CN104762375A - Application of POU5F1B in diagnosis, treatment, prognosis and relapse prediction of tumor - Google Patents

Application of POU5F1B in diagnosis, treatment, prognosis and relapse prediction of tumor Download PDF

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CN104762375A
CN104762375A CN201510112706.1A CN201510112706A CN104762375A CN 104762375 A CN104762375 A CN 104762375A CN 201510112706 A CN201510112706 A CN 201510112706A CN 104762375 A CN104762375 A CN 104762375A
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pou5f1b
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宋立兵
李隽�
张鑫
谭展瑶
吴淑
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TUMOR PREVENTION AND THERAPY CENTER ZHONGSHAN UNIV
Sun Yat Sen University Cancer Center
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Abstract

The invention discloses an application of POU5F1B in diagnosis, treatment, prognosis and relapse prediction of tumor. In the invention, it is found that the copy number and the expression level of the gene POU5F1B in various tumors are closely related to transfer, relapse, patient prognosis and dryness of the tumor cells. A research result proves that by means of high expression of the POU5F1B, formation of TIC is promoted and further the in-vivo tumorigenic capability of esophagus cancer cells is enhanced, thereby reducing the survival rate, and oppositely, by means of inhibition of the expression of the POU5F1B, the formation of the TIC is promoted and further the in-vivo tumorigenic capability of the esophagus cancer cells is reduced, thereby increasing the survival rate, so that it is obviously that by means of inhibition of the endogenous POU5F1B, the in-vivo tumorigenic capability of the esophagus cancer cells can be significantly reduced, thereby increasing the survival rate of patients, which proves that the POU5F1B can be used as a marker for diagnosis, treatment, prognosis and relapse prediction. An inhibitor of the POU5F1B has a great clinical application value in preparation of a drug composition for tumor, so that the invention provides a new drug and a new method for effective treatment of tumor.

Description

The application of POU5F1B in diagnosing tumor, treatment, prognosis and predicting recurrence
Technical field
The present invention relates to the application of POU5F1B in diagnosing tumor, treatment, prognosis and predicting recurrence.
Background technology
The esophageal carcinoma is one of common malignant tumour, and its sickness rate occupies the 8th of malignant tumour, and mortality ratio is in the 6th.The Incidence of esophageal cancer of China is high, accounts for the whole world 50%, and China has become the highest country of whole world trouble mortality rate of esophageal cancer.
At present, the methods for the treatment of for the esophageal carcinoma mainly contains operative therapy, radiotherapy and chemotherapy etc., but the outcome of patient is still poor, and the patient of 75% treats in latter 1 year dead in acceptance, only has the survival time of the patient of 5 ~ 10% can reach 5 years.
Discovery and the rationally application of tumor markers are the prerequisites of tumor recurrence prediction.Up to now, the mark of the esophageal carcinoma that world projection is recurred in early days and prognosis is not good is still immature, for new diagnosis of esophageal patient, understands progression of disease risk and guides the instrument of therapeutic process also very limited.Therefore, oneself treatment and prevention of tumour through becoming the esophageal carcinoma this molecule and Clinical heterogeneity with height of research detecting recurrence of Esophageal Carcinoma mark is badly in need of the important scientific problems of solution.
Summary of the invention
The object of the present invention is to provide a kind of lesion detection test kit.
Another object of the present invention is to the prognosis kit that a kind of tumour is provided.
Another object of the present invention is the recurrence prediction test kit providing a kind of tumour.
The technical solution used in the present invention is:
A kind of lesion detection test kit, containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
Further, in mentioned reagent box containing can the reagent of detection by quantitative gene POU5F1B expression level.
Further, the primer sequence of gene POU5F1B transcriptional level is detected in mentioned reagent box containing real time fluorescent quantitative.
Further, the primer sequence of gene POU5F1B transcriptional level is detected in mentioned reagent box containing real time fluorescent quantitative: upstream primer 5'-GCCATACGGTCACAGAGCTT-3'(SEQ ID NO:1) and downstream primer 5'-GGAAGCTTAGCCAGGTCAGA-3'(SEQ ID NO:2).
A prognosis kit for tumour, containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
Further, in mentioned reagent box containing can the reagent of detection by quantitative gene POU5F1B expression level.
Further, in mentioned reagent box containing can the reagent of detection by quantitative gene POU5F1B copy number.
A recurrence prediction test kit for tumour, containing can the reagent of detection by quantitative gene POU5F1B expression level in this test kit.
A kind of oncotherapy medicament, containing the reagent that suppressor gene POU5F1B expresses in this medicament.
Further, the reagent that above-mentioned suppressor gene POU5F1B expresses is selected from the reagent that can make gene POU5F1B silence.
The invention has the beneficial effects as follows:
Present invention finds POU5F1B significantly to increase in kinds of tumors is as the esophageal carcinoma, mammary cancer, liver cancer and ovarian cancer etc.Not perfect for clinical tumor aided diagnosis technique at present, and POU5F1B is obviously amplification in tumour compared with healthy tissues, this prompting POU5F1B has the potential becoming tumour auxiliary diagnosis marker.
The present invention is found by a series of analytical procedure: the transfer of the amplification of POU5F1B and tumour in kinds of tumors, recurrence, patient's prognosis and tumour cell dryness are closely related.Found by functional experiment in a series of body: high expression level POU5F1B can promote the formation of TIC and then strengthen in the body of esophageal cancer cell to become knurl ability, reduces the survival rate without knurl mouse.Prompting POU5F1B can be used as the target spot of esophageal carcinoma therapy.And by suppressing the expression level of POU5F1B in cancer cells and tissue, in the body that can effectively suppress the formation of TIC to reduce esophageal cancer cell, becoming knurl ability, improving survival rate without knurl mouse.Above experimental data all suffice to show that suppress endogenous POU5F1B significantly can reduce esophageal cancer cell body in become knurl ability, improve survival, this has pointed out POU5F1B to have to become the potential of marker of diagnosis, treatment, prognosis and predicting recurrence.POU5F1B inhibitor has clinical value, for effective treatment of tumour provides new medicine and method on the pharmaceutical composition of preparation treatment tumour.
Accompanying drawing explanation
Fig. 1 is the AFLP system of human genome in 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines by drawing the data analysis in Tumorscape database;
Fig. 2 is the AFLP system of human genome in 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines drawn by the data analysis in GISTIC 2.0 pairs of Tumorscape databases;
Fig. 3 is the AFLP system of human genome in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues by drawing the data analysis in TCGA database;
Fig. 4 is the AFLP system of human genome in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues drawn by the data analysis in GISTIC 2.0 pairs of TCGA databases;
Fig. 5 is the distribution collection of illustrative plates of gene in 8q24.21 section;
Fig. 6 is: the 5443 routine primary tumor tissue of sample respectively in TCGA database of the copy number increase of FAM84B, PCAT1, POU5F1B, MYC, PVT1, GSDMC, FAM49B and ASAP1 gene on 8q24.21 section, 393 example transfers organize and 63 examples recur ratio shared in tissue;
Fig. 7 is presented at the amplification (Amplification) of POU5F1B copy number in 9297 routine tumor samples in TCGA database and to increase (Gain) be all remarkable negative correlation with the overall survival of tumour patient;
The amplification (Amplification) that Fig. 8 is presented at POU5F1B copy number in 9297 routine tumor samples in TCGA database is remarkable negative correlation with patient's disease free survival rate, and the disease free survival rate that the tumour patient of POU5F1B copy number amplification (Amplification) and POU5F1B copy number increase the tumour patient of (Gain) has significant difference;
Fig. 9 shows the ratio of amplification in specific tumors type of POU5F1B;
Figure 10 display is by FISH(fluorescence in situ hybridization) confirm that POU5F1B increases in the tissue and clone of the esophageal carcinoma, mammary cancer and liver cancer;
Figure 11 shows, and Real-Time pcr analysis result shows that POU5F1B is at 3 kinds of tumor tissues, comprises in the esophageal carcinoma, mammary cancer and liver cancer and increasing;
Kaplan – Meier survival curve display in Figure 12, amplification and the patient with esophageal carcinoma disease free survival rate of POU5F1B copy number are remarkable negative correlation;
Kaplan – Meier survival curve display in Figure 13, amplification and patient with breast cancer's disease free survival rate of POU5F1B copy number are remarkable negative correlation;
Kaplan – Meier survival curve display in Figure 14, amplification and the liver cancer patient disease free survival rate of POU5F1B copy number are remarkable negative correlation;
Figure 15 analyzes the POU5F1B mRNA level in-site of cancer general in TCGA database, and the amplification of result display POU5F1B promotes the rise of POU5F1B mrna expression;
Figure 16 display is analyzed by the dependency of Gene Set Enrichment Analysis (GSEA) analytical procedure to the POU5F1B copy number of cancer general in the TCGA variation gene set relevant to tumor recurrence, result;
Figure 17 display is analyzed by the dependency of Gene Set Enrichment Analysis (GSEA) analytical procedure to the POU5F1B copy number of cancer general in the TCGA variation gene set relevant to tumor prognosis, and result shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to prognosis is negative correlation;
Figure 18 display is analyzed by the dependency of Gene Set Enrichment Analysis (GSEA) analytical procedure to the POU5F1B copy number of cancer general in the TCGA variation gene set relevant to tumour cell dryness, and result shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to tumour cell dryness is proportionate;
Figure 19 inoculates the mouse bearing tumor easier than the mouse of inoculation Eca 9706-vector of Eca 9706-POU5F1B;
Figure 20 shows, and high expression level POU5F1B significantly increases the tumour initiator cell (TIC, tumor initiated cells) in the esophageal carcinoma;
Figure 21 shows the mouse bearing tumor easier than inoculation Eca 9706-Control mouse of inoculation Eca 9706-shPOU5F1B1#2 and inoculation Eca 9706-sh POU5F1B1#3;
Figure 22 shows, and reticent POU5F1B significantly increases the tumour initiator cell (TIC, tumor initiated cells) in the esophageal carcinoma;
The high expression level that Figure 23 is presented at POU5F1B in 16 mouse with without the remarkable negative correlation of knurl mouse survival rate; In 16 mouse POU5F1B low expression with without the remarkable positive correlation of knurl mouse survival rate;
Figure 24 shows, and mouse tumor recurrence model experimental result shows that POU5F1B high expression level promotes that the recurrence POU5F1B high expression level of the esophageal carcinoma promotes the recurrence of the esophageal carcinoma.
Embodiment
A kind of lesion detection test kit, containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
Preferably, the reagent containing detection by quantitative gene POU5F1B copy number in mentioned reagent box is upstream primer 5'-GCCATACGGTCACAGAGCTT-3'(SEQ ID NO:1) and downstream primer 5'-GGAAGCTTAGCCAGGTCAGA-3'(SEQ ID NO:2).
A kind of lesion detection test kit, containing can the reagent of detection by quantitative gene POU5F1B expression level in this test kit.
Preferably, the primer sequence of gene POU5F1B transcriptional level is detected in mentioned reagent box containing real time fluorescent quantitative.
Preferably, the primer sequence of gene POU5F1B transcriptional level is detected in mentioned reagent box containing real time fluorescent quantitative: upstream primer 5'-GCCATACGGTCACAGAGCTT-3'(SEQ ID NO:1) and downstream primer 5'-GGAAGCTTAGCCAGGTCAGA-3'(SEQ ID NO:2).
A kind of lesion detection test kit, containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit, and containing can the reagent of detection by quantitative gene POU5F1B expression level.
A prognosis kit for tumour, containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
Preferably, in mentioned reagent box containing can the reagent of detection by quantitative gene POU5F1B expression level.
Preferably, in mentioned reagent box containing can the reagent of detection by quantitative gene POU5F1B copy number.
A recurrence prediction test kit for tumour, containing can the reagent of detection by quantitative gene POU5F1B expression level in this test kit.
A kind of oncotherapy medicament, containing the reagent that suppressor gene POU5F1B expresses in this medicament.
Preferably, the reagent that above-mentioned suppressor gene POU5F1B expresses is selected from the reagent that can make gene POU5F1B silence.
Preferably, the reagent of above-mentioned reticent POU5F1B is the shPOU5F1B with hairpin structure, and its sequence is: 5 '-CGGAGAAGTCCCAGGACAT-3 ' (SEQ ID NO:3).
Below in conjunction with specific embodiment, set forth content of the present invention further.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition, as the condition described in " Molecular Cloning: A Laboratory guide " (third edition), or according to the condition that manufacturer advises.
embodiment 1:8q24.21 is the focus of amplification in Oncogenome
1. Tumorscape database analysis
Method: to the genome copy numbers variation data analysis in 3138 routine tumor samples in Tumorscape database (mention below 2526 routine tumor tissues add 611 tumor cell lines), 2526 routine tumor tissues and 611 tumor cell lines.The per-cent of the sample increased by certain chromosome segment copy number shared by 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines utilizes circos software to present with the form of circle, outmost circle is chromogene group, different karyomit(e) distinct colors represents, and in every bar karyomit(e), every a line represents a locus.3, the inside circle represents the per-cent of sample shared by 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines that certain chromosome segment copy number increases from outside to inside respectively.Copy number amplification degree can be divided into Gain and Amplification, orangely represents Gain, and namely the copy number of low degree increases (the extra copy increasing by 1 to 4); Vermilion red represents Amplification, namely additionally increases more than 4 copies.By the center of circle and certain section line chromosomal, the per-cent of sample shared by 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines that certain chromosome segment copy number increases just can be obtained.
Result: as shown in Figure 1, compared with other karyomit(e)s, chromosome arm 1q, 5p, 7, the frequency that increases in tumour of gene in 8q, 17q and 20 is the highest.
Interpretation of result: above analytical results shows, compared with other karyomit(e)s, chromosome arm 1q, 5p, 7, the frequency that increases in the 3138 routine tumor samples of gene in Tumorscape database, 2526 routine tumor tissues and 611 tumor cell lines in 8q, 17q and 20 is the highest, prompting chromosome arm 1q, 5p, 7, some gene in 8q, 17q and 20 can be used as the target spot of auxiliary diagnosis tumour.
2. GISTIC analyzes
Method: utilize Genomic Identification of Significant Targets in Cancer 2.0 (GISTIC2.0) to the genome copy numbers variation data analysis in 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines in Tumorscape database.G-scores represents the degree of gene amplification.G-scores is larger, represents that the degree of gene amplification is higher.Vice versa.Q-values is false discovery rate (false positive rate).
Result: as shown in Figure 2,8q24.21 section all has highest level to increase in 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines.
Interpretation of result: 8q24.21 section all has highest level to increase in 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines, certain gene in prompting 8q24.21 section can be used as the target spot of auxiliary diagnosis tumour.
3. TCGA database analysis
Method: to the genome copy numbers variation data analysis in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues in TCGA database.The sample increased by certain chromosome segment copy number shared per-cent in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues utilizes circos software to present with the form of circle, outmost circle is chromogene group, different karyomit(e) distinct colors represents, and in every bar karyomit(e), every a line represents a locus.3, the inside circle represents that the sample that certain chromosome segment copy number increases is organized and per-cent shared in 63 example recurrence tissues in 5443 routine primary tumor tissue, 393 example transfers from outside to inside respectively.Copy number amplification degree can be divided into Gain and Amplification, orangely represents Gain, and namely the copy number of low degree increases (the extra copy increasing by 1 to 4); Vermilion red represents Amplification, namely additionally increases more than 4 copies.By the center of circle and certain section line chromosomal, the per-cent of sample shared by 3138 routine tumor samples, 2526 routine tumor tissues and 611 tumor cell lines that certain chromosome segment copy number increases just can be obtained.
Result: as shown in Figure 3, compared with other karyomit(e)s, chromosome arm 1q, 5p, 7, the frequency that increases in tumour of gene in 8q, 17q and 20 is the highest.
Interpretation of result: above analytical results shows, compared with other karyomit(e)s, chromosome arm 1q, 5p, 7, the frequency that increases in the 5443 routine primary tumor tissue of gene in TCGA database, 393 example transfer tissues and 63 example recurrence tissues in 8q, 17q and 20 is the highest, prompting chromosome arm 1q, 5p, 7, some gene in 8q, 17q and 20 can be used as the target spot of auxiliary diagnosis tumour.
4. GISTIC analyzes
Method: utilize Genomic Identification of Significant Targets in Cancer 2.0 (GISTIC2.0) to the genome copy numbers variation data analysis in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues in Tumorscape database.G-scores represents the degree of gene amplification.G-scores is larger, represents that the degree of gene amplification is higher.Vice versa.Q-values is false discovery rate (false positive rate).
Result: as shown in Figure 4,8q24.21 section all has highest level to increase in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues.
Interpretation of result: 8q24.21 section all has highest level to increase in 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues, and certain gene in prompting 8q24.21 section can be used as the target spot of auxiliary diagnosis tumour.
embodiment 2:POU5F1B is positioned at the amplified peak of 8q24.21, and relevant to tumour patient prognosis
1. the distribution of gene on No. 8 karyomit(e)s
Method: analytical results prompting before, certain gene in 8q24.21 section can be used as the target spot of auxiliary diagnosis tumour, so searched which gene distribution on 8q24.21 section.
Result: 8q24.21 section has FAM84B, PCAT1, POU5F1B, MYC, PVT1, GSDMC, FAM49B and ASAP1.
2. TCGA database analysis result shows that POU5F1B amplification degree in 8q24.21 section is the highest
Method: Fig. 6 essence is a part of Fig. 3.
Result: as shown in Figure 6, POU5F1B is the gene that amplification degree is the highest in 8q24.21 section, and in TGCA database 5443 routine primary tumor tissue, 393 example transfer tissues and 63 example recurrence tissues, the amplification frequency ratio oncogene MYC of POU5F1B is high.
Interpretation of result: have bibliographical information to cross 8q24.21 and increase in kinds of tumors, this section contains known oncogene MYC.POU5F1B is the gene that amplification degree is the highest in 8q24.21 section, and in TGCA database, the amplification frequency ratio MYC of POU5F1B is high.Known by the gene database of NCBI, POU5F1 plays crucial effect in fetal development and stem cell totipotency.And POU5F1B is the pseudogene of POU5F1, the albumen of function of encoding, this albumen is almost identical with POU5F1 transcription factor length and highly similar, the regulation and control that prompting POU5F1B may express with participation POU5F1, relevant to the function that POU5F1 plays.
3. TCGA database analysis shows that the overall survival of POU5F1B amplification degree and tumour patient is remarkable negative correlation
Method: all statistical analysis SPSS17.0 statistical softwares process.Adopt Kaplan-Meier method to draw survival analysis curve, and adopt the log-rank method of inspection to detect its statistical significance.Test coefficient P<0.05 thinks there is significant difference statistically.SPSS statistical software is used to analyze the amplification of POU5F1B copy number and the relation of tumour patient overall survival.
Result: as shown in Figure 7, the tumour patient of POU5F1B copy number amplification (Amplification) significantly reduces than the overall survival of the normal tumour patient of POU5F1B copy number; The tumour patient that POU5F1B copy number increases (Gain) significantly reduces than the overall survival of the normal tumour patient of POU5F1B copy number; The tumour patient of POU5F1B copy number amplification (Amplification) and POU5F1B copy number increase the overall survival no significant difference of the tumour patient of (Gain).Wherein the amplification degree of POU5F1B copy number can be divided into Gain and Amplification.Gain, namely the copy number of low degree increases, and copy number additionally increases by 1 to 4; Amplification, namely copy number additionally increases by more than 4.
Interpretation of result: amplification (Amplification) and the increase (Gain) of SPSS statistic analysis result display POU5F1B copy number are all remarkable negative correlation (P<0.001) with the overall survival of patient, and the increase of POU5F1B copy number clearly indicates poor prognosis.Therefore, POU5F1B can be used as the potential index of patient's prognosis.
4. TCGA database analysis shows that the increase overall survival of degree and tumour patient, disease free survival rate of POU5F1B is remarkable negative correlation
Method: all statistical analysis SPSS17.0 statistical softwares process.Adopt Kaplan-Meier method to draw survival analysis curve, and adopt the log-rank method of inspection to detect its statistical significance.Test coefficient P<0.05 thinks there is significant difference statistically.SPSS statistical software is used to analyze the amplification of POU5F1B copy number and the relation of tumour patient disease free survival rate.
Result: as shown in Figure 8, the tumour patient of POU5F1B copy number amplification (Amplification) significantly reduces (P<0.001) than the disease free survival rate of the normal tumour patient of POU5F1B copy number; The tumour patient that POU5F1B copy number increases (Gain) reduces than the disease free survival rate of the normal tumour patient of POU5F1B copy number; The disease free survival rate that the tumour patient of POU5F1B copy number amplification (Amplification) and POU5F1B copy number increase the tumour patient of (Gain) has palpability difference (P<0.001).Wherein the amplification degree of POU5F1B copy number can be divided into Gain and Amplification.Gain, namely the copy number of low degree increases, and copy number additionally increases by 1 to 4; Amplification, namely copy number additionally increases by more than 4.
Interpretation of result: the amplification (Amplification) of SPSS statistic analysis result display POU5F1B copy number is remarkable negative correlation (P<0.001) with the disease free survival rate of patient, and the increase of POU5F1B copy number clearly indicates poor prognosis.Therefore, POU5F1B can be used as the potential index of patient's prognosis.
5. POU5F1B increases at feminine proses, digestive system tumor and tumor in respiratory system camber
Method: whether increase in specific tumors to explore POU5F1B further, to the CNV data analysis of 10700 routine various tumor tissues in TCGA database.Classify according to system to 10700 routine tumor tissues, classification is respectively feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Statistics, in the tumour of these classifications, has the POU5F1B in how many tumours to be amplification (Amplification and Gain).Classify by tumour particular type to 10700 routine tumor tissues, classification is respectively the specific tumor type such as OV, ESCA, UVM, adds up in the tumour of these classifications, has the POU5F1B in how many tumours to be amplification (Amplification and Gain).
Result: as shown in Figure 9, the tumor type of classifying is carried out in 1 ~ 9 expression according to system.POU5F1B Gain light red represents, POU5F1B Amplification redness represents.Show in figure, detect that the ratio shared in feminine proses, digestive system tumor and tumor in respiratory system sample in TCGA database of the tumor sample of POU5F1B amplification (comprising Amplification and Gain) is more than 50%.As shown on the right side of Fig. 9, POU5F1B significantly increases in 14 kinds of tumours of the general cancer data set of TCGA.Detect that the ratio shared in certain specific tumors of the tumour of POU5F1B Amplification carries out descending sort in seeing, front 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 increases at feminine proses, digestive system tumor and tumor in respiratory system camber, at ovarian serous cystadenocarcinoma (OV), esophageal carcinoma (ESCA), uveal melanoma (UVM), breast invasive carcinoma (BRCA), and liver hepatocellular carcinoma (LIHC) camber increases.Prompting POU5F1B amplification may with specific tumors type, as ovarian serous cystadenocarcinoma (OV), esophageal carcinoma (ESCA), uveal melanoma (UVM), breast invasive carcinoma (BRCA), and liver hepatocellular carcinoma (LIHC) are relevant.
6. by FISH(fluorescence in situ hybridization) confirm that POU5F1B increases in the tissue and clone of the esophageal carcinoma, mammary cancer and liver cancer
Method:
Probe synthesizes:
The position of probe fragment in genome: Location:8q24.21, No. BAC: RP11-327N12, by ARES Alexa Fluor 546 DNA Labeling Kit synthesising probing needle.
FISH(fluorescence in situ hybridization) step:
1. probe sex change
By probe incubation 5 min in 75 DEG C of waters bath with thermostatic control, to set to 0 immediately DEG C, 5 ~ 10 min, make double chain DNA probe sex change.
2. sample sex change
(1) karyomit(e) slide sample roasting sheet 2 ~ 3 h in 50 DEG C of incubators will prepared.(after the sample of Giemsa dyeing need fade in advance in stationary liquid, baking sheet again).
(2) take out slide sample, be immersed in sex change 2 ~ 3 min in the sex change liquid of the volume fraction 70% methane amide/2 × SSC of 70 ~ 75 DEG C.
(3) in order sample is dewatered through volume fraction 70%, volume fraction 90% and volume fraction 100% ice ethanol series immediately, each 5 min, then dry air.
3. hybridize
The DNA probe 10 μ L of sex change or preannealing to be dripped in sex change and on the slide sample of dehydration, covered, uses Parafilm mounting, and (about 15 ~ 17 h) to be placed in moist magazine 37 DEG C of hybridized overnight.Because hybridization solution is less, and hybridization temperature is higher, and the time length is long again, and therefore in order to keep the moisture state of sample, this process is carried out in wet box.
4. wash-out
This step contributes to the probe removing non-specific binding, thus reduces background.
(1) hybridize next day, sample is taken out from 37 DEG C of incubators, gently cover glass is taken off with blade.
(2) slide sample of having hybridized is positioned in the volume fraction 50% methane amide/2 × SSC of preheating 42 ~ 50 DEG C and washs 3 times, each 5 min.
(3) wash 3 times in 1 × SSC of preheating 42 ~ 50 DEG C, each 5 min.
(4) at room temperature, by slide sample, in 2 × SSC, fine laundering is once.
(5) slide is taken out, seasoning.
(6) get 200 μ L counterstain solutions (PI/antifade or DAPI/antifade dye liquor) to drip on slide sample, covered.
5. the amplification (being applicable to use biotin labeled probe) of hybridization signal
(1) add 150 μ L confining liquid I at the hybridization position of slide, cover with preservative film, 37 DEG C of incubation 20min.
(2) remove preservative film, then add 150 μ L avidin-FITC on sample, cover with preservative film, 37 DEG C are continued incubation 40 min.
(3) take out sample, put it in the elutriant of preheating 42 ~ 50 DEG C and wash 3 times, each 5 min.
(4) add 150 μ L confining liquid II at the hybridization position of slide sample, cover preservative film, 37 DEG C of incubation 20 min.
(5) remove preservative film, add 150 μ L antiavidin on sample, cover new preservative film, 37 DEG C of incubation 40 min.
(6) take out sample, put it in the new elutriant of preheating 42 ~ 50 DEG C, wash 3 times, each 5 min.
(7) repeating step (1), (2), (3), then room temperature is cleaned in 2 × SSC.
(8) slide is taken out, seasoning.
(9) get 200 μ L PI/antifade dye liquors to drip on slide sample, covered.
6. mounting
Dissimilar mounting liquid can be adopted.If mounting liquid can not be made to produce self-enclosed effect containing Mowiol(in mounting liquid), for preventing the solution evaporation between cover plate and slide glass, nail varnish can be used cover plate periphery seal.The slide sample sealed can keep the several months long in the magazine in the refrigerator of-20 ~-70 DEG C.
7. fluorescence microscope FISH result
The probe of mark POU5F1B place section sends red fluorescence, and the probe marking No. 8 karyomit(e) the other end sections sends green fluorescence.
Result: result as shown in Figure 10, POU5F1B significantly increases (being all more than or equal to 20 copies) in the esophageal carcinoma, mammary cancer, liver cancer, significantly increase (being all more than or equal to 20 copies) in esophageal carcinoma cell line Kyse510, breast cancer cell line MDA-MB-231 and cancerous cell line HuH-7, and it only has two copies in peripheral blood lymphocytes.In contrast, karyomit(e) the other end section all only has two copies in all samples.
Interpretation of result: POU5F1B significantly increases in the esophageal carcinoma, mammary cancer, liver cancer, significantly increases in esophageal carcinoma cell line Kyse510, breast cancer cell line MDA-MB-231 and cancerous cell line HuH-7, and prompting POU5F1B can be used as the target spot of auxiliary diagnosis tumour.
7. Real-Time PCR detects the copy number of gene POU5F1B in the esophageal carcinoma, mammary cancer and liver cancer
Method: detect the POU5F1B copy number in 20 routine esophageal tissues, 243 routine human esophageal carcinomas, 20 routine mammary tissues, 238 routine breast cancer tissues, 20 routine hepatic tissues and 226 routine liver cancer tissues by Real-Time PCR.
All statistical analysis SPSS17.0 statistical softwares process.Compare between different group with independent t inspection (Chang Bianliang), between two groups, the comparison t of mean checks, classified variable relatively use χ 2inspection, compares between many groups and uses one-way analysis of variance.Numerical value mean ± SD represents.Test coefficient P<0.05 thinks there is significant difference statistically.SPSS statistical analysis method is used to compare the average of POU5F1B copy number in esophageal tissue and human esophageal carcinoma, mammary tissue and breast cancer tissue, hepatic tissue and liver cancer tissue.
Result: as shown in figure 11, copy number is that 2 genus are normal, and namely this gene neither increases and also do not lack.Copy number is greater than 2 and is defined as amplification.POU5F1B is in three kinds of tumours for the display of Real-Time pcr analysis, comprises in the esophageal carcinoma, mammary cancer and liver cancer and significantly increasing.
Interpretation of result: POU5F1B, in three kinds of tumours, comprises in the esophageal carcinoma, mammary cancer and liver cancer and significantly increasing.Therefore, with the primer (upstream primer 5'-GCCATACGGTCACAGAGCTT-3'(SEQ ID NO:1) containing specific binding POU5F1B; Downstream primer 5'-GGAAGCTTAGCCAGGTCAGA-3'(SEQ ID NO:2)) auxiliary diagnostic box the amplification of POU5F1B in clinical sample detected, more can be diagnosed as this tissue samples is tumor sample.The analysis of clinical sample points out POU5F1B to can be used as the target spot of auxiliary diagnosis further.
8. the amplification of POU5F1B copy number and patient with esophageal carcinoma disease free survival rate are remarkable negative correlation
Method: all statistical analysis SPSS17.0 statistical softwares process.Adopt Kaplan-Meier method to draw survival analysis curve, and adopt the log-rank method of inspection to detect its statistical significance.Test coefficient P<0.05 thinks there is significant difference statistically.SPSS statistical software is used to analyze the amplification of POU5F1B copy number and the relation of the esophageal carcinoma (Esophageal carcinoma) patient's disease free survival rate.
Result: as shown in figure 12, the disease free survival rate of the patient with esophageal carcinoma of red curve display POU5F1B copy number amplification, blue curve display POU5F1B copy number does not increase the disease free survival rate of patient with esophageal carcinoma of (namely copy number is normal).The disease free survival rate of the patient with esophageal carcinoma patient with esophageal carcinoma more normal than POU5F1B copy number of POU5F1B copy number amplification significantly reduces (P=0.002).
Interpretation of result: amplification and the patient with esophageal carcinoma disease free survival rate of SPSS statistic analysis result display POU5F1B copy number are remarkable negative correlation (P=0.002), and the amplification of POU5F1B copy number clearly indicates poor prognosis.Therefore, POU5F1B can be used as the potential index of Esophagus Patients prognosis.
9. the amplification of POU5F1B copy number and patient with breast cancer's disease free survival rate are remarkable negative correlation
Method: all statistical analysis SPSS17.0 statistical softwares process.Adopt Kaplan-Meier method to draw survival analysis curve, and adopt the log-rank method of inspection to detect its statistical significance.Test coefficient P<0.05 thinks there is significant difference statistically.SPSS statistical software is used to analyze the amplification of POU5F1B copy number and the relation of mammary cancer (Breast carcinoma) patient's disease free survival rate.
Result: as shown in figure 13, the disease free survival rate that POU5F1B copy number expands patient with breast cancer's (Amp curve shown in redness) patient with breast cancer (Not Amp curve blueness shown in) more normal than POU5F1B copy number that 1 increases significantly reduces (P<0.05)
Interpretation of result: amplification and patient with breast cancer's disease free survival rate of SPSS statistic analysis result display POU5F1B copy number are remarkable negative correlation (P=0.020), and the amplification of POU5F1B copy number clearly indicates poor prognosis.Therefore, POU5F1B can be used as the potential index of mammary gland patient prognosis.
10. the amplification of POU5F1B copy number and liver cancer patient disease free survival rate are remarkable negative correlation
Method: all statistical analysis SPSS17.0 statistical softwares process.Adopt Kaplan-Meier method to draw survival analysis curve, and adopt the log-rank method of inspection to detect its statistical significance.Test coefficient P<0.05 thinks there is significant difference statistically.SPSS statistical software is used to analyze the amplification of POU5F1B copy number and the relation of liver cancer (Hepatocellular carcinoma) patient's disease free survival rate.
Result: as shown in figure 14, the disease free survival rate of liver cancer patient (the Amp curve shown in the redness) liver cancer patient (Not Amp curve blueness shown in) more normal than POU5F1B copy number of POU5F1B copy number amplification significantly reduces (P<0.05)
Interpretation of result: amplification and the liver cancer patient disease free survival rate of SPSS statistic analysis result display POU5F1B copy number are remarkable negative correlation (P=0.005), and the amplification of POU5F1B copy number clearly indicates poor prognosis.Therefore, POU5F1B can be used as the potential index of liver cancer patient prognosis.
the amplification of embodiment 3:POU5F1B promotes the expression of POU5F1B mRNA, strengthens the characteristic that tumor stem cell is relevant
1. the amplification of TCGA database analysis result display POU5F1B promotes the rise of POU5F1B mrna expression
In order to explore POU5F1B role in tumour, CNV (copy number variants) and the dependency of rna level are analyzed.
Method: the general cancer data in TCGA are classified according to the degree that increases of POU5F1B in general cancer, classification has diploid (4415 example), Gain(2857 example) and Amplification(1014 example).
All statistical analysis SPSS17.0 statistical softwares process.Compare between different group with independent t inspection (Chang Bianliang), between two groups, the comparison t of mean checks, classified variable relatively use χ 2inspection, compares between many groups and uses one-way analysis of variance.Numerical value mean ± SD represents.Test coefficient P<0.05 thinks there is significant difference statistically.Using SPSS statistical analysis method to compare POU5F1B copy number is diploid (4415 example), Gain(2857 example) and (1014 is routine) general cancer in the average of POU5F1B mRNA relative expression quantity.
Result: is as shown in figure 15 2(Diploid with 4415 routine POU5F1B copy numbers) general cancer in POU5F1B mRNA level in-site compared with, the POU5F1B mRNA level in-site in the general cancer (Pan-cancers) of 2857 routine POU5F1B Gain significantly rises.Compared with being the POU5F1B mRNA level in-site in the general cancer of 2 with 4415 routine POU5F1B copy numbers, the POU5F1B mRNA level in-site in the general cancer of 1014 routine POU5F1B Amplification significantly rises.Compared with the POU5F1B mRNA level in-site in the general cancer of 2857 routine POU5F1B Gain, the POU5F1B mRNA level in-site in the general cancer of 1014 routine POU5F1B Amplification significantly rises.
The CNV of interpretation of result: POU5F1B in tumour (copy number variants) and its mRNA level in-site are remarkable positive correlation, and the amplification of POU5F1B promotes the rise of POU5F1B mrna expression.Therefore, with containing detecting the primer (upstream primer 5'-GCCATACGGTCACAGAGCTT-3'(SEQ ID NO:1) of POU5F1B mRNA level in-site by specific quantification; Downstream primer 5'-GGAAGCTTAGCCAGGTCAGA-3'(SEQ ID NO:2)) auxiliary diagnostic box detect that in clinical sample, POU5F1B mRNA level in-site is higher than normal sample, more can be diagnosed as this tissue samples is tumor sample.The analysis prompting POU5F1B of clinical sample can be used as the target spot of auxiliary diagnosis.
2. GSEA analytical results shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to tumor recurrence is proportionate
Method: classify according to system to tumor tissues in TCGA, classification is feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Degree is high, POU5F1B increases low two classes of degree above tumour to be divided into POU5F1B to increase respectively according to the amplification degree of POU5F1B.By GSEA(gene set enrichment analysis by name entirely, gene set enrichment is analyzed) analytical procedure to the POU5F1B of tumour increase degree and Recurrence up signatures, Recurrence down signatures gene set dependency analyze.
Result: as shown in figure 16,1 ~ 9 is respectively feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Normalized Enrichment Score(NES) be stdn enrichment score, NES zero-range set constant is far away, shows that dependency is larger.NES positive and negative values is for the direction of positive negative correlation.In Figure 16, if NES value is positive number, then represent by redness.NES value zero-range set constant is far away, and redness is darker, represents that positive correlation is larger.If NES value is negative, then represent by blueness.NES value zero-range set constant is far away, and blueness is darker, represents that negative correlation is larger.Result shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to tumor recurrence is proportionate.
The gene set that the amplification of interpretation of result: POU5F1B in all types of tumour is relevant to tumor recurrence is proportionate.POU5F1B can be used as the potential index of predicting tumors recurrence.
3. the gene set that GSEA analyzes the amplification of POU5F1B in all types of tumour relevant to prognosis is negative correlation
Method: classify according to system to tumor tissues in TCGA, classification is feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Degree is high, POU5F1B increases low two classes of degree above tumour to be divided into POU5F1B to increase respectively according to the amplification degree of POU5F1B.By GSEA analytical procedure to the POU5F1B of tumour increase degree and Prognosis poor signatures, Prognosis good signatures gene set dependency analyze.
Result: as shown in figure 17,1 ~ 9 is respectively feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Normalized Enrichment Score(NES) be stdn enrichment score, NES zero-range set constant is far away, shows that dependency is larger.NES positive and negative values represents the direction of dependency.In Figure 17, if NES value is positive number, then represent by redness.NES value zero-range set constant is far away, and redness is darker, represents that positive correlation is larger.If NES value is negative, then represent by blueness.NES value zero-range set constant is far away, and blueness is darker, represents that negative correlation is larger.Result shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to tumor recurrence is proportionate.
The gene set that the amplification of interpretation of result: POU5F1B in all types of tumour is relevant to tumor prognosis is negative correlation.POU5F1B can be used as the potential index of predicting tumors prognosis.
4. GSEA analytical results shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to tumour cell dryness is proportionate
Method: classify according to system to tumor tissues in TCGA, classification is feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Degree is high, POU5F1B increases low two classes of degree above tumour to be divided into POU5F1B to increase respectively according to the amplification degree of POU5F1B.By GSEA analytical procedure to the POU5F1B of tumour increase degree and Stemness up signatures, Stemness down signatures gene set dependency analyze.
Result: as shown in figure 18,1 ~ 9 is respectively feminine proses, digestive system tumor, tumor in respiratory system male reproductive system tumour, skin and soft tissue neoplasm, urinary tract system tumor, nervous system neoplasm, blood-lymphatic system tumour and endocrine system carcinoma.Normalized Enrichment Score(NES) be stdn enrichment score, NES zero-range set constant is far away, shows that dependency is larger.NES positive and negative values represents the direction of dependency.In Figure 18, if NES value is positive number, then represent by redness.NES value zero-range set constant is far away, and redness is darker, represents that positive correlation is larger.If NES value is negative, then represent by blueness.NES value zero-range set constant is far away, and blueness is darker, represents that negative correlation is larger.Result shows that the gene set that the amplification of POU5F1B in all types of tumour is relevant to tumour cell dryness is proportionate.
The gene set that the amplification of interpretation of result: POU5F1B in all types of tumour is relevant to tumour cell dryness is proportionate.POU5F1B can be used as the potential index of predicting tumors recurrence.
embodiment 4: build the cell strain stablizing high expression level POU5F1B and the cell strain building sh POU5F1B.
1. build the cell strain of stable POU5F1B high expression level
(1) build pOU5F1Bexpression plasmid
With the cDNA full length sequence of pcr amplification POU5F1B precursor, the POU5F1B full length sequence of purifying is building up to pMSCV-retro-puro expression vector (purchased from Clontech company), obtains POU5F1B process LAN plasmid pMSCV-retro-puro-POU5F1B.
(2) transfected with human esophageal cancer cell Eca 9706
Utilize Lipofectamine2000 (Invitrogen respectively, #11668) with the packaging plasmid (Invitrogen, K4975-00) optimized, the pMSCV-retro-puro-POU5F1B of acquisition and pMSCV-retro-puro empty carrier are in contrast proceeded to 293FT cell.And after 12 hours, change DMEM substratum (DMEM; Gibco BRL) be aided with 10% foetal calf serum (Gibco BRL).48 hours and 72 h before harvest Retroviral supernatant, and transduction enters esophageal cancer cell Eca 9706 cell.
With puromycin (0.5 μ g/mL; Sigma-aldrich) screening can obtain the cell strain Eca 9706-POU5F1B of stable POU5F1B high expression level for about 10 days, and compared with control cells strain is Eca 9706-Vector.
(3) cell strain of stable high expression level POU5F1B is cultivated
Use DMEM substratum (DMEM; Gibco BRL) be aided with the culture medium culturing esophageal cancer cell Eca 9706 of the foetal calf serum (Gibco BRL) of 10%, and 37 DEG C containing the steril cell incubator of 5% concentration carbonic acid gas in cultivate.
2. build the cell strain of sh POU5F1B
Prepare hairpin structure (hairpin structure) the sh POU5F1B that reticent POU5F1B expresses, after hairpin structure (hairpin structure) is incorporated into cellular genome, express siRNA.SiRNA and RISC by suppressing mRNA translation or cutting mRNA, thus reduces goal gene at intracellular expression level.Above-mentioned shPOU5F1B can be synthesized by associated biomolecule company, can the expression of reticent POU5F1B, and the shPOU5F1B used by the present invention is according to POU5F1B ORF sequences Design.Sense seq: the 5 '-CGGAGAAGTCCCAGGACAT-3 ' (SEQ ID NO:3) of shPOU5F1B, and the sequence of negative control Control is stochastic sequence.By shPOU5F1B and negative control transient transfection in Eca 9706 cell strain, obtain Eca 9706 cell strain containing shPOU5F1B (referred to as Eca 9706-shPOU5F1B; Compared with control cells strain Eca 9706-Control.Eca 9706 cell strain containing shPOU5F1B hereinafter described, compared with control cells strain Eca 9706-Control cell strain are all obtain in this way.
Use DMEM substratum (DMEM; Gibco BRL) be aided with the culture medium culturing esophageal cancer cell Eca 9706 of the foetal calf serum (Gibco BRL) of 10%, and 37 DEG C containing the steril cell incubator of 5% concentration carbonic acid gas in cultivate.
the functional study of embodiment 5:POU5F1B in esophageal cancer cell and liver cancer cell
1. by become in body knurl test draw POU5F1B process LAN strengthen esophageal cancer cell body in become knurl ability
Method: the left survey dorsal sc 100,500,1000,5000 experimental group cell Eca 9706-POU5F1B or cellular control unit Eca 9706-vector being expelled to respectively nude mice about 64 week ages.The formational situation of tumour and the size of tumour in latter 2 months is injected in monitoring.
Result: as shown in figure 19, non-oncogenic per-cent in the mouse of red square curve representation injection process LAN POU5F1B cell Eca 9706-POU5F1B, blue circle point curve represents non-oncogenic per-cent in the mouse of injection cellular control unit Eca 9706-vector.Therefrom can find out that in injection Eca 9706-POU5F1B group, the ratio shared by non-oncogenic mouse is starkly lower than the mouse of control group inoculation Eca 9706-vector.In the mouse of inoculation Eca 9706-POU5F1B, the per-cent not forming mice with tumor reduces along with the increase of inoculating cell quantity, and the Amplitude Ratio control group reduced is large.
Interpretation of result: process LAN POU5F1B enhances the body interior one-tenth knurl ability of esophageal cancer cell.Prompting POU5F1B can be used as the target spot of esophageal carcinoma therapy.
2. show that the high expression level of POU5F1B significantly increases the tumour initiator cell in the esophageal carcinoma by SP sorting experiment
Method: with trypsin digestion cell, add perfect medium, centrifugal 4 minutes of 800rpm.Then be resuspended in containing 2% foetal calf serum DMEM in, adjustment cell (Eca 9706-POU5F1B and Eca 9706-vector) concentration become 1 × 10 6individual/mL.Every class cell is divided into two groups, and the quantity of two groups of cells is equal.One group in the verapamil (Sigma-Aldrich) of 100 μMs 37 DEG C hatch 30min, to suppress ATP-binding cassette (ABC) transporters.Other one group then with the isopyknic sterilized water of verapamil in 37 DEG C hatch 30min.Then two groups of cells hatch 90min at 5 μ g/mL Hoechst 33342 (Sigma-Aldrich) in 37 DEG C, in the concussion of every 15 to 20 minutes of period once, finally add 2mL glacial phosphoric acid damping fluid (PBS) termination reaction, supernatant is abandoned after centrifugal 10 min of 1 500 r/min, 1 time is washed again with the PBS of the foetal calf serum containing 2%, resuspended with the PBS of the foetal calf serum of 2%, PI is added to final concentration 1 μ g/mL, and before carrying out fluidic cell sorting, qualification, 4 degree, cell keeps in Dark Place.Flow cytomery sorting, 350nm(375) length ultraviolet optical excitation Hoechst dyes, and measure blue light in logical lower collection of 450/20nm band, logical lower collection of 675nm band measures ruddiness.Choose linear signal, do Hoechst Red (X-axis) and Hoechst Blue (Y-axis) two-dimentional scatter diagram, by Hoechst Red and Hoechst Blue weak signal and the region of verapamil control group disappearance is set as the door of SP cell, calculate per-cent.
Result: the result of above-mentioned flow cytometer inspection as shown in figure 20, therefrom can find out that the quantity of esophageal carcinoma tumour initiator cell (TIC, tumor initiated cells) in POU5F1B overexpressing cell system Eca 9706-POU5F1B significantly increases.
Interpretation of result: high expression level POU5F1B promotes the formation of tumour initiator cell in the esophageal carcinoma.Prompting POU5F1B can be used as the target spot of esophageal carcinoma therapy.
3. by become in body knurl test draw POU5F1B low express reduce esophageal cancer cell body in become knurl ability
Method: the left survey dorsal sc 100,500,1000,5000 experimental group cell Eca 9706-shPOU5F1B#2, Eca 9706-shPOU5F1B#3 or cellular control unit Eca 9706-Control being expelled to respectively nude mice about 64 week ages.The formational situation of tumour and the size of tumour in latter 2 months is injected in monitoring.
Result: as shown in figure 21, at inoculation Eca 9706-shPOU5F1B#2(tangerine look square curve), Eca 9706-shPOU5F1B#3(black triangles sigmoid curve) mouse in, the ratio inoculation Eca 9706-Control(purple circle point curve shared by non-oncogenic mouse) mouse high.In the mouse of inoculation Eca 9706-shPOU5F1B#2, Eca 9706-shPOU5F1B#3, ratio shared by non-oncogenic mouse reduces along with the increase of inoculating cell quantity, and the mouse of the Amplitude Ratio inoculation Eca 9706-Control reduced is little.In the mouse of inoculation Eca 9706-shPOU5F1B#2, the ratio shared by non-oncogenic mouse is close with the mouse of inoculation Eca 9706-shPOU5F1B#3.
Interpretation of result: the expression of reticent POU5F1B reduces the body interior one-tenth knurl ability of esophageal cancer cell.Prompting POU5F1B can be used as the target spot of esophageal carcinoma therapy.
4. show that the silence of POU5F1B significantly reduces the tumour initiator cell in the esophageal carcinoma by the experiment of SP cell sorting
Method: specific experiment method is as shown in embodiment 5 the 2nd.
Result: the result of above-mentioned flow cytometer inspection as shown in figure 22, therefrom can find out that the silence of POU5F1B significantly significantly reduces tumour initiator cell (TIC, the tumor initiated cells) quantity in the esophageal carcinoma.
Interpretation of result: reticent POU5F1B suppresses the formation of tumour initiator cell in the esophageal carcinoma.Prompting POU5F1B can be used as the target spot of esophageal carcinoma therapy.
5. become in body knurl experimental result to show, the remarkable negative correlation of survival rate after POU5F1B high expression level and mouse tumor resection
Method: by 1 × 10 6individual experimental group cell (Eca 9706-POU5F1B, Eca 9706-shPOU5F1B#2) or cellular control unit (Eca 9706-vector, Eca 9706-Control) are expelled to the left survey dorsal sc of nude mice about 84 week ages respectively.The tumor resection grown with it by nude mice after 35 days, adds up the survival rate of nude mice for 25 days afterwards.
Result: as shown in figure 23, survival rate after the mouse tumor resection of POU5F1B high expression level (in figure Eca 9706-POU5F1B curve) is than the obvious reduction of control group, and the survival rate after the mouse tumor resection of the low expression of POU5F1B (in figure Eca 9706-shPOU5F1B#2 curve) is than the obvious rising of control group.
Interpretation of result: the remarkable negative correlation of survival rate after result display POU5F1B high expression level and mouse tumor resection, the survival rate positive correlation after the low expression of POU5F1B and mouse tumor resection, POU5F1B high expression level clearly indicates poor prognosis.Therefore, POU5F1B can be used as the potential index of patient's prognosis.
6. mouse tumor recurrence model experimental result shows that POU5F1B high expression level promotes the recurrence of the esophageal carcinoma
Method: the tumour that the excision in a upper experiment is got off is shredded respectively according to cell category (Eca 9706-POU5F1B, Eca 9706-shPOU5F1B#2, Eca 9706-vector, Eca 9706-Control), is digested to individual cells, then these cells is pressed every injected in mice 1 × 10 6individual quantity is expelled to the left survey dorsal sc of nude mice about 84 week ages respectively.Within 60 days, add up the survival rate of nude mice afterwards.
Result: as shown in figure 24, mouse all death afterwards in 24 days of the cell Eca 9706-POU5F1B of injection process LAN POU5F1B, and the mouse of injecting the cell Eca 9706-shPOU5F1B#2 of reticent POU5F1B still all survived after 60 days.
Interpretation of result: the above results illustrates the expression level of POU5F1B and the survival rate negative correlation of mouse, the survival rate positive correlation of the low expression of POU5F1B and mouse, POU5F1B high expression level clearly indicates poor prognosis, and POU5F1B high expression level promotes the recurrence of the esophageal carcinoma.Therefore, POU5F1B can be used as the potential index of prediction patient prognosis recurrence.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
<110> Zhongshan Univ. Cancer Cure Center
 
The application of <120> POU5F1B in diagnosing tumor, treatment, prognosis and predicting 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 (10)

1. a lesion detection test kit, is characterized in that: containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
2. a lesion detection test kit, is characterized in that: containing can the reagent of detection by quantitative gene POU5F1B expression level in this test kit.
3. a kind of lesion detection test kit according to claim 2, is characterized in that: the primer sequence detecting gene POU5F1B transcriptional level in this test kit containing real time fluorescent quantitative.
4. a kind of lesion detection test kit according to claim 3, is characterized in that: the primer sequence detecting gene POU5F1B transcriptional level in this test kit containing real time fluorescent quantitative: upstream primer 5'-GCCATACGGTCACAGAGCTT-3'(SEQ ID NO:1) and downstream primer 5'-GGAAGCTTAGCCAGGTCAGA-3'(SEQ ID NO:2).
5. a prognosis kit for tumour, is characterized in that: containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
6. a prognosis kit for tumour, is characterized in that: containing can the reagent of detection by quantitative gene POU5F1B expression level in this test kit.
7. a recurrence prediction test kit for tumour, is characterized in that: containing can the reagent of detection by quantitative gene POU5F1B copy number in this test kit.
8. a recurrence prediction test kit for tumour, is characterized in that: containing can the reagent of detection by quantitative gene POU5F1B expression level in this test kit.
9. an oncotherapy medicament, is characterized in that: containing the reagent that suppressor gene POU5F1B expresses in this medicament.
10. a kind of oncotherapy medicament according to claim 9, is characterized in that: the reagent that described suppressor gene POU5F1B expresses is selected from the reagent that can make gene POU5F1B silence.
CN201510112706.1A 2015-03-13 2015-03-13 Applications of the POU5F1B in diagnosing tumor, treatment, prognosis and prediction recurrence Active CN104762375B (en)

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