CN102816856B - Application of DIAPH3 gene and expression product thereof - Google Patents
Application of DIAPH3 gene and expression product thereof Download PDFInfo
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- CN102816856B CN102816856B CN201210323259.0A CN201210323259A CN102816856B CN 102816856 B CN102816856 B CN 102816856B CN 201210323259 A CN201210323259 A CN 201210323259A CN 102816856 B CN102816856 B CN 102816856B
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Abstract
The invention relates to application of a DIAPH3 gene and an expression product thereof, in particular relates to application of the DIAPH3 gene and the expression product thereof to preparation of a product for diagnosing liver cancer and preparation of a medicament for treating the liver cancer, and belongs to the technical field of biology. The DIAPH3 gene and the expression product thereof are used as markers for diagnosing the liver cancer, so that the liver cancer is diagnosed more accurately and quickly. The DIAPH3 gene and the expression product thereof, as target genes of preparing a medicament for treating the liver cancer, provide novel treatment targets and treatment ways for treating the liver cancer.
Description
Technical field
The invention belongs to biological technical field, relate to the application of DIAPH3 gene and expression product thereof.Further relate to the application in the product of preparing diagnosing liver cancer of DIAPH3 gene and expression product thereof and DIAPH3 gene and expression product thereof and preparing the application in the medicine of Hepatoma therapy.
Background technology
Liver cancer refers to the malignant tumour that betides liver, comprises two kinds of primary hepatocarcinoma and secondary liver cancers, and mostly what the liver cancer of the daily theory of people referred to is primary hepatocarcinoma.Primary hepatocarcinoma is one of modal malignant tumour clinically, and according to recent statistics, the annual new liver cancer patient approximately 600,000 in the whole world, occupies the 5th of malignant tumour.Liver cancer is that China occupies second cancer " killer ", is common in middle-aged male.Because its grade of malignancy is high, disease progression is fast, patient does not generally have any discomfort in early days, once occur that symptom is medical, has often belonged to middle and advanced stage, therefore treatment difficulty is large, weak curative effect, after general morbidity, survival time is only 6 months, person " king in cancer ".In China, there are every year 110000 people to die from liver cancer, the wherein male sex 80,000, women 30,000, account for 45% of whole world PLC mortality number, become a large killer of serious threat China people health and lives, and its danger can not look down upon.
The diagnosis of liver cancer especially early diagnosis is the key of clinic diagnosis and prognosis.It is generally acknowledged following four class biomolecules Chang Zuowei primary liver cancer markers: 1, cancer embryo and glycoprotein antigen; 2, enzyme and isozyme; 3, cytokine; 4, gene.Wherein alpha-fetoprotein (AFP) is the liver cancer tumor markers that the current whole world is most widely used, and has applied many decades.Except AFP, other blood serum designated objects that detect for liver cancer in recent years also comprise: alpha-fetoprotein variant (AFP heteroplasmon), gamma glutamyl transpeptidase isozyme II(GGT-II), alkaline phosphatase isoenzyme I, zymohexase isozyme A(ALD-A), fucosidase (AFU), antitrypsin I, abnormal prothrombin, ferritin and Acidic Ferritin etc.The application of AFP heteroplasmon and abnormal prothrombin has improved the recall rate of liver cancer patient, but early diagnosis and instruct the molecule parting diagnosis of personalized treatment to be still the very big challenge that we face.
To detect Serum AFP as main, there is the effect of establishing diagnosis, early diagnosis, differential diagnosis at the etiologic diagnosis of China's liver cancer.When normal, AFP is produced by yolk sac and embryonic liver, be born and remain low-level after 1 year, when hepatopathy, raise, obviously increase and see hepatocellular carcinoma and teratoma, more than 60% liver cancer case Serum AFP >400 μ g/L, therefore AFP is considered to the specific tumour mark of clinical diagnosis primary hepatocarcinoma always.Because AFP detects less dependence iconography equipment and new technology, therefore also there is no at present can comparing favourably with AFP of other tumor markerses.But because AFP detection sensitivity is only 40%～65%, specificity is only 76%～96%.Although AFP can raise in about 80% liver cancer patient blood serum in HAS, but occur that at germinoma AFP positive rate is 50%, also can there is rising in various degree in other enterogastric tube tumour as the patient such as carcinoma of the pancreas or lung cancer and liver cirrhosis, so the susceptibility of AFP and specificity are all unsatisfactory.Therefore find that the new tumor markers that sensitivity and specificity are high will be the key that improves hepatocarcinoma early diagnosis level.
The treatment of liver cancer has obtained very large progress in the past over 20 years, surgical resection and liver transplantation belong to " radical-ability " treatment, are still the primary selection of liver cancer treatment.Along with the raising of hepatocarcinoma early diagnosis level and the change of oncotherapy biological concept, improve day by day the status of tumor by local non-operative treatment in liver cancer treatment.Transcatheter arterial chemoembolization (TACE) has become the prefered method that can not excise liver cancer treatment, is the important means of mid and late liver cancer treatment.The method can make more than 90% liver cancer patient be benefited, but general curative effect has much room for improvement.In addition, in percutaneous transhepatic puncture knurl, ethanol treatment (PEI) is also the local chemotherapy method of commonly using, and the liver cancer to cancer diameter <3cm and the treatment of TTPV have certain values.But the chemotherapeutics of liver cancer is continued to use the medicine of other tumours substantially at present, and specific aim is poor, unsatisfactory curative effect, therefore, and in the urgent need to finding new action target spot, the special novel drugs of research and development liver cancer, specificity and the validity of raising chemotherapy.In recent years along with going deep into liver cancer fundamental research, biotherapy has obtained greater advance in the complex therapy of liver cancer, become the fourth-largest therapy of oncotherapy after operation, radiotherapy, chemotherapy, as immunotherapy, gene therapy, comprise angiogenesis inhibitor, suicide gene therapy, tumor vaccine therapy, siRNA technology etc.But the clinical efficacy of many biotherapies is still very dissatisfied, and the overwhelming majority is also in the experimental study stage, and relevant key theory and experimental technique still need further research and development.
DIAPH3 gene (Gene ID:81624; NG_032693.1RefSeqGene) be a member in transparent formin subfamily, this family member relates to Actin muscle reconstruct and regulates cell movement and adhesion.Research shows that DIAPH3 transgenation is in a disguised form associated with autosomal dominant Auditory neuropathy.But, yet there are no the diagnosis that is applied in liver cancer of DIAPH3 gene and expression product thereof and the correlative study for the treatment of aspect report.
Summary of the invention
In view of this, the object of the present invention is to provide the application aspect diagnosis and the treatment of liver cancer of DIAPH3 gene and expression product thereof.
The present invention is first by the expression of reverse transcription-polymerase chain (RT-PCR) reaction detection DIAPH3 gene in liver cancer tissue, result is presented in 24 routine liver cancer cases, in 18 routine cancerous tissues, the expression amount of DIAPH3 gene is higher than cancer beside organism, account for 75% ratio, show that the expression level of DIAPH3 gene in liver cancer tissue is apparently higher than the expression level in cancer beside organism.
Further, the present invention adopts relative quantification method to detect the DIAPH3 gene differential expression in liver cancer sample, and result shows that the expression level of DIAPH3 gene in liver cancer tissue is apparently higher than the expression level in cancer beside organism.
The present invention is also by the expression of RT-PCR reaction detection DIAPH3 gene in normal human tissue, MICE FETAL LIVER tissue, hepatoma cell strain, and result demonstration DIAPH3 gene is expressed in normal human's fetal liver, in ripe liver, does not express; DIAPH3 gene high expression in the liver of mice embryonic phase and in 3 days of just birth, and along with the continuous maturation of liver development, DIAPH3 gene expression amount obviously reduces; DIAPH3 gene is high expression level in 7701 and 7402, only has weak expression at PLC, SMMC7721 and YY8103 cell.
In sum, the above results and the AFP diagnostic result in liver cancer has similarity, proves that the generation development of DIAPH3 gene and liver cancer has very close relationship, can become the new mark of diagnosing cancer of liver.Those skilled in the art can be by the PCR primer of design DIAPH3 gene, by RT-PCR react and real-time fluorescence quantitative PCR reaction detection tumor tissues in the content diagnosing liver cancer of DIAPH3 gene RNA, rna content height illustrates that the possibility of trouble liver cancer is high, otherwise low.Therefore, the invention provides the application in the product of preparing diagnosing liver cancer of DIAPH3 gene and expression product thereof.
Wherein, the product of diagnosing liver cancer of the present invention is RT-PCR diagnosing liver cancer test kit, real-time quantitative PCR diagnosing liver cancer test kit, immunodetection diagnosing liver cancer test kit, in situ hybridization diagnosing liver cancer test kit or gene chip diagnosis liver cancer test kit.Therefore the present invention also provides a kind of RT-PCR diagnosing liver cancer test kit, a kind of real-time quantitative PCR diagnosing liver cancer test kit, a kind of immunodetection diagnosing liver cancer test kit, a kind of in situ hybridization diagnosing liver cancer test kit and a kind of gene chip diagnosis liver cancer test kit.
A kind of RT-PCR diagnosing liver cancer test kit of the present invention, at least comprises the primer sets of a specific amplified DIAPH3 gene.
A kind of real-time quantitative PCR diagnosing liver cancer test kit of the present invention, at least comprises the primer sets of a specific amplified DIAPH3 gene.
The primer sets of the specific amplified DIAPH3 gene containing in RT-PCR diagnosing liver cancer test kit of the present invention or real-time quantitative PCR diagnosing liver cancer test kit in some embodiments, is by having the upstream primer of nucleotide sequence as shown in SEQID NO:1 or its complementary sequence and having nucleotide sequence as shown in SEQ ID NO:2 or the downstream primer of its complementary sequence forms.
The test kit of a kind of immunodetection diagnosing liver cancer of the present invention, at least comprises the antibody of being combined with DIAPH3 protein-specific, and described antibody is polyclonal antibody or monoclonal antibody.Those skilled in the art can prepare pin and the special antibody of DIAPH3 albumen by a series of methods known in the art.For example, the people DIAPH3 gene product of purification or its antigen fragment are injected in animal body to produce polyclonal antibody.Equally, the cell of expression people's DIAPH3 albumen or its antigen also can be used for animal to cause immunity and produce antibody.Antibody prepared in accordance with the present invention can be monoclonal antibody, and these monoclonal antibodies can be prepared in time with hybridoma.Antibody of the present invention comprises the antibody that can prevent DIAPH3 function, can be also the antibody that does not affect people DIAPH3 function.Each antibody-like can cause immunity by the fragment to people DIAPH3 gene product or functional domain and produce, and people DIAPH3 gene product and fragment thereof can produce or synthesize with Peptide synthesizer with recombination method.With the antibody that the DIAPH3 of non-modified form gene product is combined, can utilize the gene product for example producing in E.coli at prokaryotic cell prokaryocyte carry out immune animal and obtain.With the antibody that posttranslational modification form is combined as glycosylation or phosphorylation DIAPH3 albumen or polypeptide, can utilize the gene product producing in as yeast or insect cell at eukaryotic cell carry out immune animal and obtain.
As preferably, the test kit of immunodetection diagnosing liver cancer of the present invention can be radioimmunoassay kits, Enzymoimmune reagent kit or fluorescence immunoassay kit.More preferably Enzymoimmune reagent kit.
A kind of in situ hybridization diagnosing liver cancer test kit of the present invention, at least comprises the probe with the nucleic acid array hybridizing of DIAPH3 gene.
A kind of gene chip diagnosis liver cancer test kit of the present invention, at least comprises the probe with the nucleic acid array hybridizing of DIAPH3 gene.
In the present invention, the probe of the nucleic acid array hybridizing of described and DIAPH3 gene can be DNA, RNA, DNA-RNA mosaic, PNA or other derivatives.The length of probe of the present invention does not limit, as long as complete specific hybrid, with DIAPH3 gene nucleotide series specific binding, any length can, can be as short as 25,20,15,13 or 10 base length, can grow to equally 60,80,100,150,300 base pairs or longer, even whole gene.
Because different probe length has different impacts to hybridization efficiency, signal specificity, it is at least 14 base pairs that the length of the probe of the nucleic acid array hybridizing of of the present invention and DIAPH3 gene is preferably, be the longlyest generally no more than 30 base pairs, is more preferably 15-25 base pair with the length of DIAPH3 gene nucleotide series complementation.Further, described probe self complementation is most preferably less than 4 base pairs, in order to avoid affect hybridization efficiency.
Further, the test kit of diagnosing liver cancer of the present invention also comprises other components.
In certain embodiments, described RT-PCR diagnosing liver cancer test kit or real-time quantitative PCR diagnosing liver cancer test kit also comprise other components of nucleic acid amplification, if PCR reaction buffer, reversed transcriptive enzyme, various dNTP(are that dNTP comprises dATP, dTTP, dGTP and dCTP), RNA enzyme inhibitors etc.
In certain embodiments, the test kit of described immunodetection diagnosing liver cancer also comprises other components of immunodetection, as Enzymoimmune reagent kit also comprises enzyme, the enzyme reaction substrate etc. of reaction buffer, traget antibody, wherein the enzyme of traget antibody has (horseradish peroxidase), alkaline phosphorus enzyme (alkaline phosphatase) etc.
In further embodiments, described in situ hybridization diagnosing liver cancer test kit also comprises other components of molecular hybridization, as prehybridization solution, biotinylation mouse-anti digoxin, biotinylation peroxidase etc.
On the other hand, the present invention utilizes liposome small ribonucleic acid molecules (siRNA) to be delivered to the expression of liver cancer cell internal interference DIAPH3 gene, measure the growth activity of liver cancer cell 7701,7402 by CCK-8 method, result proves that the siRNA molecule of specificity interference DIAPH3 gene can suppress the external growth of liver cancer cell, shows that DIAPH3 gene can be used as preparing the target gene of cancer treatment drug.Therefore the present invention also provides DIAPH3 gene and the application of expression product in the medicine of preparing Hepatoma therapy thereof.
As preferably, the medicine of Hepatoma therapy of the present invention, at least comprises a kind of protein of the DIAPH3 of inhibition protein-active.
As preferably, the medicine of Hepatoma therapy of the present invention, at least comprises by RNA and disturbs the siRNA that suppresses DIAPH3 genetic expression.
In some embodiments, siRNA positive-sense strand of the present invention has the nucleotide sequence shown in SEQ ID NO:7, and siRNA antisense strand has the nucleotide sequence shown in SEQ ID NO:8.
In some embodiments, siRNA positive-sense strand of the present invention has the nucleotide sequence shown in SEQ ID NO:9, and siRNA antisense strand has the nucleotide sequence shown in SEQ ID NO:10.
The present invention proves that by RT-PCR and real-time quantitative PCR the expression of DIAPH3 gene in liver cancer tissue is apparently higher than cancer beside organism, and by the expression of RT-PCR reaction detection DIAPH3 gene in normal human tissue, MICE FETAL LIVER tissue, hepatoma cell strain, proof DIAPH3 gene is expressed in fetal liver, in ripe liver, does not express.Therefore DIAPH3 gene and the application of expression product in the product of preparing diagnosing liver cancer thereof are provided.The present invention further proves that by DIAPH3 gene order specific siRNA interference experiment specific siRNA can significantly suppress the growth of liver cancer cell.Therefore DIAPH3 gene and the application of expression product in the medicine of preparing Hepatoma therapy thereof are provided.DIAPH3 gene and expression product thereof be as the mark of diagnosing liver cancer, makes diagnosing cancer of liver more accurately, fast, provides new treatment target spot and treatment approach as the target gene of preparing Hepatoma therapy medicine for Hepatoma therapy.
Brief description of the drawings
Fig. 1 shows the expression schematic diagram of embodiment 1RT-PCR checking DIAPH3 gene in 24 routine hepatocarcinoma patient cancerous tissues and cancer beside organism, and wherein, " N " refers to cancer beside organism, and " C " refers to liver cancer tissue;
Fig. 2 shows that embodiment 2 real-time quantitative PCRs detect the expression schematic diagram of DIAPH3 gene in people's liver cancer tissue and cancer beside organism, and wherein, " Non-HCC " refers to cancer beside organism, and " HCC " refers to liver cancer tissue;
Fig. 3 shows that embodiment 3RT-PCR detects the expression of DIAPH3 gene in normal adult heart, lung, spleen, testis, ovary, oesophagus, small intestine, stomach, pancreas, brain, liver and tire liver;
Fig. 4 shows that embodiment 4RT-PCR detects the expression of DIAPH3 gene in MICE FETAL LIVER;
Fig. 5 shows that embodiment 5RT-PCR detects the expression of DIAPH3 gene in human hepatoma cell strain PLC, SMMC7721,7402, YY8103 and 7701;
Fig. 6 show in embodiment 6 in liver cancer cell 7701 specific siRNA disturb after the expression of DIAPH3 gene, wherein X-coordinate is siRNA kind, ordinate zou is DIAPH3mRNA relative expression quantity;
Fig. 7 show in embodiment 7 in liver cancer cell 7701 specific siRNA disturb after the cell growth curve schematic diagram of DIAPH3 gene, wherein X-coordinate is number of days, ordinate zou is viable cell quantity under 450nm;
Fig. 8 show in embodiment 8 in liver cancer cell 7402 specific siRNA disturb after the expression of DIAPH3 gene, wherein X-coordinate is siRNA kind, ordinate zou is DIAPH3mRNA relative expression quantity;
Fig. 9 show in embodiment 9 in liver cancer cell 7402 specific siRNA disturb after the cell growth curve schematic diagram of DIAPH3 gene, wherein X-coordinate is number of days, ordinate zou is viable cell quantity under 450nm.
Embodiment
The embodiment of the invention discloses the application of DIAPH3 gene and expression product thereof.Those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Method of the present invention is described by preferred embodiment, and related personnel obviously can change method as herein described in content of the present invention, spirit and scope or suitably change and combination not departing from, and realizes and apply the technology of the present invention.
The present invention is first by the expression of reverse transcription-polymerase chain (RT-PCR) reaction detection DIAPH3 gene in liver cancer tissue.
RT-PCR refers to reverse transcription (Reverse Transcription; RT) reaction and PCR (Polymerase Chain Reaction) react the method for combining.RT-PCR combines with PCR synthetic the cDNA taking RNA as template, and a kind of method of rapid sensitive of analyzing gene expression is provided.RT-PCR is for detecting or quantitatively expressing information.RT-PCR than other RNA analytical technology including Northern trace, RNase protection analysis, in situ hybridization and the analysis of S1 nuclease sensitiveer, be easier to operation.
The template of RT-PCR can be total RNA or poly (A) selectivity RNA.Reverse transcription reaction can use reversed transcriptive enzyme, initial with the primer (GSP) of random primer, oligo (dT) or gene specific.
RT-PCR can single stage method or the form of two-step approach carry out.In two-step approach RT-PCR, each step is all carried out under top condition, and first the synthetic of cDNA carry out in reverse transcription damping fluid, then takes out 1/10 reaction product and carries out PCR.In single stage method RT-PCR, reverse transcription and PCR are under the condition optimized of reverse transcription and PCR at the same time, carry out in turn in by all means one.
The present invention adopts Trizol test kit (Invitrogen) to extract total RNA.Trizol test kit is the step extraction process based on acidic phenol, directly from cell or tissue, extracts total RNA, and it can keep the integrity of RNA in the time of broken and dissolved cell.Add after chloroform centrifugally, sample is divided into water sample layer and organic layer, and RNA is present in water sample layer.Collect after water sample layer above, RNA can reduce by isopropanol precipitating.Wherein, all need to carry out rnase inactivation treatment for extracting total RNA (RNA) vessel used and water, to ensure the environment of deoxyribonuclease in experiment.
Extracting the concrete extraction step of total RNA comprises: broken tissue → isolation of RNA → precipitated rna → washing RNA → melt RNA → preservation RNA.Wherein, broken tissue and deactivation RNA enzyme can synchronously carry out, and can use the broken tissue such as Guanidinium hydrochloride, guanidine thiocyanate, NP-40, SDS, Proteinase K, add β-ME can suppress RNA enzymic activity.Isolation of RNA is generally used the organic solvent such as phenol, chloroform, adds a small amount of primary isoamyl alcohol, and after this step, centrifugal, RNA is generally distributed in upper strata, separates with egg white layer.Precipitated rna is generally used ethanol, 3M NaAc(pH=5.2) or Virahol.Washing RNA uses 70% washing with alcohol, and for avoiding RNA to be washed off, this step can save sometimes, after washing, can dry or dry ethanol, but can not be too dry, otherwise not soluble.Melt RNA and generally use TE.Preserve the RNA low temperature of should trying one's best.In order to prevent the pollution of trace RNase, the RNA being separated to from be rich in the sample (as pancreas, liver) of RNase need to be stored in formaldehyde to preserve high-quality RNA, all the more so for long-term storage.The RNA extracting from rat liver, store a week and just substantially degraded, and the RNA extracting from Rats Spleen preserves and within 3 years, still keeps stable in water in water.In addition, it is more responsive than little transcript for the degraded of trace RNase that length is greater than the transcript of 4kb.In order to increase the stability of storage RNA sample, RNA can be dissolved in deionized methane amide, be stored in-70 DEG C.Necessarily can not contain the foreign material of degradation of rna for preserving the methane amide of RNA.The RNA that derives from pancreas at least can preserve 1 year in methane amide.In the time preparing to use RNA, can add NaAc to 0.3M12, centrifugal 5 minutes precipitated rnas of 000 × g:
After extracting total RNA, need to carry out the synthetic of cDNA, concrete steps are:
(1) ice bath centrifuge tube the inside adds template ribonucleic acid 4 μ L(0.5 μ g/ μ L), the random primer 2 μ L of 20pmol/ μ L, deionized water 5 μ L, mix, centrifugal 3-5 second;
(2) 70 DEG C of water-baths 5 minutes, ice bath 30 seconds, so that primer and template are correctly matched;
(3) add 5 × M-MLV reverse transcriptase reaction liquid, 4 μ L, RNase inhibitor 1 μ L, dNTP 2uL(100mM), mix;
(4) 37 DEG C of water-baths 5 minutes, add 1 μ LM-MLV ThermoScript II (200 units of enzyme activity/μ L), mix;
(5) 37 DEG C of water-baths 1 hour, carry out reverse transcription;
(6) 70 DEG C 10 minutes so that enzyme-deactivating, avoid subsequent experimental to produce and disturb, product is put and is carried out pcr amplification on ice ,-70 DEG C of remaining preservations.
RT-PCR need to design special primer, and desirable primer pair is only with the unique sequence of aim sequence both sides but not the annealing of other sequences, but not special primer may amplify other non-aim sequence simultaneously.Desirable primer has following common feature: typical 18 to 24 nucleosides of primer are long.Primer needs sufficiently long, ensures sequence uniqueness, and reduces the possibility that sequence is present in non-aim sequence site.But length is greater than the primer of 24 nucleosides and does not mean that higher specificity.Longer sequence may be hybridized with wrong matched sequence, has reduced specificity, and slower than short sequence hybridization, thereby has reduced output.Selecting GC content is 40% to 60% or the primer of GC content reflection template GC content.This outer primer 5' end and intermediate zone are that the primer of G or C can increase the stability of primer and the stability that primer is hybridized with aim sequence.Primer pair 3' end should avoid existing complementary sequence, otherwise can form primer dimer, suppression of amplification.Avoid 3' end to be rich in GC.When design primer, ensure in the end in 5 nucleosides, to contain 3 A or T.Avoid the mistake pairing of 3' end.3' end nucleosides need to extend for polysaccharase catalysis with template annealing.Avoid having the sequence that may produce inner secondary structure, this can destroy primer annealing stability.
For DIAPH3 gene in proved invention can be used as the target spot of preparing cancer treatment drug, the present invention is achieved through the following technical solutions:
1, chemosynthesis siRNA molecule, its sequence-specific is for DIAPH3 gene order.Utilize liposome to be delivered in liver cancer cell, disturb the expression of DIAPH3 gene, measure the growth activity of liver cancer cell 7701,7402 by CCK-8 method.
2, DIAPH3 genetic expression is disturbed in external assessment, and liver cancer cell clonality, soft-agar cloning form the change of the characteristics of cell biology such as ability, cell cycle, apoptosis.
3, utilize various carriers, comprise that DAN carrier, adenovirus, gland relevant viral vector disturb the expression of DIAPH3 gene, reach the effect of body internal interference DIAPH3 gene, detect their results for the treatment of to nude mice by subcutaneous transplanted tumor, liver in-situ inoculating knurl, thereby reality suppresses the object of liver cancer cell proliferation in vivo.
4, obtain the polypeptide, the monoclonal antibody that can specificity suppress DIAPH3 gene kinase activity, reach the object that suppresses DIAPH3 activity, thereby reality suppresses the object of liver cancer cell proliferation in vivo.
5, obtain the compound that can specificity suppresses DIAPH3 protein-active, reach the object that suppresses DIAPH3 protein-active, thereby reality suppresses the object of hepatoma cell proliferation.
Wherein, the results show specificity of the present invention disturbs the siRNA of DIAPH3 gene can suppress the external growth of liver cancer cell, illustrates that DIAPH3 gene can be used as preparing the target gene of cancer treatment drug.
In embodiment, disturb the siRNA sequence principle of design of DIAPH3 gene as follows for specificity:
(1), from the AUG initiation codon of transcript (mRNA), find " AA " two and connect sequence, and write down 19 base sequences of its 3' end, as potential siRNA target site.Have result of study show GC content at about 45%-55% siRNA than higher more effective of those GC content.
Tuschl etc. suggestion non-coding region (the untranslated regions for 5' and 3' end not in the time of design siRNA, UTRs), reason is that these places have abundant modulin calmodulin binding domain CaM, thereby and these UTR may affect siRNA endonuclease enzyme complex in conjunction with albumen or translation initiation complex affects the effect of siRNA in conjunction with mRNA.
(2) potential sequence and corresponding genome database (people, or mouse, rat etc.) are compared, get rid of the sequence of those and other encoding sequence/EST homologies, for example, use BLAST(www.ncbi.nlm.nih.gov/BLAST/).
(3) selecting suitable target sequence synthesizes.A common gene need to design the siRNA of multiple target sequences, to find the external assessment of the most effective siRNA sequence to disturb DIAPH3 genetic expression.
In order further to understand the present invention, below in conjunction with embodiment, the present invention is described in detail.Following examples are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in embodiment, conventionally according to normal condition, the people such as such as Sambrook, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1:RT-PCR detects the expression of DIAPH3 gene in liver cancer tissue
1, separate tissue: test with tissue-derived in the operation patients of primary hepatocarcinoma.The liver of excision is once in vitro, cuts rapidly focus and 5 centimeters of outer cancer beside organisms around, puts into liquid nitrogen (80 DEG C) and preserves.The diagnosis on cancer and cancer side is all taking pathological diagnosis as final foundation.
2, the extracting of Yeast Nucleic Acid (RNA): extract total RNA with Trizol test kit (Invitrogen), extracting method is with reference to Trizol test kit specification sheets.In the time preparing to use RNA, can use following method precipitated rna: add NaAc to 0.3M, centrifugal 5 minutes of 12,000 × g.
3, cDNA's is synthetic:
(1) ice bath centrifuge tube the inside adds template ribonucleic acid 4 μ L(0.5 μ g/ μ L), the random primer 2 μ L of 20pmol/ μ L, deionized water 5 μ L, mix, centrifugal 3-5 second;
(2) 70 DEG C of water-baths 5 minutes, ice bath 30 seconds;
(3) add 5 × M-MLV reverse transcriptase reaction liquid, 4 μ L, RNase inhibitor 1 μ L, dNTP 2 μ L(100mM), mix;
(4) 37 DEG C of water-baths 5 minutes, add 1 μ L M-MLV ThermoScript II (200 units of enzyme activity/μ L), mix;
(5) 37 DEG C of water-baths 1 hour;
(6) 70 DEG C, 10 minutes finish reaction, and product is put and carried out next step PCR experiment on ice ,-70 DEG C of remaining preservations.
4, the design of primers of RT-PCR and amplification
The primer of design is concrete as table 1.
Table 1RT-PCR primer
Using β-actin as internal reference.The reaction system of PCR: add respectively 1.5 μ l 10 × Buffer in each reaction tubes, add respectively 0.4 μ l dNTP(10mM), 0.3 μ l forward direction primer (10 μ M), 0.3 μ l reverse primer (10 μ M), 1 μ l cDNA template, (5u/ μ l), adds water to 15 μ l to 0.1 μ l Taq enzyme.PCR reaction conditions is as follows: 94 DEG C, and 5 minutes denaturations; 94 DEG C, sex change in 30 seconds; 55 DEG C, annealing in 30 seconds; 72 DEG C, within 30 seconds, extend; 35 circulations, agarose gel electrophoresis detects pcr amplification product, the results are shown in Figure 1.
5, experimental result: the expression level by the visible DIAPH3 gene of Fig. 1 result in liver cancer tissue is apparently higher than the expression level in cancer beside organism.In 24 routine liver cancer cases, in 18 routine cancerous tissues, the expression amount of DIAPH3 gene, higher than cancer beside organism, accounts for 75% ratio.
Embodiment 2: real-time quantitative PCR experiment
Adopt real-time quantitative PCR method to detect the differential expression of DIAPH3 gene in liver cancer sample.
Laboratory apparatus reagent: Thermal Cycler DiceTM Real Time System TP800, Takara;
premix Ex TaqTM, Takara.
The primer table 2 of design, wherein DIAPH3 the primer is consistent with sxemiquantitative RT-PCR DIAPH3 primer used.
Table 2 real-time quantitative PCR primer
Quantitative fluorescent PCR (real-time PCR) reaction system is as follows: cumulative volume 20 μ L, SYBR PremixEx Taq 10 μ L, the each 0.4 μ L of upstream and downstream primer (10 μ mol/L), cDNA1 μ L, ddH
2o 8.2 μ L, mix reagent, and putting into PCR instrument after centrifugal carries out amplified reaction.PCR condition is: 94 DEG C of 4min; 94 DEG C of 30s, 60 DEG C of annealing 20s, 40 circulations.Agarose gel electrophoresis detects pcr amplification product, the results are shown in Figure 2.
From Fig. 2 result, the expression level of DIAPH3 gene in liver cancer tissue is apparently higher than the expression level in cancer beside organism, through t inspection, P<0.05.
Embodiment 3: detect the expression of DIAPH3 gene in normal human tissue
Expression with RT-PCR detection DIAPH3 gene in normal adult tissue.Extract normal adult according to method described in embodiment 1 and respectively organize RNA, cDNA is synthesized in reverse transcription, using β-actin as internal reference, taking the each tissue cDNA of normal adult of synthesizing as template, carry out PCR reaction according to method described in embodiment 1, agarose gel electrophoresis detects pcr amplification product, the results are shown in Figure 3.
By Fig. 3 result, visible DIAPH3 expresses in fetal liver, in ripe liver, does not express.
Embodiment 4:PCR experiment detects the expression of DIAPH3 gene in MICE FETAL LIVER tissue
Expression with RT-PCR detection DIAPH3 gene in MICE FETAL LIVER tissue.Extract the MICE FETAL LIVER of different development stage according to method described in embodiment 1 and organize RNA, cDNA is synthesized in reverse transcription, using β-actin as internal reference, taking the MICE FETAL LIVER tissue cDNA of the different development stage that synthesizes as template, carry out PCR reaction according to method described in embodiment 1, agarose gel electrophoresis detects pcr amplification product, the results are shown in Figure 4.
From Fig. 4 result, DIAPH3 gene is high expression level in the liver of mice embryonic phase and in 3 days of just birth, and along with the continuous maturation of liver development, genetic expression obviously reduces.
Embodiment 5:PCR experiment detects the expression of DIAPH3 gene in hepatoma cell strain
Expression with RT-PCR detection DIAPH3 gene in hepatoma cell strain PLC, SMMC7721,7402, YY8103 and 7701.Wherein said cell strain derives from Chinese Academy of Sciences's Shanghai cell bank.
Extract different hepatoma cell strain RNA according to method described in embodiment 1, cDNA is synthesized in reverse transcription, using β-actin as internal reference, taking the different hepatoma cell strain cDNA that synthesize as template, carry out PCR reaction according to method described in embodiment 1, agarose gel electrophoresis detects pcr amplification product, the results are shown in Figure 5.
From Fig. 5 result, DIAPH3 gene is high expression level in 7701 and 7402, only has weak expression at PLC, SMMC7721 and YY8103 cell.
The small interference ribonucleic acid (siRNA) of embodiment 6:DIAPH3 gene
The features such as external chemosynthesis small interference ribonucleic acid (siRNA) has fast, simple and high specificity, have wide practical use at aspects such as antineoplastons.Utilize the impact of siRNA research DIAPH3 gene pairs 7701 and 7402 growth and proliferation of cell.
For the synthetic two pairs of siRNA sequences of DIAPH3 gene mRNA design, be respectively siRNA-1531 and siRNA-2220, irrelevant sequence is set in addition as negative control siRNA, concrete sequence is as shown in table 3.
Table 3siRNA sequence
Utilize liposome (Lipofectamine 2000) transfection reagent respectively above-mentioned 3 kinds of siRNA to be entered in 7701 and 7402 cells (being provided by Chinese Academy of Sciences's Shanghai cell bank) by final concentration 50nmol/L transfection, with the DMEM cultivation containing 10% foetal calf serum.Extract different hepatoma cell strain RNA according to method described in embodiment 1, cDNA is synthesized in reverse transcription, using β-actin as internal reference, taking the different hepatoma cell strain cDNA that synthesize as template, carry out real-time quantitative PCR experiment according to method described in embodiment 2 and detect the expression of DIAPH3 gene in hepatoma cell strain 7402 and 7701, the results are shown in Figure 6 and Fig. 8.
Every hole inoculation 3 × 10 in 96 orifice plates
3hepatoma cell line 7701 and 7402 cell (serum-free mediums, provided by Chinese Academy of Sciences's Shanghai cell bank), in the time that cell density reaches 40% left and right, utilize liposome (Lipofectamine 2000) transfection reagent respectively above-mentioned 3 kinds of siRNA to be entered in 7701 and 7402 cells by final concentration 50nmol/L transfection, after 4h, change the DMEM containing 10% foetal calf serum into.Cultivate 7d taking 24h as 1 detection unit, detect this cell density every day 1 time.In the enchylema to be measured of every hole, add 10 μ LCCK-8, hatch 1h for 37 DEG C.Utilize microplate reader under 450nm wavelength, to detect its absorbancy to represent cell survival ability, draw growth curve, the results are shown in Figure 7 and Fig. 9.
From Fig. 6-9 result, compare with control group (siRNA-NC), specificity is disturbed the small interference ribonucleic acid (siRNA) of DIAPH3 genetic expression: the mRNA level that siRNA-1531 and siRNA-2220 can effective reticent DIAPH3 genes, can suppress the growth of hepatoma cell line 7701 and 7402 cells, the survival rate of liver cancer cell is starkly lower than the cell of control group simultaneously.Result shows that the down-regulated expression of people DIAPH3 gene can suppress liver cancer cell growth to a certain extent.
Embodiment 7: a kind of RT-PCR diagnosing liver cancer test kit
A kind of RT-PCR diagnosing liver cancer test kit, comprise have the nucleotide sequence as shown in SEQ ID NO:1 or its complementary sequence upstream primer, there is nucleotide sequence as shown in SEQ ID NO:2 or downstream primer, ThermoScript II, OligoT, reaction buffer and the RNA enzyme inhibitors of its complementary sequence.
Embodiment 8: a kind of real-time quantitative PCR diagnosing liver cancer test kit
A kind of real-time quantitative PCR diagnosing liver cancer test kit, comprises and has the upstream primer of the nucleotide sequence as shown in SEQ ID NO:1 or its complementary sequence and have nucleotide sequence as shown in SEQ ID NO:2 or the downstream primer of its complementary sequence.
Embodiment 9: a kind of enzyme linked immunosorbent detection diagnosing liver cancer test kit
A kind of enzyme linked immunosorbent detection diagnosing liver cancer test kit, comprises horseradish peroxidase and the enzyme reaction substrate of the antibody of being combined with DIAPH3 protein-specific, reaction buffer, traget antibody.
Embodiment 10: a kind of in situ hybridization diagnosing liver cancer test kit
A kind of in situ hybridization diagnosing liver cancer test kit, comprises probe, prehybridization solution, biotinylation mouse-anti digoxin and biotinylation peroxidase with the nucleic acid array hybridizing of DIAPH3 gene.
Embodiment 11: a kind of medicine of Hepatoma therapy
A medicine for Hepatoma therapy, comprise have the nucleotide sequence shown in SEQ ID NO:7 siRNA positive-sense strand, there is the siRNA antisense strand of the nucleotide sequence shown in SEQ ID NO:8.
Embodiment 12: a kind of medicine of Hepatoma therapy
A medicine for Hepatoma therapy, comprise have the nucleotide sequence shown in SEQ ID NO:9 siRNA positive-sense strand, there is the siRNA antisense strand of the nucleotide sequence shown in SEQ ID NO:10.
The explanation of above embodiment is just for helping to understand product of the present invention, method and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improvement and modification also fall in the protection domain of the claims in the present invention.
Claims (3)
1. the application of the siRNA of inhibition DIAPH3 genetic expression in the medicine of preparing Hepatoma therapy.
2. application as claimed in claim 1, is characterized in that, described siRNA positive-sense strand has the nucleotide sequence shown in SEQ ID NO:7, and siRNA antisense strand has the nucleotide sequence shown in SEQ ID NO:8.
3. application as claimed in claim 1, is characterized in that, described siRNA positive-sense strand has the nucleotide sequence shown in SEQ ID NO:9, and siRNA antisense strand has the nucleotide sequence shown in SEQ ID NO:10.
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| DIAPH3 governs the cellular transition to the amoeboid tumour phenotype;Martin H. et al.;《EMBO Molecular Medicine》;20120831;第4卷(第8期);第743-760，尤其是第744页第2栏第2段及图1 C，摘要。 * |
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