CN115747337A - Application of CCT2 as glioma marker - Google Patents
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Abstract
The invention belongs to the technical field of biological medicines, and particularly relates to application of CCT2 as a glioma marker. The invention searches two databases of The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) by bioinformatics analysis, finds that the expression level of CCT2 in Glioma is increased along with the increase of the grade of Glioma, and simultaneously, the high expression of CCT2 is related to the bad prognosis of Glioma patients. The expression level of CCT2 in the glioma cell line is reduced by the small interfering RNA, so that the cell activity and the invasion and migration capacity of the glioma cell line can be obviously inhibited; constructing a glioma cell line with CCT2 stable low expression by a lentivirus technology, and then carrying out intracranial xenotransplantation on the constructed GBM cell line with CCT2 stable low expression in a nude mouse; the low expression of CCT2 is found to inhibit the growth of intracranial tumor of nude mouse by the live imaging of small animal, and the survival time of nude mouse is prolonged. CCT2 can be used as a molecular marker for diagnosing and treating glioma and plays a role in prognosis evaluation.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and particularly relates to application of CCT2 as a glioma marker.
Background
The information disclosed in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Gliomas are the most common primary central nervous system tumors, and more than half of cases are WHO 4-grade gliomas with extremely high malignancy, i.e., glioblastoma multiforme (GBM), and have the characteristics of high malignancy, strong invasive ability, easy recurrence, poor prognosis and the like. Clinical treatments for gliomas are mostly combination of postoperative Temozolomide (TMZ) chemotherapy with radiation therapy, but the overall therapeutic effect is not ideal, especially the median survival time of the most malignant glioblastoma multiforme patients is about 15 months. In recent years, with the development of scientific technology and scientific research means, particularly gene sequencing technology, more and more genes playing an important role in the occurrence and development process of tumors are identified. Some specific genes are related to the occurrence of specific tumors, so that the genes are used as targets for tumor diagnosis and treatment, and corresponding inhibitors are also applied to clinical treatment of tumors, thereby bringing good news to tumor patients. Therefore, finding glioma-related biological markers and providing new targets for diagnosis, treatment and prognosis evaluation of glioma become important directions for glioma research.
Protein is an organic macromolecule, is a basic substance constituting all life, and is a main undertaker of life activities. After the protein synthesizes peptide chains from ribosomes, the protein needs to be folded into mature protein with spatial conformation under the assistance of molecular chaperones so as to exert biological functions. The most complex chaperones are eukaryotic cytoplasmic chaperones containing TCP1[ CCT, also known as try (TCP 1 loop complex) ] which are composed of 8 different subunits ( CCT 1,2,3,4,5,6,7 and 8). CCT is critical for cell viability and has been shown to assist in folding cytoskeletal proteins (tubulin and actin) and other proteins associated with carcinogenesis in an atp-dependent manner, such as p53, STAT3, and the like. In this case, elevated expression of the CCT subunit may lead to upregulation of these oncogenic proteins, which in turn leads to carcinogenesis.
The CCT2 is reported to play a role in the malignant progression of various cancers such as breast cancer, liver cancer and the like, and no relevant literature report about the important role of the CCT2 in the diagnosis, treatment and prognosis evaluation of glioma exists at present.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide the application of CCT2 as a glioma marker. The invention searches two databases of The Cancer Genome Atlas (TCGA) and the Chinese Glioma Genome Atlas (CGGA) through bioinformatics analysis, finds that the expression level of CCT2 in Glioma increases along with the increase of the grade of Glioma, and meanwhile, the high expression of CCT2 is related to the bad prognosis of Glioma patients. The expression level of CCT2 in the glioma cell line is reduced through the small interfering RNA, so that the cell viability and the invasion and migration capacity of the glioma cell line can be obviously inhibited; constructing a glioma cell line with stable and low CCT2 expression by a lentivirus technology, and then carrying out xenografting on the constructed GBM cell line with stable and low CCT2 expression in the cranium of a nude mouse; the low expression of CCT2 is found to inhibit the growth of intracranial tumors of the nude mice through the live imaging of the small animals, and the survival time of the nude mice is prolonged. The invention proves that CCT2 can be used as a molecular marker for diagnosing and treating glioma and plays a role in prognosis evaluation.
In order to achieve the purpose, the invention is realized by the following technical scheme:
in a first aspect, the invention provides the application of CCT2 as a glioma marker in the preparation of glioma diagnosis, treatment and prognosis products, wherein the nucleotide sequence of CCT2 (NCBI Gene ID: 10576) is shown as Seq ID No. 1.
The beneficial effects obtained by one or more technical schemes of the invention are as follows:
(1) The invention proves that the expression of CCT2 gene/protein is reduced by siRNA to inhibit the cell proliferation and colony forming ability of glioma and the invasion migration ability for the first time, and the CCT2 gene/protein is proved to be a potential action target spot for treating glioma;
(2) The invention provides a tumor marker with CCT2 as glioma, the expression quantity of the CCT2 is increased along with the increase of the glioma grade, and the expression quantity of normal brain tissue in GBM is high, so that the CCT2 can be used as an index for auxiliary diagnosis of the glioma; the high expression of CCT2 is in positive correlation with the poor prognosis of glioma patients, so that the CCT2 can be used as an index for prognosis evaluation of glioma.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are included to illustrate an exemplary embodiment of the invention and not to limit the invention.
FIG. 1 is a data diagram of the bioinformatics analysis of CCT2 expression in example 1, wherein a is the expression of glioma specimen CCT2 in TCGA database, b is the expression of glioma specimen CCT2 in CGGA database, and c is the difference between the expression of CCT2 gene in normal brain tissue and glioblastoma multiforme in database of GEPIA2 website;
FIG. 2 is a Western Blot (WB) of human astrocytes (NHA) and CCT2 protein from T98, U118, A172, LN229, U251 and P3# GBM glioma cell lines from example 1;
FIG. 3 is a graph showing the effect of the expression level of glioma specimens on the survival time in the TCGA (a) and CGGA (b) databases in example 1;
fig. 4 is a graph showing the results of verifying the CCT2 protein knockdown efficiency of siRNA and lentivirus by western immunoblotting (WB) in example 2, wherein a is western immunoblotting of siCCT2# 1 and siCCT2# 2 with blank (NC) in LN229, U251, P3# GBM glioma cell line, and b is western immunoblotting of lentivirus (shCCT 2) with blank (NC) in LN229, P3# GBM glioma cell line;
FIG. 5 is a graph showing the changes in cell viability and colony forming ability of LN229, U251 and P3# GBM glioma cell lines after CCT2 protein knockdown using siCCT2# 1 and siCCT2# 2, wherein the control group is siCtrl, a is a CCK8 absorbance contrast graph, b is a cell staining graph, and c is a cell counting graph;
FIG. 6 is a comparison graph of the migration capacity of LN229 and U251 glioma cell lines after CCT2 protein knockdown using siCCT2# 1 and siCCT2# 2, the control group being siCtrl, wherein a is a cell staining graph and b is a comparison graph of relative cell migration data;
FIG. 7 is a comparison graph of the migration capacity of U251 and P3# GBM glioma cell lines after CCT2 protein knockdown using siCCT2# 1 and siCCT2# 2, wherein a is a microscopic observation graph and b is an invasion distance comparison graph;
FIG. 8 is a graph showing the results of an intracranial tumorigenesis experiment conducted by constructing a glioma cell line (shCCT 2) with low CCT2 gene/protein stability and a control group (shScramble) through lentivirus infection, wherein a is a live imaging image, b is a live imaging light flux contrast image, and c is a survival curve.
Detailed Description
In a first exemplary embodiment of the invention, CCT2 is used as a glioma marker in the preparation of glioma diagnosis, treatment and prognosis products, and the nucleotide sequence of CCT2 (NCBI Gene ID: 10576) is shown as Seq ID No. 1.
In one or more embodiments of this embodiment, the product includes, but is not limited to, a kit for diagnosing glioma and prognosis evaluation of glioma, and a drug for treating glioma.
In one or more embodiments of this embodiment, the kit uses CCT2 gene/protein as a target for detection.
In one or more embodiments of this embodiment, the kit comprises a primer sequence for detecting a CCT2 gene and/or a CCT2 antibody for detecting a CCT2 protein.
In one or more embodiments of this embodiment, the glioma-treating agent comprises a CCT2 inhibitor.
In one or more embodiments of this embodiment, the CCT2 inhibitor is one or more of an siRNA that prevents CCT2 gene/protein expression, a neutralizing antibody, or a compound that inhibits CCT2 protein function.
In one or more embodiments of this embodiment, the siRNA comprises:
In one or more embodiments of this embodiment, the compound comprises a lentivirus; the nucleotide sequence of the lentivirus is shown in Seq ID No.4.
In order to make the technical solutions of the present invention more clearly understood by those skilled in the art, the technical solutions of the present invention will be described in detail below with reference to specific embodiments.
Example 1
Clinical information of Glioma samples in two databases of The Cancer Genome Atlas (TCGA) and Chinese Glioma Genome Atlas (CGGA) is retrieved as a sample through bioinformatics analysis, and the sample is analyzed through GEPIA2 and GlioVis. As shown in FIG. 1, CCT2 Gene/protein (NCBI Gene ID: 10576) is highly expressed in GBM normal brain tissue, and the expression level of CCT2 Gene/protein is increased along with the increase of glioma grade.
Human Astrocytes (NHA) and T98, U118, A172, LN229, U251 and P3# GBM glioma cell lines were lysed with RIPA lysate and sonicated to fully lyse them. The lysate 13000 was centrifuged at high speed and the supernatant collected and the protein concentration measured by BCA kit. Then, the protein supernatant and SDS-PAGE protein loading buffer (5X) are mixed uniformly according to a certain proportion, and then heated in a metal bath to denature the protein. Proteins of different molecular weights were separated by gel electrophoresis and transferred to PVDF membranes. After the skim milk was blocked, the primary antibody was incubated overnight. After incubation of HRP-labeled secondary antibody, the exposure was developed. The results are shown in fig. 2, and the expression level of CCT2 protein in glioma cell line is higher than that in NHA cell as a whole.
And analyzing the relationship between the expression condition and the survival condition of the glioma samples in the TCGA and CGGA databases by utilizing an analysis tool of online websites GlioVis. The Kaplan-Meier method was used to plot survival curves, and the log-rank test method was used to test their statistical significance. As shown in fig. 3, high expression of CCT2 gene/protein in glioma patients resulted in poor prognosis of survival.
In conclusion, the CCT2 gene/protein is highly expressed in the GBM compared with normal brain tissue, the expression quantity of the CCT2 is increased along with the increase of the grade of glioma, the high expression of the CCT2 gene/protein is positively correlated with the poor prognosis of a glioma patient, and a foundation is laid for the CCT2 as a glioma marker to be further applied to glioma diagnosis, treatment and prognosis.
Example 2
Transfecting siRNA aiming at the CCT2 gene into LN229, U251 and P3# GBM glioma cell lines by using a transfection reagent lipo2000, and knocking down the expression quantity of the CCT2 gene/protein of the glioma cell lines by the siRNA, wherein the siRNA sequence (5 'to 3') is as follows:
siCtrl, UUCC UCC GAA CGU GUC ACG UTT, seq ID No.5, siRNA not directed against CCT2, as a control group.
LN229 and P3# GBM glioma cell lines with low CCT2 gene/protein stability are constructed by lentivirus infection and compared with a blank group (NC), and the MOI value is 10, and the sequence (5 'to 3') of lentivirus (shCCT 2) is TTATCGAGGAAGTCATGATTG, seq ID NO.4. The lentivirus negative control sequence (5 'to 3') is CCTAAGGTTAAGTCGCCCCTCG, seq ID No.6.
As shown in FIG. 4, both siRNA and lentivirus play a good role in inhibiting the expression level of CCT2 protein.
Example 3
Appropriate glioma cell lines are planted in a 96-well plate, each cell line is divided into three groups, and negative control and siCCT2# 1 and siCCT2# 2 are transfected respectively. Cell viability of glioma cell lines was measured with CCK8 at the same time each day and monitored for 4 consecutive days (0, 1,2,3, 4). And (3) replanting the glioma cell lines transfected with siRNA in the same number in a six-well plate, wherein each well contains about 1000 cells, abandoning the culture medium after about 14 days, adding polyformaldehyde for fixation, staining with crystal violet, and counting the colony formation condition. The results are shown in fig. 5, after siRNA knockdown CCT2 protein expression of glioma cell lines, cell viability and colony formation ability of glioma cell lines were inhibited.
The same number of siRNA transfected glioma cell lines were resuspended in a full culture containing 1% serum and plated in the upper chamber of a transwell chamber, with a full culture containing 30% serum in the lower chamber. After the cells migrate for a period of time, fixing the cells in the upper chamber and the lower chamber of the small chamber by using paraformaldehyde, wiping off the cells in the upper chamber, staining the cells in the lower chamber by using crystal violet, and then observing the cells under a microscope. The result is shown in fig. 6, the CCT2 knockdown group has fewer cells penetrating the bottom membrane of the chamber than the control group, which indicates that siRNA knockdown CCT2 protein expression level can inhibit the migration ability of the glioma cell line.
The glioma cell lines transfected by siRNA with the same quantity are replanted in a U-bottom low-adhesion 96-well plate, and the 96-well plate is placed in an incubator for culture after being centrifuged for 5 minutes at 300g in a centrifuge. And 2 days later, adding the matrigel to wrap the cell balls into a 96-well plate after the cells are polymerized and grown into the cell balls. After centrifugation at 300g for 5 minutes at 4 ℃ the 96-well plate was placed in an incubator to solidify matrigel, and then an appropriate amount of medium was supplemented to the 96-well plate. The cells were monitored under a microscope for invasive growth in matrigel and photographed. Image J software was used to assist in the statistical analysis of the invasive potential of the cells. The results are shown in fig. 7, and the invasion capacity of the glioma cell line is inhibited after the expression level of CCT2 is knocked down by siRNA.
Example 4
A luciferase-labeled CCT2 stably low-expressing glioma cell line (shCCT 2) was implanted intracranial in nude mice along with a control cell line (shscarmble, constructed using a lentiviral negative control sequence), and the growth size of intracranial tumors in nude mice was monitored by a mouse in vivo imager, and the survival curve of the mice was monitored. The result is shown in figure 8, the knockdown of CCT2 protein inhibits the intracranial tumorigenicity ability of glioma cell lines, and prolongs the survival time of nude mice.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
- The application of CCT2 as a glioma marker in preparing glioma diagnosis, treatment and prognosis products is characterized in that the nucleotide sequence of CCT2 is shown as Seq ID No. 1.
- 2. The use of claim 1, wherein said products include but are not limited to kits for diagnosis of glioma and prognosis evaluation of glioma, drugs for treatment of glioma.
- 3. The use of claim 2, wherein the kit uses CCT2 gene/protein as a target for detection.
- 4. The use of claim 3, wherein the kit comprises a primer sequence for detecting CCT2 gene and/or a CCT2 antibody for detecting CCT2 protein.
- 5. The use of claim 2, wherein the glioma treating drug comprises a CCT2 inhibitor.
- 6. The use of claim 5, wherein the CCT2 inhibitor is one or more of siRNA, neutralizing antibody or compound inhibiting CCT2 protein function for CCT2 gene/protein expression.
- 7. The use of claim 6, wherein the siRNA comprises:siCCT2#1, GCAAAUACUGGUAUGGAUATT, and the nucleotide sequence is shown as Seq ID No. 2;siCCT2#2, GCUUGAAGUUGCAAACUUTT, and the nucleotide sequence is shown as Seq ID No. 3.
- 8. The use of claim 6, wherein the compound comprises a lentivirus having the nucleotide sequence set forth in Seq ID No.4.
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| CN103025890A (en) * | 2010-04-06 | 2013-04-03 | 卡里斯生命科学卢森堡控股 | Circulating biomarkers for disease |
| US20150301058A1 (en) * | 2012-11-26 | 2015-10-22 | Caris Science, Inc. | Biomarker compositions and methods |
| WO2022187744A1 (en) * | 2021-03-05 | 2022-09-09 | University Of Central Florida Research Foundation, Inc. | Chaperonin-containing tcp-1 inhibitors for the treatment of cancer |
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| OKITSU Y,等: "Homo sapiens chaperonin containing TCP1 subunit 2 (CCT2), transcript variant 1, mRNA，NCBI Reference Sequence: NM_006431.3，1916bp mRNA linear", 《NCBI GENBANK》, pages 1 - 5 * |
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