CN107119118A - The application of BANCR long-chains non-coding RNA and its micromolecular inhibitor in oophoroma hepatic metastases is suppressed - Google Patents
The application of BANCR long-chains non-coding RNA and its micromolecular inhibitor in oophoroma hepatic metastases is suppressed Download PDFInfo
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Abstract
The invention discloses the application of BANCR long-chains non-coding RNA and its micromolecular inhibitor in oophoroma hepatic metastases is suppressed.Present invention discover that, Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and Pyrrolopyrimidine thion III can significantly inhibit the expression of LncRNA BANCR in HO 8910PM cells, so as to significantly inhibit the motion invasive ability of HO 8910PM cells, suppress the ability that HO 8910PM cells attack liver formation metastatic liver cancer in nude mouse.The inhibitor of Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and Pyrrolopyrimidine thion III and other LncRNA BANCR can be used for developing the medicine for being prepared into and suppressing oophoroma hepatic metastases.The present invention is progressive with prominent substantive distinguishing features and significantly.
Description
Technical field
The invention belongs to field of gene, it is related to the application of gene target and its inhibitor, and in particular to LncRNA
The application of BANCR and its micromolecular inhibitor in oophoroma hepatic metastases is suppressed.
Background technology
Liver is often one of organ of generation Malignant tumor of bonal metastasis.Surgery excision is effective treatment means of metastatic liver cancer.
However, about 80-90% metastatic liver cancer patient is because liver function can not compensatory, metastatic carcinoma be big and many, the primary disease progression of number etc.
Reason can not row surgery excision.Metastatic liver cancer for being unable to surgery excision, more using systemic chemotherapy, trans-hepatic artery PCI
Or palliative therapy, effect is unsatisfactory.Using gene therapy means, on a molecular scale to the hepatic metastases of malignant tumour
Potential is suppressed, and is reduction malignant tumour hepatic metastases incidence, is improved hope (the Exploring gene of prognosis
expression signatures for predicting disease free survival after resection of
colorectal cancer liver metastases,PLoS One,2012；Inhibition of chemokine
receptor expression on uveal melanomas by CXCR4 siRNA and its effect on uveal
melanoma liver metastases,Invest OphthalmolVis Sci,2009)。
Non-coding RNA refers to the RNA of not encoding proteins matter, including rRNA, tRNA, snRNA, snoRNA, miRNA and
Long-chain non-coding RNA (Lnc RNA).Lnc RNA are the RNA molecules that a class length is more than 200 bases, are effectively opened due to lacking
The formula reading frame of putting not encoding proteins, but possess the biological function of complexity and played an important role in various bioprocess,
Such as chromatin modification, the inactivation of X chromosome, participate in genetic transcription, translation and to albumen activity regulate and control, its make a variation and adjust
It can result in the multiple disease including tumour.The Lnc RNA of unconventionality expression can pass through different approaches and different mechanism of action
Each stage of generation, development, invasion and attack and the transfer of tumour is participated in, is the key factor of tumour progression.Recent study shows
The Lnc RNA of unconventionality expression take part in the regulation and control such as apoptosis, propagation, invasion and attack and the transfer of tumour cell by multiple pathways, with liver
The generation of the tumours such as cancer has close relationship with transfer.
The long-chain non-coding RNA that the non-coding RNA (BANCR) of BRAF activation is 693bp, first in melanoma cells
It is found, then, Lnc is found in thyroid papillary carcinoma, retinoblastoma cell cancer, lung cancer, stomach cancer, colorectal cancer and liver cancer
RNA BANCR abnormal expressions.
At present, not yet there are some researches prove LncRNA BANCR and its micromolecular inhibitor have with suppressing oophoroma hepatic metastases
Close.
The content of the invention
Present invention aims at provide LncRNA BANCR and its micromolecular inhibitor in oophoroma hepatic metastases is suppressed
Using.
Realize that above-mentioned purpose technical scheme of the present invention is as follows：
Applications of the LncRNA BANCR as drug targets in the medicine for suppressing oophoroma hepatic metastases is prepared.
Application of the LncRNA BANCR inhibitor in the medicine for suppressing oophoroma hepatic metastases is prepared.
Preferably, the inhibitor is the Pyrrolopyrimidine thion micromolecular compound with following general structure,
Wherein, R is alkyl.
Preferably, the inhibitor is selected from the compound of following structure：
One kind is used to suppress oophoroma hepatic metastases pharmaceutical composition, contains any of the above-described inhibitor.
The outstanding advantages of the present invention：
Present invention discover that Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and Pyrrolopyrimidine thion III can significantly inhibit HO-
LncRNA BANCR expression in 8910PM cells, so as to suppress the motion invasion and attack of HO-8910PM cells, suppresses HO-8910PM
Cell attacks the ability of liver formation metastatic liver cancer in nude mouse.Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and pyrrolo-
Pyrimidone III and other LncRNA BANCR inhibitor can be used for developing the medicine for being prepared into and suppressing oophoroma hepatic metastases.
Brief description of the drawings
Fig. 1 is each group HO-8910PM cell BANCR mRNA relative expression levels；
Fig. 2 is each group HO-8910PM cell E-cadherin and Vimentin relative expression levels；
Fig. 3 is each group HO-8910PM cell movements inhibiting rate (%) and invasion and attack inhibiting rate (%)；
Fig. 4 is each group metastatic liver cancer incidence (%).
Embodiment
The just in conjunction with the embodiments specific essentiality content for introducing the present invention below, due to length reason, experimentation is retouched
Stating can not accomplish very in detail, and the part not being described in detail in every experiment is conventional behaviour well known to those skilled in the art
Make.
First, experiment material
Human ovarian cancer high-transfer cell HO-8910PM is purchased from hundred fervent (Shanghai) Biotechnology Ltd. of match.4 week old
Healthy female BALB/c nude mices, body weight is 19-21g, purchased from Nanjing University's animal center.
Pyrrolopyrimidine thion I, II, III is synthesized by our company's combining unit according to literature method, and structure is confirmed through nuclear-magnetism.With
DMSO dissolves the mother liquor that 1mg/mL is made, and various concentrations are diluted to as needed.RPMI-1640 culture mediums are purchased from Gibco companies；
Hyclone is purchased from Hangzhou Chinese holly bioengineering and Materials Research Laboratories；Transwell cells are purchased from Corning companies.
2nd, experimental method
1st, cell culture and packet
Recovery culture Proliferation of Human Ovarian Cell HO-8910PM.With the RPMI-1640 culture mediums containing 10% hyclone (containing double
It is anti-) cellar culture is in 37 DEG C, 5%CO2In the constant incubator of relative humidity 95%.Cell passes through had digestive transfer culture, takes the logarithm
The cell in growth period is cultivated and tested.Ensure that cell used refuses dye rate more than 95% before experiment with trypan exclusion stain.
Administration group is grouped and administration concentration is as follows：
I group of Pyrrolopyrimidine thion：Contain final concentration of 5 μM, 20 μM of Pyrrolopyrimidine thion I in nutrient solution；
II group of Pyrrolopyrimidine thion：Contain final concentration of 5 μM, 20 μM of Pyrrolopyrimidine thion II in nutrient solution；
III group of Pyrrolopyrimidine thion：Contain final concentration of 5 μM, 20 μM of Pyrrolopyrimidine thion III in nutrient solution.
2nd, RNA is extracted and RT-PCR detection BANCR mRNA relative expression levels
6 orifice plates are added after HO-8910PM cell dissociations are counted, cell density is 2 × 105After/mL, cell attachment, plus
Enter 5 μM of Pyrrolopyrimidine thion, 20 μM of culture 24h, collect cell, determine BANCR relative expression levels.Another setting control group, it is right
According to group without Pyrrolopyrimidine thion.Total serum IgE is extracted using TRIzol reagents (Invitrogen companies).Use
The random primer of PrimeScript RT kits (TaKaRa companies) at the standard conditions, be by total serum IgE (500ng) reverse transcription
10 μ L final volume.The detection of BANCR expressions is said according to STBR Premix Ex Taq (TaKaRa companies) use
It is bright to carry out.Pass through 2-ΔΔCtMethod, according to internal reference GAPDH cubage BANCR mRNA relative expression levels.
LncRNABANCR and GAPDH RT-PCR primer is as follows：
BANCR sense primers：5’-ACAGGACTCCATGGCAAACG-3’；
BANCR anti-sense primers：5’-ATGAAGAAAGCCTGGTGCAGT-3’；
GAPDH sense primers：5’-ACCACAGTCCATGCCATCAC-3’；
GAPDH anti-sense primers：5’-TCCACCACCCTGTTGCTGTA-3’.
3rd, Western blot detect protein expression
By detecting that the concentration of E-cadherin and Vimentin albumen can draw BANCR expression indirectly.
6 orifice plates are added after HO-8910PM cell dissociations are counted, cell density is 2 × 105After/mL, cell attachment, plus
Enter 5 μM of Pyrrolopyrimidine thion, 20 μM of culture 24h, collect cell, determine E-cadherin and Vimentin albumen relative expression's water
It is flat.Another to set control group, control group is without Pyrrolopyrimidine thion.Using mammalian proteins extracts reagent RIPA, and supplement
Partial Protein Protease Inhibitor Cocktail and phenylmethyl sulfonylfluoride dissolve cell.Egg is determined with Bio-Rad analysis of protein boxes
White concentration.Take 50 μ g protein extracts to carry out 10% SDS-PAGE experiments, protein is gone into nitrocellulose filter (Sigma
Company), and use 1：E-cadherin antibody (BD companies), Vimentin antibody (the Cell Signaling of 1000 dilutions
Technology companies) it is incubated.By gel images processing system (QuantityOne softwares, Bio-Rad companies) to radiating certainly
Development picture is quantified, using GAPDH as control, determines E-cadherin and Vimentin albumen relative expression levels.
4th, Transwell methods detection HO-8910PM cell movement invasive abilities
The μ L of RPMI-1640 culture mediums 600 containing 10 μ g/mL fiber laminins are added per hole in 24 well culture plates.
The HO-8910PM cells of exponential phase are collected, use adds Pyrrolopyrimidine thion (5 μM, 20 μM) and do not add pyrrolopyrimidine respectively
The RPMI-1640 culture mediums of ketone are resuspended, and it is 1 × 10 to adjust concentration6/mL.Transwell cells are dipped in the condition of 24 orifice plates
In culture medium, each cell adds the μ L of cell suspension 100, puts 37 DEG C, 5%CO2Culture 6h in incubator.By Transwell cells
Take out, filter membrane fixes 1min, violet staining 15min with methanol, wash, cotton swab carefully wipes the cell for not wearing film, neutral tree
Glue mounting, in counting the cell number through filter membrane under 400 power microscopes.5 random different visuals field in being counted up and down per film,
Every group parallel to set 3 filter membranes.Motion inhibiting rate (%) is calculated by following formula.In Transwell cells film during invasion and attack experiment
Inner surface apply the μ g (10 μ L) of basement membrane components Matrigel 5, super-clean bench ventilation is allowed to drying, forms artificial recombination base overnight
Counterdie, remaining step is tested with cell movement.Invasion and attack inhibiting rate (%) is calculated by following formula：
Motion inhibiting rate (%)=(control group wears theca cell number-administration group and wears theca cell number)/control group wears theca cell number
× 100%；
Invasion and attack inhibiting rate (%)=(control group wears theca cell number-administration group and wears theca cell number)/control group wears theca cell number
× 100%.
5th, ovarian Cancer of Nude Mice liver metastasis model observation cancer metastasis ability
40 nude mices are raised under SPF environment, being randomly divided into I group of Pyrrolopyrimidine thion, II group of Pyrrolopyrimidine thion, pyrroles
III group of hepyramine and Normal group, carry out ovarian cancer cell and are tested into knurl by every group 10.Nude mice weighs, anaesthetize after open abdomen,
Exposure spleen.Collect exponential phase HO-8910PM cells 1 × 106It is individual, it is resuspended in after 0.1ml physiological saline, in nude mice spleen
Slow injection about 5min.Compressing injection pin hole hemostasis 3min, prevents tumour from closing abdomen after abdominal metastas, operating process follows no knurl
Principle.Nude mice plants the cancer cell same day, and the daily tail vein of each group injects Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion respectively
IIth, the 5mg/kg of Pyrrolopyrimidine thion III, control group injection normal saline, continuous injection 28 days.Last time injection 8 hours
After take nude mice liver, row 2mm thickness serial section, whether metastatic liver cancer is formed for observation, counts liver metastasis cancer nude mice number
Be averaged into knurl quantity, and metastatic liver cancer incidence (%) is calculated as follows：
Metastatic liver cancer incidence (%)=liver metastasis nude mice number/this group of nude mice sum × 100%.
In experimentation, each group is dead without nude mice, and every group of final nude mice sum is 10.
6th, statistical method
Data analysis is carried out using SPSS16.0 statistical softwares, with P<0.05 is that difference is statistically significant.
3rd, experimental result
1st, influence of the Pyrrolopyrimidine thion to HO-8910PM cell BANCR mRNA relative expression levels
Compared with control group, Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and the administration group HO- of Pyrrolopyrimidine thion III
8910PM cell BANCR mRNA relative expression levels significantly reduce (P ＜ 0.05), and high concentration group (20 μM) compares low concentration
Group (5 μM) HO-8910PM cell BANCR mRNA relative expression levels reduction is more notable.Table 1 and Fig. 1 are each group HO-
8910PM cell BANCR mRNA relative expression levels compare.
The each group HO-8910PM cell BANCR mRNA relative expression levels of table 1
2nd, influence of the Pyrrolopyrimidine thion to HO-8910PM cell BANCR expressions
Known LncRNA BANCR take part in the regulation of endothelial cell interstitial approach, and meeting is lowered in LncRNA BANCR expression
Cause under Vimentin reconcile E-cadherin up-regulation, by detect E-cadherin and Vimentin albumen concentration can between
Connect the expression for drawing BANCR.Compared with control group, Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and Pyrrolopyrimidine thion
III administration group HO-8910PM cells Vimentin expression is significantly reduced (P ＜ 0.05), E-cadherin expression water
Average significantly rise (P ＜ 0.05), and high concentration group (20 μM) is than (5 μM) reductions of low concentration group or raises more notable.The He of table 2
Fig. 2 is that each group HO-8910PM cell E-cadherin and Vimentin relative expression levels compare.
Each group HO-8910PM cell E-cadherin and the Vimentin relative expression levels of table 2
3rd, influence of the Pyrrolopyrimidine thion to HO-8910PM cell movement invasive abilities
Compared with control group, Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and the administration group HO- of Pyrrolopyrimidine thion III
The motion of 8910PM cells and invasive ability are significantly reduced (P ＜ 0.05), and high concentration group (20 μM) is than low concentration group (5 μM)
Suppressed effect is more notable.Table 3 and Fig. 3 are each group HO-8910PM cell movements inhibiting rate (%) and invasion and attack inhibiting rate (%).
The each group HO-8910PM cell movements inhibiting rate (%) of table 3 and invasion and attack inhibiting rate (%)
4th, influence of the Pyrrolopyrimidine thion to ovarian Cancer of Nude Mice hepatic metastases incidence
In observation period, each group is dead without nude mice, and every group of final nude mice sum is 10.Compared with control group, pyrrole
Coughing up hepyramine I, Pyrrolopyrimidine thion II and the administration group metastatic liver cancer incidence (%) of Pyrrolopyrimidine thion III significantly reduces (P
＜ 0.05).
Each group liver metastasis nude mice number and metastatic liver cancer incidence (%) are shown in Table 4 and Fig. 4.
The each group liver metastasis nude mice number of table 4 and metastatic liver cancer incidence (%)
| Liver metastasis nude mice number (only) | Metastatic liver cancer incidence (%) | |
| I group of Pyrrolopyrimidine thion | 1 | 10 |
| II group of Pyrrolopyrimidine thion | 1 | 10 |
| III group of Pyrrolopyrimidine thion | 1 | 10 |
| Control group | 10 | 100 |
Above-mentioned experiment is it can be found that Pyrrolopyrimidine thion I, Pyrrolopyrimidine thion II and Pyrrolopyrimidine thion III can be notable
Suppress the expression of LncRNA BANCR in HO-8910PM cells, so as to significantly inhibit the motion invasion and attack energy of HO-8910PM cells
Power, suppresses the ability that HO-8910PM cells attack liver formation metastatic liver cancer in nude mouse.Pyrrolopyrimidine thion I, pyrrolo-
Pyrimidone II and Pyrrolopyrimidine thion III and other LncRNA BANCR inhibitor can be used for exploitation and is prepared into suppression ovary
The medicine of cancer hepatic metastases.
Above-described embodiment is the embodiment to essentiality content of the present invention, for preferably explaining the present invention, but this area skill
Art personnel are it is to be understood that protection scope of the present invention should not be confined to above-mentioned specific embodiment.
Claims (5)
- Applications of the 1.LncRNABANCR as drug targets in the medicine for suppressing oophoroma hepatic metastases is prepared.
- Application of the 2.LncRNABANCR inhibitor in the medicine for suppressing oophoroma hepatic metastases is prepared.
- 3. application according to claim 2, it is characterised in that：The inhibitor is the pyrrolo- with following general structure Pyrimdinone micromolecular compound,Wherein, R is alkyl.
- 4. application according to claim 3, it is characterised in that the inhibitor is selected from the compound of following structure：
- 5. one kind is used to suppress oophoroma hepatic metastases pharmaceutical composition, it is characterised in that：Contain any suppression of claim 2-4 Agent.
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| CN110408695A (en) * | 2019-04-05 | 2019-11-05 | 辽宁省肿瘤医院 | Malignant Neoplasms Arising from Endometriosis correlation ovarian cancer diagnosis or prognostic marker and application |
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| CN106496233A (en) * | 2016-09-26 | 2017-03-15 | 东南大学 | Azolopyrimidines, Its Preparation Method And Use |
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| CN106496233A (en) * | 2016-09-26 | 2017-03-15 | 东南大学 | Azolopyrimidines, Its Preparation Method And Use |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108753828A (en) * | 2018-06-26 | 2018-11-06 | 昆明医科大学 | A kind of interference carrier, carrier system and its application of BANCR genes |
| CN110408695A (en) * | 2019-04-05 | 2019-11-05 | 辽宁省肿瘤医院 | Malignant Neoplasms Arising from Endometriosis correlation ovarian cancer diagnosis or prognostic marker and application |
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