CN104762375B - Applications of the POU5F1B in diagnosing tumor, treatment, prognosis and prediction recurrence - Google Patents
Applications of the POU5F1B in diagnosing tumor, treatment, prognosis and prediction recurrence Download PDFInfo
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Abstract
The invention discloses applications of the POU5F1B in diagnosing tumor, treatment, prognosis and prediction recurrence.Present invention discover that the copy number of gene POU5F1B, the transfer of expression and tumour, recurrence, patient's prognosis and tumour cell dryness are closely related in kinds of tumors.It finds that high expression POU5F1B can promote the formation of TIC to enhance the internal one-tenth knurl ability of esophageal cancer cell by studying, reduces survival rate.And by inhibiting the expression of POU5F1B, can effectively inhibit the formation of TIC reduces the internal one-tenth knurl ability of esophageal cancer cell, improves survival rate.It can be seen that inhibiting endogenous POU5F1B that can significantly reduce the internal one-tenth knurl ability of esophageal cancer cell, improve survival, illustrate that POU5F1B can be as the marker of diagnosis, treatment, prognosis and prediction recurrence.POU5F1B inhibitor great clinical value in the pharmaceutical composition for preparing treatment tumour, effective treatment for tumour provide new drug and method.
Description
Technical field
The present invention relates to applications of the POU5F1B in diagnosing tumor, treatment, prognosis and prediction recurrence.
Background technology
The cancer of the esophagus is one of common malignant tumour, and incidence occupies the 8th of malignant tumour, and the death rate is in the 6th
Position.The Incidence of esophageal cancer in China is high, accounts for about the whole world 50%, and China has become the whole world to suffer from mortality rate of esophageal cancer highest
Country.
At present, mainly there are operative treatment, radiotherapy and chemotherapy etc. for the therapy of the cancer of the esophagus, but patient
Outcome is still poor, and 75% patient is dead in 1 year after receiving to treat, and the life span of only 5~10% patient can
Reach 5 years.
The discovery of tumor markers and rationally application are the premises of tumor recurrence prediction.So far, world projection early stage
The marker of recurrence and the bad cancer of the esophagus of prognosis is still immature, for new diagnosis of esophageal cancer patient, understands progression of disease wind
The tool of danger and guide therapeutic process is also very limited.Therefore, the research of detection recurrence of Esophageal Carcinoma marker is own through becoming the cancer of the esophagus
The treatment and prevention of tumour of this molecule and Clinical heterogeneity with height is badly in need of the important scientific problems solved.
Invention content
The purpose of the present invention is to provide a kind of lesion detection kits.
Another object of the present invention is to provide a kind of prognosis kit of tumour.
It is still another object of the present invention to provide a kind of recurrence prediction kits of tumour.
The technical solution used in the present invention is：
A kind of lesion detection kit, containing the reagent that detects gene POU5F1B copy numbers can be quantified in the kit.
Further, the reagent that can quantify detection gene POU5F1B expressions is contained in mentioned reagent box.
Further, the primer sequence containing real time fluorescent quantitative detection gene POU5F1B transcriptional levels in mentioned reagent box
Row.
Further, the primer sequence containing real time fluorescent quantitative detection gene POU5F1B transcriptional levels in mentioned reagent box
Row：Sense primer 5'-GCCATACGGTCACAGAGCTT-3'（SEQ ID NO：1）With downstream primer 5'-
GGAAGCTTAGCCAGGTCAGA-3'（SEQ ID NO：2）.
A kind of prognosis kit of tumour, containing the examination that detects gene POU5F1B copy numbers can be quantified in the kit
Agent.
Further, the reagent that can quantify detection gene POU5F1B expressions is contained in mentioned reagent box.
Further, the reagent that can quantify detection gene POU5F1B copy numbers is contained in mentioned reagent box.
The recurrence prediction kit of a kind of tumour, containing detection gene POU5F1B expression water can be quantified in the kit
Flat reagent.
A kind of oncotherapy medicament, the reagent containing suppressor POU5F1B expression in the medicament.
Further, the reagent of above-mentioned suppressor POU5F1B expression is selected from the reagent that can make gene POU5F1B silences.
The beneficial effects of the invention are as follows：
Significantly expand in the kinds of tumors such as cancer of the esophagus, breast cancer, liver cancer and oophoroma present invention finds POU5F1B
Increase.It is not perfect currently used for clinical tumor aided diagnosis technique, and POU5F1B compared with normal structure is apparent in tumour
Amplification, this prompting POU5F1B has the potential as tumour auxiliary diagnosis marker.
The present invention is had found by a series of analysis methods：The amplification of POU5F1B and tumour shifts, is multiple in kinds of tumors
Hair, patient's prognosis and tumour cell dryness are closely related.It is tested and found by a series of in vivo functionalities：Height expression POU5F1B can
To promote the formation of TIC and then enhance the internal one-tenth knurl ability of esophageal cancer cell, the survival rate of tumor-free mice is reduced.Prompting
POU5F1B can be as the target spot of esophageal carcinoma therapy.And by inhibiting expressions of the POU5F1B in cancer cell and tissue, it can
The internal one-tenth knurl ability of esophageal cancer cell is reduced with the formation for effectively inhibiting TIC, improves the survival rate of tumor-free mice.Above experiment
Data suffice to show that the internal one-tenth knurl ability for inhibiting endogenous POU5F1B that can significantly reduce esophageal cancer cell, improve patient's life
Rate is deposited, this has prompted the potential of marker that POU5F1B has as diagnosis, treatment, prognosis and prediction recurrence.POU5F1B presses down
Preparation great clinical value in the pharmaceutical composition for preparing treatment tumour, effective treatment for tumour provide new medicine
Object and method.
Description of the drawings
Fig. 1 is that the human genome obtained by the way that the data in Tumorscape databases are carried out with analysis swells at 3138
AFLP system in knurl sample, 2526 tumor tissues and 611 tumor cell lines；
Fig. 2 is that by GISTIC 2.0 data in Tumorscape databases are carried out with the human genome that analysis obtains
AFLP system in 3138 tumor samples, 2526 tumor tissues and 611 tumor cell lines；
Fig. 3 is by analyze the human genome obtained in 5443 primary tumo(u)rs to the data in TCGA databases
AFLP system in tissue, 393 transfer tissues and 63 recurrence tissues；
Fig. 4 is analyze the human genome obtained 5443 to the data in TCGA databases by GISTIC 2.0
AFLP system in example primary tumor tissue, 393 transfer tissues and 63 recurrence tissues；
Fig. 5 is the distribution collection of illustrative plates of gene in 8q24.21 sections；
Fig. 6 is：FAM84B, PCAT1, POU5F1B, MYC, PVT1, GSDMC, FAM49B on 8q24.21 sections and
The increased sample of copy number of ASAP1 genes 5443 primary tumor tissues in TCGA databases, 393 transfer groups respectively
It knits and ratio shared in 63 recurrence tissues；
Fig. 7 is shown in the amplification of POU5F1B copy numbers in 9297 tumor samples in TCGA databases
（Amplification）And increase（Gain）With the overall survival of tumor patient in significantly negatively correlated；
Fig. 8 is shown in the amplification of POU5F1B copy numbers in 9297 tumor samples in TCGA databases
（Amplification）With patient's disease-free survival rate in significantly negatively correlated, POU5F1B copy numbers amplification（Amplification）
Tumour patient and POU5F1B copy numbers increase（Gain）Tumour patient disease-free survival rate have significant difference；
Fig. 9 shows ratio of the amplification of POU5F1B in specific tumors type；
Figure 10, which is shown, passes through FISH（Fluorescence in situ hybridization）Confirm tissues of the POU5F1B in the cancer of the esophagus, breast cancer and liver cancer
It is expanded in cell line；
Figure 11 shows, Real-Time PCR analysis results show POU5F1B in 3 kinds of tumor tissues, including the cancer of the esophagus, breast
It is expanded in gland cancer and liver cancer；
Kaplan-Meier survivorship curves are shown in Figure 12, and the amplification of POU5F1B copy numbers and patient with esophageal carcinoma are without diease occurrence
Rate is deposited in significantly negatively correlated；
Kaplan-Meier survivorship curves are shown in Figure 13, and the amplification of POU5F1B copy numbers and patient with breast cancer are disease-free
Survival rate is in significantly negatively correlated；
Kaplan-Meier survivorship curves are shown in Figure 14, amplification and the liver cancer patient disease-free survival of POU5F1B copy numbers
Rate is in significantly negatively correlated；
As a result Figure 15 shows POU5F1B's to analyze the POU5F1B mRNA level in-sites of general cancer in TCGA databases
Amplification promotes the up-regulation of POU5F1B mRNA expression；
Figure 16 is shown through Gene Set Enrichment Analysis (GSEA) analysis methods to cancer general in TCGA
The correlation of POU5F1B copy number variation and the relevant gene set of tumor recurrence is analyzed, as a result；
Figure 17 is shown through Gene Set Enrichment Analysis (GSEA) analysis methods to cancer general in TCGA
The correlation of POU5F1B copy number variation and the relevant gene set of tumor prognosis is analyzed, the results showed that POU5F1B is all
Amplification and the relevant gene set of prognosis in the tumour of type is negatively correlated；
Figure 18 is shown through Gene Set Enrichment Analysis (GSEA) analysis methods to cancer general in TCGA
The correlation of POU5F1B copy number variation and the relevant gene set of tumour cell dryness is analyzed, the results showed that POU5F1B exists
Amplification in all types of tumours is proportionate with the relevant gene set of tumour cell dryness；
The mouse of Figure 19 inoculation Eca 9706-POU5F1B is easier long swell than being inoculated with the mouse of Eca 9706-vector
Knurl；
Figure 20 shows that height expression POU5F1B dramatically increases the tumour initiator cell in the cancer of the esophagus（TIC, tumor
initiated cells）；
Figure 21 displays inoculation Eca 9706-shPOU5F1B1#2 and the mouse ratio for being inoculated with Eca 9706-sh POU5F1B1#3
Inoculation Eca 9706-Control mouse are easier bearing tumor；
Figure 22 shows that silence POU5F1B dramatically increases the tumour initiator cell in the cancer of the esophagus（TIC, tumor
initiated cells）；
The high expression that Figure 23 is shown in POU5F1B in 16 mouse is significantly negatively correlated with tumor-free mice survival rate；At 16
The low expression of POU5F1B and the notable positive correlation of tumor-free mice survival rate in mouse；
Figure 24 shows, mouse tumor recurrence model the experimental results showed that POU5F1B high expressing promotings into the cancer of the esophagus recurrence
POU5F1B high expressing promotings are into the recurrence of the cancer of the esophagus.
Specific embodiment
A kind of lesion detection kit, containing the reagent that detects gene POU5F1B copy numbers can be quantified in the kit.
Preferably, the reagent containing quantitatively detection gene POU5F1B copy numbers in mentioned reagent box is sense primer 5'-
GCCATACGGTCACAGAGCTT-3'（SEQ ID NO：1）With downstream primer 5'- GGAAGCTTAGCCAGGTCAGA-3'（SEQ
ID NO：2）.
A kind of lesion detection kit, containing the examination that detects gene POU5F1B expressions can be quantified in the kit
Agent.
Preferably, the primer sequence containing real time fluorescent quantitative detection gene POU5F1B transcriptional levels in mentioned reagent box.
Preferably, the primer sequence containing real time fluorescent quantitative detection gene POU5F1B transcriptional levels in mentioned reagent box：
Sense primer 5'-GCCATACGGTCACAGAGCTT-3'（SEQ ID NO：1）With downstream primer 5'-
GGAAGCTTAGCCAGGTCAGA-3'（SEQ ID NO：2）.
A kind of lesion detection kit, containing the reagent that detects gene POU5F1B copy numbers can be quantified in the kit,
And contain the reagent that can quantify detection gene POU5F1B expressions.
A kind of prognosis kit of tumour, containing the examination that detects gene POU5F1B copy numbers can be quantified in the kit
Agent.
Preferably, the reagent that can quantify detection gene POU5F1B expressions is contained in mentioned reagent box.
Preferably, the reagent that can quantify detection gene POU5F1B copy numbers is contained in mentioned reagent box.
The recurrence prediction kit of a kind of tumour, containing detection gene POU5F1B expression water can be quantified in the kit
Flat reagent.
A kind of oncotherapy medicament, the reagent containing suppressor POU5F1B expression in the medicament.
Preferably, the reagent of above-mentioned suppressor POU5F1B expression is selected from the reagent that can make gene POU5F1B silences.
Preferably, the reagent of above-mentioned silence POU5F1B is the shPOU5F1B with hairpin structure, and sequence is：5’-
CGGAGAAGTCCCAGGACAT-3’ （SEQ ID NO：3）.
With reference to specific embodiment, content that the present invention is further explained.Actual conditions are not specified in the following example
Experimental method, usually according to normal condition, such as《Molecular Cloning:A Laboratory guide》Condition described in (third edition) or according to system
Make the condition proposed by manufacturer.
Embodiment 1：8q24.21 is the hot spot of amplification in Oncogenome
1. Tumorscape database analysis
Method：To 3138 tumor samples in Tumorscape databases（2526 tumor tissues being subsequently noted add
611 tumor cell lines）, the genome copy numbers variation data in 2526 tumor tissues and 611 tumor cell lines carry out
Analysis.By certain increased sample of chromosome segment copy number in 3138 tumor samples, 2526 tumor tissues and 611 tumours
Shared percentage is presented using circos softwares in the form of circle in cell line, and outmost circle is chromogene
Group, different chromosome are showed with different colors, and a locus is represented per a line in every chromosome.The circle of the inside 3
Represent respectively from outside to inside certain increased sample of chromosome segment copy number 3138 tumor samples, 2526 tumor tissues and
Shared percentage in 611 tumor cell lines.Copy number amplification degree can be divided into Gain and Amplification, orange generation
The copy number of table Gain, i.e. low degree increase（The additional copy for increasing by 1 to 4）；Vermilion represents Amplification, i.e.,
It is additional to increase more than 4 copies.By some of the center of circle and chromosome section lines, the increasing of certain chromosome segment copy number can be obtained
The sample added percentage shared in 3138 tumor samples, 2526 tumor tissues and 611 tumor cell lines.
As a result：As shown in Figure 1, compared with other chromosomes, the gene in chromosome arm 1q, 5p, 7,8q, 17q and 20 exists
The frequency highest expanded in tumour.
Interpretation of result：More than analysis result shows compared with other chromosomes, chromosome arm 1q, 5p, 7,8q, 17q and
3138 tumor samples, 2526 tumor tissues and 611 tumour cells of the gene in Tumorscape databases in 20
Frequency highest is expanded in system, prompts certain genes in chromosome arm 1q, 5p, 7,8q, 17q and 20 that can swell as auxiliary diagnosis
The target spot of knurl.
2. GISTIC is analyzed
Method：Utilize Genomic Identification of Significant Targets in Cancer 2.0
(GISTIC2.0) to 3138 tumor samples, 2526 tumor tissues and 611 tumor cell lines in Tumorscape databases
In genome copy numbers variation data analyzed.G-scores represents the degree of gene magnification.G-scores is bigger, table
Show that the degree of gene magnification is higher.Vice versa.Q-values is false discovery rate（False positive rate）.
As a result：As shown in Fig. 2, 8q24.21 sections are in 3138 tumor samples, 2526 tumor tissues and 611 tumours
There is highest level amplification in cell line.
Interpretation of result：8q24.21 sections are in 3138 tumor samples, 2526 tumor tissues and 611 tumor cell lines
In have a highest level amplification, prompt some gene in 8q24.21 sections can be as the target spot of auxiliary diagnosis tumour.
3. TCGA database analysis
Method：To in 5443 primary tumor tissues, 393 transfer tissues and 63 recurrence tissues in TCGA databases
Genome copy numbers variation data analyzed.By certain increased sample of chromosome segment copy number in 5443 primary tumo(u)rs
Shared percentage is presented using circos softwares in the form of circle in tissue, 393 transfer tissues and 63 recurrence tissues,
Outmost circle is DNA sequence, and different chromosome is showed with different colors, per a line generation in every chromosome
One locus of table.The circle of the inside 3 represents certain increased sample of chromosome segment copy number at 5443 respectively from outside to inside
Shared percentage in primary tumor tissue, 393 transfer tissues and 63 recurrence tissues.Copy number amplification degree can be divided into
Gain and Amplification, orange to represent Gain, i.e. the copy number of low degree increases（The additional copy for increasing by 1 to 4）；
Vermilion represents Amplification, i.e., additional to increase more than 4 copies.By some of the center of circle and chromosome section lines, just
Certain increased sample of chromosome segment copy number be can obtain in 3138 tumor samples, 2526 tumor tissues and 611 tumours
Shared percentage in cell line.
As a result：As shown in figure 3, compared with other chromosomes, the gene in chromosome arm 1q, 5p, 7,8q, 17q and 20 exists
The frequency highest expanded in tumour.
Interpretation of result：More than analysis result shows compared with other chromosomes, chromosome arm 1q, 5p, 7,8q, 17q and
Expand in 5443 primary tumor tissues in TCGA databases of gene in 20,393 transfer tissues and 63 recurrence tissues
Increase frequency highest, prompt certain genes in chromosome arm 1q, 5p, 7,8q, 17q and 20 can be as the target of auxiliary diagnosis tumour
Point.
4. GISTIC is analyzed
Method：Utilize Genomic Identification of Significant Targets in Cancer 2.0
(GISTIC2.0) to 5443 primary tumor tissues, 393 transfer tissues and 63 recurrence tissues in Tumorscape databases
In genome copy numbers variation data analyzed.G-scores represents the degree of gene magnification.G-scores is bigger, table
Show that the degree of gene magnification is higher.Vice versa.Q-values is false discovery rate（False positive rate）.
As a result：As shown in figure 4,8q24.21 sections are multiple in 5443 primary tumor tissues, 393 transfer tissues and 63
There is highest level amplification in hair tissue.
Interpretation of result：8q24.21 sections are in 5443 primary tumor tissues, 393 transfer tissues and 63 recurrence tissues
In have a highest level amplification, prompt some gene in 8q24.21 sections can be as the target spot of auxiliary diagnosis tumour.
Embodiment 2：POU5F1B is located at the amplified peak of 8q24.21, and related to tumor patient prognosis
1. the distribution of gene on No. 8 chromosomes
Method：Analysis result before is prompted, some gene in 8q24.21 sections can be as the target of auxiliary diagnosis tumour
Point, in being to look for which gene distribution on 8q24.21 sections.
As a result：Have on 8q24.21 sections FAM84B, PCAT1, POU5F1B, MYC, PVT1, GSDMC, FAM49B and
ASAP1。
2. TCGA database analysis is the result shows that POU5F1B expands degree highest in 8q24.21 sections
Method：Fig. 6 is substantially a part of Fig. 3.
As a result：As shown in fig. 6, POU5F1B is the amplification highest gene of degree in 8q24.21 sections, in TGCA data
The amplification frequency of POU5F1B is than cancer base in 5443 primary tumor tissues, 393 transfer tissues and 63 recurrence tissues in library
Because of MYC high.
Interpretation of result：There is document report to cross 8q24.21 to expand in kinds of tumors, this section contains known oncogene
MYC.POU5F1B is the amplification highest gene of degree in 8q24.21 sections, the amplification frequency of POU5F1B in TGCA databases
Rate is than MYC high.By the gene database of NCBI it is found that POU5F1 plays pass in embryonic development and stem cell totipotency
The effect of key.And POU5F1B is the pseudogene of POU5F1, can encode functional albumen, this albumen and POU5F1 transcribe because
Sub- length is almost the same and height is similar, and the regulation and control for prompting POU5F1B that may be expressed with participating in POU5F1 are played with POU5F1
Function it is related.
3. TCGA database analysis shows that the overall survival of POU5F1B amplification degree and tumour patient is in significantly negatively correlated
Method：All statistical analysis are handled with SPSS17.0 statistical softwares.It is painted using Kaplan-Meier methods
Survival analysis curve processed, and its statistical significance is detected using the log-rank methods of inspection.Test coefficient P<0.05 thinks uniting
Meter learns upper significant difference.With the amplification of SPSS statistical softwares analysis POU5F1B copy numbers and tumor patient overall survival
Relationship.
As a result：As shown in fig. 7, POU5F1B copy numbers expand（Amplification）Tumour patient copied than POU5F1B
The overall survival of the normal tumour patient of shellfish number significantly reduces；POU5F1B copy numbers increase（Gain）Tumour patient ratio
The overall survival of the normal tumour patient of POU5F1B copy numbers significantly reduces；POU5F1B copy numbers expand
（Amplification）Tumour patient and POU5F1B copy numbers increase（Gain）Tumour patient overall survival without apparent
Difference.The amplification degree of wherein POU5F1B copy numbers can be divided into Gain and Amplification.Gain's, i.e. low degree
Copy number increases, and copy number additionally increases by 1 to 4；Amplification, i.e. copy number additionally increase by 4 or more.
Interpretation of result：SPSS statistic analysis results show the amplification of POU5F1B copy numbers（Amplification）And increase
（Gain）With the overall survival of patient in significantly negatively correlated（P<0.001）, POU5F1B copy number increases are explicitly indicated poor
Prognosis.Therefore, POU5F1B can be as the potential index of patient's prognosis.
4. TCGA database analysis shows POU5F1B amplification degree and overall survival, the disease-free survival rate of tumour patient
In notable negative correlation
Method：All statistical analysis are handled with SPSS17.0 statistical softwares.It is painted using Kaplan-Meier methods
Survival analysis curve processed, and its statistical significance is detected using the log-rank methods of inspection.Test coefficient P<0.05 thinks uniting
Meter learns upper significant difference.With the amplification of SPSS statistical softwares analysis POU5F1B copy numbers and tumor patient disease-free survival
The relationship of rate.
As a result：As shown in figure 8, POU5F1B copy numbers expand（Amplification）Tumour patient copied than POU5F1B
The disease-free survival rate of the normal tumour patient of shellfish number significantly reduces（P<0.001）；POU5F1B copy numbers increase（Gain）It is swollen
Knurl patient reduces than the disease-free survival rate of the normal tumour patient of POU5F1B copy number；POU5F1B copy numbers expand
（Amplification）Tumour patient and POU5F1B copy numbers increase（Gain）The disease-free survival rate of tumour patient have
Apparent sex differernce（P<0.001）.The amplification degree of wherein POU5F1B copy numbers can be divided into Gain and Amplification.
The copy number of Gain, i.e. low degree increase, and copy number additionally increases by 1 to 4；Amplification, i.e. copy number additionally increase
Add 4 or more.
Interpretation of result：SPSS statistic analysis results show the amplification of POU5F1B copy numbers（Amplification）With patient
Disease-free survival rate in significantly negatively correlated（P<0.001）, poor prognosis is explicitly indicated in POU5F1B copy number increases.Therefore,
POU5F1B can be as the potential index of patient's prognosis.
5. POU5F1B is highly expanded in feminine proses, digestive system tumor and tumor in respiratory system
Method：In order to further explore whether POU5F1B expands in specific tumors, to 10700 in TCGA databases
The CNV data of various tumor tissues are analyzed.Classify to 10700 tumor tissues according to system, classification is respectively female
Sexual reproduction system tumor, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm are secreted
Urethral system tumour, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.It counts in the swollen of these classifications
In knurl, the POU5F1B in how many tumour is amplification（Amplification and Gain）.To 10700 tumor tissues by swollen
Knurl specific type is classified, and classification is respectively the specific tumor type such as OV, ESCA, UVM, counts the tumour in these classifications
In, the POU5F1B in how many tumour is amplification（Amplification and Gain）.
As a result：As shown in figure 9,1 ~ 9 represents the tumor type classified according to system.POU5F1B Gain light reds
It represents, POU5F1B Amplification are represented with red.It is shown in figure, detects that POU5F1B is expanded（Including
Amplification and Gain）Tumor sample feminine proses, digestive system tumor and exhaled in TCGA databases
Shared ratio is more than 50% in desorption system tumor sample.Shown on the right side of Fig. 9, POU5F1B is at 14 kinds of the general cancer data sets of TCGA
It is significantly expanded in tumour.The ratio shared in certain specific tumors in the tumour for detecting POU5F1B Amplification is seen
Descending sort is carried out, preceding 5 kinds of tumours are ovarian serous cystadenocarcinoma (OV) respectively,
Esophageal carcinoma (ESCA), uveal melanoma (UVM), breast invasive carcinoma
(BRCA), and liver hepatocellular carcinoma (LIHC).
Interpretation of result：POU5F1B highly expands in feminine proses, digestive system tumor and tumor in respiratory system
Increase, in ovarian serous cystadenocarcinoma (OV), esophageal carcinoma (ESCA),
Uveal melanoma (UVM), breast invasive carcinoma (BRCA), and liver
Height expands in hepatocellular carcinoma (LIHC).Prompt POU5F1B amplification may with specific tumors type,
Such as ovarian serous cystadenocarcinoma (OV), esophageal carcinoma (ESCA), uveal
Melanoma (UVM), breast invasive carcinoma (BRCA), and liver hepatocellular
Carcinoma (LIHC) is related.
6. pass through FISH（Fluorescence in situ hybridization）Confirm tissues and cell of the POU5F1B in the cancer of the esophagus, breast cancer and liver cancer
It is expanded in system
Method：
Probe synthesizes：
Position of the probe fragment in genome：Location:8q24.21, No. BAC：RP11-327N12 passes through ARES
546 DNA Labeling Kit synthesising probing needles of Alexa Fluor.
FISH（Fluorescence in situ hybridization）Step：
1. probe is denaturalized
Probe in 75 DEG C of waters bath with thermostatic control is incubated into 5 min, is set to 0 immediately DEG C, 5~10 min become double chain DNA probe
Property.
2. sample is denaturalized
（1）The chromosome slide sample prepared is baked into 2~3 h of piece in 50 DEG C of incubators.（The mark dyed through Giemsa
This need bakes piece again after fading in fixer in advance）.
（2）Slide sample is taken out, is immersed in 70~75 DEG C of the denaturing liquid of 70% formamide/2 of volume fraction × SSC
It is denaturalized 2~3 min.
（3）Immediately in order by sample through 100% ice ethanol series of volume fraction 70%, volume fraction 90% and volume fraction
Dehydration, 5 min, is then air-dried every time.
3. hybridization
It will be denaturalized or 10 μ L drops of the DNA probe of preannealing be on the slide sample for being denaturalized and being dehydrated, close the lid glass
Piece with Parafilm mountings, is placed in 37 DEG C of hybridized overnights in moist magazine（About 15~17 h）.Since hybridization solution is less, and
Hybridization temperature is higher, and the duration is again long, therefore for the moisture state for keeping sample, this process carries out in wet box.
4. elution
This step helps to remove the probe of non-specific binding, so as to reduce background.
（1）Hybridize next day, sample from 37 DEG C of incubators is taken out, is gently taken off coverslip with blade.
（2）The slide sample hybridized is positioned over to warmed-up 42~50 DEG C of 50% formamide/2 of volume fraction × SSC
Middle washing 3 times, every time 5 min.
（3）Wash 3 times in warmed-up 42~50 DEG C of 1 × SSC, every time 5 min.
（4）At room temperature, slide sample is rinsed in 2 × SSC.
（5）Slide is taken out, is spontaneously dried.
（6）Take 200 μ L counterstain solutions（PI/antifade or DAPI/antifade dye liquors）It is added dropwise on slide sample,
Covered.
5. the amplification of hybridization signal（Suitable for using the probe of biotin labeling）
（1）Add 150 μ L confining liquid I at the hybridization position of slide, covered with preservative film, 37 DEG C of incubation 20min.
（2）Remove preservative film, then 150 μ L avidin-FITC is added to be covered on sample with preservative film, 37 DEG C are continued temperature
Educate 40 min.
（3）Sample is taken out, puts it into and washs 3 times in warmed-up 42~50 DEG C of eluent, every time 5 min.
（4）Add 150 μ L confining liquid II at the hybridization position of slide sample, cover preservative film, 37 DEG C of 20 min of incubation.
（5）Remove preservative film, adding 150 μ L antiavidin, 37 DEG C incubate 40 on sample, covering new preservative film
min。
（6）Taking-up sample, puts it into warmed-up 42~50 DEG C of new eluent, washing 3 times, every time 5 min.
（7）Repeat step（1）、（2）、（3）, room temperature is cleaned in 2 × SSC.
（8）Slide is taken out, is spontaneously dried.
（9）200 μ L PI/antifade dye liquors is taken to be added dropwise on slide sample, covered.
6. mounting
Different types of mounting liquid can be used.If Mowiol is not contained in mounting liquid（It can generate mounting liquid self-enclosed
Effect）, to prevent the solution evaporation between cover plate and slide glass, nail polish can be used by cover plate periphery seal.The slide mark sealed
Originally it can be kept for the several months long in the magazine in -20~-70 DEG C of refrigerator.
7. fluorescence microscope FISH results
The probe of section where label POU5F1B sends out red fluorescence, the probe hair of No. 8 another end sections of chromosome of label
Go out green fluorescence.
As a result：The results are shown in Figure 10, and POU5F1B is significantly expanded in the cancer of the esophagus, breast cancer, liver cancer（It is all higher than being equal to
20 copies）, significantly expand in esophageal carcinoma cell line Kyse510, breast cancer cell line MDA-MB-231 and cancerous cell line HuH-7
Increase（It is all higher than being equal to 20 copies）, and its in peripheral blood mononuclear cells only there are two copy.As control, chromosome is another
Only there are two copy in all samples for end section.
Interpretation of result：POU5F1B is significantly expanded in the cancer of the esophagus, breast cancer, liver cancer, esophageal carcinoma cell line Kyse510,
It is significantly expanded in breast cancer cell line MDA-MB-231 and cancerous cell line HuH-7, prompts POU5F1B that can be used as auxiliary diagnosis tumour
Target spot.
7. Real-Time PCR detect copy numbers of the gene POU5F1B in the cancer of the esophagus, breast cancer and liver cancer
Method：By Real-Time PCR detect 20 esophageal tissues, 243 human esophageal carcinomas, 20 breast tissues,
POU5F1B copy numbers in 238 breast cancer tissues, 20 hepatic tissues and 226 liver cancer tissues.
All statistical analysis are handled with SPSS17.0 statistical softwares.Different comparison among groups examined with independent t (
Chang Bianliang), the comparison of mean is examined with t between two groups, the comparison χ of classified variable2 It examines, more comparison among groups single factor test sides
Difference is analysed.Numerical value is represented with mean ± SD.Test coefficient P<0.05 thinks statistically significant difference.With SPSS
Statistical analysis method compares in esophageal tissue and human esophageal carcinoma, breast tissue and breast cancer tissue, hepatic tissue and liver cancer tissue
The mean value of POU5F1B copy numbers.
As a result：As shown in figure 11, copy number belongs to normal for 2, i.e., the gene is neither expanded nor lacked.Copy number is more than
2 are defined as expanding.Real-Time PCR analysis shows that POU5F1B in three kinds of tumours, including in the cancer of the esophagus, breast cancer and liver cancer
Significantly amplification.
Interpretation of result：POU5F1B is in three kinds of tumours, including significantly being expanded in the cancer of the esophagus, breast cancer and liver cancer.Therefore, it uses
Primer containing specific binding POU5F1B（Sense primer 5'-GCCATACGGTCACAGAGCTT-3'（SEQ ID NO：1）；
Downstream primer 5'- GGAAGCTTAGCCAGGTCAGA-3'（SEQ ID NO：2））Auxiliary diagnostic box detect clinical sample
The amplification of POU5F1B in this can more be diagnosed as the tissue samples as tumor sample.The analysis of clinical sample is further prompted
POU5F1B can be as the target spot of auxiliary diagnosis.
8. the amplification of POU5F1B copy numbers is with patient with esophageal carcinoma disease-free survival rate in significantly negatively correlated
Method：All statistical analysis are handled with SPSS17.0 statistical softwares.It is painted using Kaplan-Meier methods
Survival analysis curve processed, and its statistical significance is detected using the log-rank methods of inspection.Test coefficient P<0.05 thinks uniting
Meter learns upper significant difference.Amplification and the cancer of the esophagus with SPSS statistical softwares analysis POU5F1B copy numbers（Esophageal
carcinoma）The relationship of patient's disease-free survival rate.
As a result：As shown in figure 12, red curve shows the disease-free of the patient with esophageal carcinoma of POU5F1B copy numbers amplification
Survival rate, blue curve show that POU5F1B copy numbers do not expand（I.e. copy number is normal）Patient with esophageal carcinoma without diease occurrence
Deposit rate.The disease-free survival rate of the patient with esophageal carcinoma patient with esophageal carcinoma more normal than POU5F1B copy number of POU5F1B copy numbers amplification
It significantly reduces（P=0.002）.
Interpretation of result：SPSS statistic analysis results show the amplification of POU5F1B copy numbers and patient with esophageal carcinoma disease-free survival
Rate is in significantly negatively correlated（P=0.002）, POU5F1B copy numbers amplification poor prognosis is explicitly indicated.Therefore, POU5F1B can make
Potential index for Esophagus Patients prognosis.
9. the amplification of POU5F1B copy numbers is with patient with breast cancer's disease-free survival rate in significantly negatively correlated
Method：All statistical analysis are handled with SPSS17.0 statistical softwares.It is painted using Kaplan-Meier methods
Survival analysis curve processed, and its statistical significance is detected using the log-rank methods of inspection.Test coefficient P<0.05 thinks uniting
Meter learns upper significant difference.Amplification and breast cancer with SPSS statistical softwares analysis POU5F1B copy numbers（Breast
carcinoma）The relationship of patient's disease-free survival rate.
As a result：As shown in figure 13, POU5F1B copy numbers expand 1 patient with breast cancer increased（Amp curves shown in red）Than
The normal patient with breast cancer of POU5F1B copy numbers（Not Amp curves shown in blue）Disease-free survival rate significantly reduce（P<
0.05）
Interpretation of result：SPSS statistic analysis results show the amplification of POU5F1B copy numbers and patient with breast cancer's disease-free survival
Rate is in significantly negatively correlated（P=0.020）, POU5F1B copy numbers amplification poor prognosis is explicitly indicated.Therefore, POU5F1B can make
Potential index for mammary gland patient's prognosis.
10. the amplification of POU5F1B copy numbers is with liver cancer patient disease-free survival rate in significantly negatively correlated
Method：All statistical analysis are handled with SPSS17.0 statistical softwares.It is painted using Kaplan-Meier methods
Survival analysis curve processed, and its statistical significance is detected using the log-rank methods of inspection.Test coefficient P<0.05 thinks uniting
Meter learns upper significant difference.Amplification and liver cancer with SPSS statistical softwares analysis POU5F1B copy numbers
（Hepatocellular carcinoma）The relationship of patient's disease-free survival rate.
As a result：As shown in figure 14, the liver cancer patient of POU5F1B copy numbers amplification（Amp curves shown in red）Than
The normal liver cancer patient of POU5F1B copy numbers（Not Amp curves shown in blue）Disease-free survival rate significantly reduce（P<
0.05）
Interpretation of result：SPSS statistic analysis results show the amplification of POU5F1B copy numbers and liver cancer patient disease-free survival rate
In notable negative correlation（P=0.005）, POU5F1B copy numbers amplification poor prognosis is explicitly indicated.Therefore, POU5F1B can conduct
The potential index of liver cancer patient prognosis.
Embodiment 3：The amplification of POU5F1B promotes the expression of POU5F1B mRNA, enhances the relevant characteristic of tumor stem cell
1. TCGA database analysis result shows that the amplification of POU5F1B promotes the up-regulation of POU5F1B mRNA expression
In order to explore effects of the POU5F1B played in tumour, to CNV (copy number variants) and RNA water
Flat correlation is analyzed.
Method：The general cancer data in TCGA are expanded degree according to POU5F1B in general cancer to classify, classification has diploid
（4415）、 Gain（2857）And Amplification（1014）.
All statistical analysis are handled with SPSS17.0 statistical softwares.Different comparison among groups examined with independent t (
Chang Bianliang), the comparison of mean is examined with t between two groups, the comparison χ of classified variable2It examines, more comparison among groups single factor test sides
Difference is analysed.Numerical value is represented with mean ± SD.Test coefficient P<0.05 thinks statistically significant difference.With SPSS
Statistical analysis method compares POU5F1B copy numbers for diploid（4415）、 Gain（2857）With（1014）General cancer in
The mean value of POU5F1B mRNA relative expression quantities.
As a result：As shown in figure 15, it is 2 with 4415 POU5F1B copy numbers（Diploid）General cancer in POU5F1B
MRNA level in-site is compared, the general cancer of 2857 POU5F1B Gain（Pan-cancers）In POU5F1B mRNA level in-sites it is notable on
It rises.Compared with the POU5F1B mRNA level in-sites in the general cancer that 4415 POU5F1B copy numbers are 2,1014 POU5F1B
POU5F1B mRNA level in-sites in the general cancer of Amplification significantly rise.With in the general cancer of 2857 POU5F1B Gain
POU5F1B mRNA level in-sites compare, the POU5F1B mRNA level in-sites in the general cancer of 1014 POU5F1B Amplification
Significantly rise.
Interpretation of result：CNVs (copy number variants) of the POU5F1B in tumour is with its mRNA level in-site in notable
Positive correlation, the amplification of POU5F1B promote the up-regulation of POU5F1B mRNA expression.Therefore, with containing can be detected with specific quantification
The primer of POU5F1B mRNA level in-sites（Sense primer 5'-GCCATACGGTCACAGAGCTT-3'（SEQ ID NO：1）；Draw in downstream
Object 5'- GGAAGCTTAGCCAGGTCAGA-3'（SEQ ID NO：2））Auxiliary diagnostic box detect in clinical sample
POU5F1B mRNA level in-sites are higher than normal sample, can more be diagnosed as the tissue samples as tumor sample.The analysis of clinical sample carries
Show that POU5F1B can be as the target spot of auxiliary diagnosis.
2. GSEA analysis results show amplifications of the POU5F1B in all types of tumours and the relevant base of tumor recurrence
Because collection is proportionate
Method：Classify to tumor tissues in TCGA according to system, classification is feminine proses, digestive system
Tumour, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, the urinary tract system tumor, nervous system swell
Knurl, blood-lymphatic system tumour and endocrine system carcinoma.More than tumour is divided into respectively according to the amplification degree of POU5F1B
POU5F1B amplification degree is high, low two class of POU5F1B amplifications degree.Pass through GSEA（Complete entitled gene set enrichment
Analysis, gene set enrichment analysis）Analysis method is to the POU5F1B amplification degree of tumour and Recurrence up
Signatures, Recurrence down signatures gene sets correlation analyzed.
As a result：As shown in figure 16,1 ~ 9 is respectively feminine proses, digestive system tumor, tumor in respiratory system man
Sexual reproduction system tumor, skin and soft tissue neoplasm, the urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour
And endocrine system carcinoma.Normalized Enrichment Score（NES）Score, NES zero-range set constants are enriched with for standardization
It is more remote, show that correlation is bigger.NES positive and negative values are for the direction of positive negative correlation.In Figure 16, if NES values are positive number, with red
It represents.NES value zero-range set constants are more remote, red deeper, represent that positive correlation is bigger.If NES values are negative, represented with blue.
NES value zero-range set constants are more remote, and blue is deeper, represent that negative correlation is bigger.The result shows that POU5F1B is in all types of tumours
Amplification be proportionate with the relevant gene set of tumor recurrence.
Interpretation of result：Amplifications of the POU5F1B in all types of tumours is in positive with the relevant gene set of tumor recurrence
It closes.POU5F1B can be as the potential index of prediction tumor recurrence.
It is negatively correlated that 3. GSEA analyzes amplifications and prognosis relevant gene set of the POU5F1B in all types of tumours
Method：Classify to tumor tissues in TCGA according to system, classification is feminine proses, digestive system
Tumour, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, the urinary tract system tumor, nervous system swell
Knurl, blood-lymphatic system tumour and endocrine system carcinoma.More than tumour is divided into respectively according to the amplification degree of POU5F1B
POU5F1B amplification degree is high, low two class of POU5F1B amplifications degree.Journey is expanded to the POU5F1B of tumour by GSEA analysis methods
It spends and is divided with the correlation of Prognosis poor signatures, Prognosis good signatures gene sets
Analysis.
As a result：As shown in figure 17,1 ~ 9 is respectively feminine proses, digestive system tumor, tumor in respiratory system man
Sexual reproduction system tumor, skin and soft tissue neoplasm, the urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour
And endocrine system carcinoma.Normalized Enrichment Score（NES）Score, NES zero-range set constants are enriched with for standardization
It is more remote, show that correlation is bigger.NES positive and negative values represent the direction of correlation.In Figure 17, if NES values are positive number, with red table
Show.NES value zero-range set constants are more remote, red deeper, represent that positive correlation is bigger.If NES values are negative, represented with blue.NES
It is more remote to be worth zero-range set constant, blue is deeper, represents that negative correlation is bigger.The result shows that POU5F1B is in all types of tumours
Amplification is proportionate with the relevant gene set of tumor recurrence.
Interpretation of result：Amplifications of the POU5F1B in all types of tumours is in negative with the relevant gene set of tumor prognosis
It closes.POU5F1B can be as the potential index of prediction tumor prognosis.
4. GSEA analysis results show that amplifications of the POU5F1B in all types of tumours is related to tumour cell dryness
Gene set be proportionate
Method：Classify to tumor tissues in TCGA according to system, classification is feminine proses, digestive system
Tumour, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, the urinary tract system tumor, nervous system swell
Knurl, blood-lymphatic system tumour and endocrine system carcinoma.More than tumour is divided into respectively according to the amplification degree of POU5F1B
POU5F1B amplification degree is high, low two class of POU5F1B amplifications degree.Journey is expanded to the POU5F1B of tumour by GSEA analysis methods
It spends and is analyzed with the correlation of Stemness up signatures, Stemness down signatures gene sets.
As a result：As shown in figure 18,1 ~ 9 is respectively feminine proses, digestive system tumor, tumor in respiratory system man
Sexual reproduction system tumor, skin and soft tissue neoplasm, the urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour
And endocrine system carcinoma.Normalized Enrichment Score（NES）Score, NES zero-range set constants are enriched with for standardization
It is more remote, show that correlation is bigger.NES positive and negative values represent the direction of correlation.In Figure 18, if NES values are positive number, with red table
Show.NES value zero-range set constants are more remote, red deeper, represent that positive correlation is bigger.If NES values are negative, represented with blue.NES
It is more remote to be worth zero-range set constant, blue is deeper, represents that negative correlation is bigger.The result shows that POU5F1B is in all types of tumours
Amplification is proportionate with the relevant gene set of tumour cell dryness.
Interpretation of result：Amplifications of the POU5F1B in all types of tumours be in the relevant gene set of tumour cell dryness
Positive correlation.POU5F1B can be as the potential index of prediction tumor recurrence.
Embodiment 4：Structure stablizes the cell strain of high expression POU5F1B and the cell strain of structure sh POU5F1B.
1. the cell strain of the stable POU5F1B high expression of structure
（1）Build the expression plasmid of POU5F1B
With the cDNA full length sequences of PCR amplification POU5F1B precursors, the POU5F1B full length sequences of purifying are building up to
PMSCV-retro-puro expression vectors（Purchased from Clontech companies）, obtain POU5F1B and be overexpressed plasmid pMSCV-retro-
puro-POU5F1B。
（2）Transfect human esophagus cancer cell Eca 9706
It is utilized respectively Lipofectamine2000 (Invitrogen, #11668) and the packaging plasmid of optimization
(Invitrogen, K4975-00) is using the pMSCV-retro-puro-POU5F1B of acquisition and as the pMSCV- compareed
Retro-puro empty carriers are transferred to 293FT cells.And DMEM culture mediums (DMEM was replaced after 12 hours；Gibco
BRL) it is aided with 10% fetal calf serum (Gibco BRL).Retroviral supernatant is collected after 48 hours and 72 hours, and is turned
It leads into 9706 cells of esophageal cancer cell Eca.
With puromycin (0.5 μ g/mL；Sigma-aldrich stabilization can be obtained for 10 days or so by) screening
The cell strain Eca 9706- POU5F1B of POU5F1B high expression, control cell strain are Eca 9706-Vector.
（3）The cell strain of the stable high expression POU5F1B of culture
Use DMEM culture mediums（DMEM；Gibco BRL）It is aided with 10% fetal calf serum（Gibco BRL）Culture medium training
Esophageal cancer cell Eca 9706 is supported, and is cultivated in 37 DEG C of the steril cell incubator containing 5% concentration carbon dioxide.
2. build the cell strain of sh POU5F1B
Prepare the hairpin structure of silence POU5F1B expression（hairpin structure）Sh POU5F1B, hairpin structure
（hairpin structure）After being integrated into cellular genome, siRNA is expressed.SiRNA and RISC by inhibit mRNA translate or
MRNA is cut, so as to reduce the expression of target gene in the cell.Above-mentioned shPOU5F1B can be carried out by associated biomolecule company
Synthesis, can silence POU5F1B expression, the present invention used in shPOU5F1B be according to POU5F1B ORF sequence designs.
The sense seq of shPOU5F1B：5’-CGGAGAAGTCCCAGGACAT-3’ （SEQ ID NO：3）, and negative control
The sequence of Control is random sequence.ShPOU5F1B and negative control are transiently transfected into 9706 cell strains of Eca, obtained
9706 cell strains of Eca containing shPOU5F1B（Referred to as Eca 9706-shPOU5F1B；Control cell strain Eca 9706-
Control.9706 cell strains of Eca containing shPOU5F1B described below, control cell strain Eca 9706-Control are thin
Born of the same parents' strain is all to obtain in this way.
Use DMEM culture mediums（DMEM；Gibco BRL）It is aided with 10% fetal calf serum（Gibco BRL）Culture medium training
Esophageal cancer cell Eca 9706 is supported, and is cultivated in 37 DEG C of the steril cell incubator containing 5% concentration carbon dioxide.
Embodiment 5：Functional studies of the POU5F1B in esophageal cancer cell and liver cancer cells
1. the internal one-tenth knurl ability of the overexpression enhancing esophageal cancer cell by obtaining POU5F1B into knurl experiment in vivo
Method：By 100,500,1000,5000 experimental groups cell Eca 9706-POU5F1B or cellular control unit Eca
9706-vector is injected into the left survey dorsal sc of 64 week old or so nude mice respectively.Tumour in 2 months after monitoring injection
The size of formational situation and tumour.
As a result：As shown in figure 19, red square curve represents that injection is overexpressed POU5F1B cell Eca 9706-POU5F1B
Mouse in non-oncogenic percentage, blue circle point curve represents that injection cellular control unit Eca 9706-vector's is small
Non-oncogenic percentage in mouse.It can be seen that in injection Eca 9706-POU5F1B groups, non-oncogenic mouse
Mouse of the shared ratio than being significantly lower than control group inoculation Eca 9706-vector.Inoculation Eca 9706-POU5F1B's
In mouse, do not form the percentage of mice with tumor reduces with the increase of inoculating cell quantity, and the Amplitude Ratio pair reduced
According to the big of group.
Interpretation of result：It is overexpressed the internal one-tenth knurl ability that POU5F1B enhances esophageal cancer cell.Prompting POU5F1B can make
Target spot for esophageal carcinoma therapy.
2. the high expression for obtaining POU5F1B by SP sorting experiments dramatically increases the tumour initiator cell in the cancer of the esophagus
Method：With trypsin digestion cell, complete medium is added in, 800rpm is centrifuged 4 minutes.Then it is resuspended in containing 2%
Fetal calf serum DMEM in, adjust cell（Eca 9706-POU5F1B and Eca 9706-vector）Concentration is into 1 × 106A/
mL.Two groups will be divided into per class cell, the quantity of two groups of cells is equal.One group in 100 μM of verapamil (Sigma-
Aldrich 37 DEG C of incubation 30min in), to inhibit ATP-binding cassette (ABC) transporters.Other one
Organize then 37 DEG C of incubation 30min in the sterile water isometric with verapamil.Then two groups of cells are in 5 μ g/mL Hoechst
33342 (Sigma-Aldrich) are incubated 90min in 37 DEG C, primary in the concussion of every 15 to 20 minutes of period, are eventually adding 2mL
Glacial phosphoric acid buffer solution（PBS）Reaction is terminated, 1 500 r/min abandon supernatant, then with containing 2% fetal calf serum after centrifuging 10 min
PBS wash 1 time, be resuspended with the PBS of 2% fetal calf serum, PI added in 1 μ g/mL of final concentration, is carrying out fluidic cell point
4 degree of cell is kept in dark place before choosing, identification.Flow cytomery sorts, 350nm（375）Length ultraviolet light excitation Hoechst dyes
Color is collected under 450/20nm band logicals and measures blue light, is collected under 675nm band logicals and measures feux rouges.Linear signal is chosen, is done
The two-dimentional scatter plots of Hoechst Red (X-axis) and Hoechst Blue (Y-axis), Hoechst Red and Hoechst Blue is weak
The region of signal and Verapamil control group missing is set as the door of SP cells, calculates percentage.
As a result：The result of above-mentioned flow cytometer inspection is as shown in figure 20, it can be seen that POU5F1B overexpressing cells system
Oesophagus tumor initiator cell in Eca 9706-POU5F1B（TIC, tumor initiated cells）Quantity significantly increase
Add.
Interpretation of result：Height expression POU5F1B promotes the formation of tumour initiator cell in the cancer of the esophagus.Prompting POU5F1B can make
Target spot for esophageal carcinoma therapy.
3. the internal one-tenth knurl ability of esophageal cancer cell is reduced by the low expression for obtaining POU5F1B into knurl experiment in vivo
Method：By 100,500,1000,5000 experimental group cell Eca 9706-shPOU5F1B#2, Eca 9706-
ShPOU5F1B#3 or cellular control unit Eca 9706-Control is injected into the left survey back of 64 week old or so nude mice respectively
Subcutaneously.The size of the formational situation of tumour and tumour in 2 months after monitoring injection.
As a result：As shown in figure 21, in inoculation Eca 9706- shPOU5F1B#2（Tangerine color square curve）、Eca 9706-
shPOU5F1B#3（Black triangle curve）Mouse in, ratio shared by non-oncogenic mouse is than inoculation Eca 9706-
Control（Purple circle point curve）Mouse it is high.In inoculation Eca 9706- shPOU5F1B#2, Eca 9706-
In the mouse of shPOU5F1B#3, the ratio shared by non-oncogenic mouse is reduced with the increase of inoculating cell quantity, and
And the mouse of Amplitude Ratio inoculation Eca 9706-Control reduced is small.In the mouse of inoculation Eca 9706- shPOU5F1B#2
In, the ratio shared by non-oncogenic mouse is close with the mouse for being inoculated with Eca 9706- shPOU5F1B#3.
Interpretation of result：The expression of silence POU5F1B reduces the internal one-tenth knurl ability of esophageal cancer cell.Prompt POU5F1B
It can be as the target spot of esophageal carcinoma therapy.
4. show that the silence of POU5F1B significantly reduces the tumour initiator cell in the cancer of the esophagus by the experiment of SP cell sortings
Method：Specific experiment method is as shown in the 2nd point of embodiment 5.
As a result：The result of above-mentioned flow cytometer inspection is as shown in figure 22, it can be seen that the silence of POU5F1B is notable
Significantly reduce the tumour initiator cell in the cancer of the esophagus（TIC, tumor initiated cells）Quantity.
Interpretation of result：Silence POU5F1B inhibits the formation of tumour initiator cell in the cancer of the esophagus.Prompt POU5F1B can conduct
The target spot of esophageal carcinoma therapy.
5. in body into knurl the experimental results showed that, POU5F1B high expression and the notable negative of survival rate after mouse tumor resection
It closes
Method：By 1 × 106A experimental group cell（Eca 9706-POU5F1B、Eca 9706- shPOU5F1B#2）It is or right
According to a group cell（Eca 9706-vector、Eca 9706-Control ）It is injected into the left survey of 84 week old or so nude mice respectively
Dorsal sc.The tumor resection that will be grown with nude mice after 35 days counts the survival rate of nude mice after 25 days.
As a result：As shown in figure 23, POU5F1B high is expressed（Eca 9706-POU5F1B curves in figure）Mouse tumor resection
Survival rate afterwards is substantially reduced than control group, POU5F1B low expressions（Eca 9706- shPOU5F1B#2 curves in figure）It is small
Survival rate after mouse tumor resection is than the apparent increase of control group.
Interpretation of result：As a result show that POU5F1B high expression is significantly negatively correlated with the survival rate after mouse tumor resection,
Poor prognosis is explicitly indicated in POU5F1B low expressions and the survival rate positive correlation after mouse tumor resection, POU5F1B high expression.
Therefore, POU5F1B can be as the potential index of patient's prognosis.
6. mouse tumor recurrence model the experimental results showed that POU5F1B high expressing promotings into the cancer of the esophagus recurrence
Method：By the tumour cut off in upper one experiment according to cell category（Eca 9706-POU5F1B、Eca
9706- shPOU5F1B#2、Eca 9706-vector、Eca 9706-Control ）It shreds respectively, be digested to individual cells,
Then these cells are injected 1 × 10 by every mouse6A quantity is injected into the left survey of 84 week old or so nude mice respectively
Dorsal sc.The survival rate of nude mice is counted after 60 days.
As a result：As shown in figure 24, the mouse of the cell Eca 9706-POU5F1B of injection overexpression POU5F1B is complete after 24 days
Portion is dead, and injects mouse still all survivals after 60 days of the cell Eca 9706- shPOU5F1B#2 of silence POU5F1B.
Interpretation of result：The above results illustrate the expression of POU5F1B and the survival rate negative correlation of mouse, and POU5F1B is low
Poor prognosis, and POU5F1B high expressing promotings is explicitly indicated in expression and the survival rate positive correlation of mouse, POU5F1B high expression
Into the recurrence of the cancer of the esophagus.Therefore, POU5F1B can be as the potential index of prediction patient's prognosis recurrence.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
<110>Zhongshan Univ. Cancer Cure Center
<120>Applications of the POU5F1B in diagnosing tumor, treatment, prognosis and prediction recurrence
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence
<400> 1
gccatacggt cacagagctt 20
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ggaagcttag ccaggtcaga 20
<210> 3
<211> 19
<212> DNA
<213>Artificial sequence
<400> 3
cggagaagtc ccaggacat 19
Claims (8)
1. application of the reagent of detection gene POU5F1B copy numbers in lesion detection kit is prepared can be quantified；It is described swollen
Knurl is selected from oophoroma, breast cancer, cervical carcinoma, carcinoma of endometrium, the cancer of the esophagus, hepatocellular carcinoma, cancer of pancreas, cholangiocarcinoma, colon cancer, straight
Intestinal cancer.
2. application of the reagent of detection gene POU5F1B expressions in lesion detection kit is prepared can be quantified；It is described
Tumour be selected from oophoroma, breast cancer, cervical carcinoma, carcinoma of endometrium, the cancer of the esophagus, hepatocellular carcinoma, cancer of pancreas, cholangiocarcinoma, colon cancer,
The carcinoma of the rectum.
3. application according to claim 2, it is characterised in that：In the kit gene is detected containing real time fluorescent quantitative
The primer sequence of POU5F1B transcriptional levels.
4. application according to claim 3, it is characterised in that：In the kit gene is detected containing real time fluorescent quantitative
The primer sequence of POU5F1B transcriptional levels：Sense primer 5'-GCCATACGGTCACAGAGCTT-3' and downstream primer 5'-
GGAAGCTTAGCCAGGTCAGA-3'。
5. application of the reagent of detection gene POU5F1B copy numbers in the prognosis kit for preparing tumour can be quantified, it is described
Tumour be selected from oophoroma, breast cancer, cervical carcinoma, carcinoma of endometrium, the cancer of the esophagus, hepatocellular carcinoma, cancer of pancreas, cholangiocarcinoma, colon cancer,
The carcinoma of the rectum.
6. application of the reagent of detection gene POU5F1B expressions in the prognosis kit for preparing tumour, institute can be quantified
It states tumour and is selected from oophoroma, breast cancer, cervical carcinoma, carcinoma of endometrium, the cancer of the esophagus, hepatocellular carcinoma, cancer of pancreas, cholangiocarcinoma, colon
Cancer, the carcinoma of the rectum.
7. application of the reagent of detection gene POU5F1B copy numbers in the recurrence prediction kit for preparing tumour can be quantified,
The tumour is selected from oophoroma, breast cancer, cervical carcinoma, carcinoma of endometrium, the cancer of the esophagus, hepatocellular carcinoma, cancer of pancreas, cholangiocarcinoma, knot
Intestinal cancer, the carcinoma of the rectum.
8. the reagent of detection gene POU5F1B expressions answering in the recurrence prediction kit for preparing tumour can be quantified
With, the tumour be selected from oophoroma, breast cancer, cervical carcinoma, carcinoma of endometrium, the cancer of the esophagus, hepatocellular carcinoma, cancer of pancreas, cholangiocarcinoma,
Colon and rectum carcinoma.
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| An Expressed Retrogene of the Master Embryonic Stem Cell Gene POU5F1 Is Associated with Prostate Cancer Susceptibility;Joan P. Breyer，et al;《The American Journal of Human Genetics》;20140306;第94卷;395-404 * |
| OCT4 在食管鳞癌组织中的表达及其临床意义;张如楠等;《中国医学工程》;20120531;第20卷(第5期);82-83，85 * |
| Oct4基因的研究进展;郑鹏生等;《西安交通大学学报（医学版）》;20100930;第31卷(第5期);521-526 * |
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