CN114262732A - Application of reagent for detecting expression level of C11orf16 and kit - Google Patents
Application of reagent for detecting expression level of C11orf16 and kit Download PDFInfo
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- CN114262732A CN114262732A CN202111610775.7A CN202111610775A CN114262732A CN 114262732 A CN114262732 A CN 114262732A CN 202111610775 A CN202111610775 A CN 202111610775A CN 114262732 A CN114262732 A CN 114262732A
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Abstract
The invention belongs to the technical field of biology, and relates to application of a reagent for detecting the expression level of C11orf16 and a kit. More particularly, the invention relates to application of a reagent for detecting the expression level of C11orf16 in preparation of a preparation for auxiliary diagnosis and treatment of hepatocellular carcinoma and/or prognosis of patients with hepatocellular carcinoma, and a kit for auxiliary diagnosis and treatment of hepatocellular carcinoma and/or prognosis of patients with hepatocellular carcinoma. The invention proves that the expression of C11orf16 in hepatocellular carcinoma is remarkably increased, the high expression of C11orf16 is respectively associated with the overall survival time (OS) and the disease-related survival rate (DSS) of hepatocellular carcinoma patients to be reduced, which shows that C11orf16 can be used for the auxiliary diagnosis and treatment of hepatocellular carcinoma and/or the prognosis judgment of hepatocellular carcinoma patients, and a C11orf16 detection kit is designed based on the method.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a reagent for detecting the expression level of C11orf16 in preparation of a preparation for auxiliary diagnosis and treatment of hepatocellular carcinoma and/or prognosis of a patient with hepatocellular carcinoma, a kit for auxiliary diagnosis and treatment of hepatocellular carcinoma and/or prognosis of a patient with hepatocellular carcinoma, and application of the kit.
Background
Hepatocellular Carcinoma (LIHC) is one of the most common malignancies of the digestive system, with an increasing global mortality rate. Many risk factors, including viral infection, fibrosis, alcohol use and metabolic disorders, induce genetic or phenotypic changes and contribute to tumor progression. The complexity of the etiology increases the difficulty of the research of the molecular mechanism of the hepatocellular carcinoma. The early treatment method of the liver cell liver cancer comprises liver transplantation, tumor resection or ablation. Since prognosis of hepatocellular carcinoma is poor and 5-year survival rate of patients is particularly low, early discovery and more personalized treatment of tumors are new targets for hepatocellular carcinoma treatment. Identification of biomarkers and exploration of molecular mechanisms are key steps. Therefore, there is a need to explore deeper cellular mechanisms, which may provide potential diagnostic targets for the treatment of hepatocellular carcinoma.
Disclosure of Invention
The invention aims to provide a novel hepatocyte liver cancer marker (C11orf16), and further provides application of a reagent for detecting the expression level of C11orf16 in preparation of a preparation for adjuvant diagnosis and treatment of hepatocyte liver cancer and/or prognosis of a hepatocyte liver cancer patient, a kit for adjuvant diagnosis and treatment of hepatocyte liver cancer and/or prognosis of a hepatocyte liver cancer patient and application of the kit.
In order to achieve the above object, the first aspect of the present invention provides an application of a reagent for detecting an expression level of C11orf16 in preparing a preparation for adjuvant diagnosis and treatment of hepatocellular carcinoma and/or prognosis of a patient with hepatocellular carcinoma.
Further, the detecting the expression level of C11orf16 includes detecting the expression level of mRNA of C11orf16 and/or detecting the expression level of protein of C11orf 16.
More specifically, the method for detecting the expression level of C11orf16 comprises the following steps: detecting the expression quantity of C11orf16mRNA in liver cancer and para-cancer tissues of liver cells by an RT-qPCR method; detecting the expression quantity of C11orf16mRNA in liver cancer and para-cancer tissues of liver cells by a molecular probe technology; detecting the expression level of C11orf16 protein in liver cancer and para-carcinoma tissues of liver cells by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of C11orf16 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C11orf16 gene, a PCR primer, or an antibody targeting C11orf 16.
According to the present invention, preferably, the reagent for detecting the expression level of C11orf16 is a nucleic acid 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’-CCTTGGGAAGCCAGGAACAA-3’，SEQ ID NO：1
5’-GCTTTCGAGACCAGCTCCTT-3’，SEQ ID NO：2
The second aspect of the present invention provides a kit for assisted diagnosis and treatment of hepatocellular carcinoma and/or prognosis of hepatocellular carcinoma patients, the kit comprising: reagents for detecting the expression level of C11orf 16.
Further, the reagent for detecting the expression level of C11orf16 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C11orf16 gene, a PCR primer, or an antibody targeting C11orf 16.
Specifically, the reagent for detecting the expression level of C11orf16 is a reagent having the 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 invention discloses a protein coding gene C11orf16, which proves that the expression of C11orf16 in hepatocellular carcinoma is remarkably increased, the high expression of C11orf16 is respectively associated with the overall survival time (OS) and the disease-related survival rate (DSS) of hepatocellular carcinoma patients, which indicates that C11orf16 can be used for the auxiliary diagnosis and treatment of hepatocellular carcinoma and/or the prognosis judgment of hepatocellular carcinoma patients, and a C11orf16 detection kit is designed based on the C11orf 16.
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 expression change of the target gene C11orf16 in hepatocellular carcinoma. (A) Results of unpaired samples show that C11orf16 expression is significantly up-regulated in hepatocellular carcinoma tissues; (B) the matched sample results show that the expression of C11orf16 in the liver cancer tissue of the liver cell is obviously up-regulated.
Fig. 2 shows the correlation of C11orf16 expression with Overall Survival (OS) in patients in hepatocellular carcinoma. High expression of C11orf16 was associated with decreased OS in patients with hepatocellular carcinoma.
FIG. 3 shows the correlation of C11orf16 expression in hepatocellular carcinoma with patient Disease-related survival (DSS). High expression of C11orf16 was associated with decreased DSS in patients with hepatocellular carcinoma.
Fig. 4 shows the expression change of C11orf16 in hepatocellular 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 demonstrates that expression of the target gene C11orf16 is significantly down-regulated in hepatocellular carcinoma.
Differential analysis of C11orf16 expression in hepatocellular carcinoma:
1. data downloading and sorting:
the UCSC XENA website (https:// xenoxybronower. net/datapages /) is used for downloading TCGA liver cell liver cancer expression profile data and clinical data, and the C11orf16 expression profile data is subjected to log2 transformation and then subjected to group comparison.
2. Differential analysis of C11orf16 expression in hepatocellular carcinoma
The difference in C11orf16mRNA levels between paired and unpaired samples of hepatocellular carcinoma patients was analyzed using the R software package, and the ggplot2 package was used for visualization, as shown in fig. 1.
FIG. 1 shows the expression change of the target gene C11orf16 in hepatocellular carcinoma. (A) Results of unpaired samples show that C11orf16 expression is significantly up-regulated in hepatocellular carcinoma tissues; (B) the matched sample results show that the expression of C11orf16 in the liver cancer tissue of the liver cell is obviously up-regulated. P < 0.001.
Example 2
This example demonstrates that high expression of C11orf16 in hepatocellular carcinoma correlates with decreased overall survival in patients.
Correlation analysis of C11orf16 expression in hepatocellular carcinoma with overall patient survival:
the optimal cutoff value of C11orf16 was calculated using the R software package maxstat, the patients were divided into two groups with high and low expression of C11orf16, the prognosis difference of the two groups was further analyzed using the survivfit function of the R software package survival, and the significance of the prognosis difference between the samples of the different groups was evaluated using the logrank test method, as shown in FIG. 2.
Fig. 2 shows the correlation of C11orf16 expression with Overall Survival (OS) in patients in hepatocellular carcinoma. High expression of C11orf16 was associated with decreased OS in patients with hepatocellular carcinoma.
Example 3
This example is used to verify that high expression of C11orf16 in hepatocellular carcinoma is associated with decreased survival rates associated with patient disease.
Correlation analysis of C11orf16 expression in hepatocellular carcinoma with survival rates associated with patient disease:
the optimal cutoff value of C11orf16 was calculated using the R software package maxstat, based on which patients were divided into two groups of C11orf16 high and low expression groups, the prognosis difference of the two groups was analyzed using the survivfit function of the R software package survival, and the significance of the prognosis difference between the samples of the different groups was evaluated using the logrank test method. As shown in fig. 3.
FIG. 3 shows the correlation of C11orf16 expression in hepatocellular carcinoma with patient Disease-related survival (DSS). As can be seen, high expression of C11orf16 was associated with decreased DSS in hepatocellular carcinoma patients.
Example 4
This example illustrates the preparation of a kit for detecting the expression level of C11orf 16.
1. Primer design
PCR primers were designed based on the CDS region sequence of the transcript of the human C11orf16 Gene (Gene ID:56673), and the sequences of the primers 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 C11orf16 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. RT-PCR identification of expression change of C11orf16 in hepatocellular carcinoma
(1) Extraction of total RNA from tissues: the true bookThe assay 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 NSMCE 2: 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), the relative expression of NSMCE2 in each group was calculated using the Power function in the Excell table. The expression difference between 9 hepatoma liver cells (LIHC) samples and 9 paracarcinoma samples (normal), P, was analyzed by using software GraphPad Prism 6.0 for mapping and T test<A difference of 0.05 is statistically significant. The results are shown in FIG. 4.
Fig. 4 shows the expression change of C11orf16 in hepatocellular carcinoma. It can be seen that C11orf16 expression is significantly reduced in hepatocellular 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> New countryside medical college
<120> application of reagent for detecting expression level of C11orf16 and kit
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gctttcgaga ccagctcctt 20
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ggcacccagc acaatgaaga 20
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actcctgctt gctgatccac 20
Claims (10)
1. The application of the reagent for detecting the expression level of C11orf16 in preparing a preparation for auxiliary diagnosis and treatment of liver cell cancer and/or prognosis judgment of liver cell cancer patients.
2. The use of claim 1, wherein the detecting the expression level of C11orf16 comprises detecting the mRNA expression level of C11orf16 and/or detecting the protein expression level of C11orf 16.
3. The use of claim 1, wherein the method of detecting the expression level of C11orf16 comprises: detecting the expression quantity of C11orf16mRNA in liver cancer and para-cancer tissues of liver cells by an RT-qPCR method; detecting the expression quantity of C11orf16mRNA in liver cancer and para-cancer tissues of liver cells by a molecular probe technology; detecting the expression level of C11orf16 protein in liver cancer and para-carcinoma tissues of liver cells by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the reagent for detecting the expression level of C11orf16 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C11orf16 gene, a PCR primer, or an antibody targeting C11orf 16.
5. The use of claim 4, wherein the reagent for detecting the expression level of C11orf16 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.
6. A kit for auxiliary diagnosis and treatment of hepatocellular carcinoma and/or prognosis of patients with hepatocellular carcinoma comprises: reagents for detecting the expression level of C11orf 16.
7. The kit according to claim 6, wherein the reagent for detecting the expression level of C11orf16 is an oligonucleotide probe targeting a DNA sequence or an mRNA sequence of the C11orf16 gene, a PCR primer, or an antibody targeting C11orf 16.
8. The kit according to claim 7, wherein the reagent for detecting the expression level of C11orf16 is a nucleic acid 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 the kit according to any one of claims 6 to 9 for the preparation of a preparation for the adjuvant treatment of hepatocellular carcinoma and/or the prognosis of patients with hepatocellular carcinoma.
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