CN107129987A - Application of TIMELESS gene as target in preparation of medicine for treating cervical cancer - Google Patents
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- CN107129987A CN107129987A CN201710520388.1A CN201710520388A CN107129987A CN 107129987 A CN107129987 A CN 107129987A CN 201710520388 A CN201710520388 A CN 201710520388A CN 107129987 A CN107129987 A CN 107129987A
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Abstract
The invention belongs to the technical field of biomedicine, and provides a function of a DNA damage repair related gene in cervical cancer, wherein a molecular biology approach is adopted to confirm that the expression level of a gene TIMELESS has obvious correlation with the cervical cancer progress for the first time, and the gene TIMELESS is hardly expressed in normal cervical tissues. The invention screens siRNA taking TIMELESS as a target spot at the gene level, and provides an siRNA sequence capable of efficiently and specifically knocking down TIMELESS expression. The invention also relates to application of the TIMELESS siRNA and the pharmaceutical composition in treating cervical cancer. The pharmaceutical composition refers to the siRNA nucleotide fragment containing effective dose and pharmaceutically acceptable excipient. The TIMELESS siRNA can obviously inhibit the proliferation and the cloning formation capability of cervical cancer cells, promote the apoptosis and the aging of the cervical cancer cells and promote the cis-platinum sensitivity of the cervical cancer SiHa cells. The siRNA in the invention can up-regulate the expression of clear Caspase-3 and p21, and finally promote the apoptosis and the aging of cervical cancer cells.
Description
Technical field
The invention belongs to biomedicine technical field, it is related to TIMELESS genes and is preparing treatment of human cervical cancer medicine for target spot
In application.
Background technology
Cervical carcinoma is the common malignant tumour of women second, is the 4th of women generation tumour associated death in world wide
Principal element.With the popularization of cervical carcinoma screening, the incidence of Advanced Cervical Carcinoma is substantially reduced, but due to Chinese population radix
Greatly, still suffering from a large amount of cervical cancer patients needs to carry out radical radiation therapy or Post operation supplement Radiotherapy chemotherapy, Advanced Cervical Carcinoma and recurrence
Property cervical cancer patient prognosis is still undesirable.By finding effective targeted therapy molecule to strengthen cervical carcinoma to the quick of Radiotherapy chemotherapy
Perception, the curative effect for improving cervical carcinoma, the prognosis for improving patient has very important significance.
The treatment method of cervical carcinoma includes operative treatment, platinum-based chemotherapy medicine and radiotherapy, and then both are current late periods
And the conventional treatment means of Recurrent Cervical Cancer, the purpose of tumour cell is killed by causing DNA damage to reach.Platinum medicine
It is main to cause DNA crosslinkings and DNA replication dna fork to be obstructed；Radioactive ray then directly contribute the single-stranded or double-stranded fractures of DNA.Medicine is serious
Side effect, tumor drug resistance and chemotherapeutics poor selectivity are the main causes of Endodontic failure.And DNA Related to repair gene can be with
Influence patient to the sensitiveness of chemotherapy, Patients with Non-small-cell Lung negative such as ERCC1 is better than to the reactivity of cisplatin chemotherapy
Patient positive ERCC1.The key enzyme repaired by the DNA of targeted inhibition tumour cell, improves the sensitiveness of radiation and chemotherapy,
The always study hotspot of therapeutic field of tumor.But the research in cervical carcinoma about DNA repair functions and mechanism is still disputable：Have
There is homologous recombination repair in document report and nonhomologous end combines the defect repaired in cervical carcinoma, also have document report to exist
Occur the high expression of PARP and PAR genes in the HeLa cells of cisplatin resistance, point out the enhancing of DNA repair abilities.
TIMELESS genes are located at chromosome 12q13 .3, and highly conserved in evolution, research is found, in drosophila
TIMELESS is primarily involved in biological clock regulation, and mammal TIMELESS genes are initially cloned from neuronal cell, but are being fed
Do not find that TIMELESS has obvious biological clock regulatory function in newborn animal, but participate in maintaining DNA replication dna fork stability.In recent years
Come, increasing research shows TIMELESS in colon cancer, breast cancer, small cell lung cancer, liver cancer, carcinoma of urinary bladder kinds cancer
Middle overexpression, and high-caliber TIMELESS expression is related to low survival, this shows TIMELESS in tumour
Played an important role in progress, but still lack the research of correlation in cervical carcinoma.
TIMELESS and PARP1, which interacts, participates in homologous recombination repair and the non-homology end of regulating DNA double-strand break
End, which is combined, repairs, and this is two kind important repair modes of the cell to DNA damage caused by platinum medicine and radioactive ray.TIMELESS
Participate in maintaining DNA replication dna fork stability, also regulate and control homologous recombination repair and nonhomologous end is combined and repaired, but TIMELESS is adjusted
The specific mechanism of control DNA damage reparation is still not clear.Research display：TIMELESS DNA damage generation after several seconds in i.e.
It can raise to damage location, and dependent on PARP1 effect, TIMELESS has just left damage location in 1 hour, points out
TIMELESS participates in the early reaction of DNA double chain damage.Homologous recombination repair relied in early stage MRN compounds (by Mre11,
The compound of tri- albumen compositions of Rad50, NBS1) to raise and arrive injury site, non-homogeneous cohesive end connection, which is repaired, then to be relied on
Recruitment of the Ku70/80 dimers in injury site.Immunofluorescence common location shows TIMELESS with Mre11, Ku70/80 in cis-platinum
Intracellular common location after processing, and TIMELESS strikes and subtracts rear Mre11, Ku70/80 recruitment and then significantly reduce prompting
TIMELESS may be by promoting MRN compounds, Ku70/80 in the recruitment of DNA double chain breaking part, so as to promote homologous recombination to repair
Multiple and nonhomologous end, which is combined, to be repaired.In addition, homologous recombination repair is with DNA replication dna, the two molecular events are often coupled at one
Rise, many important gene such as RPA, Rad51 etc. simultaneously participate in homologous recombination repair and DNA replication dna fork progress, DNA replication dna fork
Stability directly affect the DNA repair functions such as homologous recombination repair, TIMELESS clpp genes subtract after DNA replication dna fork retardance
It is one of impaired possible cause of homologous recombination repair.
However, up to the present still without any document report TIMELESS overexpressions in uterine neck carcinogenesis progress
Effect, and TIMELESS siRNA can significantly inhibit the propagation of cervical cancer cell, promote its aging and apoptosis, promote its right
The sensitiveness of cis-platinum.There is not yet targeting TIMELESS siRNA is used for the treatment of cervical carcinoma.Targeting TIMELESS simultaneously combines tradition
Chemotherapeutics perhaps can produce synergistic therapeutic effect, make reduction chemotherapeutics consumption, mitigate toxic side effect and improve and control
Selectivity is treated to be possibly realized.
The content of the invention
For above-mentioned technical problem, the present invention proposes a kind of new opplication of TIMELESS genes.
Application of the TIMELESS genes as target spot in the medicine for treating cervical carcinoma is prepared, the TIMELESS genes
NCBI Serial No. NM_003920.4；
In the TIMELESS genes as the sequence of target spot for GCCGCATCATCAAGAACAATA or
GCAGCTGTTGCGCAAGCTAGG。
A kind of siRNA for TIMELESS gene targets, it is characterised in that：The siRNA be siTIM 1392,
siTIM 3181。
Described siRNA, it is characterised in that：The sequence for the TIMELESS gene targets that siTIM 1392 is directed to is
GCCGCATCATCAAGAACAATA；
The sense strand sequence of the siTIM 1392 is GCCGCAUCAUCAAGAACAAUA, and the siTIM's 1392 is anti-
Adopted chain-ordering is UUGUUCUUGAUGAUGCGGCUG.
Described siRNA, it is characterised in that：The sequence for the TIMELESS gene targets that siTIM 3181 is directed to is
GCAGCTGTTGCGCAAGCTAGG；
The sense strand sequence of the siTIM 3181 is GCAGCUGUUGCGCAAGCUAGG, and the siTIM's 3181 is anti-
Adopted chain-ordering is UAGCUUGCGCAACAGCUGCUG.
It is TGGTTTACATGTCGACTAATT as the Non-targeting siRNA targeting sequences of negative control；Non-
Targeting siRNA sense strand sequence is UGGUUUACAUGUCGACUAATT, the Non-targeting siRNA's
Antisense strand sequence is UUAGUCGACAUGUAAACCATT.
Applications of the described siRNA in the medicine for preparing treatment cervical carcinoma.
A kind of pharmaceutical composition, it is characterised in that：SiRNA described in claim 2 containing the upper effective dose for the treatment of, or
And contain its pharmaceutically acceptable excipients or additional dose.
Application of the described pharmaceutical composition in the medicine for preparing treatment cervical carcinoma.
Present invention firstly provides a kind of function of DNA damage Related to repair gene in cervical carcinoma, using molecular biosciences
Learning to do section and confirming gene TIMELESS expressions and cervical carcinoma progress first has significant correlation, and in normal cervical tissues
Hardly express.
The present invention has screened in siRNA of the gene level using TIMELESS as target spot that there is provided can be efficiently and special
Property strike drop TIMELESS expression siRNA sequence.
The invention further relates to the purposes of above-mentioned TIMELESS siRNA and pharmaceutical composition in treatment of human cervical cancer.
Pharmaceutical composition refers to above-mentioned siRNA nucleotide fragments and pharmaceutically acceptable tax containing effective dose
Shape agent.
TIMELESS siRNA in the present invention can significantly inhibit the propagation and clonality of cervical cancer cell, promote
Enter cervical carcinoma apoptosis and aging, and promote the cisplatin sensitivity of Intrauterine device bleeding.
The expression of siRNA up-regulation Cleaved Caspase-3 and p21 in the present invention, it is final to promote cervical cancer cell to wither
Die and aging.
The present invention carries out bioinformatics by the four cervical cancer gene mRNA express spectras storehouses provided ncbi database
Analysis, find it is a variety of directly participate in the genes that DNA repair is presented in cervical carcinoma it is high express, such as TIMELESS, FANCI,
FOXM1, RFC4, MSH6, BRCA1, point out the abnormal activation that there are some DNA repair pathways in cervical cancer cell.In these DNA
In Related to repair gene, differential expression most significantly TIMELESS genes.And then, the clinical tissue mark of present invention small sample
The immunohistochemical experiment of sheet and cervical cancer tissues chip confirms TIMELESS overexpressions in cervical cancer tissues, and table
There is correlation with cervical carcinoma progress up to level.Real-time quantitative PCR equally confirms TIMELESS mRNA in cervical cancer tissues
Average expression level is significantly higher than cancer beside organism, and strong indication TIMELESS may play a significant role in cervical carcinoma.
In order to evaluate TIMELESS roles in cervical carcinoma, the present invention, which is devised, efficiently targets TIMELESS's
SiRNA, and CCK-8 methods in cervical cancer cell are transferred to, flow cytometer detection Annexin-V/7-AAD double-stainings survey apoptosis, gram
Grand to form experiment, beta galactosidase decoration method prompting TIMELESS siRNA are exogenous, which to strike drop TIMELESS expression, can suppress palace
Neck cancer cell multiplication and clonality, promote cervical cancer cell to occur apoptosis and aging, it was demonstrated that the TIMELESS siRNA
The TIMELESS silences effect of mediation can disturb the growth final result of cervical cancer cell.Meanwhile, Western Blot results are shown
Apoptosis mark Cleaved Caspase-3 and aging mark in cervical cancer cell can be raised after TIMELESS siRNA transfections
The expression of p21 albumen, it is final to promote Hela Cell Apoptosis and aging.In addition, TIMELESS siRNA and Cisplatin
Synergy can be produced, the 503nhibiting concentration of Intrauterine device bleeding is reduced, promotes cisplatin sensitivity.Therefore propose to prepare treatment
The stylish drug target TIMELESS of the medicine of cervical carcinoma.TIMELESS siRNA compare other treatment methods, intervene target
Point is definitely.
The beneficial effects of the present invention are：Present invention prompting TIMELESS high expression level and the development of cervical carcinoma are in just
Correlation, and hardly expressed in normal cervical tissues, it is thin that the siRNA for targetting TIMELESS using high-efficient characteristic transfects cervical carcinoma
Born of the same parents, can significantly inhibit the propagation and clonality of cervical carcinoma SiHa, Ca Ski and HeLa cells, promote cervical cancer cell
Generation apoptosis and aging；And the cis-platinum 503nhibiting concentration of Intrauterine device bleeding can be significantly reduced, promotes it to control cis-platinum
The sensitiveness for the treatment of.Therefore the stylish drug target TIMELESS of the medicine for preparing treatment cervical carcinoma is proposed.TIMELESS
SiRNA compares other treatment methods, intervenes target spot definitely.In view of TIMELESS hardly tables in normal cervical tissues
Reach, therefore TIMELESS siRNA have clear and definite selectivity, the mRNA produced in the cervical cancer tissues that selectively targeted can degrade
Play a role, selectivity and security are good.This aspect is significantly better than traditional chemical and radiotherapy.Meanwhile, join with cis-platinum
The cooperation used time, the cisplatin sensitivity of cervical cancer cell can be promoted so that reduction chemotherapeutics usage amount and mitigation side effect into
For possibility.It is experimentally confirmed, TIMELESS siRNA influence cervical cancer cell propagation, apoptosis and the sensitiveness to cis-platinum,
There is potential application value in terms of oncotherapy and antineoplastic research and development.
Brief description of the drawings
Fig. 1：For cervical intraepithelial neoplasia and the immunohistochemical study picture (A) of each 20 of cervical cancer tissues, (B) is carried
Show：TIMELESS is incremented by obvious from CIN I, II, III, expression gradually rises grade into the progression of cervical carcinoma
Trend.(C) in cervical cancer tissues chip (including 40 cervical carcinomas and 10 normal cervix) TIMELESS immunohistochemical analysis,
Prompting TIMELESS is positive high expression in 70% cervical carcinoma.(D) 10 pairs of cervical carcinomas and pairing cancer beside organism in TIMELESS
MRNA expression.MRNA average levels of the real-time quantitative PCR result prompting TIMELESS in cervical cancer tissues, which is significantly higher than, matches somebody with somebody
To cancer beside organism；
Fig. 2：For TIMELESS Mrna (A) and Western after TIMELESS siRNA transfection cervical squamous cell carcinoma SiHa cell lines
Horizontal expression (B) situation map, wherein Control siRNA represent negative control, siTIM 1392, siTIM 3181 and siTIM
3454 represent TIMELESS 3 siRNA fragments；
Fig. 3：For CCK-8 methods detection cervical squamous cell carcinoma cell line SiHa (A), Ca Ski (B) and gland cell system HeLa (C)
The result of proliferation activity：Compared with cellular control unit, TIMELESS siRNA transfection groups cell propagation is substantially suppressed, difference
With statistical significance (p<0.05)；
Fig. 4：The result of SiHa and Ca Ski Apoptosis is detected for Annexin-V/7-AAD double-stainings：With control group
Compare, TIMELES siRNA transfection group Hela Cell Apoptosis level rise, difference has statistical significance (p<0.05).
Fig. 5：The Clone formation result of SiHa and HeLa cells is detected for crystal violet staining assay：Compared with control group,
TIMELESS siRNA transfection group cervical cancer cells clonality declines, and difference has statistical significance (p<0.01)；
Fig. 6：The result of Intrauterine device bleeding cisplatin sensitivity is detected for CCK-8 methods：It is after 48 hours plus different after transfection
The cisplatin effect of concentration gradient 24 hours, the cis-platinum 503nhibiting concentration of control group SiHa cells is 8.362ug/ml, and
TIMELESS siRNA transfection group cell cis-platinum 503nhibiting concentrations are reduced to 5.422ug/ml and 3.112ug/ml respectively, and difference has
Statistical significance (p<0.01)；
Fig. 7：For beta galactosidase method detection cell SiHa cell ageings figure (A) and quantitative analysis figure (B)：With compareing
Group is compared, TIMELESS siRNA transfection groups senile cell number after transfecting 72 hours.Transfection adds cis-platinum (5ug/ after 24 hours
Ml) handle 48 hours, TIMELESS siRNA transfection group cell ageings level is higher than control group, while also above independent
TIMELESS siRNA transfection group levels, difference has statistical significance (p<0.001)；
Fig. 8：For apoptosis mark Cleaved Caspase-3's in SiHa and Ca Ski cells and aging mark p21
Expression change Western blot figures (A) and quantitative analysis figure (B)：Compared with control group, TIMELESS siRNA transfections
Cleaved Caspase-3 and p21 expression are raised in SiHa and Ca Ski cells, and difference has statistical significance (p
<0.05)。
Embodiment
The present invention is further described below by embodiment, but present disclosure is not limited thereto.
Fig. 1：For cervical intraepithelial neoplasia and the immunohistochemical study picture (A) of each 20 of cervical cancer tissues, (B) is carried
Show：TIMELESS is incremented by obvious from CIN I, II, III, expression gradually rises grade into the progression of cervical carcinoma
Trend.(C) in cervical cancer tissues chip (including 40 cervical carcinomas and 10 normal cervix) TIMELESS immunohistochemical analysis,
Prompting TIMELESS is positive high expression in 70% cervical carcinoma.(D) 10 pairs of cervical carcinomas and pairing cancer beside organism in TIMELESS
MRNA expression.MRNA average levels of the real-time quantitative PCR result prompting TIMELESS in cervical cancer tissues, which is significantly higher than, matches somebody with somebody
To cancer beside organism；
Fig. 2：For TIMELESS Mrna (A) and Western after TIMELESS siRNA transfection cervical squamous cell carcinoma SiHa cell lines
Horizontal expression (B) situation map, wherein Control siRNA represent negative control, siTIM 1392, siTIM 3181 and siTIM
3454 represent TIMELESS 3 siRNA fragments；
Fig. 3：For CCK-8 methods detection cervical squamous cell carcinoma cell line SiHa (A), Ca Ski (B) and gland cell system HeLa (C)
The result of proliferation activity：Compared with cellular control unit, TIMELESS siRNA transfection groups cell propagation is substantially suppressed, difference
With statistical significance (p<0.05)；
Fig. 4：The result of SiHa and Ca Ski Apoptosis is detected for Annexin-V/7-AAD double-stainings：With control group
Compare, TIMELES siRNA transfection group Hela Cell Apoptosis level rise, difference has statistical significance (p<0.05).
Fig. 5：The Clone formation result of SiHa and HeLa cells is detected for crystal violet staining assay：Compared with control group,
TIMELESS siRNA transfection group cervical cancer cells clonality declines, and difference has statistical significance (p<0.01)；
Fig. 6：The result of Intrauterine device bleeding cisplatin sensitivity is detected for CCK-8 methods：It is after 48 hours plus different after transfection
The cisplatin effect of concentration gradient 24 hours, the cis-platinum 503nhibiting concentration of control group SiHa cells is 8.362ug/ml, and
TIMELESS siRNA transfection group cell cis-platinum 503nhibiting concentrations are reduced to 5.422ug/ml and 3.112ug/ml respectively, and difference has
Statistical significance (p<0.01)；
Fig. 7：For beta galactosidase method detection cell SiHa cell ageings figure (A) and quantitative analysis figure (B)：With compareing
Group is compared, TIMELESS siRNA transfection groups senile cell number after transfecting 72 hours.Transfection adds cis-platinum (5ug/ after 24 hours
Ml) handle 48 hours, TIMELESS siRNA transfection group cell ageings level is higher than control group, while also above independent
TIMELESS siRNA transfection group levels, difference has statistical significance (p<0.001)；
Fig. 8：For apoptosis mark Cleaved Caspase-3's in SiHa and Ca Ski cells and aging mark p21
Expression change Western blot figures (A) and quantitative analysis figure (B)：Compared with control group, TIMELESS siRNA transfections
Cleaved Caspase-3 and p21 expression are raised in SiHa and Ca Ski cells, and difference has statistical significance (p
<0.05)。
Embodiment
1. cell culture and transfection
SiHa and Ca Ski cells, which are used, contains 10% hyclone (Bio-sera companies) and 1% penicillin/streptomycin (green cloud
Its company) RPMI1640 (Hyclone companies) culture be based on 37 DEG C, 5%CO2Cultivated in saturated humidity environment, HeLa cells
With DMEM (Hyclone) growth medium culture, growth cell of taking the logarithm is tested.
2.TIMELESS siRNA sequence design, synthesis：
With a variety of siRNA design softwares such as Ambion, Qiagen, Dharmacon, people is obtained according to GenBank
TIMELESS Mrna sequence (NM_003920.4) search AA sequences simultaneously record 19 adjacent nucleotides of each AA3 ' ends, sieve
Select siRNA of the G/C content between 30%-55%.Further screened according to siRNA basic design principles, and will screening
The siRNA sequence gone out retrieves its homology in GenBank genome database with BLAST, has 3 from non-homogeneous gene
The sequence of base mispairing more than individual, it is final true on the basis of conditions above is met to exclude the possibility of non-specific suppression
Fixed 3 TIMELESS siRNA sequence (table 1).SiRNA sequence is synthesized by Shanghai Ai Bosi bio tech ltd, simultaneously
One out of order and any mammalian genes of nonrecognition sequence of synthesis are used as negative control.
Table 1：SiRNA sequence used
3. cell transfecting
The day before transfection, after cervical cancer cell is counted with pancreatin digestion, by 2 × 105Individual/hole is laid in six orifice plates, training
Support base and be free of antibiotic.Lipofectamine is utilized when cell growth converges to about 40%~50%TM 3000
(Invitrogen companies) is transfected；Liposome and the configuration of siRNA mixtures are as follows：1. 250 μ l serum free mediums+
200pmol siRNA (10 μ l)/Ctrl siRNA, are stored at room temperature 5 minutes；2. the μ l liposomes of 250 μ l serum free mediums+5, room
Temperature stands 5 minutes.It will 1. and 2. mix, be incubated at room temperature 20 minutes, be then slowly added into cell.1.5ml bases are added per hole
Basal culture medium.In 37 DEG C, 5%CO2After being cultivated 6 hours in incubator, it is replaced by the complete medium without antibiotic and (contains 10%
FBS) continue to cultivate.Experiment is repeated 3 times.
1. the screening of high efficiency siRNA sequence
TIMELESS siRNA transfection SiHa cells collect cell after 48 hours, extract total serum IgE, wherein 28S：18S bar
Band is 2：1, extract RNA OD260：280 be 1.9-2.1.TIMELESS mRNA expressions are detected with real-time quantitative PCR,
Meanwhile, cell extraction total protein is collected after transfecting 72 hours, passes through Western Blot after BCA standard measures and detects TIMELESS
The expression of albumen, as a result shows compared with cellular control unit, and TIMELESS 3 siRNA sequences, which can significantly strike, to be subtracted
TIMELESS mRNA and albumen expression.Wherein siTIM 1392 and 3,181 two sequences of siTIM are highly efficient, therefore selection
The two sequences carry out follow-up test.This experiment is repeated 3 times.
5.CCK-8 methods detect cell proliferation activity
SiHa is chosen in the present invention, multiplication capacity is detected after Ca Ski and HeLa cells are transfected.Each cell line is real
Test points four groups：1. Ctrl siRNA negative control groups, 2. transfection groups of siTIM 1392；3. transfection groups of siTIM 3181；4. blank
Control group.Every group sets 3 repeating holes.In 24h after transfection, 48h, 72h, 96h, 120h detects 450nm extinctions with ELIASA respectively
Degree.Comprise the following steps that：
(a) SiHa of fast growing period, Ca Ski, HeLa cell, digest to form single cell suspension through pancreatin, adjust respectively
Concentration is 8 × 104Individual/ml and 3 × 104Individual/ml.Cell is inoculated in cell culture 24h in 96 orifice plates respectively with every hole 100ul；
(b) according to the specifications of Lipofectamine 3000, corresponding siRNA is transfected into 96 orifice plates, serum-free transfection
The growth medium without antibiotic is replaced by after 6h.
(c) in taking out culture plate after culture 24,48,72,96,120h respectively, supernatant is suctioned out, 90 μ l 1640 are added per hole
Basic culture solution and 10 μ l CCK-8 reagents continue to cultivate 1h；The daily set time determines the suction for adding 1h 450nm after CCK8
Luminosity (OD values).Cell growth curve is drawn after blank control group zeroing.
6.PE-AnnexinV/7-AAD double-stainings detect Apoptosis
The present invention uses SiHa and Ca Ski cell detections, and experiment is divided into three groups：1. Ctrl siRNA negative control groups；②
The transfection groups of siTIM 1392；3. transfection groups of siTIM 3181；4. positive controls.Positive controls cell adds in detection the previous day
Cis-platinum (5ug/ml) promotees apoptosis, for constructing plan adjustment fluorescence compensation.Comprise the following steps that：
(a) after transfection 72h, cell conditioned medium is suctioned out to a suitable centrifuge tube, PBS washings attached cell once, adds
Appropriate pancreatin (containing 0.02% EDTA) vitellophag.It is incubated at room temperature about 5min, Microscopic observation.
(b) to when gently piping and druming can be such that the attached cell blow and beat, the cell culture fluid of collection in step (a) is added slightly
Mix, be transferred in centrifuge tube, 1000rpm centrifugation 5min are abandoned and cell is collected after supernatant, cell is gently resuspended with PBS and counts；
Note：The cell culture fluid added in step (a) can both collect the cell for occurring apoptosis or necrosis suspended, again can be effective
Suppress or neutralize the pancreatin of residual；The pancreatin of residual, which can digest and degrade the PE-Annexin V subsequently added, causes dyeing to be lost
Lose.
(c) 1 × 10 is taken6Supernatant is abandoned after the cell that/ml is resuspended, 1000rpm centrifugations 5min, 100ul Annexin V- are added
Cell is gently resuspended in FITC combination liquid；
(d) 5 μ l PE-Annexin V and 5ul 7-AAD dyeing liquors are added, are gently mixed；
Note：Positive controls cell point is equally divided into 4 pipes, respectively the mono- dyes of PE-Annexin V, the mono- dyes of 7-AAD, Quan Buran,
And double dyes.Preceding 3 manage for building scheme and adjustment compensation, and the 4th pipe is used as positive control
(e) room temperature (20-25 DEG C) lucifuge is incubated 15min.Or carry out lucifuge using aluminium foil；Often pipe is added after the completion of incubation
400ul Annexin V combinations liquid is mixed.
(f) flow cytomery is carried out immediately, and Annexin V-FITC are green fluorescence, and 7-AAD is red fluorescence；Stream
The passband filter detection FITC fluorescence that formula cell instrument excitation wavelength 488nm is 515nm with a wavelength, another wavelength is more than
560nm filter detection 7-AAD.
(g) result judges：Apoptotic cell pair can identify that the dyestuff 7-AAD of non-viable non-apoptotic cell has an anti-metachromia, and non-viable non-apoptotic cell is then not
Energy.The DNA that cell membrane has the cell of damage can dye by 7-AAD and produce red fluorescence, and the intact cell of cell membrane then will not
There is red fluorescence.Therefore, Apoptosis early stage 7-AAD will not dye without produce red fluorescent.Normal live cells with
This is similar.On the scatter diagram of bivariate flow cytometer, the non-living cells of left lower quadrant is (FITC-/7-AAD-)；Upper right as
Limit is non-living cell, i.e. non-viable non-apoptotic cell, is (FITC+/7-AAD+)；And right lower quadrant is apoptotic cell, show (FITC+/
7AAD-)。
7. plate clone detects Cell growth ability
Cell inoculation survival rate represents number adherent after cell inoculation, but attached cell not necessarily can breed and be formed
Clone, the cell for forming clone must be cell that is adherent and having proliferation activity, i.e. cloning efficiency reflection cell colony dependence
And multiplication capacity.This research cervical cancer cell SiHa and HeLa, each cell line set three experimental groups：1. Ctrl siRNA are cloudy
Property control group, 2. transfection groups of siTIM 1392；3. transfection groups of siTIM 3181.Comprise the following steps that：
(a) bed board, is transfected (the same)
(b) 48h after transfecting, cell dissociation is counted and gradient dilution, and with the amount of every 500 cells in hole, (Ca Ski are per hole
1000) it is inoculated in 6 orifice plates, 3 repeating holes are set.37 DEG C, 5%CO are positioned over after gently mixing2Incubator in cultivate；
(c) not good liquor was changed every 3 days；Micro- Microscopic observation clone size after 7-10 days；
(d) culture medium is abandoned, PBS is washed 3-4 times；15min is fixed with the methanol room temperature of -20 DEG C of precoolings；
(e) PBS is washed after twice, and dyeing 30min is carried out with crystal violet；
(f) ventilation drying, the clone for taking pictures and counting cell number ﹥ 50, analysis result are placed in after deionized water washing.
Experiment is repeated three times.
8. beta galactosidase decoration method detects cell ageing
The usual volume of senile cell cell becomes big, and expression has the beta galactosidase of enzymatic activity high.Cell ageing β-gala
Glucosides enzyme staining reagent kit can generate navy blue production using X-Gal as substrate under the beta galactosidase catalysis of senescence-specific
Thing, shows as becoming under an optical microscope the cell or tissue of the expression beta galactosidase of au bleu.Cell ageing is also swollen
A kind of suppressed embodiment of knurl.
This experiment selects cervical cancer cell lines SiHa to detect, every plant of cell is divided into six groups：1. Ctrl siRNA negative control groups,
2. transfection groups of siTIM 1392；3. transfection groups of siTIM 3181；4. Ctrl siRNA+ cis-platinums (5ug/ml) group；⑤siTIM
1392+ cis-platinums (5ug/ml) group；6. siTIM 3181+DDP (5ug/ml) group.Experiment is comprised the following steps that：
(a) cell of 72h after transfection is taken, culture medium is abandoned, is washed with PBS one time, β is added and adds galactoside enzyme dyeing to fix
Liquid, fixes 15min at room temperature.
(b) fixer is sucked, 3 times/3min of cell is washed with PBS.
(c) 1ml dyeing working solution (being prepared using polypropylene or glass container) is added per hole.
Dye Working solution prescription such as following table：
| Composition | Volume (ul) |
| Beta galactosidase dyeing liquor A | 10 |
| Beta galactosidase dyeing liquor B | 10 |
| Beta galactosidase dyeing liquor C | 930 |
| X-gal | 50 |
(d) 37 DEG C are free of CO2Incubator in be incubated overnight, sealed with preservative film six orifice plates prevent evaporation；
(e) micro- Microscopic observation is taken pictures.
9. cisplatin sensitivity is tested
This hair selects SiHa cell detections.Experiment is divided into three groups：1. Ctrl siRNA+ cis-platinums group；2. siTIM 1392+ cis-platinums
Group；3. siTIM 3181+DDP groups.Comprise the following steps that：
(a) bed board, transfection procedure is with cell proliferation experiment part.
(b) after transfection 48h, supernatant is abandoned.Every group of DDP for being separately added into the various concentrations that 100ul complete mediums dilute is molten
Liquid, concentration gradient is 0.097,0.195,0.391,0.781,1.563,3.125,6.25,12.5,25 (ug/ml)；Treat 24h.
(c) supernatant is abandoned, is incubated in the CCK8 reagent incubators that the basal mediums of 90ul 1640 and 10ul are added per hole after 1h
Determined with ELIASA and add the absorbance (OD values) that CCK8 is incubated 450nm after 1h.Inhibiting rate is calculated after blank control group zeroing.Press
According to formula：Cell inhibitory rate (%)=[(cell controls group A values-experimental group A values)/(cell controls group A values-blank control group A
Value)] × 100%.Experiment is repeated 3 times.
(d) the IC50 values of every group of cell are calculated with GraphPad Prism5 Software on Drawing curves and.
10. total serum IgE is extracted
(a) pending cell is taken out, PBS is abandoned after supernatant twice；
(b) 1ml Trizol are added, piping and druming repeatedly is mixed, room temperature places cracking 15min.Tissue sample is extracted before total serum IgE,
Fritter tissues need to be put into the EP pipes of no RNase, be homogenized rapidly after adding Trizol.Homogenate is fully then operated with cell such as
Under；
(c) 0.2ml chloroforms are added, 30s is acutely vibrated, mixed；It is stored at room temperature 30min；
(d) 4 DEG C, 12000rpm centrifugation 15min, transfer 400ul supernatants are into another 1.5ml EP pipes without enzyme；
(e) 0.5ml isopropanols are added.Turn upside down mixing.Room temperature places 20min；
(f) 4 DEG C, 12000rpm abandons supernatant after centrifuging 10min；
(g) ethanol of 1ml 75% is added, slight concussion is mixed.4 DEG C, 7500rpm centrifugations 5min；
(h) abandon most supernatant to dry after superclean bench, until precipitation becomes translucent；Add the dissolving of 20ul DEPC water；
(i) take 2ul RNA to dilute 50 times, OD260/OD280 (normal range (NR) 1.7-2.1) and OD230/ is measured respectively
OD260 (normal range (NR) 1.8-2.2)；
(j) in -80 DEG C of refrigerator freezings, save backup.
11.Real-time PCR detect TIMELESS mRNA expressions
(A) reverse transcription is into cDNA
A) 200 treated μ lPCR pipes will be dezymotized to put on ice, sequentially adds following reagent：
Mix, 3000rpm is centrifuged 30 seconds.
B) reverse transcription is carried out by following condition：
| 25℃ | 30min |
| 42℃ | 1h |
| 70℃ | 15min |
Product cDNA for Real-time PCR or is put into -20 DEG C of refrigerator preservations.
(B) Real-time PCR react
TIMELESS in cervical cancer tissues and pairing cancer beside organism, four kinds of cell lines of cervical carcinoma is detected with Real-time
Expression.Actin, GAPDH are used as internal reference.Using the cDNA after reverse transcription as template.Primer sequence is as follows：
(a) Real-time PCR reaction systems are as follows：
(b) Real-time PCR reaction conditions are as follows：
Analysing amplified curve and solubility curve, TIMELESS relative expression levels are represented with Ct, according to 2-△△CtMethod is calculated
The expression of target gene.
11. protein immunization imprinting
(A) configuration of sds page
(a) selection of separation gel
The separation gel of various concentrations is selected according to surveyed molecular weight of albumen size.
(b) separation gel and concentration glue formula table
Each concentration separation gel (15ml) and 5% concentration glue (6ml) are prepared according to following table formula.
Separation gel formula table (ml)
| Composition/concentration | 8% | 10% | 12% | 15% |
| dd H2O | 5 | 4 | 3 | 1.5 |
| Acr-Bis (30%) | 4 | 5 | 6 | 7.5 |
| 1M Tris, pH8.8 | 5.7 | 5.7 | 5.7 | 5.7 |
| 10%SDS | 0.15 | 0.15 | 0.15 | 0.15 |
| 10% ammonium persulfate | 0.15 | 0.15 | 0.15 | 0.15 |
| TEMED | 0.009 | 0.006 | 0.006 | 0.006 |
5% concentration glue formula table (ml)
| Components Name | Add volume (ml) |
| ddH2O | 4.1 |
| Acr-Bis (30%) | 1 |
| 1M Tris, pH6.8 | 0.75 |
| 10%SDS | 0.06 |
| 10% ammonium persulfate | 0.06 |
| TEMED | 0.006 |
(c) appropriate volume is poured into after separation gel is prepared, and between double-deck electrophoresis plate, (liquid level is left apart from glass plate top 4cm
It is right), carefully spread into isopropanol 1ml to completely cut off air on liquid level upper strata.It is stored at room temperature solidification 30min.
(d) after gelling to be separated is solid, upper strata isopropanol is carefully discarded, is blotted with filter paper.The concentration glue configured is poured into, directly
To filling up between double-deck electrophoresis plate, the comb matched is rapidly inserted into, it is to avoid bubble is produced.It is stored at room temperature 30min.
(e) glue to be concentrated polymerize completely, the electrophoresis plate containing gel is loaded into electrophoresis tank (comb towards inner side), to inside groove
The electrophoresis liquid (Running buffer) of Fresh is poured into until diffusing out the height of water jacket 2/3.Comb is extracted, makes soak
In electrophoresis liquid.
(B) protein electrophorese
(a) take out protein sample boil after place on ice, respectively loading and record loading order.Every block of plate reserves a swimming lane
Plus pre-dyed molecular weight of albumen marker is to determine target protein position.
(b) voltage stabilizing electrophoresis：Concentrate glue 80V, about 30min.Separation gel 120V, about 1h.
(c) stop electrophoresis when at bromophenol blue electrophoresis to electrophoresis tank lower end about 1cm, take out electrophoresis plate, be soaked in precooling
In transferring film liquid (Transfer buffer).
(C) protein electrotransfer
(a) according to the filter paper of pvdf membrane 6 of size clip 1 of gel；
(b) pvdf membrane is placed in absolute methanol and activates 5min, filter paper is soaked in stand-by in transferring film buffer solution；
(c) by sponge, filter paper, pvdf membrane, gel is positioned on transferring film clamping plate in the following order：Black clamping plate → sponge
→ 3 filter paper → gel → pvdf membrane → 3 filter paper → sponge → white clamping plates, buckle, are put into electrotransfer groove, transferring film clamping plate
Black flour pours into transferring film liquid to flooding transferring film clamping plate completely against the black side of transfer groove.100V, transfer about 90min (needs basis
Target protein size suitably adjusts transfer time and voltage).
(D) immunoblotting assay
(a) after the completion of shifting, take out pvdf membrane and twice of removal transferring film liquid is rinsed in TBST solution.
(b) close.Pvdf membrane is transferred in 5% skimmed milk power (Blocking buffer) shaken up in advance, is placed in and shakes
Room temperature slowly rocks closing 1-1.5h on bed, or 4 DEG C of closings are stayed overnight.After the completion of closing, film is washed 3 times with 0.1%TBST, every time
10min, shaking table washing.Carry out cutting film with reference to molecular weight of albumen marker.
(c) primary antibody is incubated.According to primary antibody specification, destination protein antibody is carried out suitably with Western primary antibodies dilution
Dilution proportion, adds and (is advisable in antibody incubation box so that the pvdf membrane containing target stripe is completely covered).4 DEG C are incubated overnight or room
Warm slow rock is incubated 2h.After the completion of incubation, primary antibody is reclaimed, film is washed 3 times with 0.1%TBST, each 10min, shaking table washing.
(d) secondary antibody is incubated.According to secondary antibody specification, proper proportion is carried out to required secondary antibody with Western secondary antibodies dilution
After dilution, add in corresponding incubation box, room temperature slowly shakes incubation 1h.After the completion of incubation, secondary antibody is reclaimed, washing film, (step is same
Before).
(f) luminescent solution configuration and development are taken pictures.Precooling gel imager is stand-by.Take equivalent ECL A liquid and B liquid to mix (to press
According to every after development film 200ul calculate) after be laid on pvdf membrane develop, room temperature reaction 1min.Film is put into imager darkroom
Middle development is taken pictures, and preserves experimental data.
(g) analysis of experimental results.Western band gray scale scannings are carried out using ImageJ softwares, GAPDH are chosen as interior
Reference.
Example given above is to realize the present invention preferably example, and the invention is not restricted to above-described embodiment.This area
Technical staff any nonessential addition, the replacement made according to the technical characteristic of technical solution of the present invention, belong to this
The protection domain of invention.
Sequence table
<110>University Of Suzhou
<120>Application of the TIMELESS genes as target spot in the medicine for treating cervical carcinoma is prepared
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213>Non-targeting siRNA target sequence
<400> 1
TGGTTTACATGTCGACTAATT 21
<210> 2
<211> 21
<212> DNA
<213>The sequence for the TIMELESS gene targets that siTIM 1392 is directed to
<400> 2
GCCGCATCATCAAGAACAATA 21
<210> 3
<211> 21
<212> DNA
<213>The sequence for the TIMELESS gene targets that siTIM 3181 is directed to
<400> 3
GCAGCTGTTGCGCAAGCTAGG 21
<210> 4
<211> 21
<212> RNA
<213>Non-targeting siRNA positive-sense strand
<400> 4
UGGUUUACAUGUCGACUAATT 21
<210> 5
<211> 21
<212> RNA
<213>Non-targeting siRNA antisense strand
<400> 5
UUAGUCGACAUGUAAACCATT 21
<210> 6
<211> 21
<212> RNA
<213>SiTIM 1392 positive-sense strand
<400> 6
GCCGCAUCAUCAAGAACAAUA 21
<210> 7
<211> 21
<212> RNA
<213>SiTIM 1392 antisense strand
<400> 7
UUGUUCUUGAUGAUGCGGCUG 21
<210> 8
<211> 21
<212> RNA
<213>SiTIM 3181 positive-sense strand
<400> 8
GCAGCUGUUGCGCAAGCUAGG 21
<210> 9
<211> 21
<212> RNA
<213>SiTIM 3181 antisense strand
<400> 9
UAGCUUGCGCAACAGCUGCUG 21
Claims (7)
- Application of the 1.TIMELESS genes as target spot in the medicine for treating cervical carcinoma is prepared, the TIMELESS genes NCBI Serial No. NM_003920.4；In the TIMELESS genes as the sequence of target spot for GCCGCATCATCAAGAACAATA or GCAGCTGTTGCGCAAGCTAGG。
- 2. a kind of siRNA for TIMELESS gene targets, it is characterised in that：The siRNA is siTIM 1392, siTIM 3181。
- 3. siRNA according to claim 2, it is characterised in that：The TIMELESS gene targets that siTIM 1392 is directed to Sequence is GCCGCATCATCAAGAACAATA；The sense strand sequence of the siTIM 1392 is GCCGCAUCAUCAAGAACAAUA, the antisense strand of the siTIM 1392 Sequence is UUGUUCUUGAUGAUGCGGCUG.
- 4. the siRNA described in claim 2, it is characterised in that：The sequence for the TIMELESS gene targets that siTIM 3181 is directed to For GCAGCTGTTGCGCAAGCTAGG；The sense strand sequence of the siTIM 3181 is GCAGCUGUUGCGCAAGCUAGG, the antisense strand of the siTIM 3181 Sequence is UAGCUUGCGCAACAGCUGCUG.
- 5. applications of the siRNA in the medicine for preparing treatment cervical carcinoma described in claim 2.
- 6. a kind of pharmaceutical composition, it is characterised in that：SiRNA described in claim 2 containing the upper effective dose for the treatment of, or simultaneously Contain its pharmaceutically acceptable excipients or additional dose.
- 7. application of the pharmaceutical composition in the medicine for preparing treatment cervical carcinoma described in claim 6.
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| CN114058641A (en) * | 2022-01-17 | 2022-02-18 | 苏州大学 | Carrier system, application and method for degrading target protein through carrier system |
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