CN103751806B - Interference SIRT1 expresses the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression - Google Patents
Interference SIRT1 expresses the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression Download PDFInfo
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Abstract
The invention belongs to chemical field, specifically disclose interference SIRT1 and express the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression, by suppressing the expression of dryness transcription factor in liver-cancer stem cell, reduce the multiplication capacity of liver-cancer stem cell, clonality and balling-up ability, thus suppress the self renewal of liver-cancer stem cell; Therefore interference SIRT1 can be expressed the medicine of reagent for the preparation of targeted therapy of liver cancer.
Description
Technical field
The invention belongs to chemical field, particularly disturb SIRT1 to express the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression.
Background technology
Tumor stem cell (cancer stem cells, CSCs) is the cell colony that the sub-fraction be present in tumor has stem cell properties, and having the cell of infinite multiplication, self renewal and multi-lineage potential, is the root of tumorigenesis.Large quantifier elimination shows, after stem cell produces by multipotency transcription factor inherently as Oct4, SOX2 and Nanog regulates external signal, thus in the microenvironment (niche) stem cell being resided in can maintain " dryness " characteristic, and then maintenance self-renewal capacity, these multipotency transcription factor have vital effect for effects such as maintaining the potential versatility of stem cell and mediates cell proliferation.Research confirms, form a Feedback adjusting loop between multipotency transcription factor Oct4, SOX2 and Nanog three, activate and promote stem cell growth and self renewal, the expression of Inhibited differentiation gene simultaneously, but excite stem cell Forming Mechanism to be still not clear for above-mentioned transcription factor.In addition, research finds multiple protein coding gene, and their product take part in the generation of tumor stem cell " dryness " maintenance and tumor.Therefore, study the interaction of these genes and multipotency transcription factor, the mechanism of action for research tumor stem cell self renewal has important effect.
The enzyme modifying histone in cell is also often the enzyme modifying transcription factor, and the important transcription factor maintaining versatility is also subject to the regulation and control of post translational modification.Deacetylase SIRT1 is by the function of regulator gene and transcribe thus determine the destiny of cell; the function of its regulator gene and transcribe and mainly regulate histone activity (mainly H4K16 and H3K9 site) and acetylation modification to regulate the transcription factor of nonhistones expression particularly some keys as p53 in the mode of epigenetic; FOXO; c-MYC, HIF1 α, HIF2 α etc.Recently study discovery, SIRT1 has important effect in maintenance Mus embryonic stem cell (ESC) and human embryo stem cell (HSC) self renewal.SIRT1 by acetylation in p53, thus regulate the multipotency transcription factor Nanog of stem cell, the expression of Oct4 and SOX2.In people and Mus hematopoietic stem cell, SIRT1 by the activity of the change adjustment transcription factor of energy metabolism, and then maintains the self renewal of hematopoietic stem cell.But the effect regulating multipotency transcription factor to maintain stem cell self renewal in solid tumor stem cell about SIRT1 there is not yet bibliographical information.
Summary of the invention
In view of this, interference SIRT1 is the object of the present invention is to provide to express the novelty teabag of reagent, for the targeted therapy of hepatocarcinoma is significant.
For achieving the above object, the invention provides following technical scheme:
Interference SIRT1 expresses the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression.
Preferably, described interference SIRT1 expresses the slow virus or Tenovin-6 that reagent is interference SIRT1 expression.
Preferred, the concentration of described Tenovin-6 is 2.5 ~ 10 μMs.
Preferably, the slow virus that described interference SIRT1 expresses by after Lv-sh-SIRT1 plasmid transfection 293T cell, through packaging, purification and obtaining.
Preferably, described dryness transcription factor is SOX2, Oct4, c-Myc or Nanog.
Preferably, described dryness transcription factor is SOX2 or Nanog.
Beneficial effect of the present invention is: the invention discloses interference SIRT1 and express the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression, by suppressing dryness transcription factor in hepatoma liver cells, thus the propagation of suppression liver-cancer stem cell, Clone formation, balling-up ability and subcutaneous transplantation neoplasia rate, delay tumor growth in vivo, suppress the self renewal of liver-cancer stem cell.Therefore interference SIRT1 can be expressed reagent and be used for targeted therapy of liver cancer, significant to prolongation hepatocarcinoma life span.
Accompanying drawing explanation
In order to make object of the present invention, technical scheme and beneficial effect clearly, the invention provides following accompanying drawing:
Fig. 1 is the expression detecting each dryness transcription factor after interference SIRT1 expresses.
Fig. 2 is the expression that after interference SIRT1 expresses, different time detects dryness transcription factor.
Fig. 3 is the expression of variable concentrations TV-6 process cell detection Nanog and SOX2.
Fig. 4 is the expression that after 5 μMs of TV-6 process liver-cancer stem cells, different time points detects Nanog, SOX2.
Fig. 5 is the expression of SIRT1 and SOX2 in liver cancer tissue and hepatoma cell line.
Fig. 6 is protein expression situation (the A:western-blot detection of interference SIRT1 process LAN SOX2; B: Immunofluorescence test).
Fig. 7 is interference SIRT1 process LAN SOX2 on the impact of liver-cancer stem cell Clone formation and balling-up ability.
Fig. 8 is that interference SIRT1 process LAN SOX2 is on the impact (A: transplanted tumor observed result of NOD/SCID mice-transplanted tumor; B: the weight and volume of transplanted tumor).
Fig. 9 is the expression that immunofluorescence and SABC detect GFP or SIRT1 and SOX2 in transplanted tumor respectively.
Figure 10 is Nanog
posliver-cancer stem cell in disturb SIRT1 after express SOX2 affect Nanog expression (A:western-blot detection at Nanog
posliver-cancer stem cell in disturb SIRT1 expression of GFP in the cell of expressing SOX2; B:western-blot detects the expression at interference SIRT1 GFP in the transplanted tumor expressing SOX2).
Figure 11 is at Nanog
posliver-cancer stem cell in disturb SIRT1 after express SOX2 cell in detect in Luciferase reporter system Nanog promoter expression change.
Figure 12 is Nanog
posliver-cancer stem cell in disturb SIRT1 to detect SOX2 in conjunction with the expression of Nanog after the cell of expressing SOX2 by CHIP.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described in detail.The experimental technique of unreceipted actual conditions in embodiment, usual conveniently condition, the such as Molecular Cloning: A Laboratory guide (third edition, J. Pehanorm Brooker etc. work) described in condition, Shan et al.2012, the condition that conditioned disjunction described in hepatology, 56:1014-1014 is advised according to manufacturer.
The liver-cancer stem cell model that the present invention uses is built by this laboratory, this model contains the Lentiviral (Nanog/GFP reporting system) that Nanog promoter regulation GFP reporter gene builds, its construction method see Shan et al.2012, hepatology, 56:1014-1014; Lv-sh-Scramble plasmid and Lv-sh-SIRT1 plasmid, AD-GFP are built by this laboratory, specifically see Shan et al.2012, and hepatology, 56:1014-1014; HEK293 cell is preserved by this laboratory.AD-SIRT1 is so kind as to give by Zhu Weiguo professor, and its construction method is shown in Liu et al.2011, and Proc Natl Acad Sci U S A.108:1925-1930.
Human primary liver cancer cell line PLC/PRF/5 and hepatoma cell line Huh7 is purchased from Shanghai cell institute of the Chinese Academy of Sciences; Anti-SIRT1 antibody purchased from American Santa Cruz company, anti-Albumin antibody purchased from American Abcam company.Envision
tMwith DAB nitrite ion purchased from DAKO company of Denmark; PNANOG-Luc and Lv-Over-SOX2 plasmid purchased from American Addgene company; Transfection reagent box purchased from American QIGEN company; SOX2 antibody and CHIP rank rabbit anti-human SOX2 purchased from American Milliopore company.
TV-6(Tenovin-6) configuration of solution: use DMSO dissolving TV-6 dry powder to be configured to the storage liquid of 20 μMs respectively, use complete medium to be configured to the working solution of 0.5 μM, 2.5 μMs, 5.0 μMs, 7.5 μMs and 10 μMs during pending cell.
Embodiment 1
One, the expression of expression impact " dryness " transcription factor SOX2 of SIRT1
Get human primary liver cancer cell line PLC/PRF/5, hepatoma cell line Huh7 and hepatoma carcinoma cell T1224, (be designated as Nanog with flow cytometer difference sorting Nanog masculine liver cancer stem cell
pos) and the negative non-liver cancer stem cell of Nanog (be designated as Nanog
neg).
The packaging of slow virus Lv-sh-SIRT1 and Lv-sh-Scramble: at the culture dish middle berth 2 ~ 5 × 10 of 10cm
6individual 293T cell (preferably before 20 generations, the state of cell is required good, the speed of growth is very fast, otherwise it is very large on viral yield impact), culture fluid is containing the DMEM(v/v of 10%FBS), when cell confluency to 70%, renew the fresh DMEM(v/v containing 10%FBS) culture fluid, for transfection after 3-4h.
Get Lv-sh-SIRT1 or the Lv-sh-Scramble slow virus expression plasmid of 15-20 μ g, drip the CaCl of 50 μ L2.5M respectively
2mixing, sterilized water is added respectively to cumulative volume 450 μ L in mixed liquor, 500 μ L2 × HBS buffer (2 × HBS buffer shakes before using on turbula shaker) are added after mixing, then be placed on turbula shaker and shake more than 10s, drip after terminating, continue concussion, after room temperature places 5 minutes, liquid is added in the 293T of cultivation, after adding, rocks mixing all around, add the DMEM(v/v that rear 6-12 hour renews fresh 10%FBS) culture fluid.
The collection of slow virus Lv-sh-SIRT1 and Lv-sh-Scramble and purification: start respectively to collect a cell culture fluid in latter 48 hours, 72 hours from transfection, after 48 hr collections, supplement the culture fluid of 15mL again, the virus of collecting centrifugal 10min under 1000rpm condition is removed cell debris, collect supernatant, and with the membrane filtration of 0.45 μm, filtrate is transferred to the evaporating column of Millopore, is concentrated into 1mL(to the liquid volume in the groove of upper strata and can adds several times 5000rpm is centrifugal).By concentrated solution subpackage, be placed in-80 DEG C of cryogenic refrigerators and preserve.
At Nanog
posliver-cancer stem cell infect Lv-shRNA-SIRT1 or Lv-shRNA-Scramble slow virus: get human primary liver cancer cell line PLC/PRF/5, hepatoma cell line Huh7 and hepatoma carcinoma cell T1224, by selected by flow cytometry apoptosis Nanog
posliver-cancer stem cell, be then seeded in 6 orifice plates, at 37 DEG C, 5%CO
2incubator is cultivated, and treats that cell grows to 5 × 10
5time, liquid in sucking-off hole, adds the 10%FBS/DMEM(v/v of 1mL) culture fluid, then every hole is with 10 MOI values, and add Lv-shRNA-SIRT1 or Lv-shRNA-Scramble slow virus, blank group does not add slow virus, then in 37 DEG C, 5%CO
2incubator is cultivated, and after 6-8 hour, liquid in sucking-off hole, adds the 10%FBS/DMEM culture fluid of 2mL, after 72h, detects Nanog
posthe expression of dryness transcription factor SOX2, Nanog, Oct4 and c-Myc in cell, testing result as shown in Figure 1.Result shows, and after interference SIRT1 expresses, the trend that SOX2 lowers is the most remarkable, is secondly Nanog and Oct4, and c-Myc lowers not obvious.
According to the Nanog of identical method by Lv-shRNA-SIRT1 slow virus infection PLC/PRF/5
poscell, respectively at infection latter 0 hour, 12 hours, 24 hours and 36 hr collections cells, detects Nanog
posthe expression of cell dryness transcription factor SOX2, Nanog, Oct4 and c-Myc, result as shown in Figure 2.Result shows, and when disturbing SIRT1 to express 24h, SOX2 protein expression significance ground is lowered; When interference SIRT1 expresses 36h, the expression of c-Myc albumen is significantly lowered, and Nanog, Oct4 protein expression changes not obvious in time.
By the PLC/PRF/5Nanog of sorting
posand Huh7Nanog
posbe planted in 6 orifice plates respectively, treat that cell grows to about 80%, dripping concentration is respectively after the TV-6 of 0.0 μM, 0.5 μM, 2.5 μMs, 5.0 μMs, 7.5 μMs, 10 μMs cultivates 48 hours, then collecting albumen utilizes western-blot method to detect the expression of SOX2 and Nanog, be simultaneously internal reference with GAPDF, testing result as shown in Figure 3.Result shows, and after adding the inhibitor TV-6 of SIRT1, the TV-6 effect of 5 μMs is remarkable to the downward of SOX2 albumen in liver-cancer stem cell.
By the PLC/PRF/5Nanog of sorting
posbe planted in 6 orifice plates, treat that cell grows to about 80%, drip the TV-6 that concentration is 5.0 μMs, collect the albumen of 0h, 4h, 8h, 12h, 24h and 36h after dripping respectively, western-blot detects the expression of SOX2 and Nanog, and be internal reference with GAPDF, its result as shown in Figure 4 simultaneously.Result shows, and after the TV-6 of 5.0 μMs acts on 4 hours, the expression of SOX2 albumen sharply declines, and decline just appears in Nanog albumen expression after the TV-6 of 5.0 μMs acts on 36 hours.
The above results shows, and in liver-cancer stem cell, the expression of interference SIRT1 not only significantly can suppress the expression of SOX2, and is in early days event occurs.
Two, the dependency of SIRT1 and SOX2 protein expression in hepatoma cell line and clinical liver cancer tissue sample is detected
Get 6 routine hepatoma cell line and 22 routine clinical liver cancer tissue samples, detected the expression of SIRT1 and SOX2 by Western blot, then calculate Person value, result as shown in Figure 5.Result shows, and in 6 routine hepatoma cell line, the dependency of SIRT1 and SOX2 and Person value are 0.708; In 22 routine clinical hepatocarcinoma samples, its Person value is 0.825, shows that SIRT1 and Sox2 has positive correlation.The above results shows in liver-cancer stem cell, and SIRT1 can by activating the expression of SOX2 and then maintaining liver-cancer stem cell self renewal.
Embodiment 2
One, in liver-cancer stem cell, disturb SIRT1 to cross SOX2 to Clone formation, balling-up and the impact becoming tumor ability simultaneously
The pivotal role in liver-cancer stem cell self renewal process is regulated and controled, by the Huh7Nanog of sorting at SOX2 at SIRT1 for clear and definite
poswhile infecting slow virus Lv-sh-SIRT1, transfection Lv-Over-SOX2 slow virus (its preparation method is identical with slow virus Lv-sh-SIRT1), after combined effect 24h, 48h, 72h, detect the expression of SIRT1 and SOX2 albumen, then by the expression of SIRT1 and SOX2 albumen after Immunofluorescence test combined effect 48h, result as shown in Figure 6.As shown in Figure 6, disturbing SIRT1 while during process LAN SOX2 combined effect 48h, the protein expression of SOX2 is in the trend increased.
Then after Lv-sh-SIRT1 slow virus and Lv-Over-SOX2 slow virus combined effect 72h, observe clonality and the balling-up ability of liver-cancer stem cell, result as shown in Figure 7.Result shows, and compared with matched group, at interference SIRT1 process LAN SOX2 simultaneously, its Clone formation (p=0.0009) and balling-up are formed and manifest outstanding property increase (p=0.0054), have significant difference.
Two, disturb in liver-cancer stem cell SIRT1 simultaneously process LAN SOX2 transplanted tumor is become to the impact of tumor ability
Carry out subcutaneously injecting 1 × 10 respectively in NOD/SCID Mus body
6control(transfection slow virus of quantity, containing expression plasmid), shSIRT1(disturb SIRT1) and shSIRT+SOX2(disturb SIRT1 process LAN SOX2 simultaneously) PLC/PRF/5Nanog masculine liver cancer stem cell, often only inject 1 × 10
6cell number, point 10 some injections, after surrounding, put to death mice, peel off tumor, observe it and become tumor situation, then add up the weight and volume of tumor, result as shown in Figure 8.Result shows, and it is 100%(10/10 that control forms ratio of outflow), it is 60.0%(6/10 that shSIRT1 forms ratio of outflow), it is 83% (10/12) that shSIRT+SOX2 forms ratio of outflow; Compare shSIRT1 group, its transplanted tumor tumor weight of shSIRT+SOX2 group moves tumor volume in significantly rising with planting.Then transplanted tumor paraffin embedding is cut into slices, after immunohistochemical staining, use confocal laser scanning microscope.Concrete grammar is: by fixing NOD/SCID Mus subcutaneous transplantation tumor, dewater and use paraffin embedding, H & E dyes and serial section, often open 4-5 μm thick, sheet is mounted with SABC slide, room temperature is placed for subsequent use, then by the roasting sheet instrument baking section 20min of section, put into dimethylbenzene, dewaxing 10-20min, be 70%-100% graded ethanol aquation by volume fraction again, distilled water rinsing is cut into slices 3 times, each 5min, then section to be placed in by 41mL citric acid and 9mL sodium citrate and the pH of standardize solution most 500mL is the liquor sodii citratis of 6.0, under 800W condition after Microwave method 3.30min to solution boiling, under 150W condition, 16min maintains fluidized state again, to cold water, room temperature is cooled to after reparation, distilled water rinsing is cut into slices 3 times, each 5min, then the H of mass fraction 3% is dripped by 50 μ L/ sheets
2o
2solution, hatches 20min for 37 DEG C, after distilled water rinsing 5min, then cuts into slices 2 times with PBS rinsing, each 5min, with dripping 1:300(v/v) the anti-SIRT1 antibody of rabbit that dilutes, 4 DEG C of overnight incubation, taking-up next day PBS rinsing is cut into slices 3 times, each 5min, then drips EnVision by 50 μ L/ sheets
tMtwo resist, and hatch 30min for 37 DEG C, PBS rinsing is cut into slices 3 times, each 5min, DAB nitrite ion is dripped, tap water cessation reaction, then with haematoxylin lining dye 45s after rinsing, hydrochloride alcohol breaks up, tap water, oil blackeite, finally use volume fraction 70%-100% gradient alcohol dehydration, dimethylbenzene is transparent, resinene mounting, result as shown in Figure 9.Result shows, and compares shSIRT1 group, and the GFP of process LAN SOX2 group expresses and strengthens in significance, and immunohistochemical method shows that process LAN SOX2 can increase the expression of SOX2 in transplanted tumor equally.The display of above result, no matter in vivo or external, process LAN SOX2 not only can recover Clone formation, balling-up ability to liver-cancer stem cell after interference SIRT1, and can increase into tumor ability.Therefore, what stem cell transcription factor SOX2 maintained liver-cancer stem cell self renewal at SIRT1 plays crucial effect on.
Embodiment 3
One, disturb in liver-cancer stem cell and transplanted tumor SIRT1 simultaneously process LAN SOX2 affect the expression of Nanog
Find in the research of embodiment 1, interference SIRT1 can not only lower the expression of SOX2, and can lower the expression of Nanog.In order to study SIRT1 Nanog role in maintenance liver-cancer stem cell self renewal process further, first can recover Nanog detect interference SIRT1 process LAN SOX2 in liver-cancer stem cell after to express, be specially, control(transfection slow virus, containing expression plasmid), shSIRT1(disturb SIRT1) and shSIRT+SOX2(disturbs SIRT1 while process LAN SOX2) PLC/PRF/5Nanog and Huh7Nanog
postransfection in masculine liver cancer stem cell, be internal reference with GAPD, result is as shown in A in Figure 10.Result shows, and interference SIRT1 while, process LAN SOX2 significantly can promote the up-regulated of GFP.The liver-cancer stem cell of process LAN SOX2 while of interference SIRT1 is migrated to NOD/SCID mice subcutaneous, every only injection 1 × 10
6cell number, point 10 some injections, after surrounding, put to death mice, then detect the expression of GFP in transplanted tumor, and be internal reference with GAPD, structure is as shown in B in Figure 10.Result shows, and in the NOD/SCID mice-transplanted tumor tissue of interference SIRT1 simultaneously process LAN SOX2, detection also demonstrate that external result, namely facilitates the expression of Nanog or GFP.These results show that, in liver-cancer stem cell, Nanog maintains liver-cancer stem cell self renewal at SIRT1 and occupies critical role equally on.
Two, at liver-cancer stem cell, interference SIRT1 simultaneously process LAN SOX2 affects Nanog promoter activity
Multipotency transcription factor Oct4, SOX2 with Nanog are the important transcription factor regulating the series of genes relevant with stem cell to express.In order to study the effect of Nanog at SIRT1 maintenance liver-cancer stem cell self renewal further, by the Nanog of selected by flow cytometry apoptosis
poscell seeding, in 6 orifice plates, treats that cell grows to about 60%, infects Lv-shRNA-SIRT1 and Lv-Over-SOX2 respectively, then at 37 DEG C, 5%CO
2incubator continue cultivate 36h, digestion, centrifuge cell, and be planted in 24 orifice plates cultivate, transfection in second day.Transfection method for add PBS successively in cell, and luciferase reporter gene carrier pGL31.0 μ g, internal reference plasmid Renillia0.5 μ g, amounts to 50 μ L, then in 37 DEG C, 5%CO
2continue in incubator to cultivate, use Dual-luciferase reporter gene detection system (purchased from American Promega company) examining report gene activity after transfection 48h and detect Nanog promoter activity, detection method is carried out see description, and testing result as shown in figure 11.Result shows, and when disturbing SIRT1 while when process LAN SOX2, uciferase activity strengthens, and shows that Nanog promoter activity strengthens.Then Nanog primer is designed, chromatin immune co-precipitation (ChIP) technology for detection (Promega company of the test kit U.S.) SOX2 is utilized to be attached to the ability of Nanog promoter region, detection method is undertaken by test kit description, and the concrete sequence of primer is: Nanog1: forward primer: gggatagacaagaaaccaaac(SEQ ID NO.1); Downstream primer: caactagctccattttcctc(SEQ ID NO.2); Nanog2: forward primer: cggcctcccaatttactg(SEQ ID NO.3); Downstream primer: tctaggttcaccacgtttc(SEQ ID NO.4); Nanog3: forward primer: tggaaacgtggtgaacctag(SEQ ID NO.5); Downstream primer: agtctcaccaaggccattg(SEQ ID NO.6)
Then interpretation of result is carried out,
(1) amount (Normalize DNA) of first standardization DNA
△Ct
[normalized CHIP]=Ct
[ChIP]-(Ct
[Input]-Lg(2×Input Diluttion Factor))
Input Dilution Factor=(fraction of the input control chromation saved)
-1
(2) DNA fragmentation calculated in ChIP sample accounts for the percentage ratio % in Input Control
Input=2
（-△Ct[normalized CHIP]）
Analysis result as shown in figure 12.Result shows, and the expression of interference SIRT1 can reduce the ability that SOX2 is attached to Nanog promoter region; Correspondingly significantly can strengthen the ability that SOX2 is attached to Nanog promoter region after process LAN SOX2.
Above result shows, in liver-cancer stem cell, SIRT1 expresses by maintaining SOX2 thus strengthens its transcriptional control to Nanog, and then maintains the characteristic of liver-cancer stem cell self renewal.But how SIRT1 regulates the expression of SOX2 and molecular mechanism thereof still unclear in liver-cancer stem cell.
What finally illustrate is, above preferred embodiment is only in order to illustrate technical scheme of the present invention and unrestricted, although by above preferred embodiment to invention has been detailed description, but those skilled in the art are to be understood that, various change can be made to it in the form and details, and not depart from claims of the present invention limited range.
Claims (6)
1. disturb SIRT1 to express the application of reagent in the reagent of preparation suppression liver-cancer stem cell dryness transcription factor expression.
2. application according to claim 1, is characterized in that: described interference SIRT1 expresses the slow virus or Tenovin-6 that reagent is interference SIRT1 expression.
3. application according to claim 2, is characterized in that: the concentration of described Tenovin-6 is 2.5 ~ 10 μMs.
4. application according to claim 2, is characterized in that: the slow virus that described interference SIRT1 expresses by after Lv-sh-SIRT1 plasmid transfection 293T cell, through packaging, purification and obtaining.
5. the application according to any one of claim 1-4, is characterized in that: described dryness transcription factor is SOX2, Oct4, c-Myc or Nanog.
6. application according to claim 5, is characterized in that: described dryness transcription factor is SOX2 or Nanog.
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