CN114277153A - Application of reagent for detecting expression level of ZFP92 and kit - Google Patents
Application of reagent for detecting expression level of ZFP92 and kit Download PDFInfo
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- CN114277153A CN114277153A CN202210056904.0A CN202210056904A CN114277153A CN 114277153 A CN114277153 A CN 114277153A CN 202210056904 A CN202210056904 A CN 202210056904A CN 114277153 A CN114277153 A CN 114277153A
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Abstract
The invention belongs to the technical field of biology, and relates to application of a reagent for detecting ZFP92 expression level and a kit. More particularly, the invention relates to application of a reagent for detecting the expression level of ZFP92 in preparation of a preparation for renal clear cell carcinoma auxiliary diagnosis and treatment and/or renal clear cell carcinoma patient prognosis, and a kit for renal clear cell carcinoma auxiliary diagnosis and/or renal clear cell carcinoma patient prognosis. The invention proves that the expression of ZFP92 in renal clear cell carcinoma is remarkably reduced, the low expression of ZFP92 is respectively associated with the reduction of the overall survival time (OS), the progression-free survival time (PFI) and the specific survival rate (DSS) of renal clear cell carcinoma patients, which shows that ZFP92 can be used for auxiliary diagnosis and treatment of renal clear cell carcinoma and/or prognosis judgment of renal clear cell carcinoma patients, and a ZFP92 detection kit is designed based on the ZFP 3578 expression.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a reagent for detecting ZFP92 expression level in preparation of a preparation for renal clear cell carcinoma auxiliary diagnosis and treatment and/or renal clear cell carcinoma patient prognosis judgment, a kit for renal clear cell carcinoma auxiliary diagnosis and/or renal clear cell carcinoma patient prognosis judgment, and application of the kit.
Background
Renal clear cell carcinoma (KIRC) is one of the most common and relatively poor prognosis types of renal tumors. With the development of times and the progress of modern high-throughput sequencing technologies, renal clear cell carcinoma is understood more deeply at a molecular level, but at present, the occurrence, progression and transfer mechanisms are still unclear, and effective biomarkers are lacked. The heterogeneity of renal clear cell carcinoma presents a challenge to predict patient prognosis. Some traditional prediction indexes cannot accurately predict prognosis, and the search of new prognosis prediction factors and potential treatment targets is very necessary.
Disclosure of Invention
The invention aims to provide a novel kidney clear cell carcinoma marker (ZFP92), and further provides application of a reagent for detecting the expression level of ZFP92 in preparation of a preparation for auxiliary diagnosis and treatment of kidney clear cell carcinoma and/or prognosis judgment of a patient with kidney clear cell carcinoma, a kit for auxiliary diagnosis and treatment of kidney clear cell carcinoma and/or prognosis judgment of the patient with kidney clear cell carcinoma and application of the kit.
In order to achieve the above object, the first aspect of the present invention provides a use of a reagent for detecting the expression level of ZFP92 in the preparation of a preparation for renal clear cell carcinoma-assisted diagnosis and/or renal clear cell carcinoma patient prognosis.
Further, the detecting the expression level of ZFP92 includes detecting the mRNA expression level of ZFP92 and/or detecting the protein expression level of ZFP 92.
More specifically, the method for detecting the expression level of ZFP92 comprises: detecting the expression quantity of ZFP92mRNA in renal clear cell carcinoma and para-carcinoma tissues by an RT-qPCR method; detecting the expression quantity of ZFP92mRNA in renal clear cell carcinoma and para-carcinoma tissues by a molecular probe technology; the expression level of ZFP92 protein in renal clear cell carcinoma and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of the ZFP92 is an oligonucleotide probe, a PCR primer or an antibody targeting the DNA sequence or the mRNA sequence of the ZFP92 gene, or the ZFP92 gene.
According to the present invention, preferably, the reagent for detecting the expression level of ZFP92 is a reagent having the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
5’-AAAAGGTCCTCTACAAGCGGG-3’，SEQ ID NO：1
5’-GTCTGCTACCCAGGGTCCTT-3’，SEQ ID NO：2
The second aspect of the present invention provides a kit for auxiliary diagnosis and treatment of renal clear cell carcinoma and/or prognosis of renal clear cell carcinoma patients, comprising: agents that detect the level of expression of ZFP 92.
Further, the reagent for detecting the expression level of the ZFP92 is an oligonucleotide probe or a PCR primer of a DNA sequence or an mRNA sequence of a ZFP92 gene, or an antibody of a ZFP92 gene.
Specifically, the reagent for detecting the expression level of ZFP92 is a reagent with the nucleotide sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
According to the present invention, the kit may further contain other conventional reagents for real-time fluorescence quantification, and preferably, the kit further comprises at least one of the following components: trizol, isopropanol, chloroform, absolute ethanol, RNase-free water, random primers, 5 XM-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, a primer having the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and (4) ACTB real-time fluorescence quantitative PCR specific primers of the nucleotide sequence shown in the specification.
5’-GGCACCCAGCACAATGAAGA-3’，SEQ ID NO：3；
5’-ACTCCTGCTTGCTGATCCAC-3’，SEQ ID NO：4。
The ZFP92 is a protein coding gene, the research of the invention proves that the expression of ZFP92 in renal clear cell carcinoma is obviously reduced, the low expression of ZFP92 is respectively associated with the reduction of the overall survival time (OS), the progression-free survival time (PFI) and the specific survival rate (DSS) of renal clear cell carcinoma patients, which indicates that ZFP92 can be used for auxiliary diagnosis and treatment of renal clear cell carcinoma and/or prognosis judgment of renal clear cell carcinoma patients, and a ZFP92 detection kit is designed based on the ZFP92 detection kit.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
Fig. 1 shows the change in expression of the target gene ZFP92 in renal clear cell carcinoma. (A) Unpaired sample results show significant down-regulation of ZFP92 expression in renal clear cell carcinoma tissues; (B) paired sample results showed significant down-regulation of ZFP92 expression in renal clear cell carcinoma tissue.
Fig. 2 shows the correlation of ZFP92 expression with overall patient survival (OS) in renal clear cell carcinoma. Low expression of ZFP92 was associated with decreased OS in renal clear cell carcinoma patients.
Figure 3 shows the correlation of ZFP92 expression with patient progression free survival (PFI) in renal clear cell carcinoma. Low expression of ZFP92 was associated with decreased PFI in renal clear cell carcinoma patients.
Fig. 4 shows the correlation of ZFP92 expression with patient disease-specific survival (DSS) in renal clear cell carcinoma. Low expression of ZFP92 was associated with decreased DSS in renal clear cell carcinoma patients.
Fig. 5 shows the expression changes of ZFP92 in renal clear cell carcinoma.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples, in which the specific conditions are not specified, were conducted under the conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Example 1
This example serves to demonstrate significant down-regulation of the expression of the target gene ZFP92 in renal clear cell carcinoma.
Differential analysis of ZFP92 expression in renal clear cell carcinoma:
1. data downloading and sorting:
the UCSC XENA website (https:// xenoxybronser. net/datapages /) is used for downloading TCGA renal clear cell carcinoma expression profile data and clinical data, and ZFP92 expression profile data is transformed by log2 and then is compared among groups.
2. Differential analysis of expression of ZFP92 in renal clear cell carcinoma
The R software package was used to analyze the ZFP92mRNA level differences between paired and unpaired samples of renal clear cell carcinoma patients, and the ggplot2 was used for visualization, as shown in fig. 1.
Fig. 1 shows the change in expression of the target gene ZFP92 in renal clear cell carcinoma. ZFP92 expression was significantly down-regulated in renal clear cell carcinoma; p <0.05, p < 0.001.
Example 2
This example serves to illustrate that low expression of ZFP92 in renal clear cell carcinoma correlates with poor overall patient prognosis.
Analysis of association of ZFP92 expression in renal clear cell carcinoma with overall patient prognosis:
the optimal cutoff value of ZFP92 was calculated using the R software package maxstat, patients were divided into two groups of ZFP92 high and low expression, the overall prognosis (OS) difference of the two groups was further analyzed using the survivfit function of the R software package survivval, and the significance of the prognosis difference between the samples of the different groups was evaluated using logrank test method, as shown in fig. 2.
Fig. 2 shows the correlation of ZFP92 expression in renal clear cell carcinoma with Overall Survival (OS) in patients. Low expression of ZFP92 was associated with decreased OS in renal clear cell carcinoma patients.
Example 3
This example was used to verify that low expression of ZFP92 in renal clear cell carcinoma is associated with a decrease in progression free survival in patients.
Analysis of association of ZFP92 expression in renal clear cell carcinoma with patient progression-free survival:
the optimal cutoff value of ZFP92 was calculated using the R software package maxstat, based on which patients were divided into two groups of ZFP92 expression levels, the two groups were analyzed for Progression-free interval (PFI) differences using the survivat function of the R software package survival, and the significance of prognostic difference between samples of different groups was evaluated using the logrank test method. As shown in fig. 3.
Figure 3 shows the correlation of ZFP92 expression with patient progression free survival (PFI) in renal clear cell carcinoma. Low expression of ZFP92 was associated with decreased PFI in renal clear cell carcinoma patients.
Example 4
This example was used to verify that low expression of ZFP92 in renal clear cell carcinoma is associated with a decrease in patient-specific survival.
Analysis of ZFP92 expression in renal clear cell carcinoma correlation with patient-specific survival:
the optimal cutoff value of ZFP92 was calculated using R software package maxstat, based on which patients were divided into two groups of ZFP92 expression high and low, Disease-specific survival (DSS) differences were analyzed using the subvert function of R software package subvalr, and prognostic significance was evaluated between samples of different groups using logrank test method. As shown in fig. 4.
Fig. 4 shows the correlation of ZFP92 expression with patient disease-specific survival (DSS) in renal clear cell carcinoma. Low expression of ZFP92 was associated with decreased DSS in renal clear cell carcinoma patients.
Example 5
This example illustrates the preparation of a kit for detecting the expression level of ZFP 92.
1. Primer design
PCR primers were designed based on the CDS region sequence of the transcript of the human ZFP92 Gene (Gene ID:139735), and the primer sequences are shown in Table 1.
2. Kit Components
(1)Trizol 50.0mL；
(2) 50.0mL of isopropanol;
(3) 50.0mL of chloroform;
(4) 50.0mL of absolute ethyl alcohol;
(5) 5.0mL of RNase-free water;
(6) 1.0. mu.M Random primers (Random primers) 50.0. mu.L;
(7) 2.0mL of 5 XM-MLV buffer;
(8)10.0mM base triphosphate deoxynucleotides (dNTPs) 100.0. mu.L;
(9) 40U/. mu.L RNase inhibitor 50.0. mu.L;
(10) 200U/. mu. L M-MLV reverse transcriptase 50.0. mu.L;
(11)ABI 2×PCR Mix 2.0mL；
(12)10.0 mu M ZFP92 real-time fluorescent quantitative PCR specific primer 30.0 mu L, and the primer sequence is shown in Table 1;
(13) 10.0. mu.M ACTB real-time fluorescent quantitative PCR specific primers 30.0. mu.L, and the primer sequences are shown in Table 1.
TABLE 1 fluorescent quantitative RT-PCR primer sequences
3. Identification of expression changes of ZFP92 in renal clear cell carcinoma by RT-PCR
(1) Extraction of total RNA from tissues: the experiment was performed in an ice bath. Placing 30-50 mg of tissue (fresh or tissue preserved at-70 ℃ in liquid nitrogen) into a 1.5mL centrifuge tube, adding 1mL Trizol, homogenizing fully, and standing at room temperature for 5 min; adding 200 μ L chloroform into each tube, mixing vigorously for 30sec, standing for 15min, and centrifuging at 4 deg.C and 12000rpm for 15 min; gently sucking 400 μ L of the supernatant liquid into another new centrifuge tube, adding isopropanol with the same volume, gently inverting and mixing, and centrifuging at 12000rpm at 4 deg.C for 10 min; discarding the supernatant, adding 1ml of 75% alcohol to wash the precipitate, and centrifuging at 4 ℃ and 12000rpm for 10 min; the supernatant was discarded as much as possible, air-dried at room temperature for 10min, and dissolved by adding 10. mu.L of RNase-free water to each tube (10-15 min. lysis-promoting at 65 ℃). Determination of OD260And calculating the RNA concentration.
RNA(mg/mL)＝40×OD260X dilution multiple (n)/1000
(2) Reverse transcription: each 25. mu.L reverse transcription system contained 100pmol of random primer, 2. mu.g of total RNA, 1. mu.L of M-MLV reverse transcriptase, 0.625. mu.L of RNase inhibitor, 1.25. mu.L of dNTPs (10mM), 5. mu.L of 5 XM-MLV buffer, and 25. mu.L of RNase-free water. The reaction conditions are as follows: 1h at 37 ℃ and 5min at 95 ℃.
(3) Quantitative PCR: each 20. mu.L reaction system contained 2 XPCR Mix 10. mu.L, upstream and downstream primers 0.4. mu.L each, cDNA 1. mu.L, ddH2O8.2. mu.L. The reaction conditions are as follows: 94 ℃ for 2min, 94 ℃ for 15s, 60 ℃ for 40s, 40 cycles.
(4)2-ΔΔCTCalculating the relative expression of ZFP 92: ACTB as reference gene, target gene C determined by qPCRTC of reference gene ACTB with the same value as that of the tissue sourceTSubtracting the values to obtain Δ CTThen, Δ C is addedTAnd control group Δ CTSubtracting to obtain Δ Δ CT(taking a paracarcinoma sample Δ CTHas an average value of Δ CTControl), relative expression of ZFP92 for each group was calculated using the Power function in Excell tables. Expression difference between 9 clear cell renal carcinoma samples (KIRC) and 9 paracarcinoma samples (Normal), P-test, was analyzed by using software GraphPad Prism 6.0 mapping, T-test<A difference of 0.05 is statistically significant. The results are shown in FIG. 5.
Fig. 5 shows the expression changes of ZFP92 in renal clear cell carcinoma. As can be seen, ZFP92 expression was significantly reduced in renal clear cell carcinoma.
Having described embodiments of the present invention, the foregoing description is intended to be exemplary, not exhaustive, and not limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments.
Sequence listing
<110> Jiangsu medical profession college
NCPC GENETECH BIOTECHNOLOGY DEVELOPMENT Co.,Ltd.
<120> application of reagent for detecting expression level of ZFP92 and kit
<130> 2103255
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<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaaaggtcct ctacaagcgg g 21
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
gtctgctacc cagggtcctt 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ggcacccagc acaatgaaga 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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actcctgctt gctgatccac 20
Claims (10)
1. The application of the reagent for detecting the expression level of ZFP92 in preparing a preparation for auxiliary diagnosis and treatment of renal clear cell carcinoma and/or prognosis judgment of renal clear cell carcinoma patients.
2. The use of claim 1, wherein the detecting the level of ZFP92 expression comprises detecting the level of mRNA expression of ZFP92 and/or detecting the level of protein expression of ZFP 92.
3. The use of claim 1, wherein the method of detecting the expression level of ZFP92 comprises: detecting the expression quantity of ZFP92mRNA in renal clear cell carcinoma and para-carcinoma tissues by an RT-qPCR method; detecting the expression quantity of ZFP92mRNA in renal clear cell carcinoma and para-carcinoma tissues by a molecular probe technology; the expression level of ZFP92 protein in renal clear cell carcinoma and paracarcinoma tissues is detected by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the agent for detecting the expression level of ZFP92 is an oligonucleotide probe targeting the DNA sequence or mRNA sequence of ZFP92 gene, a PCR primer, or an antibody targeting ZFP 92.
5. The use of claim 4, wherein the agent that detects the expression level of ZFP92 is a nucleic acid molecule having the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
6. A kit for auxiliary diagnosis and treatment of renal clear cell carcinoma and/or prognosis of renal clear cell carcinoma patients, the kit comprising: agents that detect the level of expression of ZFP 92.
7. The kit of claim 6, wherein the reagent for detecting the expression level of ZFP92 is an oligonucleotide probe targeting the DNA sequence or mRNA sequence of ZFP92 gene, a PCR primer, or an antibody targeting ZFP 92.
8. The kit of claim 7, wherein the agent that detects the expression level of ZFP92 is a nucleic acid molecule having the sequence of SEQ ID NO: 1 and SEQ ID NO: 2 in the presence of a primer for real-time fluorescent quantitative PCR.
9. The kit of claim 6, wherein the kit further comprises at least one of the following components: trizol, isopropanol, chloroform, absolute ethanol, RNase-free water, random primers, 5 XM-MLV buffer, dNTPs, RNase inhibitor, M-MLV reverse transcriptase, a primer having the sequence of SEQ ID NO: 3 and SEQ ID NO: 4, and (4) ACTB real-time fluorescence quantitative PCR specific primers of the nucleotide sequence shown in the specification.
10. Use of a kit according to any one of claims 6 to 9 for the preparation of a preparation for the assisted diagnosis and/or prognosis of a patient with renal clear cell carcinoma.
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