CN114457164A - Application of reagent for detecting expression level of chromosome 10 open reading frame 81 and kit - Google Patents

Application of reagent for detecting expression level of chromosome 10 open reading frame 81 and kit Download PDF

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CN114457164A
CN114457164A CN202210336170.1A CN202210336170A CN114457164A CN 114457164 A CN114457164 A CN 114457164A CN 202210336170 A CN202210336170 A CN 202210336170A CN 114457164 A CN114457164 A CN 114457164A
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c10orf81
pancreatic cancer
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张小蒙
杨恒月
吴红雁
卓微伟
李艳萍
刘子义
李凤
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Jiangsu Vocational College of Medicine
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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 C10orf81 and a kit. More particularly, the application of the reagent for detecting the expression level of C10orf81 in preparing a preparation for the auxiliary diagnosis of pancreatic cancer and/or the prognosis of a pancreatic cancer patient and a kit for the auxiliary diagnosis of pancreatic cancer and/or the prognosis of a pancreatic cancer patient are provided. The detection result of a clinical sample shows that the expression of C10orf81 in pancreatic cancer is obviously increased compared with that in a para-carcinoma tissue; and high expression of C10orf81 is detrimental to overall survival of pancreatic cancer patients. The reagent for detecting the change of the gene expression can be used for pancreatic cancer prognosis or diagnosis and treatment.

Description

Application of reagent for detecting expression level of chromosome 10 open reading frame 81 and kit
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 C10orf81 in preparation of a preparation for auxiliary diagnosis of pancreatic cancer and/or prognosis of a pancreatic cancer patient, and a kit for auxiliary diagnosis of pancreatic cancer and/or prognosis of a pancreatic cancer patient.
Background
Pancreatic cancer is one of the common malignant tumors in the digestive tract, and is called the king of cancer in the field of tumors. According to the records of the J.Lancet, the five-year survival rate of pancreatic cancer after diagnosis is about 10%, which is one of the worst malignant tumors.
Pancreatic cancer is clinically insidious and atypical, and is a difficult malignancy of the digestive tract to diagnose and treat, with about 90% ductal adenocarcinomas originating from the epithelium of the gland duct. Its morbidity and mortality has increased dramatically in recent years. The early diagnosis rate of pancreatic cancer is low, the operative mortality rate is high, and the cure rate is low. The incidence rate of pancreatic cancer is higher for men than for women, the ratio of men to women is 1.5-2: 1, male patients are more common than for women before menopause, and the incidence rate of postmenopausal women is similar to that of men.
Pancreatic cancer has a high malignancy, a low surgical resection rate, and a poor prognosis. Although surgery remains the primary treatment, combined treatment of pancreatic cancer is required because pancreatic cancer is often found late and the chance of radical cure is lost. To date, as with most tumors, there is no comprehensive treatment regimen that is highly effective and fully applicable. The existing comprehensive treatment still takes surgical treatment as the main part and radiotherapy and chemotherapy as the auxiliary part, and a new method combining biological treatment of immunity, molecules and the like is discussed.
Chromosome 10 open reading frame 81, also known as C10orf81, encodes a protein localized to the cytoplasm and nucleus. At present, no report is found about the role of the C10orf81 gene in the development of pancreatic cancer. The inventor discovers that The high expression of C10orf81 is not beneficial to The prognosis of pancreatic Cancer patients through high-throughput data mining of Cancer and tumor gene maps (TCGA), and further verifies The prognostic effect of The expression difference of C10orf81 on pancreatic Cancer patients through collecting clinical pancreatic Cancer patient samples and follow-up information.
Therefore, based on the above discussion, there is an urgent need to develop more targeted pancreatic cancer prognostic markers to meet clinical needs. With the rapid increase of multigroup high-throughput data, some molecular biological markers are found to be related to pancreatic cancer occurrence and prognosis, which makes it possible to diagnose pancreatic cancer more accurately and effectively.
Disclosure of Invention
The invention aims to discover a novel pancreatic cancer prognostic marker C10orf81, and further provides application of a reagent for detecting the expression level of C10orf81 in preparation of a preparation for pancreatic cancer auxiliary diagnosis and/or pancreatic cancer patient prognosis judgment, and a kit for pancreatic cancer auxiliary diagnosis and/or pancreatic cancer patient prognosis judgment.
The first aspect of the invention provides application of a reagent for detecting the expression level of C10orf81 in preparing a preparation for auxiliary diagnosis of pancreatic cancer and/or prognosis of patients with pancreatic cancer.
Specifically, the expression level of C10orf81 includes detecting the gene expression level of C10orf81 and/or detecting the protein expression level of C10orf 81.
More specifically, the method for detecting the expression level of C10orf81 comprises the following steps: detecting the expression amount of C10orf81 in pancreatic cancer and paracarcinoma tissues by using an RT-qPCR method; detecting the expression quantity of C10orf81 mRNA in pancreatic cancer and paracarcinoma tissues by a molecular probe technology; the change of the expression level of C10orf81 protein in pancreatic cancer tissues was detected by immunohistochemistry or Western-Blot.
More specifically, the reagent for detecting the expression level of C10orf81 is an oligonucleotide probe targeting a DNA sequence encoding C10orf81, a PCR primer, or an antibody targeting C10orf 81.
More specifically, the reagent for detecting the expression level of C10orf81 is a real-time fluorescent quantitative PCR specific primer with the nucleotide sequences shown in SEQ ID NO. 1 and SEQ ID NO. 2.
SEQ ID NO 1 is 5'-AAGGTGTAGATGATGCTAAGGC-3'.
SEQ ID NO 2 is 5'-TGGATACTCTGAGGCAGCAT-3'.
The second aspect of the present invention provides a kit for auxiliary diagnosis of pancreatic cancer and/or prognosis of pancreatic cancer patients, which comprises: a reagent for detecting the expression level of C10orf 81.
Specifically, the reagent for detecting the expression level of C10orf81 is an oligonucleotide probe targeting a DNA sequence encoding C10orf81, a PCR primer, or an antibody targeting C10orf 81.
More specifically, the reagent for detecting the expression level of C10orf81 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.
More specifically, the kit may also contain other conventional reagents for real-time fluorescence quantification.
More specifically, the kit further comprises at least one of the following components: trizol, isopropanol, chloroform, absolute ethyl alcohol, RNase-free water, a random primer, a 5 XM-MLV buffer solution, dNTPs, an RNase inhibitor, M-MLV reverse transcriptase and an ACTB real-time fluorescent quantitative PCR specific primer having nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4.
SEQ ID NO 3 is 5'-GGCACCCAGCACAATGAAGA-3'.
SEQ ID NO. 4 is 5'-ACTCCTGCTTGCTGATCCAC-3'.
The detection result of a clinical sample shows that the expression of C10orf81 in pancreatic cancer is obviously increased compared with that in a para-carcinoma tissue; and high expression of C10orf81 is detrimental to overall survival of pancreatic cancer patients. The reagent for detecting the change of the gene expression can be used for pancreatic cancer prognosis or diagnosis and treatment.
The invention has the beneficial effects that: a novel pancreatic cancer prognostic marker C10orf81 is discovered, so that the application of the reagent for detecting the expression level of C10orf81 in preparing a preparation for pancreatic cancer auxiliary diagnosis and/or pancreatic cancer patient prognosis and a kit for pancreatic cancer auxiliary diagnosis and/or pancreatic cancer patient prognosis are further provided.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
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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 is a TCGA high throughput data analysis of C10orf81 expression in pancreatic cancer. C10orf81 expression was significantly higher in pancreatic cancer tissues than in normal tissues.
FIG. 2 is a TCGA high throughput data analysis of the effect of high expression of C10orf81 on survival in pancreatic cancer patients. High expression of C10orf81 was detrimental to overall survival in pancreatic cancer patients (fig. 2A); high expression of C10orf81 was detrimental to patient disease-free survival (fig. 2B).
FIG. 3 is the expression of C10orf81 in pancreatic cancer tissue. C10orf81 expression was significantly higher in pancreatic cancer tissues than in normal tissues.
FIG. 4 is a graph of the effect of high expression of C10orf81 in pancreatic cancer tissues on survival of pancreatic cancer patients. High expression of C10orf81 is detrimental to overall survival of pancreatic cancer patients.
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 conventional products which are not indicated by manufacturers and are commercially available.
Example 1: this example demonstrates TCGA high throughput data analysis of expression changes in C10orf81 in pancreatic cancer.
1. TCGA high throughput data analysis process.
The user logs in the UALCAN (http:// UALCAN. path. uab. edu/index. html) first page of the TCGA portal, clicks "Analysis", inputs the gene name "C10 orf 81", selects "pancreatetic cancer" under TCGA dataset to search, clicks "Expression", and records the result. And (4) mapping by using GraphPad 6.0 software, wherein the statistical method is T test, and the difference is statistically significant when P is less than 0.05.
2. And (6) obtaining the result.
Expression of C10orf81 was significantly elevated in pancreatic cancer tissues compared to normal tissues, as shown in fig. 1.
Example 2: this example illustrates the relationship of high expression of C10orf81 in TCGA high throughput data analysis with pancreatic cancer prognosis.
1. TCGA high throughput data analysis process.
The TCGA portal site cBioPortal (http:// www.cbioportal.org /), tumor dataset "functional Adenocercinoma" was selected, omics data "mRNA Expression z-Scores (RNA Seq V2 RSEM)" was selected, and gene input "C10 orf 81: and EXP > 2 ", then searching, clicking 'survivval' in a pop-up page, and recording the result. The Kaplan-Meier method draws a survival curve, and log-rank tests the difference of the survival curve, wherein the difference has statistical significance when P is less than 0.05.
2. And (6) obtaining the result.
High expression of C10orf81 was detrimental to overall survival in pancreatic cancer patients, as shown in fig. 2A; high expression of C10orf81 is detrimental to patient disease-free survival, as shown in fig. 2B.
Example 3: this example illustrates the preparation of a reagent for detecting the expression level of C10orf81 for use in preparing a kit (50 reactions) for prognosis of patients with pancreatic cancer.
1）Trizol 50.0mL；
2) 50.0mL of isopropanol;
3) 50.0mL of chloroform;
4) 50.0mL of absolute ethyl alcohol;
5) RNase-free water 5.0mL
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) 50.0 μ L of 200U/. mu.L-MLV reverse transcriptase;
11）ABI 2×PCR Mix 2.0ml；
12) 10.0 mu M C10 and 10orf81 real-time fluorescent quantitative PCR specific primers 30.0 mu L, and the primer sequences are shown in Table 1;
13) the real-time fluorescent quantitative PCR specific primer of 10.0 mu M ACTB is 30.0 mu L, and the primer sequence is shown in the table 1;
table 1: fluorescent quantitative RT-PCR primer sequence
Figure DEST_PATH_IMAGE001
Example 4: this example illustrates the detection of clinical pancreatic cancer tissue sample C10orf 81.
1. And preparing a specimen.
The study was performed with patient informed consent. Clinical information from patients for 48 pancreatic cancer patients was obtained from patient visit records. Pancreatic cancer specimens are divided into two parts: one fraction was frozen immediately in liquid nitrogen and stored at-80 ℃ until RNA extraction was performed, and the other fraction was used for histopathological evaluation.
2. Extraction of total RNA from the tissue.
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 12000rpm at 4 ℃ 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. for dissolution promotion at 65 ℃). OD260 was measured and the RNA concentration was calculated.
3. And (5) reverse transcription.
Each 25. mu.L of reverse transcription system contained 100 pmol 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 ℃.
4. And (5) 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.
5、2-ΔΔCTThe relative expression level of C10orf81 was calculated.
This experiment detected C10orf81 in 48 pancreatic cancer tissues and 20 paracarcinoma tissuesThe relative expression amount was varied. ACTB as reference Gene, the target gene C10orf 81C was 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 level of C10orf81 in each group was calculated using the Power function in Excell tables. Differential expression of pancreatic and paracancerous C10orf81, P, was analyzed using a software GraphPad Prism 6.0 mapping, T-test<A difference of 0.05 is statistically significant.
6. High expression of C10orf81 was prognostic for pancreatic cancer patients.
The follow-up time of the patients is 1-40 months, and the number of successfully received follow-up patients is 48. The expression level of C10orf81 was high when the expression level was 2 times higher than the average expression level of the para-carcinoma tissues, and 16 cases were total, while the expression level of C10orf81 was low, and 32 cases were total. The Kaplan-Meier method draws a survival curve, and log-rank tests the difference of the survival curve, wherein the difference has statistical significance when P is less than 0.05.
7. And (6) obtaining the result.
The detection result of a clinical sample shows that the expression of C10orf81 in pancreatic cancer is obviously increased compared with that in a tissue beside cancer, as shown in figure 3; and high expression of C10orf81 is detrimental to overall survival of pancreatic cancer patients, as shown in fig. 4.
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
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Claims (5)

1. Application of reagent for detecting expression level of chromosome 10 open reading frame 81 (C10 orf 81) in preparation of preparation for prognosis judgment of pancreatic cancer patients.
2. The use of claim 1, wherein the detecting the expression level of C10orf81 comprises detecting the gene expression level of C10orf81 and/or detecting the protein expression level of C10orf 81.
3. The use of claim 1, wherein the method of detecting the expression level of C10orf81 comprises: detecting the expression amount of C10orf81 in pancreatic cancer and paracarcinoma tissues by using an RT-qPCR method; detecting the expression quantity of C10orf81 mRNA in pancreatic cancer and paracarcinoma tissues by a molecular probe technology; the change of the expression level of C10orf81 protein in pancreatic cancer tissues was detected by immunohistochemistry or Western-Blot.
4. The use of claim 1, wherein the agent for detecting the expression level of C10orf81 is an oligonucleotide probe targeting a C10orf81 encoding DNA sequence, a PCR primer, or an antibody targeting C10orf 81.
5. The use of claim 4, wherein the reagent for detecting the expression level of C10orf81 is a real-time fluorescent quantitative PCR specific primer having the nucleotide sequence shown in SEQ ID NO. 1 and SEQ ID NO. 2.
CN202210336170.1A 2022-04-01 2022-04-01 Application of reagent for detecting expression level of chromosome 10 open reading frame 81 and kit Pending CN114457164A (en)

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Application publication date: 20220510