CN114717321B - Application of CD29, DKK2 and BMI1 expression level detection reagent in preparation of ovarian cancer tissue dryness identification reagent - Google Patents

Application of CD29, DKK2 and BMI1 expression level detection reagent in preparation of ovarian cancer tissue dryness identification reagent Download PDF

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CN114717321B
CN114717321B CN202210477309.4A CN202210477309A CN114717321B CN 114717321 B CN114717321 B CN 114717321B CN 202210477309 A CN202210477309 A CN 202210477309A CN 114717321 B CN114717321 B CN 114717321B
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ovarian cancer
dkk2
bmi1
expression
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CN114717321A (en
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杨牧垚
郎廷元
周琦
李蕴哲
杨玲玲
崔晨曦
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Chongqing University Cancer Hospital
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Abstract

The invention discloses an application of a CD29, DKK2 and BMI expression level detection reagent in preparation of an ovarian cancer tissue dryness identification reagent. The ovarian cancer tissue can be used as a material source for separating and culturing ovarian cancer stem cells, and the dryness degree of the ovarian cancer tissue determines the success rate of separating and culturing the ovarian cancer stem cells, so that the method can be used for identifying the dryness degree of the ovarian cancer tissue, thereby improving the success rate of separating and culturing the ovarian cancer stem cells.

Description

Application of CD29, DKK2 and BMI1 expression level detection reagent in preparation of ovarian cancer tissue dryness identification reagent
Technical Field
The invention relates to the fields of medical science, life science research and production, in particular to application of a detection reagent for detecting expression levels of CD29, DKK2 and BMI1 in preparation of an ovarian cancer tissue dryness identification reagent.
Background
Ovarian Cancer (OC) is the second most common gynaecological malignancy, with mortality rate in the first place in gynaecological malignancy, and despite the current advances in Ovarian cancer treatment regimens, ovarian cancer survival rates of about 30% in 5 years are still present. Ovarian cancer stem cells refer to a cell subset of ovarian cancer cells with stem cell characteristics and capacity, and are main driving factors for occurrence, development, recurrence, metastasis and drug resistance of ovarian cancer, and are main causes of high malignancy of ovarian cancer. Thus, the study of ovarian cancer stem cells is advantageous for developing an effective treatment for ovarian cancer. The separation and culture of ovarian cancer stem cells from ovarian cancer tissues is an important link for the research of ovarian cancer. In addition, the ovarian cancer stem cells may become future cell factories after being modified, so the separation and extraction of the ovarian cancer stem cells may be an important link in the production of future biological products.
At present, the lack of an effective detection method capable of identifying the tissue dryness of ovarian cancer results in low success rate of separating and culturing ovarian cancer stem cells from an ovarian cancer sample. The research of the applicant finds that CD29, DKK2 and BMI1 can be used as markers in other human stem cells, and can be negatively related to survival of ovarian cancer patients and can be used as a stem marker for detecting ovarian cancer tissues, so that the success rate of separating and culturing ovarian cancer stem cells from an ovarian cancer sample is improved. Therefore, the invention provides application of a detection reagent for the expression levels of three genes of CD29, DKK2 and BMI1 in preparation of an ovarian cancer tissue dryness identification reagent, which is used for improving the success rate of separating and culturing ovarian cancer stem cells in scientific research and production.
Disclosure of Invention
The invention aims to provide an application of a reagent for detecting the expression levels of three genes of CD29, DKK2 and BMI1 in preparing a reagent for identifying the dryness of an ovarian cancer sample.
In order to achieve the above purpose, the present invention provides the following technical solutions:
application of CD29, DKK2 and BMI3 expression level detection reagent in preparation of ovarian cancer tissue dryness identification reagent.
Preferably, the CD29 is also called ITGB1, which is generally described as Integrin subunit beta, the ID in the Pubmed data is 3688, and the location in the human Chromosome is Chromosome 10, NC_000010.11 (32900318-32958230).
In the preferred embodiment of the present invention, the DKK2 is described in its entirety as Dickkopf WNT signaling pathway inhibitor, the ID in the Pubmed data is 27123, and the location in the human Chromosome is Chromosome 4, NC_000004.12 (106921802-107036313).
In the preferred embodiment of the invention, BMI1 is described in its entirety as BMI1 proto-oncogene, polycomb ring finger, ID 648 in Pubmed data, and position Chromosome 10, NC_000010.11 in human Chromosome (22321099-22331484).
In the present invention, the expression level of CD29, DKK2, BMI1 refers to the mRNA expression level and/or protein expression level of CD29, DKK2, BMI 1.
In the present invention, the method for detecting the mRNA expression levels of CD29, DKK2 and BMI1 preferably comprises a probe method, a qRT-PCR method and a sequencing method.
More preferably, the detection method of the mRNA expression amounts of the CD29, the DKK2 and the BMI1 is a qRT-PCR method, and primers used by the qRT-PCR are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6.
Preferably, the protein expression detection method of the CD29, DKK2 and BMI1 comprises western blot, immunohistochemistry and mass spectrometry.
Preferably, the ovarian cancer tissue refers to in-situ tissue, metastatic tissue, ascites and peritoneal tissue and cells of ovarian cancer.
Preferably, the ovarian cancer tissue dryness refers to the microsphere-forming capacity of the isolated ovarian cancer primary cells in an in vitro suspension culture environment.
Preferably, the strong dry ovarian cancer tissue refers to poor survival ability of the ovarian cancer patient corresponding to the positive sample.
More preferably, the high expression judgment criterion is ΔCT (CD29-GAPDH) CD29 high expression less than or equal to 7, delta CT (DKK2-GAPDH) DKK2 high expression is less than or equal to 7, delta CT (BMI1-GAPDH) And BMI3 high expression is less than or equal to 7.
The invention has the beneficial effects that: the invention discloses a reagent for detecting the expression levels of three genes of CD29, DKK2 and BMI1 for the first time, which can be applied to the preparation of an ovarian cancer tissue dryness identification reagent. The ovarian cancer tissue can be used as a material source for separating and culturing ovarian cancer stem cells, and the stem degree of the ovarian cancer tissue determines the success rate of separating and culturing the ovarian cancer stem cells, so that the method can be used for identifying the stem degree of the ovarian cancer tissue, thereby improving the success rate of separating and culturing the ovarian cancer stem cells, and the selectable detection method is various and has good market prospect.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 is a graph showing the results of a correlation analysis of CD29 expression levels with survival of ovarian cancer patients;
FIG. 2 is a graph showing the results of a correlation analysis between DKK2 expression level and survival of ovarian cancer patients;
FIG. 3 is a graph showing the results of a correlation analysis between BMI1 expression levels and survival of ovarian cancer patients;
figure 4 is a fold increase analysis of the success rate of isolating ovarian cancer stem cells from CD29, DKK2, BMI1 positive tissues.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, in which specific experimental methods are not specified, typically under conventional conditions or under conditions recommended by the manufacturer, so that those skilled in the art will better understand the invention and practice it. The examples are not to be construed as limiting the invention.
Example 1, high CD29 expression indicates poor prognosis for ovarian cancer patients
The CD29 expression data and corresponding survival data of the ovarian cancer patients are downloaded from a TCGA (The Cancer Genome Atlas) database, the correlation between the expression level of CD29 and the survival of the ovarian cancer patients is analyzed through a Kaplan-meier formula, and the result is shown in figure 1, and the survival difference between the CD29 high expression group patients and the CD29 low expression group patients has statistical significance And high expression of CD29 is indicative of a poor prognosis for the patient.
Example 2 high DKK2 expression indicates poor prognosis for ovarian cancer patients
Downloading DKK2 expression data and corresponding from TCGA (The Cancer Genome Atlas) database for ovarian cancer patientsSurvival data, the correlation between DKK2 expression level and survival of ovarian cancer patients is analyzed by Kaplan-meier formula, and the result is shown in figure 2, and the survival difference between DKK2 high expression group patients and DKK2 low expression group patients has statistical significance And high DKK2 expression is predictive of poor prognosis for patients.
Example 3 high BMI1 expression indicates poor prognosis for ovarian cancer patients
The BMI1 expression data and the corresponding survival data of the ovarian cancer patients are downloaded from a TCGA (The Cancer Genome Atlas) database, the correlation between the expression level of the BMI1 and the survival of the ovarian cancer patients is analyzed through a Kaplan-meier formula, and the result is shown in figure 3 that the survival difference between the BMI1 high expression group patients and the BMI1 low expression group patients has statistical significance And high BMI1 expression is indicative of a poor prognosis for the patient.
Example 4, fold increase analysis of success rate of isolated ovarian cancer stem cells from CD29, DKK2, BMI1 positive tissues.
Ovarian cancer tissues are obtained from a xenograft mouse model, mRNA expression levels of CD29, DKK2 and BMI1 are identified through qRT-PCR method, and delta CT is obtained (CD29-GAPDH) CD29 high expression less than or equal to 7, delta CT (DKK2-GAPDH) DKK2 high expression is less than or equal to 7, delta CT (BMI1-GAPDH) And 7. Ltoreq.7 is BMI1 high expression, the success rate is calculated by the number of samples successfully cultured compared with the total number of samples cultured, and the result is shown in figure 4, wherein the success rate of the culture of the all high expression group is more than 4 times higher than that of the culture of the all low expression group. The results show that the ovarian cancer tissues with high expression of CD29, DKK2 and BMI1 have higher dryness and higher success rate of separating and culturing ovarian cancer stem cells.
The primers used for the RT-PCR detection were as follows:
CD29 F：5'-cctacttctgcacgatgtgatg-3'(SEQ ID NO.1)；
CD29 R：5'-cctttgctacggttggttacatt-3'(SEQ ID NO.2)；
DKK2 F：5'-tgtaccaaggactggcattcg-3'(SEQ ID NO.3)；
DKK2 R:5'-ctgtggcaatacctcccaact-3'(SEQ ID NO.4)；
BMI1 F：5'-tggactgacaaatgctggaga-3'(SEQ ID NO.5)；
BMI1 R：5'-gaagattggtggttaccgctg-3'(SEQ ID NO.6)；
GAPDH F：5’-ctgggctacactgagcacc-3’(SEQ ID NO.7)；
GAPDH R：5’-aagtggtcgttgagggcaatg-3’(SEQ ID NO.8)。
the above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of Chongqing affiliated tumor Hospital
Application of <120> CD29, DKK2 and BMI1 expression level detection reagent in preparation of ovarian cancer tissue dryness identification reagent
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 1
cctacttctg cacgatgtga tg 22
<210> 2
<211> 23
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 2
cctttgctac ggttggttac att 23
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 3
tgtaccaagg actggcattc g 21
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 4
ctgtggcaat acctcccaac t 21
<210> 5
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 5
tggactgaca aatgctggag a 21
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 6
gaagattggt ggttaccgct g 21
<210> 7
<211> 19
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 7
ctgggctaca ctgagcacc 19
<210> 8
<211> 21
<212> DNA
<213> Artificial sequence (Artificial sequences)
<400> 8
aagtggtcgt tgagggcaat g 21

Claims (10)

1. Use of a reagent for simultaneously detecting expression levels of CD29, DKK2 and BMI1 in the preparation of a reagent for identifying dryness of ovarian cancer tissue.
2. The use according to claim 1, characterized in that: said CD29, also known as ITGB1, is fully described as Integrin subunit beta, with an ID of 3688 in Pubmed data and a position of chromosome10 in the human Chromosome, NC 000010.11 (32900318-32958230);
the DKK2 is fully described as Dickkopf WNT signaling pathway inhibitor, ID 27123 in Pubmed data, chromosome 4 in the human Chromosome, NC 000004.12 (106921802-107036313);
the BMI1 is described in its entirety as BMI1 proto-oncogene, polycomb ring finger, ID 648 in the Pubmed data, and position Chromosome 10, NC_000010.11 (22321099-22331484) in the human Chromosome.
3. The use according to claim 1, characterized in that: the expression level of CD29, DKK2 and BMI1 refers to mRNA expression level and/or protein expression level of CD29, DKK2 and BMI 1.
4. A use according to claim 3, characterized in that: the detection method of the mRNA expression quantity of the CD29, the DKK2 and the BMI1 comprises a probe method, a qRT-PCR method and a sequencing method.
5. A use according to claim 3, characterized in that: the detection method of the mRNA expression amounts of the CD29, the DKK2 and the BMI1 is a qRT-PCR method, and primers used by the qRT-PCR are respectively shown as SEQ ID NO.1 and SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and SEQ ID NO.5 and SEQ ID NO.6.
6. A use according to claim 3, characterized in that: the protein expression detection method of CD29, DKK2 and BMI1 comprises western blot, immunohistochemistry and mass spectrometry.
7. The use according to claim 1, characterized in that: the ovarian cancer tissue refers to in-situ tissue, metastatic tissue, ascites and peritoneal tissue and cells of ovarian cancer.
8. The use according to claim 1, characterized in that: the ovarian cancer tissue dryness refers to the microsphere forming capability of the isolated ovarian cancer primary cells in an in vitro suspension culture environment.
9. The use according to claim 1, characterized in that: the ovarian cancer tissue dryness refers to poor viability of ovarian cancer patients corresponding to high-expression samples of CD29, DKK2 and BMI 3.
10. The use according to claim 9, characterized in that: the high expression judgment standard is delta CT (CD29 - GAPDH) CD29 high expression less than or equal to 7, delta CT (DKK2 - GAPDH) DKK2 high expression is less than or equal to 7, delta CT (BMI1 - GAPDH) And the expression of BMI1 is less than or equal to 7.
CN202210477309.4A 2022-05-04 2022-05-04 Application of CD29, DKK2 and BMI1 expression level detection reagent in preparation of ovarian cancer tissue dryness identification reagent Active CN114717321B (en)

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* Cited by examiner, † Cited by third party
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US20090098538A1 (en) * 2006-03-31 2009-04-16 Glinsky Gennadi V Prognostic and diagnostic method for disease therapy
WO2007114896A2 (en) * 2006-03-31 2007-10-11 Ordway Research Institute Prognostic and diagnostic method for cancer therapy

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* Cited by examiner, † Cited by third party
Title
Ghader Babaei 等.EMT, cancer stem cells and autophagy The three main axes of metastasis.《Biomedicine & Pharmacotherapy》.2020,第133卷第1-11页. *
Role of BMI1 in epithelial ovarian cancer: investigated via the CRISPR/Cas9 system and RNA sequencing;Qianying Zhao等;《Journal of Ovarian Research 》;第11卷(第31期);第1-9页 *
癌基因 Bm-i1与干细胞及肿瘤发生;顾军等;《肿瘤防治研究》;第第34卷卷(第第1期期);第78-79页 *

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