CN104630218A - Use of PAX6 in stomach cancer pathogenesis - Google Patents
Use of PAX6 in stomach cancer pathogenesis Download PDFInfo
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- CN104630218A CN104630218A CN201310559735.3A CN201310559735A CN104630218A CN 104630218 A CN104630218 A CN 104630218A CN 201310559735 A CN201310559735 A CN 201310559735A CN 104630218 A CN104630218 A CN 104630218A
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- pax6
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Abstract
A potential cancer promoting gene (PAX6) highly expressed in stomach cancer can promote stomach cancer cell proliferation and inhibit stomach cancer cell apoptosis. Two pairs of siRNAs and lentiviruses are invented, can be chemically synthesized, have a high PAX6 expression silencing efficiency, and has a potential treatment effect on the stomach cancer.
Description
Technical field
Invention herein relates to the effect of genetic expression in pathogenesis of cancer mechanism, particularly relates to cancer of the stomach
Background technology
Cancer of the stomach is one of malignant tumour occurred frequently in worldwide, prognosis is poor, mortality ratio is in the second in all malignant tumours, Most patients has lost the chance of surgical radical treatment when making a definite diagnosis, current treating malignant tumor often adopts operation, chemotherapy, radiotherapy, biological response modifier to treat the complex therapy combined, but general curative effect is still unsatisfactory.Therefore, people are constantly finding new methods for the treatment of.Along with the establishment of new theory and continuing to bring out of new technology, for the appearance of malignant tumour new therapy lays the first stone.Pairing box gene 6 (Paired box6, PAX6) in retina tumor, carcinoma of the pancreas, mammary cancer, high expression level is presented, can promote that malignant tumour is bred, suppress malignant tumour apoptosis, but the research of PAX6 in cancer of the stomach, and suppress the mechanism of malignant tumour apoptosis to yet there are no report.
Summary of the invention
Present invention finds PAX6 to express in cancer of the stomach and increase, there is the effect promoting propagation and apoptosis inhibit, and invented two couples of siRNA, can chemosynthesis, there is reticent PAX6 expression efficiency high, have potential therapeutic action to cancer of the stomach.
The siRNA that reticent PAX6 expresses is to the short propagation of stomach cancer cell and apoptosis inhibit research
1 cell cultures and transfection
1.1 cell cultures
Cell culture incubator is set to 37 DEG C, 5%CO
2, under saturated humidity condition, people cancer of the stomach HGC27 and MKN45 cell go down to posterity foster in the RPMI-1640 nutrient solution containing 10% foetal calf serum.
1.2 cell transfecting
1.2.1 day before transfection, will be in 1.5 × 10 of logarithmic phase
6individual ags cell is seeded on 6 orifice plates, and 1.5ml is containing the RPMI-1640 culture medium culturing of 10%FBS, and 24h cell density reaches 30-50%.
1.2.2200pmol PAX6si-1 serum-free Opti-MEM cell culture medium becomes final volume 185ul, mixes gently.
1.2.3 in the serum-free Opti-MEM cell culture medium of 24ul, add liposome OligofectamineTM6ul, mix ambient temperatare gently and put 5min.
PAX6si-1 and the OligofectamineTM reagent mix of 1.2.4 will dilute, mixes room temperature gently and places 20min, to form mixture.
1.2.5 added by the mixture of 215ul in 6 orifice plates of 800ul serum-free Opti-MEM cell culture medium, cumulative volume is the final concentration of 1015ul, siRNA is 100pmol/ml, 37 DEG C, 5%CO
2incubator in continue cultivate 4-6h.
1.2.64-6h exhaust old nutrient solution afterwards in 6 orifice plates, adds the RPMI-1640 fresh culture of 2ml containing 10%FBS, 37 DEG C, 5%CO
2incubator, continue cultivate, carry RNA or albumen respectively at 24h, 48h, 72h.
1.2.7PAX6si-2 operate the same with negative control group transfection NC, final concentration is 100pmol/ml; Blank group Mock group adds 1ml serum-free Opti-MEM cell culture medium (containing 6ul OligofectamineTM), changes the RPMI-1640 fresh culture of 2ml10%FBS after 4-6h into.
2. the RNA extracting of cell: adopt Trizol reagent single stage method to extract stomach cancer cell RNA.
3.RT-PCR detects PAX6 genetic expression
Experimental procedure
3.1 reverse transcription reaction
3.1.1 get thin wall centrifugal tube (RNase free) and add following reaction system, 70 DEG C of sex change 5min, immediately 4 DEG C of coolings.
3.1.2 each reagent is added successively by order below reaction system, 37 DEG C of reaction 5min
3.1.3 in PCR instrument, reverse transcription reaction is carried out, 42 DEG C of incubation 60min, 70 DEG C of 15min termination reactions, 4 DEG C of coolings.The cDNA of reverse transcription is for subsequent use in-20 DEG C of storages.
3.2PCR amplified reaction
3.2.1 according to the cDNA sequence of the PAX6 of the Genbank mankind, application primer-design software Primers designs primer, and is synthesized by Invitrogen company.Because Oligo DNA is membranaceously attached on tube wall in very light, first brief centrifugation before use, then open pipe lid gently, after being dissolved as 10pmol/ul with deionized water, store-20 DEG C for subsequent use.Each primer sequence is as follows:
The each primer sequence of table 2:PCR
3.2.2 take out the cDNA increased to dissolve on ice, because the similar length of internal reference GAPDH and object band PAX6, so PCR reaction will be carried out respectively.
3.2.3PCR reaction system is as follows:
3.2.4PCR amplification program
94 DEG C of 5min warm starts, 94 DEG C of 30sec, 57 DEG C of 35sec, 72 DEG C of 45sec, 35 circulations of increasing.72 DEG C extend 7min, rear-20 DEG C of storages.
3.2.5DNA electrophoresis
Get in 1.5% sepharose that 10ul pcr amplification product adds containing EB, be about 30min with 100v/cm electrophoresis, the people PAX6 amplified band of visible corresponding length under uviolizing, with gel electrophoresis Image analysis system scanning analysis electrophoretic image.
4. the protein extraction of cell sample
After cell culture medium is discarded, after cleaning three times with PBS liquid, every 1-5 × 10
5individual cell is resuspended in 50ul lysate, and piping and druming evenly, is scraped with cell under ice bath, moved to by enchylema in EP pipe after scraping, and leaves standstill 30min on ice, centrifugal in 4 DEG C, and 20000g × 30min collects supernatant, measures protein content through Bradford method, store-80 DEG C for subsequent use.
5. protein content determination (Bradford method)
5.1 the preparation of protein standard curve
Get the bovin serum albumin (BSA) 0,5ul, 10ul that concentration is 1ug/ul respectively, 15ul, 20ul and 25ul put in test tube, and add water 100ul respectively, 95ul, 90ul, 85ul, 80ul, 75ul, then add Bradford working fluid 1ml respectively in every test tube, 2min is placed after mixing, with the solution containing BSA0ug/ul for blank, measure each pipe in 1h in 595nm place absorption value, do typical curve.
5.2 sample protein content determinations
Get 1ul albumen sample solution, add 99ul ddH
2o and 1ml Bradfordl working fluid mixes, and places 2min, with the solution containing BSA0ug/ul for blank, measures 595nm place absorption value.Absorption value is brought into the typical curve that step 6.1 obtains, calculate sample protein content.
The sex change of 5.3 protein samples
In 1: 1 ratio biased sample and 5 × SDS-PAGE sample buffer, boil 5min, room temperature cooling 5min.
6. immunoblotting (Western blot) detects the expression of PAX6
6.1SDS-PAGE electrophoresis
6.1.1 recording of separation gel/concentrated glue
Prepare acrylamide separating gel solution according to table 1, after joined solution is poured into sheet glass, overing number flattens liquid level, and polymerized at room temperature, discards covering liquid.
Table 312% resolving polyacrylamide gel formula
Prepare propylene phthalein amine according to table 4 and concentrate glue, joined solution is added in immediately on the separation gel be polymerized, insert comb, polymerized at room temperature.
Table 45% polypropylene phthalein amine concentrates glue formula
6.1.2SDS-PAG protein electrophorese
Protein example loading: adjustment sample protein matter concentration, makes the protein of each swimming lane loading be 20ug.Contrast using albumen Marker as molecular weight.Electrophoresis carries out in Bio-Rad electrophoresis chamber, and sample running voltage in concentrated glue is 60v, and when arriving separation gel interface, voltage increases to 80v, continues electrophoresis until bottom bromjophenol blue to separation gel.
6.2 electrotransfer
6.2.1 excision discards concentrated glue.In separation gel bottom right, corner cut is marked, in transfering buffering liquid, balance 15min.Clip is identical with separation gel size and carry out pvdf membrane and two pieces of 3mm filter paper of mark.Pvdf membrane first soaks into about 15sec in methyl alcohol, then proceeds to ddH
2balance 5min in O, after taking out, immerse transfering buffering liquid 5-10min.Filter paper also soaks 5-10min in transfering buffering liquid.Stack filter paper, film, gel, an other filter paper from bottom to top successively, when stacking, each step all will be noted catching up with bubble removing.
6.2.2 be placed on the half-dried electroporation positive plate of Bio-Rad, cover the upper cover of band negative plate.Constant voltage 10v is set, 45min-1h.
6.2.3 transfer terminate after, pvdf membrane is immersed methyl alcohol 10sec, after proceed to ddH
25min is balanced in O.
6.3 immunodetection and ECL chemoluminescence
6.3.1 the pvdf membrane 1h after transfer is closed under using confining liquid (TBST of 5% skim-milk and 0.5%Tween) room temperature.
6.3.2 according to the PAX6 that albumen Marker indicates, pvdf membrane is cut off by the position of α-Tubulin, then put into the homemade hybridization bag of plastic thin-film gloves respectively, add the anti-human PAX6 polyclonal antibody of rabbit diluted with confining liquid 1: 200 respectively, mouse anti human α-Tubulin the monoclonal antibody of 1: 1000 dilution, catch up with bag mouth sealing after bubble removing, 4 DEG C of incubator overnight.
6.3.3TBST pvdf membrane is washed 3 times, each 10min.
6.3.4 goat against murine and the goat anti-rabbit antibodies of the HRP mark that corresponding confining liquid 1: 5000 dilutes is added respectively, room temperature reaction 1h.
6.3.5TBST pvdf membrane is washed 3 times, each 10min.
6.3.6ECL chemiluminescence detection: the A liquid in balanced mix ECL and B liquid (reacting the amount configuration of glue/cm2PVDF according to 0.1ml) are laid on sealed membrane, then the Western blot of pvdf membrane is faced down lid thereon, drive bubble away gently, incubated at room reaction 3-5min.
6.3.7 drain ECL reagent unnecessary on pvdf membrane, pvdf membrane be placed between two-layer preservative film, bubble of carefully rushing, the Western blot of film towards on put into magazine.In the exposure of dark intraventricular pressure X egative film, development, fixing, washing film dries in the air after thousand and preserves.
7. tetrazolium bromide (MTT) method detects cell proliferation
7.1 experimental procedure
7.1.1 inoculating cell: be made into individual cells suspension, with every hole 3.5 × 10 with obtaining RPMI-1640 nutrient solution containing 10% tire calf serum
3individual cell is inoculated into 96 orifice plates, every pore volume 100ul, and three multiple holes established by each sample.
7.1.2 culturing cell and transfection: next day, cell was in logarithmic phase, gets 100nmol/L concentration PAX6si-1 respectively, PAX6si-2, NC transfection cancer of the stomach HGC27 and MKN45 cell.
7.1.3 colour generation: transfection is incubated at 24h, after 48h, 72h, every hole adds MTT solution (5mg/ml PBS, pH value 7.4) 20ul.At 37 DEG C of CO
2incubator continues to hatch 4h, stops cultivating, and careful suction abandons culture supernatant in hole, inhales again and abandon culture supernatant in hole after centrifugal for suspension cell needs.Every hole adds 100ul DMSO, and vibration 5min, makes crystallisate fully melt.
7.1.4 colorimetric: select 492nm wavelength, enzyme linked immunological monitor measures each hole absorbance value (A), record result take time as X-coordinate, and light absorption value is that ordinate zou draws cell growth curve.
8. cell clonal formation experiment
Experimental procedure:
First 8.1 carry out 6 orifice plate cell transfectings, and concrete operations, with method 1.3, stop cultivating after transfection 48h, PBS washes twice, add 0.25% appropriate tryptic digestive juice digestion 1-2min, the nutrient solution of injection about 1.5ml stops digestion, and suction pipe piping and druming is to being unicellular.
8.2, with after cell counting count board counting, are inoculated in 6 well culture plates by 500 cells/well, supply 2ml nutrient solution, put 37 DEG C, 5%CO
2cultivate in incubator.
8.3 cultivations discarded nutrient solution after 13 days, wash 2 times, add methyl alcohol about 3ml and fix 15min, discard stationary liquid with PBS, the dyeing of a Ji's nurse Sa 40min, ddH
2o slowly washes away staining fluid, dry air, is inverted by 6 orifice plates and slight stroke of upper mesh lines, counting clone number under ordinary optical microscope.
8. flow cytometry surveys apoptosis
8.1.1 inoculating cell: being made into individual cells suspension with obtaining RPMI-1640 nutrient solution containing 10% tire calf serum, being inoculated into six orifice plates, three multiple holes established by each sample.
8.1.2 culturing cell and transfection: next day, cell was in logarithmic phase, gets 100nmol/L concentration PAX6si-1 respectively, PAX6si-2, NC transfection cancer of the stomach HGC27 and MKN45 cell.
8.1.3 colour generation and detection: after transfection is incubated at 48h, the trysinization with 0.25% is made into single celled suspension, afterwards the concentration of cell is adjusted to 1 × 10
7individual/ml, often pipe 10ul, use annexin A5-green fluorescent protein-propidium iodide (AnnexinV) to dye afterwards and put into early apoptosis situation flow cytometer detecting each group of cell afterwards in 30 minutes.
9.JC-1 mitochondrial membrane potential
In the human stomach cancer cell line HGC27 that reticent PAX6 expresses, add JC-1 dye work also (5mg/L), put into cell culture incubator 37 DEG C and hatch 20 minutes, put into JC-1 dye solution afterwards again and wash 2 times, then put into cell culture fluid.Under fluorescent microscope, detect intensity level that is green, red fluorescence, represent the change of mitochondrial membrane potential with ratio that is red and green fluorescence intensity value.
10. statistical procedures
Application SPSS13.0 statistical software analyzes, and measurement data represents with mean ± standard deviation, compares and adopt one-way analysis of variance (one-way ANOVA) between group, and the comparison in difference between two factors and correlation analysis adopt χ2-test,chi-square test; P < 0.05 is for there being statistical significance.
Three, result display
The albumen of PAX6 and mRNA present high expression level in most stomach organization, and in the patients with gastric cancer of PAX6 high expression level, total survival time and Sulfurless fixative are all shorter compared with the patients with gastric cancer of the low expression of PAX6; In the proliferation of human gastric cancer cell ability of PAX6 overexpression, Cell clonality is significantly increased; And the volume of the knurl body that the cell strain injecting reticent PAX6 expression in the middle of nude mice is formed and weight are all significantly less than the nude mice of the not reticent PAX6 expression cell line of injection; Flow cyctometry and mitochondrial membrane potential detect the apoptosis all showing the cell strain of PAX6 high expression level obviously to be reduced, we also find that the apoptotic effect of PAX6 T suppression cell is mainly by Akt signal path, can suppress the activation of Caspase-3 and PARP further.
The above results shows that PAX6 can become the tumor marker of cancer of the stomach, and PAX6 can promote the propagation of cancer of the stomach and suppress the apoptosis of cancer of the stomach.
The design of PAX6siRNA
For mankind PAX6 gene mRNA sequence, design PAX6siRNA and negative control (NC) siRNA following (siRNA oligonucleotide sequence is formed by 19 bases), is synthesized by Chinese Rui Bo company.
PAX6si-1：
Target sequence: GCTTCACCATGGCAAATAA
Justice 5 ' GCUUCACCAUGGCAAAUAA dTdT3 '
Antisense 3 ' dTdT CGAAGUGGUACCGUUUAUU5 '.
PAX6si-2：
Target sequence: GCAGACGGCATGTATGATA
Justice 5 ' GCAGACGGCAUGUAUGAUA dTdT3 '
Antisense 3 ' dTdT CGUCUGCCGUACAUACUAU5 '.
Claims (4)
1. (totally two to) reticent PAX6 siRNA, by they called after PAX6si-1 and PAX6si-2 respectively.Its feature is:
PAX6si-1：
Target sequence: GCTTCACCATGGCAAATAA
Justice 5 ' GCUUCACCAUGGCAAAUAA dTdT3 '
Antisense 3 ' dTdT CGAAGUGGUACCGUUUAUU5 '.
PAX6si-2：
Target sequence: GCAGACGGCATGTATGATA
Justice 5 ' GCAGACGGCAUGUAUGAUA dTdT3 '
Antisense 3 ' dTdT CGUCUGCCGUACAUACUAU5 '.
2. slow virus: interference sequence is GCTTCACCATGGCAAATAA.
The proliferation of 3.PAX6 in cancer of the stomach
Experiment in vitro: in the middle of MKN45 and HGC27 human stomach cancer cell line, after PAX6 is disturbed by siRNA, 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (MTT) absorbance significantly reduces, and after we also find that PAX6 is disturbed by siRNA further in cell clonal formation experiment (Colony formation assay), number of cell clones significantly reduces.
In vivo test: after stable interference PAX6 expression slow-virus transfection is entered human stomach cancer cell line (MKN45 and HGC27), be injected into mouse subcutaneous, after 6 weeks, find the mouse subcutaneous tumors volume and all remarkable mouse lower than the strain of injection cellular control unit of weight that are injected into stable interference PAX6 expression cell line.
The apoptosis inhibit effect of 4.PAX6 in cancer of the stomach
In the middle of MKN45 and HGC27 human stomach cancer cell line, after PAX6 is disturbed by siRNA, the expression of Caspase-3, PARP and Bcl-xl significantly increases, and the expression of BAD significantly reduces; Meanwhile, we also find that mitochondrial membrane potential obviously declines by after PAX6 interference; And we find that PAX6 apoptosis inhibit is played a role by Akt signal path, after being disturbed by PAX6, Akt expresses indifference, and the expression of PAkt significantly reduces.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021155825A1 (en) * | 2020-02-08 | 2021-08-12 | 北京大学第三医院（北京大学第三临床医学院） | Use of pax6 gene or expression product thereof in preparation of drug for inhibiting fibrosis |
| WO2022242756A1 (en) * | 2021-05-21 | 2022-11-24 | The University Of Hong Kong | Compositions and methods of targeting the pax6 signaling pathway to reduce formation of amyloid βeta plaques and neurofibrillary tangles |
-
2013
- 2013-11-11 CN CN201310559735.3A patent/CN104630218A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| QI YANG ET AL.: "Concomitant PIK3CA amplification and RASSF1A and PAX6 hypermethylation predict worse survival in gastric cancer", 《CLINICAL BIOCHEMISTRY》 * |
| SATOSHI YAMASHITA ET AL.: "Chemical genomic screening for methylation-silenced genes in gastric cancer lines using 5-aza-2"-deoxycytidine treatment and oligonucleotide microarray", 《CANCER SCI.》 * |
| YVAN GOSMAIN ET AL.: "Pax6 controls the expression of critical genes involved in pancreatic α cell differentiation and function", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021155825A1 (en) * | 2020-02-08 | 2021-08-12 | 北京大学第三医院（北京大学第三临床医学院） | Use of pax6 gene or expression product thereof in preparation of drug for inhibiting fibrosis |
| WO2022242756A1 (en) * | 2021-05-21 | 2022-11-24 | The University Of Hong Kong | Compositions and methods of targeting the pax6 signaling pathway to reduce formation of amyloid βeta plaques and neurofibrillary tangles |
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Application publication date: 20150520 |