CN106668863A - Medicine targeting KTN1 to treat skin squamous cell carcinoma - Google Patents
Medicine targeting KTN1 to treat skin squamous cell carcinoma Download PDFInfo
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- CN106668863A CN106668863A CN201710093598.7A CN201710093598A CN106668863A CN 106668863 A CN106668863 A CN 106668863A CN 201710093598 A CN201710093598 A CN 201710093598A CN 106668863 A CN106668863 A CN 106668863A
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- ktn1
- cell carcinoma
- squamous cell
- siktn1
- skin squamous
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
- A61K31/713—Double-stranded nucleic acids or oligonucleotides
Abstract
The invention discloses a medicine targeting KTN1 to treat skin squamous cell carcinoma. An inventor uses an EGFR (epidermal growth factor receptor) highly-expressed cell strain A431 with the skin squamous cell carcinoma characteristic as an experiment object; after proofing by an experiment, the targeting reduction of KTN1 can inhibit the expression of the EGFR which influences the proliferation, transfer and invasion ability of tumor cells, so as to finally improve or treat the skin squamous cell carcinoma.
Description
Technical field
The present invention relates to the new technique of a kind of improvement or treatment cutaneous squamous cell carcinoma.
Background technology
Cutaneous squamous cell carcinoma（SCC）It is derived from the malignant tumor of keratinocyte in epithelial tissue.According to statistics, SCC
Sickness rate be only second to basal cell carcinoma in all malignant tumour of skin, occupy second.The SCC causes of disease are complicated, UV irradiations,
The factors such as HPV infection, organ transplantation, chemicals are closely related with the morbidity of SCC.
The clinical manifestation of SCC is widely different, and relevant with the pathogenetic position of disease and typing.SCC typically occurs in skin
Skin exposure portion, but other any positions of body also can occur, and about 55% betides incidence, and about 18% betides the back of the hand and forearm
Side is stretched, 13% betides lower limb, and 4% betides shoulder back, and 3% betides other positions such as upper limb, lip, crissum and genitals accounts for
7%.Generally skin lesion is unclear from the beginning of little and hard red tubercle, boundary is shown as, and easily develops into excipuliform or papilloma, in
Easily there is ulcer in centre, tumor can spread to surrounding tissue.
SCC clinical manifestations generally need histopathology to be diagnosed without explicitly specificity.In recent years to the research day of SCC
Benefit increases, and has certain understanding to its moleculess mechanism.
The general first-selection operative treatments of SCC, other Therapeutic Method include photodynamic therapy, radiotherapy, interferon and tretinoin
Deng.But these methods have certain limitation, restricted by different factors.As operative treatment is difficult to be not suitable for operation
Carry out in the special population for the treatment of meanss, the crowd that such as part old people and part have higher requirements to appearance.Operative treatment is tied
Close chemicotherapy to be still difficult to utterly destroy Skin Squamous Cell Carcinoma, there is higher relapse rate.In view of Skin Squamous Cell Carcinoma have stronger transfer and
Invasive ability.It is necessary to study to reduce Skin Squamous Cell Carcinoma cell propagation, the neoplasm medicine of transfer and invasive ability as target.
Drive associated proteins（Kinectin1, KTN1）It is a kind of membrane receptor protein on endoplasmic reticulum, mRNA total lengths
4.6kb gene mapping is primarily located within endoplasmic reticulum on No. 14 chromosome q22.1, and molecular weight of albumen is 160KD, Zhang etc.
（Journal of Cell Science, 2010,123:3901–3912）Research worker is by focal adhension (FAs) to cell
Moulding and motor capacity, it is found that KTN1 relies on the recruitment of FA albumen by mediation endoplasmic reticulum, so as to the extension for participating in endoplasmic reticulum is made
With.
Additionally, also small part KTN1 is positioned on mitochondrion, fragments molecules amount is 120KD, and it can be with driving albumen
（kinesin）Interact, mitochondrial dynamic metabolism function can be affected（Journal of Cell Science, 2004,
117, 4537-4549）.There is dependency in KTN1, but few to its research with disease, mainly to Behcet disease, amyotrophy
The research of the diseases such as disease, hepatocarcinoma, aplastic anemia, cervical cancer.KTN1 participates in intracellular vesicles by being combined with kinesin
Transhipment and film form and structure maintenance, while KTN1 also can be fixed on EF-1 complexs on endoplasmic reticulum with reference to EF-1 δ,
The translation of modulin.Inducing cell occurs adjoint during rapid apoptosis to be found to cervical cancer and acute leukemia cellses research
The fracture and degraded of KTN1, it may be possible to due to what is caused induction of caspase 7（FEBS Letters, 1998,436:51–
54）.Additionally, the distribution of intracellular KTN1 also receive endogenouss RhoG Active Regulations, so as to affect cell surface characteristic, propagation and
Motor capacity, and then develop with the generation of tumor closely related.
In sum, KTN1 has important function in the generation development and transfer of tumor.However, many exists for KTN1
Research in tumor is still in the elementary step.Such as, KTN1 is the current nothing how to play a role in cutaneous squamous cell carcinoma
Research report.Meanwhile, its potential the Molecular Biology Mechanism is unclear.
The content of the invention
It is an object of the invention to provide a kind of targeting KTN1 treats the medicine of cutaneous squamous cell carcinoma.
The technical solution used in the present invention is：
Targeted silent KTN1 lowers application of the reagent of KTN1 expressions in treatment cutaneous squamous cell carcinoma medicine is prepared.
Further, targeted silent KTN1 or lower KTN1 expressions reagent be forKTN1SiRNA.
Particularly, the sequence of siRNA is：
siKTN1_1: GAGAUUGUGUUGAAAGAAA（SEQ ID NO：1）；
And siKTN1_2: CAGGAAAGCUACAGCAAGA（SEQ ID NO：2）In at least one.
More preferably, at least part of nucleic acid in siRNA is modified, to improve its adhesion.It is at least part of in siRNA
Nucleic acid is modified using LNA.
The invention has the beneficial effects as follows：
Inventor is found through experiments and passes through with the cell strain A431 of the high expression of the distinctive EGFR of Skin Squamous Cell Carcinoma as experimental subject
Targeting lowers KTN1, can finally be improved or controlled with the expression of the EGFR of inhibitory effect tumor cell proliferation, transfer and invasive ability
Treat Skin Squamous Cell Carcinoma.
Description of the drawings
Fig. 1 is the impact that KTN1 breeds to A431 cells；（A）SiNC, siKTN1_1 or siKTN1_2 silence model is set up
Success（B）0h, 24h, 48h and 72h CCK8 detects A431 ability of cell proliferation situations after siKTN1_1 or siKTN1_2 is processed；
Fig. 2 is KTN1 to A431 cell migration and the impact of invasion and attack；（A）48h after siKTN1_1 or siKTN1_2 process
Transwell detects A431 cell migration capabilities mights（Right side is cartogram）（B）48h after siKTN1_1 or siKTN1_2 process
Transwell detects A431 cell invasion capabilities mights（Right side is cartogram）；
Fig. 3 is the expression that silence KTN1 lowers EGFR.
Specific embodiment
The reagent of targeted silent KTN1 or downward KTN1 expressions answering in treatment cutaneous squamous cell carcinoma medicine is prepared
With.
Further, targeted silent KTN1 or lower KTN1 expressions reagent be forKTN1SiRNA.
Particularly, the sequence of siRNA is：
siKTN1_1: GAGAUUGUGUUGAAAGAAA（SEQ ID NO：1）；
And siKTN1_2: CAGGAAAGCUACAGCAAGA（SEQ ID NO：2）In at least one.
More preferably, at least part of nucleic acid in siRNA is modified, to improve its adhesion.It is at least part of in siRNA
Nucleic acid is modified using LNA.
With reference to experiment, technical scheme is further illustrated.
Materials and methods：
1. application on human skin squamous cell carcinoma line A431 cells are chosen, in 37 DEG C, 5% CO2Under condition of culture, using 10% FBS
DMEM culture medium Secondary Cultures.When A431 cell confluency degree length is to 70%-80%, at random cell is divided into into 3 groups：SiNC groups,
SiKTN1_1 groups and siKTN1_2 groups.SiRNAs sequences are：
siKTN1_1: 5’- GAGAUUGUGUUGAAAGAAA-3’；
siKTN1_2: 5’- CAGGAAAGCUACAGCAAGA-3’.Washed twice with PBS, using 15 μ L lipofectamine
SiNC, siKTN1_1 and siKTN1_2 of the μ L of 2000 liposome 50nM 15, serum-free antibiotic-free DMEM culture medium is dilute
Release, cumulative volume is 400 μ L, mixed liquor room temperature places 15-20min.Subsequently each 10cm culture dish adds the depletion of blood of volume 4.4mL
Clear antibiotic-free DMEM culture medium, by the siRNAs after liposome, progressively Deca enters each culture dish, by each after 3-5hr
Liquid is discarded in ware, adds the DMEM culture medium containing serum and antibiotic, 37 DEG C, 5% CO2Continue to cultivate under condition of culture.
2. cell is collected after 48 hours, is washed twice using PBS, each culture dish adds 1ml PBS to scrape using cell
Trizol methods extract intracellular total serum IgE after cell, to determine and carry out reverse transcription reaction after concentration.Reverse transcription reaction system：
| DNase I buffer（10x） | 1μL |
| RNA sample | 1μg |
| DEPC-treated H2O | 7μL |
| Dnase I | 1μL |
| Total | 10μL |
37 DEG C, 30min；
The μ L of 25mM EDTA 1 to mixed liquor, 65 DEG C of heat shock response 10min；
50 μM of 0.5 μ L Oligo dT and 4uM 1ul dNTP is added in mixed liquor；
72 DEG C, it is placed on ice after 10min.
RNA reverse transcriptions synthesize cDNA
| 5×first strand buffer | 4μL |
| 0.1 M DTT | 1μL |
| RNase inhibitior | 0.5μL |
| M-MLV | 1μL |
| DEPC Water | 1μL |
| Total | 7.5μL |
PCR:37 DEG C, 60min；70 DEG C, 15min.
3. qRT-PCR reactions：KTN1 primer sequences：
| SYBR reaction buffer | 5μL |
| cDNA | 3μL |
| Primer（R+F） | 0.2μL |
| DEPC Water | 1.8μL |
| Total | 10μL |
4. protein extraction：Each culture dish adds 100 μ L -130 μ L RIPA protein lysates and 25 × PIC albumen enzyme levels
Agent, is placed in 80 DEG C of frozen cracking of ﹣ after 10min on ice.Next day, it is egg to collect supernatant after 12,000rpm, 4 DEG C of centrifugation 15min of sample
White lysate, determines concentration, carries out Western blot Western blot analysis.
5. statistical analysis use the version softwares of SPSS 17.0, perform independent sample one factor analysis of variance, all data
Using double tail detections and homogeneity of variance analysis, *p<0.05, * *p<0.01, * * *p<0.001 has statistical significance.
Experimental result：
KTN1 regulation and control Skin Squamous Cell Carcinoma cell propagation
First, inventor sets up siNC, siKTN1_1 or siKTN1_2 silence model A431 is intracellular first.Using qRT-
The expression change of KTN1 mRNA level in-sites after PCR method detection transfection siRNA, silence model is successfully established shown in Fig. 1-A.Use
CCK8 methods have detected the change bred in silence KTN1 descendant Skin Squamous Cell Carcinoma cell A431, find and normal skin squamous cell carcinoma
Compare, after transfection siKTN1_1 or siKTN1_2, with the prolongation of incubation time, A431 ability of cell proliferation weakens, such as Fig. 1-B
（*p<0.05, * *p<0.01, * * *p<0.001）.
Silence KTN1 significantly suppresses Skin Squamous Cell Carcinoma cell migration and invasion and attack
Secondly, inventor uses the migration of A431 cells and invasive ability in Transwell technology for detection KTN1 silence models
（Fig. 2-A/B）.After transfection siNC, siKTN1_1 or siKTN1_2 48h（Fig. 2-A）, the A431 cell subcultivations after process are existed
In Transwell cells, 12-16h collects cell, and basis of microscopic observation finds, two groups of A431 cell migration energy after silence KTN1
Power is reduced, and compared with matched group, cell number reduces about 3-4 times；Similarly, Fig. 2-B show the invasive ability of cell, siKTN1
Treatment group cell invasion ability is reduced, and compared with matched group, attacking the cell number of matrigel reduces 5-6 times（*p<0.05, * *p
<0.01, * * *p<0.001）.
The expression analysis of KTN1 and EGFR in Skin Squamous Cell Carcinoma cell
Inventor have detected the expression of KTN1 and EGFR in Skin Squamous Cell Carcinoma cell A431 after silence KTN1 and find that Fig. 3 shows,
KTN1 down-regulated expressions in A431 cells after siKTN1 process, while EGFR expression is also lowered.As a result show that KTN1 is likely to be logical
Cross regulation and control EGFR to regulate and control the propagation of Skin Squamous Cell Carcinoma, migration, invasive ability.
It is contemplated that, KTN1 is lowered by targeting, can be with inhibitory effect tumor cell proliferation, transfer and invasive ability
The expression of EGFR, it is final to improve or treat Skin Squamous Cell Carcinoma.
SEQUENCE LISTING
<110>Nanfang Medical Univ
<120>Targeting KTN1 treats the medicine of cutaneous squamous cell carcinoma
<130>
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> DNA
<213>Artificial sequence
<400> 1
gagauugugu ugaaagaaa 19
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
caggaaagcu acagcaaga 19
Claims (5)
1. application of the reagent of targeted silent KTN1 or downward KTN1 expressions in treatment cutaneous squamous cell carcinoma medicine is prepared.
2. application according to claim 1, it is characterised in that：Targeted silent KTN1 or lower KTN1 expressions reagent be
ForKTN1SiRNA.
3. application according to claim 2, it is characterised in that：The sequence of siRNA is：
siKTN1_1: GAGAUUGUGUUGAAAGAAA；
And siKTN1_2:In CAGGAAAGCUACAGCAAGA at least one.
4. the application according to Claims 2 or 3, it is characterised in that：At least part of nucleic acid in siRNA is modified, with
Improve its adhesion.
5. application according to claim 4, it is characterised in that：At least part of nucleic acid in siRNA is modified using LNA.
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| CN201710093598.7A CN106668863B (en) | 2017-02-21 | 2017-02-21 | Target the drug of KTN1 treatment cutaneous squamous cell carcinoma |
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| CN106668863B CN106668863B (en) | 2019-04-23 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110201172A (en) * | 2019-06-20 | 2019-09-06 | 深圳市人民医院 | Application of the YY1 expression inhibiting agent in preparation treatment breast cancer medicines |
| CN110229817A (en) * | 2019-06-20 | 2019-09-13 | 深圳市人民医院 | Target siRNA and its application of KTN1 treatment breast cancer |
| CN114350796A (en) * | 2021-12-17 | 2022-04-15 | 南方医科大学 | Application of LINP1 in diagnosis and treatment of skin squamous cell carcinoma |
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| WO2004045543A2 (en) * | 2002-11-14 | 2004-06-03 | Dharmacon, Inc. | Functional and hyperfunctional sirna |
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2017
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| WO2004045543A2 (en) * | 2002-11-14 | 2004-06-03 | Dharmacon, Inc. | Functional and hyperfunctional sirna |
| CN101939005A (en) * | 2007-12-14 | 2011-01-05 | 诺瓦提斯公司 | Kinesin inhibitors as cancer therapeutics |
| CN104211814A (en) * | 2013-05-29 | 2014-12-17 | 三星电子株式会社 | Composition for target membrane protein depletion |
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| Title |
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| CAO ET AL.: "A three-lncRNA signature derived from the Atlas of ncRNA in cancer (TANRIC) database predicts the survival of patients with head and neck squamous cell carcinoma", 《ORAL ONCOLOGY》 * |
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| THOMAS ET AL.: "Caspase 7-induced cleavage of kinectin in apoptotic cells", 《FEBS LETTERS》 * |
| WANG ET AL.: "Overexpression of Kif2a promotes the progression and metastasis of squamous cell carcinoma of the oral tongue", 《ORAL ONCOLOGY》 * |
| 孙花等: "人类驱动结合蛋白及其与某些疾病的相关性", 《生命的化学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110201172A (en) * | 2019-06-20 | 2019-09-06 | 深圳市人民医院 | Application of the YY1 expression inhibiting agent in preparation treatment breast cancer medicines |
| CN110229817A (en) * | 2019-06-20 | 2019-09-13 | 深圳市人民医院 | Target siRNA and its application of KTN1 treatment breast cancer |
| CN110229817B (en) * | 2019-06-20 | 2020-04-24 | 深圳市人民医院 | Small interfering RNA for targeted KTN1 treatment of breast cancer and application thereof |
| CN114350796A (en) * | 2021-12-17 | 2022-04-15 | 南方医科大学 | Application of LINP1 in diagnosis and treatment of skin squamous cell carcinoma |
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