) is thin Born of the same parents' supernatant；In 4 DEG C, 4000g centrifugation 10min, cell fragment is removed；Exceeded the speed limit with 0.45 μm of filter filtering supernatant in 40ml In centrifuge tube；Trim sample, the ultracentrifugation pipe with vial supernatant is put into Beckman ultracentrifuges one by one respectively Interior, setting parameter of noncentricity is 25000rpm, and centrifugation time is 2h, and centrifuging temperature is controlled at 4 DEG C；After centrifugation terminates, supernatant discarding, The liquid remained on tube wall is removed as far as possible, is added virus and is preserved liquid (can guard against cell culture medium with PBS to substitute), gently blows repeatedly Beat and be resuspended；After fully dissolving, after high speed centrifugation 10000rpm, 5min, supernatant is taken to dispense.
Embodiment 2RT-PCR detects the silence efficiency of PYCR1 genes
Human liver cancer BEL-7404, SMMC-7721 cell in exponential phase carries out pancreatin digestion, and cell suspension is made (cell number is about 5 × 104/ ml) 6 orifice plates are inoculated in, culture to cell fusion degree reaches about 30%.Add appropriate embodiment 1 The virus of preparation, culture changes culture medium after 6 hours, after time of infection reaches 3 days, collects cell.
RNA is extracted and cDNA synthesis：1. sample, Trizol cracking are collected:Cell is collected, 2000rpm centrifugation 5min are gone Clearly, 1mLTrizol is added in cell precipitation, is stored at room temperature 5min after fully mixing, is then transferred in new 1.5mL EP pipes； 2. often pipe adds 200 μ L chloroforms, is turned upside down EP pipe 15s with hand, is stored at room temperature 10min.3. 4 DEG C, 12800rpm, centrifugation 15min.4. draw supernatant liquid and move to new 1.5mLEP pipes, add the isopropanol of isometric precooling, 4 DEG C of standings after mixing 10min.5. 4 DEG C, 12800rpm centrifugation 12min after, abandon supernatant.6. 1mL, 75% ethanol (with DEPC water Fresh) are added, Washing precipitation.7. 4 DEG C, 11800rpm centrifugation 5min, discard most of supernatant.8. 4 DEG C, 11800rpm centrifuge 5min again, abandon Remove supernatant, drying at room temperature.9. when RNA precipitates substantially transparent, add RNase-free water (add volume regard RNA precipitate amount and It is fixed) to being completely dissolved, Nanodrop 2000/2000C spectrophotometric analysis determines the concentration and quality of institute's extracting RNA.10. will RNA reverse transcriptions acquisition cDNA (then above-mentioned system inactivates RT enzymes in 42 DEG C of water-bath 1h in 70 DEG C of water-bath 10min, will The reverse transcription product cDNA arrived).(reverse transcription reaction system is shown in Table 4).
Table 4RNA reverse transcription reaction systems
Reagent Every pipe addition
5×RT buffer 5μl
10mM dNTPs 2μl
Rnasin(40U/μL) 0.4μl
M-MLV-RTase(200U/μl) 1μl
RNase-Free H2O 5.6μl
RT-PCR:Real_time quantitative detection is carried out using the Real time PCR of the MX3000p types of Agilent companies. The primer of PYCR1 genes is as follows：- the GGCTGCCCACAAGATAATGGC-3 ' of sense primer 5 ' and anti-sense primer 5 '- CAATGGAGCTGATGGTGACGC-3’.Using house-keeping gene GAPDH as internal reference, primer sequence is as follows：Sense primer 5 '- TGACTTCAACAGCGACACCCA-3 ' and-the CACCCTGTTGCTGTAGCCAAA-3 ' of anti-sense primer 5 '.In the ratio in table 5 Configure reaction system.
Table 5RT-PCR reaction systems
Reagent Every pipe addition
SYBR premix ex taq 10.0μL
Sense primer (2.5 μM) 0.5μL
Anti-sense primer (2.5 μM) 0.5μL
cDNA 1.0μL
RNase-Free H2O 8.0μL
Setting program is two-step method Real-time PCR：95 DEG C of pre-degeneration, 30s, afterwards each 95 DEG C of step denaturation, 5s； 60 DEG C of annealing extension, 30s；45 circulations are carried out altogether.Every time light absorption value is read in the extension stage.After PCR terminates, 95 DEG C of denaturation 1min, is subsequently cooled to 55 DEG C, DNA double chain is fully combined.To 95 DEG C since 55 DEG C, each step increases by 0.5 DEG C, keeps 4s, while reading light absorption value, makes melting curve.Using 2-ΔΔCtAnalytic approach calculates the gene expression abundance for infecting PYCR1 mRNA.It is real Test result to show, PYCR1 mRNA expression has lowered 77.9% (Fig. 5) in BEL-7404 human liver cancer cell；Human liver cancer PYCR1 mRNA expression has lowered 95.7% (Fig. 6) in SMMC-7721 cells.
Embodiment 3 detects the multiplication capacity for the tumour cell for infecting PYCR1-siRNA slow virus
Human liver cancer BEL-7404, SMMC-7721 cell in exponential phase carries out pancreatin digestion, and cell suspension is made (cell number is about 5 × 104/ ml) 6 orifice plates are inoculated in, culture to cell fusion degree reaches about 30%.Add appropriate embodiment 1 The virus of preparation, culture changes culture medium after 6 hours, after time of infection reaches 5 days, collects cell.Complete medium is resuspended Into cell suspension (2 × 104/ ml), it is about 2000/hole with cell density, is inoculated in 96 orifice plates.Every group of 3 multiple holes, per hole 100ul.Complete after plate, put cell culture incubator culture.Since after bed board second day, read plate is detected once with Celigo daily, Continuous detection read plate 3-5 days；By adjusting analysis settings input parameter, each scanning holes is calculated exactly The quantity of the cell with green fluorescence in plate；Statistics drawing is carried out to data, the cell growth curve of 5 days is drawn.
According to cell counts and time point, the cell growth curve based on cell counts is drawn.Test result indicates that, Slow virus infects group tumour cell and cultivated in vitro after 5 days, and growth rate is significantly slowed, far below the increasing of control group tumour cell Speed is grown, the vigor cell number of BEL-7404 human liver cancer cell has lowered 64.0% (Fig. 7), as shown in fig. 7, slow through shRNA After virus infection 3 days, cell is laid on 96 orifice plates, and bed board number is 2000.Celigo is continuously detected 5 days, finds experimental group BEL- The multiplication rate of 7404 cells is significantly inhibited.Point out the multiplication capacity of PYCR1 genes and BEL-7404 cells significantly correlated. As shown in figure 8, after shRNA slow-virus infections 3 days, cell is laid on 96 orifice plates, bed board number is 2000.Celigo continuously detections 5 My god, it is found that the multiplication rate of experimental group SMMC-7721 cells is significantly inhibited.Point out PYCR1 genes and SMMC-7721 cells Multiplication capacity it is significantly correlated.The vigor cell number of human hepatocarcinoma BEL-7402 has lowered 61.4% (Fig. 8).
Embodiment 4 detects the ability for the tumor cell clone formation for infecting PYCR1-siRNA slow virus
Human liver cancer BEL-7404, SMMC-7721 cell in exponential phase carries out pancreatin digestion, and cell suspension is made (cell number is about 5 × 104/ ml) 6 orifice plates are inoculated in, cell is inoculated in into each experimental group in 6 orifice plate culture plates is inoculated with 400- 1000 cells/wells (being determined according to cell growth status), each experimental group sets 3 multiple holes.By the cell being inoculated with culture Continue to cultivate in case untill cell number is more than 50 by 14 days or in most single clones, carried out changing liquid simultaneously every 3 days halfway Observe cell state.Cell clone is taken pictures under fluorescence microscope before experiment is terminated, PBS washings cell 1 time.Added per hole 1mL4% paraformaldehydes, fixed cell 30-60min, PBS washs cell 1 time.Clean, free from admixture GIEMSA dye liquors are added per hole 500 μ L, dye cell 10-20min.ddH2O washings cell for several times, dries, taken pictures, colony count.
The result that liver cancer BEL-7404, the SMMC-7721 cell clonal formation as shown in Fig. 9,10 is tested, as shown in figure 9, After shRNA slow-virus infections 3 days, cell is laid on 6 orifice plates, and bed board amount is 1000.After 11 days, observation clone's number finds experimental group BEL-7404 cell colonies number is reduced, and points out the clonality of PYCR1 genes and BEL-7404 cells significantly correlated. After shRNA slow-virus infections as shown in Figure 10 3 days, cell is laid on 6 orifice plates, and bed board amount is 1000.After 11 days, observation clone's number, It was found that experimental group SMMC-7721 cell colonies number is reduced, the Clone formation energy of PYCR1 genes and SMMC-7721 cells is pointed out Power is significantly correlated.Oncocyte infection PYCR1-siRNA rear clone Forming abilities are significantly reduced, and are compared with control group, RNA interference groups Oncocyte formation clone's spot number is substantially reduced, and the volume of clone's spot is obviously reduced.Show that PYCR1 silences cause tumour cell to be formed The ability of clone declines.
Embodiment 5 detects the apoptosis of tumor cells level for infecting PYCR1-siRNA slow virus
When each orifice plate cell growth to coverage rate of experimental group 6 is about 70%, the PYCR1-siRNA inductions of interference group are withered Die；Pancreatin digests, and complete medium is resuspended into cell suspension, is collected in supernatant cell in same 5mL centrifuge tubes, and every group sets three Individual multiple holes (to ensure upper machine cell number enough, cell number >=5 × 105/ is handled)；1300rmp centrifuges 5min, abandons supernatant, 4 DEG C D-Hanks (pH=7.2~7.4) the washing cell precipitations of precooling；1 × binding buffer wash cell precipitation once, 1300 rmp, 3min are centrifuged, and collect cell；Cell precipitation is resuspended in 200 μ 1 × binding of L buffer；Add 10 μ L Annexin V-APC are dyed, room temperature lucifuge 10-15min；According to cell concentration, 1 × binding of 400-800 μ L are added Buffer, upper machine testing.
As a result show, as shown in figure 11, detect that the BEL-7404 of apoptosis occurs for experimental group within 3 days after shRNA slow-virus infections Cell is dramatically increased, and points out the apoptosis of PYCR1 genes and BEL-7404 cells significantly correlated.As shown in figure 12, shRNA slow virus Detect that the SMMC-7721 cells that apoptosis occurs for experimental group are dramatically increased within 3 days after infection, point out PYCR1 genes and SMMC-7721 The apoptosis of cell is significantly correlated.PYCR1-siRNA interference group apoptosis of tumor cells rates are significantly higher than control group, are shown in liver In cancer BEL-7404 cells (Figure 11) and SMMC-7721 cells (Figure 12), silence PYCR1 expression, apoptosis rate is significantly higher than Control group.Show that apoptosis of tumor cells can be caused by lowering PYCR1 expressions.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should It is considered as protection scope of the present invention.
SEQUENCE LISTING
<110>No. 7 People's Hospital, Shanghai City
<120>The shRNA interference sequences and its pharmaceutical applications of people's PYCR1 genes
<130>Sa Da
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 58
<212> DNA
<213>Artificial sequence
<400> 1
ccggcacagt ttctgctctc aggaactcga gttcctgaga gcagaaactg tgtttttg 58
<210> 2
<211> 58
<212> DNA
<213>Artificial sequence
<400> 2
aattcaaaaa cacagtttct gctctcagga actcgagttc ctgagagcag aaactgtg 58
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
cagtttctgc tctcaggaa 19
<210> 4
<211> 610
<212> DNA
<213>Artificial sequence
<400> 4
gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc 60
gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccggcacagt ttctgctctc 120
aggaactcga gttcctgaga gcagaaactg tgtttttgaa ttctcgacct cgagacaaat 180
ggcagtattc atccacgaat tcggatccat taggcggccg cgtggataac cgtattaccg 240
ccatgcatta gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg 300
gagttccgcg ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc 360
cgcccattga cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat 420
tgacgtcaat gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat 480
catatgccaa gtacgccccc tattgacgtc aatgacggta aatggcccgc ctggcattat 540
gcccagtaca tgaccttatg ggactttcct acttggcagt acatctacgt attagtcatc 600
gctattacca 610
<210> 5
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cctatttccc atgattcctt cata 24
<210> 6
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<212> DNA
<213>Artificial sequence
<400> 6
gtaatacggt tatccacgcg 20

Claims (10)

1. a kind of shRNA interference sequences of people PYCR1 genes, including DNA oligo positive strand sequences and anti-chain sequence, wherein, institute Stating positive strand sequence is：
5’-CCGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAACTGTGTTTTTG-3’；
The anti-chain sequence is：
5’-AATTCAAAAACACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAACTGTG-3’。
2. shRNA interference sequences according to claim 1, it is characterised in that the DNA oligo positive strand sequences and anti-chain Sequence includes siRNA target sequences：
5’-CAGTTTCTGCTCTCAGGAA-3’。
3. a kind of shRNA slow virus carriers of people PYCR1 genes, it is characterised in that the shRNA of the people PYCR1 genes is sick slowly Poisonous carrier is for the siRNA target sequences of people's PYCR1 genes：
5’-CAGTTTCTGCTCTCAGGAA-3’。
4. a kind of people PYCR1 gene siRNAs, it is characterised in that the coding people PYCR1 gene siRNAs include being directed to people PYCR1 The siRNA target sequences of gene：
5’-CAGTTTCTGCTCTCAGGAA-3’。
5. a kind of people PYCR1 gene siRNA nucleic acid carriers, it is characterised in that the people PYCR1 gene siRNAs nucleic acid carrier bag Include the siRNA target sequences for people's PYCR1 genes：
5’-CAGTTTCTGCTCTCAGGAA-3’。
6. a kind of people PYCR1 gene siRNA nucleic acid carriers, it is characterised in that the people PYCR1 gene siRNA nucleic acid carriers are：
5’-GTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGG CTTTATATATCTTGTGGAAAGGACGAAACACCGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAA CTGTGTTTTTGAATTCTCGACCTCGAGACAAATGGCAGTATTCATCCACGAATTCGGATCCATTAGGCGGCCGCGTG GATAACCGTATTACCGCCATGCATTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGG AGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATA ATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC A-3’。
7. a kind of people PYCR1 genes disturb the construction method of slow virus, it is characterised in that comprise the following steps：
1) synthesis is for the DNA oligo normal chains and anti-chain of the RNA interfered target sequences of people's PYCR1 genes, the positive strand sequence For：
5’-CCGGCACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAACTGTGTTTTTG-3’；
The anti-chain sequence is：
5’-AATTCAAAAACACAGTTTCTGCTCTCAGGAACTCGAGTTCCTGAGAGCAGAAACTGTG-3’；
2) double-stranded DNA of positive strand sequence and anti-chain the sequence anneals formation with cohesive end；
3) vector linearization being made by digestion, makes linearized vector and step 1) obtained double-stranded DNA is connected；
4) connection product is converted into competent escherichia coli cell, performing PCR identification is entered to obtained positive colony；
5) plasmid extraction, virus packaging.
8. method according to claim 7, it is characterised in that the step 3) in endonuclease reaction for add AgeI and EcoRI double digestions.
9. method according to claim 7, it is characterised in that the step 4) connection product conversion E. coli competent The specific method of cell is：
Connection product is added in competent escherichia coli cell, ice bath；Heat shock, ice bath；Add the LB liquid of antibiotic-free Culture medium, shaking table concussion and cultivate；Bacterium solution is taken uniformly to be applied on the LB solid mediums containing Amp, the mistake in 37 DEG C of incubators Night cultivates；Positive colony enters in performing PCR qualification process
What is used identifies primer for identification primer-F：5 '-CCTATTTCCCATGATTCCTTCATA-3 ' and identification primer-R:5’ GTAATACGGTTATCCACGCG-3 ', detailed process is：Picking single bacterium colony enters performing PCR amplification, electrophoresis, sequencing.
10. the shRNA interference sequences of people PYCR1 genes according to claims 1 to 6, people's PYCR1 genes RNA are built Body, people PYCR1 genes interference slow virus are preparing the growth of suppression tumour cell, promote apoptosis of tumor cells, prevent or treat Application in the medicine of tumour.
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EP3406253A1 (en) * 2017-05-24 2018-11-28 Bayer Aktiengesellschaft Inhibitors and antagonists of human pycr1
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