CN108103063A - A kind of saRNA and application thereof - Google Patents

A kind of saRNA and application thereof Download PDF

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CN108103063A
CN108103063A CN201711276454.1A CN201711276454A CN108103063A CN 108103063 A CN108103063 A CN 108103063A CN 201711276454 A CN201711276454 A CN 201711276454A CN 108103063 A CN108103063 A CN 108103063A
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张灏
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Abstract

The invention discloses a kind of saRNA, the saRNA includes the nucleotide sequence with 3000 to the 200 site areas complementation of PTPRO gene promoter areas；The invention also discloses purposes of the saRNA in the drug for treating tumour is prepared.The SaRNA of the targeting PTPRO genes of the present invention can make the Tyr123 site phosphorylations of PD L1 are horizontal to reduce, and block PD 1/PD L1 accesses, inhibit growth of tumour cell, make T cell activity recovery；Meanwhile saRNA of the invention plays synergistic function with PD L1 inhibitor in the treatment of tumour, significantly improves the therapeutic effect of PD L1 inhibitor.Therefore, saRNA of the present invention can be used for preparing antitumor drug.

Description

A kind of saRNA and application thereof
Technical field
The present invention relates to the immunotherapy techniques of oncomolecularbiology technical field, more particularly to tumour, especially one The saRNA of kind effectively treatment tumour.
Background technology
Tumour cell treatment is the immune system by transferring body, enhances anti-tumor immunity, so as to inhibit and kill Tumour cell.Tumour cell treatment is most one of research direction of prospect in current cancer therapies field.PD-1 (programmeddeath-1) it is to be obtained in the T cell hybridoma of apoptosis, is named since it is related to Apoptosis For programmed death-1 receptor, PD-1 programmed death receptors are a kind of important immunosuppression molecules, be CD28 superfamilies into Member.PD-1 is mainly expressed in the T cell and B cell of activation, is a kind of surface receptor of activated form T cell, there are two PD-1 Ligand is PD-L1 respectively
(B7-H1) and PD-L2 (B7-DC).The in vivo tumor microenvironment of machine can induce PD-1 points of the T cell of infiltration high expression Son, the ligand PD-L1 and PD-L2 of the high expression PD-1 of tumour cell meeting, causes PD-1 accesses sustained activation in tumor microenvironment, After PD-L1 couples with PD-1, T cell function is suppressed, it is impossible to the signal of attack tumour is sent to immune system.PD-1/PD-L1 Inhibitor can block the combination of PD-1 and PD-L1, block negative regulation signal, make T cell activity recovery, immune so as to enhance Response.At present, prove that they treat the effect of tumour in animal model there are many PD-1/PD-L1 inhibitor, Pembrolizumab is the humanization IgG4-kappa antibody of the combination PD-1 of high-affinity a kind of.U.S.'s food and medicine management Office (FDA) have been approved by pembrolizumab for treat late period or can not surgery excision to other drugs without the black of response Melanoma.Nivolumab is a kind of monoclonal drug of anti-PD-1, is only developed as two wires or three line medicines. Nivolumab is by FDA approvals for the second line treatment of prognosis of squamous cell lung cancer.At present, other PD-1/PD-L1 inhibitor are very Mostly all in I/I clinical trial phase stage.PD-1/PDL1 cell therapies are a kind of inhibiting tumor cell therapies currently to get most of the attention, It is intended to resist tumour using the immune system of human body itself, by the way that PD-1/PD-L1 signal paths is blocked to make death of neoplastic cells, Suitable for polytype tumour.
Based on limited clinical data and Follow-up Data at present, to the effective patient of PD-1/PD-L1 antibody, some Disease is final or can be in progress.So, what potential resistance mechanism isDrug resistance caused by how overcoming immunization therapyPD-1/ It is good whom PD-L1 inhibitor therapeutic alliance partner selectsIt is the Three Difficult Issues faced in Current cancer cell therapy.Current research is recognized For may be because other immunologic escape access compensatories it is active.But specific mechanism and the accuracy of prediction are gone back at present It is unclear.For the drug of these new accesses, there are many basic research and clinical test needs to do, therefore also need to grow very much Time.
It is existing the study found that in cell PD-L1 albumen phosphorylation level for PD-1/PD-L1 signal paths accurate tune Control has very important significance.The occurrence and development of tumour are frequently accompanied by the modification of PD-L1 protein phosphorylations and are abnormal.PD- The phosphorylation modification of phosphorylation site is to cause Lymphocyte Apoptosis in L1 amino acid sequences, and immunologic escape PD-1/ occurs for tumour An important factor for PD-L1 inhibitor is resisted.Set about from protein phosphorylation modification angle, find in PD-L1 amino acid sequences and play The phosphorylation site of key regulatory effect, finds and plays the part of important angle in the phosphorylation modification of PD-1/PD-L1 signal paths The key enzyme of color, it will provide new solution for the Three Difficult Issues in the cancer cell therapy that currently faces.
Receptor type Protein-tyrosine-phosphatase O (PTPRO) is one of member of PTPs families, and glomerulus foot is screened at one It is found and clones to come for the first time in the research of cell-specific proteins, therefore also referred to as GLEPP1.PTPRO the mankind, rat, Have between each species such as mouse well-conserved.In human genome, PTPRO genes are located at chromosome 12p13.3-p13.2, Comprising 6 known mRNA Isoforms, wherein 2 main expression products respectively by overall length PTPRO (PTPRO-FL) cDNA and Truncated-type PTPRO (PTPROt) cDNA is encoded.In tumor research, the expression of PTPRO genes and the occurrence and development of tumour It is closely related, it has been found that PTPRO has important cancer suppressing action in a variety of cancers, such as：Breast cancer, prostate cancer, the cancer of the esophagus, Colon cancer, liver cancer, lung cancer etc..The gene regulates and controls the activity of promotion sensitivity gene by dephosphorylation, makes these promotion sensitivity genes So as to influence signal path downstream, inhibitory action is played to the occurrence and development of tumour for activity down-regulation.How safety is high in vivo The expression that effect activates the gene is of great significance to the treatment of tumour.
In recent years research finds that non-coding RNA (ncRNA) molecule can participate in the regulatory mechanisms of gene expressions in multiple levels, Its small molecular double-stranded RNA (dsRNA) has been carried out the specific inhibitory effect of correlative protein expression extensive and deep Research.Current research find dsRNA can specifically by the gene activation of some silences or low expression the phenomenon that, and be referred to as " RNA Activate (RNA activation, RNAa) ", the small dsRNA with activation is referred to as small activation RNA (saRNA).With RNAi compares, and RNAa does not have to consider in terms of tumour is treated whether a certain tumour has specific oncogene as target gene, Thus there is its unique advantage, can such as utilize RNAa activation tumor suppressor gene, Cyclin-dependent kinase gene, Apoptosis base Because etc. treat tumour.
The content of the invention
Based on this, it is an object of the invention to overcome in place of above-mentioned the deficiencies in the prior art and PD-L1 can be made by providing one kind Dephosphorylation so as to block PD-1/PD-L1 accesses, makes T cell activity recovery, realizes the saRNA of tumor immunotherapy.
To achieve the above object, the technical solution taken of the present invention is：A kind of saRNA, the saRNA includes and PTPRO The nucleotide sequence of gene promoter area -3000 to -200 site areas complementation.PTPRO genes are targeted in the present invention as a result, SaRNA can make the Tyr123 site phosphorylations of PD-L1 are horizontal to reduce, and block PD-1/PD-L1 accesses, make T cell activity recovery, So as to enhance immune response；The SaRNA of targeting PTPRO genes may act on the cell or tissue of any high expression PD-L1 albumen, Including malignant tumour, tumor-infiltrating dentritic cells, tumor infiltrating lymphocyte, tumor infiltrating macrophage etc..
Preferably, the saRNA includes the nucleotide to -238 site areas with -220 site of PTPRO gene promoter areas Sequence.
Preferably, the saRNA includes the nucleotide to -676 site areas with -658 site of PTPRO gene promoter areas Sequence.
Preferably, the saRNA is by sequence such as SEQ ID NO:Positive-sense strand and sequence such as SEQ ID NO shown in 2:3 institutes The antisense strand shown forms or by sequence such as SEQ ID NO:Positive-sense strand and sequence such as SEQ ID NO shown in 8:It is anti-shown in 9 Adopted chain composition.
On the other hand, the purposes the present invention also provides above-mentioned saRNA in the drug for treating tumour is prepared.It is preferred that Ground, the saRNA administering modes include intra-tumoral injection, and intraperitoneal injection, is subcutaneously injected and systemic vein is injected.
On the other hand, the present invention also provides being used in combination for above-mentioned saRNA and PD-1/PD-L1 inhibitor to prepare use Purposes in the drug for the treatment of tumour.
Preferably, the PD-1/PD-L1 inhibitor is anti-PD-1 antibody or PD-L1 antibody.
Preferably, the PD-1/PD-L1 inhibitor is avelumab.Present inventor is it was unexpectedly observed that ought be simultaneously During using saRNA and avelumab in the present invention, inhibit the significant effect of tumour growth better than being used alone in the present invention SaRNA or avelumab, illustrate that the saRNA and avelumab that target PTPRO have between the two and synergistic inhibit tumour The effect of growth.
Preferably, which is characterized in that the tumour for NSCLC, melanoma, clear-cell carcinoma, prostate cancer, breast cancer, Glioma, the cancer of the esophagus, Hodgkin lymphoma, malignant pleural mesothelioma or H/N tumors.
In conclusion beneficial effects of the present invention are：
Existing PD-L1 inhibitor can only block PD-1/PD-L1 accesses, once after phosphorylation occurs for PD-L1, PD-L1 The effect of inhibitor will substantially reduce, and the SaRNA of the targeting PTPRO genes of the present invention exactly compensates for this defect, can Make the phosphorylation level of PD-L1 reduce, block PD-1/PD-L1 accesses, make T cell activity recovery, so as to enhance immune response； Meanwhile saRNA of the invention plays synergistic function with PD-L1 inhibitor in the treatment of tumour, significantly improves PD-L1 The therapeutic effect of inhibitor.
Description of the drawings
Fig. 1 is the effect contrast figure that 5 couples of saRNA activate PTPRO gene expressions in the embodiment of the present invention 1；
Fig. 2 Westen blot experimental verifications saPTPRO-220 can improve PTPRO expressions, while reduce PD-L1 phosphorus Acidifying is horizontal；
Fig. 3 is esophageal cancer cell TE1 in MTT experiment three groups of processing groups of detection to PD-L1 monoclonal antibody avelumab medicaments insensitives Property；
Fig. 4 is immunosuppressive factor IL-10, TGF-β and the immune activation factor in three groups of co-cultivation supernatants of ELISA method detection The content of IL-12, IL-23；
The tumor growth curve figure of tumor-bearing mice, the results show saRNA and Avelumab are same when Fig. 5 is different disposal measure When in use, having synergistic antitumor action between the two.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with the drawings and specific embodiments pair The present invention is described further.It should be noted that the chemicals involved in the present invention, cell, consumptive material etc., such as nothing are especially said Bright, from market, purchase obtains.
The design of embodiment 1saRNA molecules and its influence to PTPRO expression
First, the design of saRNA
In website NCBI (http://www.ncbi.nlm.nih.gov/) obtain PTPRO promoter region sequences.
According to the design principle according to RNA sequence, design obtain PTPRO 5 pairs of double-strand tiny RNA activation sequences and this 5 PTPRO promoter regions site is answered sequence pair.Sequence (the SEQ ID NO in -3000 site of PTPRO promoter regions site to -1 site: 1) it is as follows：
TGATTTGGAGTCTTGAAAATAGCATAATAAGATTTATCATACTTTGGAAGTATTGTATTGAAAAACCAGTCAATAGC TCAAAGAAACACAAAACATGCTCTATGAATTGAAAACCCCACACTGTGGATGACACAGCATTCACATTCTTTATGAG AATCTCTTCTAGGACACTGTTATGGTTTAAGTGCAATAAAAACAAATGAAAGTATTTTATCCAGCAATAGCAATGTA AAATACTTTTCTCTAGAGAGGAAATTTTCTGTGATTATAAAATAATACTTTCAGTCTTCAGCCCATCTAACCACAAT GTTACTAATAAAATAACAACAATGCCAATTACTAATGCTTTACTACTTACTGTTTACTGTTATTGTTCCTCCAAAGT GGTCCACATAATATATATATATATATATATATATATACATATATATATATATGCACAAAGACAGAAAGAGCTGAACA AATTGTGATGTATGACTAAGAGAAAAACAGAAAGACGCAGCAGAATATGATTATTTAAAAGGGAGCCTCATTGTGAA AGTTCTTTTAGCATTTACAAGATTAATTTATGATCAGAACTGCTTTAAACGCCCTACGCACATCAGGCAAGGCTATA TCCATGTATACACACAGACATATGCATACACACAAATGAATATCATCATACAGACCCATAATTCACAGACACATTTT AAAATTAAATGCTACTCCAAAGAGAAATTGTTGGCATCCTGTGAGTGTGATTGTTGCCCTTGGCCTATATATATCTT ATGTTCTAGAGATTAGATCACTTTACAGCCACTTCTGAGGGCGAGTGGGAATAAAATGCTGCTTCAGGAGCGTCAAA ATAAAAAGAAAACATATTAAACCAAAGTTCCTATAAGTGCAATCCCAAGGATTAAATGTTCAGATAGCCCGTAAGTC TAACCCAGAGGGAGGGAGGAGCAGTTAACATTTTCTCAAAAGAAAGAAAATGTCCAAACCAATGATGAGTGGACATG AGGGACCTGAAGAGAGCATGTGATGGGAATGTGAAAACAAAAGAAGCTTCTAAAAGAAGACACCAAGGATAATATTC TCACAAAAATTCAGACCATCATTCTATATTTTCATGCATATGAATTTTGGTACATATTTCATGCATATGAAATTTGG TACATATATACATATATGTACCATGCATATACATATGCGTACATATACATATACGTATGCATACACATATGTATCTA TGTACACACATACACATATGTGTACACACATATGTACATGCACACACATATGTGTACACATATGTACATGCACACAC ATGTGTGTACACATATGTACATGTACACATATGTGTACATGTACACATGTGTATATATACATATTCACACTTATGTA AACATATTCAATCTAAAGCTTCCTTAAGACACATACAAACAACAACCAAATGATTAGTGTTTGGCCATTGCTCATGC AGCAAAAGCTGAGTAAACACAGCTCTGGATCCTTTTCTCAGGCCACCACTCCCTAGCTGTGTGACTGACAAAGTCTA TGCCTGAACACCTACATTCATGACCAGGGTGGCCTTTCCTGTCTCCTGTTGCCCAAGCATGTCTCAGGTATAAATAA GTTCCAATACTGACAGGGCACAGCAAGGGTAATTTACATGACAATAAGAAATGATTTCTATCTGAACAGTGCATACC AGAGTTGATTTCGACAGACTTATCTTTAAAAAAATACACATAACAAAAAAGGAGACAAGTAGTAGATTGGGGACCCA CAGCTTGAAAGTCGTTGCTTGTGATTCTAAAACATCCCAGTAAAATTATTCACAGATAATTGTTTAAAAATATAACT GTACATACCGTTGACACTTGGACAACACAGAGAATCACGTAGTTGAAAATCCACATATAACTTTGAACTCATCAAAA CCTTAACTACTAATAGCCTACTCTTGACCGGAAACCTTACTGATAACATAATAAACAGTTGATTAACACACATTTTG TGTTGTATGTATTGTATACTGTATTCTTACAATAAAGTACGCTAGAGAACAGAAAATATTATTAAGAAAACCATAAA GAGGAGAAAATACATTTATATTCATTGAGTGGAAGTGAATTATCATAAAAGTCTTTAACGTCATCCTTTTCAGGCTG AGCAGGCAGAGGAGGAGGAAGAGGGTTTGGTCTTGCTGTCTCAAGGCTGGCAAAGGTGGAAGAAAATCTGCATATAA CTGGACCCAACAGTTCAAACCCGTGTTGTTCAAGGGTCATACATGTAAAATACTGTGATTTTTCCCCCTTCTATATT CAGCTTCAGGTGACCCGACACACTTTGGTATCAAAAGAGAATCTGAAATGTACAAGAACTGCGGATTTCAAATGGAA AAGGTGCATAATTGTGCTATTTGTTCCTGGGTGAGTGTGGGACGGAGACGGTGAGAGTGTTGAAATGGGATGGAGAT AATGGAAGCAGTGGGGAAGGAGAGAAAATACCCTTCCTATCACACACACTCACACACTCACACTACACACTATTTCT ACAGTCACAACTACCCAACTGTTATTGATCCTTTATAACTGCAATTGAGTACAGATGTAGGAAGATTGAGAGGGAAC TGGGATCTGGCGCCTGGATTGCTCAAGAGAGGTCAGGGAAACCCCTCAGAACTCCTGAGACCCAGAGATTGAGGGAG GGGTTGAGGCGGAGTCTGCAATGGGGGCTGTCCAGCAGTAGCAAGCAGCGGGCCGATCCTGGTGGAGGGTTGGGAGG CTGCTGTCATTTTATGGGTCGGCAGCCAGAGTGAGAGTGTCCCTGCTGCCAGAGGACTACGGCGGGCTGGGCGCGGG GTCCCCGCCTCTCGCTCACCACACAGACCCCGCGCCTCCTCTGGCAGCCGCGGTGGTGGCGGCGGCAGAGCCTCGCC CACTCCAATCCCCACCCTCTCCATCCTTAGTCATTAAAGAACAGCAGCGCCTGGCACGTTCTTGGAGGACCCCG。
saPTPRO-220：
Positive-sense strand：5’-GGU UGG GAG GCU GCU GUC A[dT][dT]-3’(SEQ ID NO:2)
Antisense strand：5’-UGA CAG CAG CCU CCC AAC C[dT][dT]-3’(SEQ ID NO:3)
(- 220 site of PTPRO promoter regions to -238 sites).
saPTPRO-398：
Positive-sense strand：5’-AUU GAG UAC AGA UGU AGG A[dT][dT]-3’(SEQ ID NO:4)
Antisense strand：5’-UCC UAC AUC UCU ACU CAA U[dT][dT]-3’(SEQ ID NO:5)
(- 398 site of PTPRO promoter regions to -416 sites).
saPTPRO-550：
Positive-sense strand：5’-AGA CGG UGA GAG UGU UGA A[dT][dT]-3’(SEQ ID NO:6)
Antisense strand：5’-UUC AAC ACU CUC ACC GUC U[dT][dT]-3’(SEQ ID NO:7)
(- 550 site of PTPRO promoter regions to -568 sites).
saPTPRO-658：
Positive-sense strand：5’-CCG ACA CAC UUU GGU AUC A[dT][dT]-3’(SEQ ID NO:8)
Antisense strand：5’-UGA UAC CAA AGU GUG UCG G[dT][dT]-3’(SEQ ID NO:9)
(- 658 site of PTPRO promoter regions to -676 sites).
saPTPRO-1026：
Positive-sense strand：5’-AAA CCU UAC UGA UAA CAU A[dT][dT]-3’(SEQ ID NO:10)
Antisense strand：5’-UAU GUU AUC AGU AAG GUU U[dT][dT]-3’(SEQ ID NO:11)
(- 1026 site of PTPRO promoter regions to -1044 sites).
2nd, the influence that above-mentioned saRNA expresses PTPRO
Breast cancer cell line SKBR3 is incubated in the DMEM/F12 complete mediums containing 10% embryo cow's serum, is placed in 37 DEG C, 5%CO2, saturated humidity cell incubator in cultivate, the cell in growth period of taking the logarithm is inoculated in 6 holes by 2 × 105/ml Culture plate overnight incubation.Next day more than saRNAs and dscontrol is harvested with the final concentration of 50nM, transfectional cell, transfection after 5 days Cell, TRIzol reagents extraction cell total rna, and the PTPROmRNA of target gene is analyzed in reverse transcription for cDNA fluorescence quantitative PCR methods Differential expression.Primer see the table below 1 used in quantitative fluorescent PCR:
Primer used in 1 quantitative fluorescent PCR of table
The results are shown in Figure 1, and saPTPRO-220, saPTPRO-658 activation effect are substantially better than other 3 couples, and SaPTPRO-220 effects are optimal, other three couples of saPTPRO-398, saPTPRO-550, saPTPRO-1026 effect unobvious.
Embodiment 2westen blot experimental verifications SaPTPRO reduces the phosphorylation level of PD-L1 albumen
By the cell inoculation in exponential phase in 6 orifice plates, cell incubator continues to cultivate, Cell abundance 50%- SaRNA is transfected during 60＆.Specification is transfected according to lipofectamine2000 to be transfected, transfection concentrations 50nmol/L.48 Leach protein after hour, BAC methods survey protein concentration, and western blot detect the expression of PTPROp-PD-L1PD-L1 albumen.
SDS-PAGE electrophoresis：A certain amount of protein extract (5 μ g containing albumen) and co-immunoprecipitation product are taken, it is appropriate to add in 5 × SDS sample-loading buffers, boiling water bath, which boils 10min, makes protein denaturation, 10000 × g centrifugations 10min；SDS-PAGE electrophoresis point It is 10% from glue, concentration glue is 5%；In a predetermined order using sample loading gun loading, in no sample well plus isometric 5 × Sds gel sample-loading buffer；100V about 70min are changed to after 60V 20min electrophoresis, until bromophenol blue reaches the bottom of separation gel, Close power supply.
Transferring film：Constant current 300mA, time 120min.
Closing：5% skimmed milk power is closed, 37 DEG C of shaking table one hour.It is shaken after having closed with TBST buffer solutions and washes 10min, weight It is 3 times multiple.
Primary antibody is incubated：Primary antibody used is respectively anti-PTPRO, anti-PD-L1, anti-p-PD-L1 (Y123) antibody, by 1:2000 ratios Example dilution, level slowly shake up, and 4 DEG C overnight.
Secondary antibody is incubated：37 DEG C of effect 20min of next day, abandon primary antibody solution, 1 × TBST washes film 10min, is repeated 4 times.Film is put In with 1 × TBST press 1:In the secondary antibody dilution of the goat anti-rabbit igg of 2000 diluted HRP marks, 37 DEG C of shaking table, 2h.
Two corresponding anti-solution is abandoned, 1 × TBST washes film 10min, is repeated 3 times.Illustrate to develop using ECL kits according to producer, clap According to record.
The results are shown in Figure 2, the results show that after saRNA transfections, the rise of PTPRO protein expression levels, PD-L1 phosphorylations Level reduces.
Embodiment 3MTT detects influence and ELISA of three groups of processing to TE1 cell growths and detects three groups of tumour cells and T Cell co-cultures the content of immunosuppressive factor IL-10, TGF-β and immune activation factor IL-12, IL-23 in supernatant
Step 1：The preparation of ripe DC cells
Density-gradient centrifugation method separates PBMC：
(1) healthy premenopausal volunteers 10m1 anticoagulant heparin peripheric venous bloods are taken under germ-free condition.
(2) by venous blood and the abundant mixing of isometric sterile saline, it is slowly dropped into 5ml syringes along tube wall On Ficoll-Paque human lymphocyte separating liquid liquid levels.2000rpm centrifuges 20min.
(3) liquid in pipe is divided into four layers after centrifuging：Upper strata is yellow plasma layer, and the second layer is white cloud narrow band, Third layer is lymphocyte separation medium and sterile saline, and the 4th layer is red blood cell layer
(4) white cloud narrow band is slowly drawn with 5ml syringes, inserted in new 15ml centrifuge tubes, add in 5 times of bodies Long-pending physiological saline, 1800rpm centrifugation 10min, abandons supernatant, after coming again.PBMCs is harvested to count.
(6) plant in culture dish, culture medium selects the U.S. giblo companies culture mediums of RPMI -1640 (not increase serum, double It is anti-).
(7) at 7~10 days, cell concentration l × 106A/ml only adds in GM-CSF (800U/mL), IL-4 in culture medium (1000U/mL) promotes immature DC to be proliferated.Then TNF-a is added in promote DC ripe, cultivates 3-5 days harvest cells.
Step 2：The preparation of cytotoxic T cell (CTL)：
(1) cancer of the esophagus TE1 cells are collected, rinses, be resuspended.
(2) and by cell count concentration adjust to 1 × 107A/mL, quick freeze is into liquid nitrogen container several minutes, Ran Houyu 37 DEG C of water-baths are thawed, multigelation 3 times；
(3) 1000r/min turns low-speed centrifugal 20min, collects supernatant filtration sterilization, 4 DEG C of preservations；
(4) DC cell culture to the 7th day when add in Tumor lysate, while add in TNF-a (15ng/mL)；
At (5) the 7th days, the DC (1 × 10 of tumour lysates antigen will be loaded6A/mI) with T lymphocytes press 1：50 ratios are mixed It amounts to being incubated 3 days, it is tumor-specific CTL to collect gained cell.
Step 3：Tumor cell proliferation detects and co-cultures immunosuppressive factor IL-10 in supernatant, TGF-β and is immunized The detection of activity factor IL-12, IL-23.
(1) experiment is divided into three groups：Blank control group, dscontrol groups, dsPTPRO groups before the treatment, will be in logarithm In 6 orifice plates, cell incubator continues to cultivate the cell inoculation in growth period, and Cell abundance transfects saRNA when being 50%-60＆.According to Lipofectamine2000 transfection specifications are transfected, transfection concentrations 50nmol/L.Cell state is observed after 6h, is replaced Culture solution, every group of cell adds in the tumor-specific CTL of isoconcentration and tumour cell co-cultures.Add in the processing of PD-1 blocking agents 72 it is small when.It is every to change a subculture and drug for 24 hours.
(2) esophageal cancer cell TE1 increments detection：Esophageal cancer cell TE1 and T cell co-culture system are established, TE1 cells are done Three kinds of processing, are divided into：Blank control group, dscontrol groups, dsPTPRO groups configure the cell suspension of 100ul in 96 orifice plates. 3 multiple holes are all provided in experiment for every group.By culture plate be placed on incubator preculture 24 it is small when, it is mono- to add in 10u1 anti-PD-1 to culture plate Anti- avelumab, concentration 0ug, 0.01ug, 0.1ug, 1.0ug, 10ug are small for 72 in incubator incubation time by culture plate When, per hole add in l0ul CCK-8 solution, in incubator be incubated 2 it is small when after microplate reader measure absorbance (450nm).
As shown in figure 3, the results show dsPTPRO is remarkably improved the drug susceptibility of PD-L1 monoclonal antibodies avelumab.
(3) inspection of immunosuppressive factor IL-10, TGF-β and immune activation factor IL-12, IL-23 in supernatant are co-cultured It surveys：Exemplified by detecting TGF-β：By 1000pg/ml, 500pg/ml, 250pg/ml, 125pg/ml, 62.5pg/ml, 31.3pg/ Each 0.1ml of standard items of ml, 15.6pg/ml are once added in kit in elisa plate hole, and 1 hole only adds sample diluting liquid conduct Zero hole, 1 hole is as TMB blank colour developing hole；Sequentially add supernatant, 100 μ l/ holes；ELISA Plate is capped, 37 DEG C of reaction 90min；Reaction The anti-human IFN-γ antibody working solution of ready biotin is sequentially added by 100 μ l/ holes afterwards, 37 DEG C of reaction 60min； 0.01MPBS is washed 3 times, is impregnated 1 minute every time；Ready ABC working solutions are sequentially added by 100 μ l/ holes, 37 DEG C of reactions 30min；0.01M PBS are washed 5 times, impregnate 1-2min or so every time；TMB developing solutions are sequentially added by every 90 μ l of hole, 37 DEG C are kept away Light reaction 25min；TMB terminate liquids are sequentially added by 100 μ l/ holes, blueness is vertical at this time turns yellow；With microplate reader in 450nm wavelength Place measures A450 values.Using IFN-γ concentration as abscissa, A450nm values are ordinate, draw standard curve, calculate detection sample The content of middle TGF-β.
Result of the test：As shown in figure 4, the results show dsPTPRO is remarkably improved immune activation factor IL-12, IL-23 Content reduces immunosuppressive factor IL-10, the content of TGF-β.
SaPTPRO-220 is verified in 4 Mice Body of embodiment and its with PD-L1 inhibitor use in conjunction to the shadow of tumour growth It rings
6 week old are chosen, weight 18-20g, health status is similar, the good nude mice of growth conditions 40, random point four groups, It is raised in SPF grades of animal houses；
Logarithmic phase growth TE1 cells are chosen, PBS, which is resuspended, to be counted, and 2x10 is made6The cell suspension of a/200ul, in mouse It is subcutaneously injected on the outside of groin, each injection site injection volume is 200ul；The human peripheral blood mononuclear cell PBMC of separator well is taken, It is resuspended into cell suspension, is injected in nude mouse with 1640 cell culture fluids.It is small to four groups respectively after PBMC is injected first day Tail vein injection PBS, avelumab, saRNA, saRNA+avelumab, wherein saRNA injection dosages are according to the weight 50nmol/kg/kg weight, injection in every 7 days is once.The injection dosage of avelumab is 10mg/kg weight, and injection in every 7 days is once.
When experiment was carried out to the 5th week, mouse is put to death, take knurl measurement size and is weighed.Statistical analysis saPTPRO-220 and Itself and influence of the PD-L1 inhibitor use in conjunction to tumour growth.It draws tumor growth curve and is analyzed with statistical method The difference of four groups of mice tumors grews.
The results are shown in Figure 5, four groups of growth curve differences show saRNA (i.e. saPTPRO-220), avelumab and SaRNA+avelumab plays the role of inhibiting gross tumor volume growth compared with PBS, wherein, saRNA, avelumab is used alone When, almost identical to the inhibition of tumour growth, when using saRNA+avelumab simultaneously, gross tumor volume growth rate is shown Writing reduces, and even, while using saRNA+avelumab after about 15 days, gross tumor volume starts to reduce, illustrate saRNA and Avelumab simultaneously in use, have synergistic antineoplastic action between the two.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected The limitation of scope is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should Understand, technical scheme can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention And scope.
<110>Zhang Hao
<120>A kind of saRNA and application thereof
<160> 11
<170> PatentIn version 3.3
<210> 1
<211> 3000
<212> DNA
<213>Homo sapiens（Homo sapiens）
<400> 1
tgatttggag tcttgaaaat agcataataa gatttatcat actttggaag tattgtattg 60
aaaaaccagt caatagctca aagaaacaca aaacatgctc tatgaattga aaaccccaca 120
ctgtggatga cacagcattc acattcttta tgagaatctc ttctaggaca ctgttatggt 180
ttaagtgcaa taaaaacaaa tgaaagtatt ttatccagca atagcaatgt aaaatacttt 240
tctctagaga ggaaattttc tgtgattata aaataatact ttcagtcttc agcccatcta 300
accacaatgt tactaataaa ataacaacaa tgccaattac taatgcttta ctacttactg 360
tttactgtta ttgttcctcc aaagtggtcc acataatata tatatatata tatatatata 420
tacatatata tatatatgca caaagacaga aagagctgaa caaattgtga tgtatgacta 480
agagaaaaac agaaagacgc agcagaatat gattatttaa aagggagcct cattgtgaaa 540
gttcttttag catttacaag attaatttat gatcagaact gctttaaacg ccctacgcac 600
atcaggcaag gctatatcca tgtatacaca cagacatatg catacacaca aatgaatatc 660
atcatacaga cccataattc acagacacat tttaaaatta aatgctactc caaagagaaa 720
ttgttggcat cctgtgagtg tgattgttgc ccttggccta tatatatctt atgttctaga 780
gattagatca ctttacagcc acttctgagg gcgagtggga ataaaatgct gcttcaggag 840
cgtcaaaata aaaagaaaac atattaaacc aaagttccta taagtgcaat cccaaggatt 900
aaatgttcag atagcccgta agtctaaccc agagggaggg aggagcagtt aacattttct 960
caaaagaaag aaaatgtcca aaccaatgat gagtggacat gagggacctg aagagagcat 1020
gtgatgggaa tgtgaaaaca aaagaagctt ctaaaagaag acaccaagga taatattctc 1080
acaaaaattc agaccatcat tctatatttt catgcatatg aattttggta catatttcat 1140
gcatatgaaa tttggtacat atatacatat atgtaccatg catatacata tgcgtacata 1200
tacatatacg tatgcataca catatgtatc tatgtacaca catacacata tgtgtacaca 1260
catatgtaca tgcacacaca tatgtgtaca catatgtaca tgcacacaca tgtgtgtaca 1320
catatgtaca tgtacacata tgtgtacatg tacacatgtg tatatataca tattcacact 1380
tatgtaaaca tattcaatct aaagcttcct taagacacat acaaacaaca accaaatgat 1440
tagtgtttgg ccattgctca tgcagcaaaa gctgagtaaa cacagctctg gatccttttc 1500
tcaggccacc actccctagc tgtgtgactg acaaagtcta tgcctgaaca cctacattca 1560
tgaccagggt ggcctttcct gtctcctgtt gcccaagcat gtctcaggta taaataagtt 1620
ccaatactga cagggcacag caagggtaat ttacatgaca ataagaaatg atttctatct 1680
gaacagtgca taccagagtt gatttcgaca gacttatctt taaaaaaata cacataacaa 1740
aaaaggagac aagtagtaga ttggggaccc acagcttgaa agtcgttgct tgtgattcta 1800
aaacatccca gtaaaattat tcacagataa ttgtttaaaa atataactgt acataccgtt 1860
gacacttgga caacacagag aatcacgtag ttgaaaatcc acatataact ttgaactcat 1920
caaaacctta actactaata gcctactctt gaccggaaac cttactgata acataataaa 1980
cagttgatta acacacattt tgtgttgtat gtattgtata ctgtattctt acaataaagt 2040
acgctagaga acagaaaata ttattaagaa aaccataaag aggagaaaat acatttatat 2100
tcattgagtg gaagtgaatt atcataaaag tctttaacgt catccttttc aggctgagca 2160
ggcagaggag gaggaagagg gtttggtctt gctgtctcaa ggctggcaaa ggtggaagaa 2220
aatctgcata taactggacc caacagttca aacccgtgtt gttcaagggt catacatgta 2280
aaatactgtg atttttcccc cttctatatt cagcttcagg tgacccgaca cactttggta 2340
tcaaaagaga atctgaaatg tacaagaact gcggatttca aatggaaaag gtgcataatt 2400
gtgctatttg ttcctgggtg agtgtgggac ggagacggtg agagtgttga aatgggatgg 2460
agataatgga agcagtgggg aaggagagaa aatacccttc ctatcacaca cactcacaca 2520
ctcacactac acactatttc tacagtcaca actacccaac tgttattgat cctttataac 2580
tgcaattgag tacagatgta ggaagattga gagggaactg ggatctggcg cctggattgc 2640
tcaagagagg tcagggaaac ccctcagaac tcctgagacc cagagattga gggaggggtt 2700
gaggcggagt ctgcaatggg ggctgtccag cagtagcaag cagcgggccg atcctggtgg 2760
agggttggga ggctgctgtc attttatggg tcggcagcca gagtgagagt gtccctgctg 2820
ccagaggact acggcgggct gggcgcgggg tccccgcctc tcgctcacca cacagacccc 2880
gcgcctcctc tggcagccgc ggtggtggcg gcggcagagc ctcgcccact ccaatcccca 2940
ccctctccat ccttagtcat taaagaacag cagcgcctgg cacgttcttg gaggaccccg 3000
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<400> 2
gguugggagg cugcugucat t 21
<210> 3
<211> 21
<212> DNA
<213>Artificial sequence
<400> 3
ugacagcagc cucccaacct t 21
<210> 4
<211> 21
<212> DNA
<213>Artificial sequence
<400> 4
auugaguaca gauguaggat t 21
<210> 5
<211> 21
<212> DNA
<213>Artificial sequence
<400> 5
uccuacaucu cuacucaaut t 21
<210> 6
<211> 21
<212> DNA
<213>Artificial sequence
<400> 6
agacggugag aguguugaat t 21
<210> 7
<211> 21
<212> DNA
<213>Artificial sequence
<400> 7
uucaacacuc ucaccgucut t 21
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence
<400> 8
ccgacacacu uugguaucat t 21
<210> 9
<211> 21
<212> DNA
<213>Artificial sequence
<400> 9
ugauaccaaa gugugucggt t 21
<210> 10
<211> 21
<212> DNA
<213>Artificial sequence
<400> 10
aaaccuuacu gauaacauat t 21
<210> 11
<211> 21
<212> DNA
<213>Artificial sequence
<400> 11
uauguuauca guaagguuut t 21

Claims (9)

1. a kind of saRNA, which is characterized in that the saRNA includes and -3000 to -200 site area of PTPRO gene promoter areas The nucleotide sequence of domain complementation.
2. saRNA as described in claim 1, which is characterized in that the saRNA includes and PTPRO gene promoter areas -220 The nucleotide sequence of site extremely -238 site areas.
3. saRNA as described in claim 1, which is characterized in that the saRNA includes and PTPRO gene promoter areas -658 The nucleotide sequence of site extremely -676 site areas.
4. saRNA according to claim 1, which is characterized in that the saRNA is by sequence such as SEQ ID NO:Shown in 2 Positive-sense strand and sequence such as SEQ ID NO:Antisense strand shown in 3 forms or by sequence such as SEQ ID NO:Positive-sense strand shown in 8 With sequence such as SEQ ID NO:Antisense strand composition shown in 9.
5. purposes of the saRNA in the drug for treating tumour is prepared as described in claim 1-4 is any.
6. saRNA and PD-1/PD-L1 inhibitor as described in claim 1-4 is any is used in combination preparing for treating Purposes in the drug of tumour.
7. purposes as claimed in claim 6, which is characterized in that the PD-1/PD-L1 inhibitor is anti-PD-1 antibody or PD- L1 antibody.
8. purposes according to claim 6, which is characterized in that the PD-1/PD-L1 inhibitor is avelumab.
9. purposes according to claim 6, which is characterized in that the tumour is NSCLC, melanoma, clear-cell carcinoma, preceding Row gland cancer, breast cancer, glioma, the cancer of the esophagus, Hodgkin lymphoma, malignant pleural mesothelioma or H/N tumors.
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