CN110257514B - Novel esophageal cancer blood miRNA marker and application thereof - Google Patents

Abstract

The invention belongs to the field of genetic engineering and tumor medicine, and particularly discloses a blood miRNA marker for auxiliary diagnosis of esophageal cancer and application thereof, wherein the blood miRNA marker is a combination of miR-30d and miR-10; the blood miRNA marker can be used for auxiliary diagnosis of esophageal cancer. The invention also discloses a kit for auxiliary diagnosis of esophageal cancer, which is used for detecting miR-30d and miR-10 in blood and comprises a detection reagent for detecting miR-30d and miR-10, wherein the detection reagent comprises an RNA reverse transcription primer and/or a DNA amplification primer used in a QPCR (quantitative polymerase chain reaction) experiment. The blood miRNA marker combination can be used for screening and diagnosing esophageal cancer, has high detection success rate and good technical reproducibility, is beneficial to reflecting the disease state of asymptomatic high-risk groups of esophageal cancer, and provides support for clinicians to quickly and accurately master the state of illness of patients and timely adopt more personalized prevention and treatment schemes.

Description

Novel esophageal cancer blood miRNA marker and application thereof

Technical Field

The invention belongs to the field of genetic engineering and oncology, and relates to a novel esophageal cancer blood miRNA marker and application thereof.

Background

Esophageal cancer is one of the six most common malignant tumors in the world (incidence and mortality account for 6 th), and China is the country with the highest incidence and mortality of esophageal cancer (incidence of 6 th and mortality of 4 th nationwide). More than half of 50 new esophageal cancer patients occur in China every year worldwide. Different from western people, the epidemiology of esophagus cancer in China is characterized by obvious regional distribution difference, and obvious high and low incidence areas (the incidence rate is different by 100 times) are formed. The Taihang mountain area of the junction of Henan, Hebei and Shanxi provinces, particularly the areas of forest county, Anyang, Hui county and the like in Henan, are China and are also the areas with the highest incidence and death rate of esophageal cancer in the world. The prognosis of cancer is very poor, and the 5-year survival rate of patients in middle and advanced stages is only about 15%. Although the 5-year survival rate of early cancer (T1N0M0) is obviously higher than that of the middle and late stages, the early cancer accounts for about 5% of the patients clinically diagnosed for the first time, and the main reasons are that the patients have no specific symptoms in the early stage and lack of effective indexes and methods for early warning screening and early discovery of high risk groups. Obviously, the establishment of a molecular typing and early-stage discovery molecular marker screening system for high-risk population improves the early-stage esophageal cancer discovery rate, and is the key to reducing the death rate of esophageal cancer.

Esophageal cancer is generally diagnosed by clinical manifestations. In the early stage, professor Shenqiong provides exfoliative cytology examination, brings gospel to current esophageal cancer patients, improves the detection rate of clinical esophageal cancer, and enables a large number of esophageal cancer patients to benefit. With the development of the technology, methods such as X-ray barium meal examination, B-mode ultrasonic examination, CT, magnetic resonance imaging and endoscopy are generated in succession. However, these methods all have some drawbacks in early diagnosis: the imaging examination has certain limitation on the diagnosis of esophageal cancer, and misdiagnosis or missed diagnosis can be caused by improper material taking or insufficient human experience in endoscopy and exfoliative cytology examination. Although some tumor markers, such as carcinoembryonic antigen (CEA) and carbohydrate antigen (e.g., CA19-9, CA74-2, etc.), have been found, they have limited sensitivity and specificity in diagnosis of esophageal cancer, especially in the discovery of early esophageal cancer and in the identification of invasive, painless tumors. Therefore, the search for new markers with high specificity and good sensitivity is increasingly receiving attention at home and abroad.

In recent years, MicroRNAs (i.e., miRNAs) have become a hotspot in the field of tumor molecular biology, and are evolutionarily very highly conserved, small single-stranded RNA molecules with a composition of about 20-25 nucleotides. mirnas do not have an Open Reading Frame (ORF) per se, are a group of short sequence RNAs that do not encode proteins, and are widely present in eukaryotes. miRNA has high time sequence, conservation and tissue specificity, and can regulate the expression of genes related to the growth, development and disease occurrence process of organisms by combining with a non-coding region at the 3' end of target mRNA, degrading or inhibiting the translation of mRNA to cause the post-transcriptional silencing of target genes. Since the discovery of lin-4 and let-7, which are involved in the regulation of nematode timing development, mirnas were introduced in the Science journal of the ten-year major scientific breakthrough in 2002 and 2003, respectively. miRNAs were predicted to regulate 5300 human genes, at least 30% of all genes, in 2005. As research progresses, more and more miRNAs are continuously discovered. The focused mirnas have been primarily free of DNA light masking, changing from "parietal" to "principal", and have brought new challenges to the central position of DNA. In recent years, the relationship between miRNA and various tumors has become a hot spot and a focus of research of scientists, and it has been found that the expression of miRNA through negative regulation gene is highly related to the onset of lung cancer, breast cancer, stomach cancer, pancreatic cancer, etc.

Researches show that the serum/plasma miRNA is very stable, the loss of the serum/plasma miRNA cannot be caused in acid-base environment, boiling, long-term storage, repeated freeze thawing and the like, the micromolecule RNAs are rich in content and easy to quantitatively detect, and the expression of the miRNA in peripheral blood also has tumor correlation and tissue specificity. The serum/plasma miRNA used as a tumor diagnosis and prognosis molecular marker not only has the advantages of small wound, high sensitivity, good repeatability, low cost and convenience, but also can improve the precision of disease diagnosis, tumor classification, prognosis estimation, curative effect and recurrence prediction, and has good clinical application prospect as a tumor marker. In recent years, some miRNAs which are abnormally expressed in the serum/plasma of patients with esophageal cancer have been researched and discovered, wherein 7 serum miRNAs have the potential to become noninvasive biomarkers for diagnosing esophageal cancer, which makes the assumption that serum free miRNA is used as a tumor marker specific to esophageal cancer possible, and whether the expression of the miRNAs in the serum/plasma is consistent with that in tissues or not is still needed to be further researched as the specific marker of esophageal cancer for diagnosing esophageal cancer.

At present, no stable biomarker for diagnosing esophageal cancer is reported, and if serum/plasma miRNAs which are abnormally expressed in esophageal cancer can be screened out as the biomarker and a corresponding diagnostic kit is developed, the diagnosis of esophageal cancer in China can be powerfully promoted.

Disclosure of Invention

Aiming at the problems and the defects in the prior art, the inventor searches a group of miRNAs with high specificity and sensitivity which are highly related to esophageal cancer by separating and researching miRNAs in an esophageal cancer patient and healthy human control serum/plasma matched with the age and the sex of the esophageal cancer patient, develops an esophageal cancer diagnosis kit which can be conveniently applied clinically, provides data support for the screening and the diagnosis of esophageal cancer and provides data support for finding a novel small molecule drug with potential therapeutic value.

In order to realize the purpose of the invention, the technical scheme adopted by the invention is as follows:

the invention provides a blood miRNA marker for auxiliary diagnosis of esophageal cancer, wherein the blood miRNA marker is a combination of miR-30d and miR-10, the sequence of miR-30d is shown in SEQ ID No.8, and the sequence of miR-10 is shown in SEQ ID No. 9.

The invention also provides application of the blood miRNA marker in preparation of an auxiliary diagnosis kit or reagent for esophageal cancer.

According to the application, the kit preferably comprises a detection reagent for detecting miR-30d and miR-10.

The invention also provides application of the detection reagents of miR-30d and miR-10 in preparation of an auxiliary diagnosis reagent or kit for esophageal cancer.

According to the above-mentioned use, preferably, the detection reagent comprises an RNA reverse transcription primer and/or a DNA amplification primer.

According to the above-mentioned use, preferably, the RNA reverse transcription primer is an Oligo (dt) primer; the nucleotide sequences of the DNA amplification primers for detecting miR-30d are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the DNA amplification primers for detecting miR-10 are shown as SEQ ID NO.3 and SEQ ID NO. 4.

The invention also provides a kit for auxiliary diagnosis of esophageal cancer, and the kit is used for detecting miR-30d and miR-10 in blood.

According to the kit, preferably, the kit comprises a detection reagent for detecting miR-30d and miR-10.

According to the above kit, preferably, the detection reagent comprises an RNA reverse transcription primer and/or a DNA amplification primer used in a QPCR experiment, the RNA reverse transcription primer being an Oligo (dt) primer; the nucleotide sequences of the DNA amplification primers for detecting miR-30d are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the DNA amplification primers for detecting miR-10 are shown as SEQ ID NO.3 and SEQ ID NO. 4.

According to the above kit, preferably, the kit further comprises a detection reagent for detecting an internal reference gene; the internal reference gene is SnRNA U6, the detection reagent of the internal reference gene comprises RNA reverse transcription primer and/or DNA amplification primer used in QPCR experiment, the nucleotide sequence of the RNA reverse transcription primer is shown as SEQ ID NO.5, and the nucleotide sequence of the DNA amplification primer used for detecting SnRNA U6 is shown as SEQ ID NO.6 and SEQ ID NO. 7.

According to the above kit, preferably, the kit further comprises a reverse transcription reaction reagent and a PCR amplification reagent, such as reverse transcriptase, a buffer, dNTPs, MgCl₂DEPC water and Taq enzyme, etc.; standards and/or controls may also be included.

Specifically, the technical solution of the present invention to solve the problem includes:

(1) establishing a unified specimen library and a database: standard procedures (SOP) were used to collect blood samples meeting the standards and the system collected complete demographic and clinical data.

(2) Analyzing differential expression profiles of blood miRNA: selecting an esophageal cancer case, comparing the esophageal cancer case with a healthy person with age and gender matched, detecting the expression profile and content of the serum/plasma miRNA, analyzing the commonality and characteristics of the serum/plasma miRNA between the esophageal cancer case and the healthy person comparison, and screening differential expression miRNAs; the selected differentially expressed miRNAs were used for further validation to determine serum/plasma miRNAs associated with the onset of esophageal cancer.

(3) Development of serum/plasma miRNA screening and diagnostic kits: miRNAs diagnostic kits were developed based on specific serum/plasma miRNAs in esophageal cancer cases and healthy human controls.

The quantitative analysis of the serum/plasma miRNA in the step (2) can be completed by RT-PCR, QPCR, Solexa sequencing technology, Tapman Low Densitylarray (TLDA) chip detection and the like. In a specific embodiment of the invention, verification is performed using QPCR.

Verification of differential expression miRNA is carried out by QPCR, and the specific operation steps are as follows:

(1) extracting total RNA of the sample;

(2) reverse transcribing the RNA obtained in step (1) into cDNA;

(3) performing amplification detection on miRNA and a reference gene on a fluorescent real-time quantitative PCR instrument;

(4) the band of interest was determined by melting curve analysis and electrophoresis, and relative quantification was performed by Δ Δ CT.

Compared with the prior art, the invention has the following positive beneficial effects:

(1) the invention adopts miRNAs chip to detect and analyze the miRNA expression spectra of the blood serum/blood plasma of esophagus cancer case and healthy human contrast, obtains the miRNAs differentially expressed in the blood serum/blood plasma between the esophagus cancer case and the healthy human contrast, and the differential expression miRNAs are verified in a large sample group, and a blood miRNA marker combination (combination of miR-30d and miR-10) stably associated with esophageal cancer is obtained by screening, the diagnosis sensitivity of the blood miRNA marker combination to esophageal cancer reaches 90 percent, the specificity reaches 81 percent, and the method is far higher than the existing screening and diagnosis method of esophageal cancer, therefore, the blood miRNA marker combination can be used for clinical screening diagnosis of esophageal cancer, is helpful for reflecting disease states of asymptomatic high risk groups of esophageal cancer, and provides support for clinicians to quickly and accurately master patient conditions and timely adopt more personalized prevention and treatment schemes.

(2) The blood miRNAs are novel biomarkers, are stable, minimally invasive, easy to detect and accurate in quantification, so that the sensitivity and specificity of esophageal cancer diagnosis can be greatly improved by detecting miR-30d and miR-10 expression in serum/plasma, a brand new situation is created for diagnosis and treatment of esophageal cancer, and reference is provided for development of biomarkers for other diseases.

(3) The kit is prepared by detecting the RNA reverse transcription primer and the DNA amplification primer of the blood miRNA marker combination (combination of miR-30d and miR-10) related to the auxiliary diagnosis of the esophageal cancer, has high sensitivity, strong specificity, simple operation and convenient use, can be used for the auxiliary diagnosis of the esophageal cancer and the screening of asymptomatic high risk groups of the esophageal cancer, ensures that the diagnosis of the esophageal cancer is more convenient and easier, lays a foundation for the clinical treatment effect evaluation of the clinician to quickly and accurately screen and diagnose the asymptomatic high risk groups of the esophageal cancer, and provides help for finding a novel small molecular drug target with potential treatment value.

Drawings

Fig. 1 is a box-line graph showing the expression of mirnas in the esophageal cancer case group and the healthy human control group.

FIG. 2 is a ROC curve showing the esophageal cancer case group against a healthy human control group.

Detailed Description

The present invention will be described in further detail with reference to specific examples, but the scope of the present invention is not limited thereto.

The experimental methods in the following examples, which do not indicate specific conditions, all employ conventional techniques in the art, or follow the conditions suggested by the manufacturers; the reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

The first embodiment is as follows: screening of human esophageal cancer-associated miRNA

1. Sample collection and sample data collation

The cases are new cases of esophageal cancer collected in an esophageal cancer focus open laboratory (1973-2018) of Henan province, the first subsidiary hospital of Zhengzhou university, and are confirmed by histopathology. Controls were healthy individuals contemporaneously screened for community disease, matched with case frequency by gender and age (+ -5 years). Samples for research are collected at the same period, and the conditions of sampling, subpackaging and storing are uniform. Through the arrangement of sample data, 200 samples (100 esophageal cancer patients and 100 healthy control groups) meeting the standard are selected by the inventor as experimental samples for miRNA chip detection, and the demographic data, clinical data and other conditions of the samples are systematically collected. Wherein, the selection standard of the sample is as follows:

(1) new cases of esophageal cancer;

(2) before blood sampling, no operation, radiotherapy and chemotherapy is performed;

(3) healthy human controls matched to age, gender of the case.

2. MiRNA chip detection

(1) Extraction of total RNA from peripheral blood:

the total RNA extraction is carried out on the blood of 200 selected samples, and the specific steps are as follows:

firstly, taking frozen blood, fully thawing, adding 3 times volume of erythrocyte lysate, uniformly mixing, standing at room temperature for 10 minutes, and centrifuging for 1 minute at 10000 rmp;

completely absorbing and discarding the supernatant, and collecting the leukocyte precipitate;

③ adding 1ml Trizol into every 100-200 mul of the white blood cell sediment collected by the blood, and standing for 5min at room temperature to ensure that the sample is fully cracked.

fourthly, centrifuging for 10min at 12000rpm at 4 ℃ and taking the supernatant;

adding Trizol, and storing for 5min at room temperature;

sixthly, adding 0.2ml of chloroform, forcibly oscillating the centrifugal tube, fully and uniformly mixing, and standing for 5min-10min at room temperature;

seventhly, after centrifuging at 12000rpm for 15min at a high speed, sucking the upper water phase (sucking 70%) into another new centrifugal tube, taking care not to suck protein substances between the two water phases, transferring into the new tube, adding equal volume of precooled isopropanol at-20 ℃, fully reversing and uniformly mixing, and placing on ice for 10 min;

eighthly, after 15min of high-speed separation at 12000rpm, carefully removing supernatant, adding 75 percent DEPC ethanol according to the proportion of 1ml/ml Trizol to wash and precipitate (preservation at 4 ℃), washing and mixing precipitates, and centrifuging at 12000rpm at 4 ℃ for 5 min;

ninthly, removing the ethanol liquid, placing for 5min at room temperature to fully air-dry the precipitate, and adding DEPC treated water to dissolve the precipitate;

the RNA purity and concentration are measured in Nanodrop2000 ultraviolet spectrophotometer and frozen at-70 deg.C.

(2) And (3) miRNA chip operation:

the miRNA chip is purchased from ABI company, and the miRNA expression profile is detected according to the instruction of the miRNA chip specification.

(3) As a result:

according to the detection result of the miRNA chip, the expressions of miR-30d and miR-10 in the blood of the esophageal cancer patient group are found to be remarkably higher than those of a healthy control group.

Example two: QPCR validation of differentially expressed miRNAs

According to the detection result of the miRNA chip in the first embodiment, miR-30d and miR-10 are selected for further QPCR verification. 100 samples of esophageal cancer and control groups were selected according to the manner of sample collection and sample data organization in example one.

1. Extraction of total RNA from peripheral blood

The specific operation steps of the RNA extraction process are the same as those in the first embodiment, and are not described herein again.

2. Reverse transcription

The total RNA template (10 pg-1. mu.g) extracted in step 1 above was mixed with 2. mu.l of 10 Xbuffer, 2. mu.l of dATP (10mM), 0.5. mu.l of polyA polymerase, 0.5. mu.l of RNase inhibitor and RNase free water (RNase freewater) in a total volume of 20. mu.l, and incubated at 37 ℃ for 1 h. Mu.l of 0.5. mu.g/. mu.l Oligo (dT) RT primer was then added to the reaction tube, and after incubation for 5min at 70 ℃ immediately followed by incubation for at least 2min on ice, the secondary structure of RNA and primer was disrupted. Finally, 20. mu.l of the above reaction mixture was mixed with 4. mu.l of 5 Xbuffer, 1. mu.l of dNTP (10mM), 0.5. mu. l M-MLV reverse transcriptase, 0.5. mu.l of ribonuclease (RNase) inhibitor, 10. mu.l of polyA reaction mixture and 4. mu.l of ribonuclease free water (RNase free water), and incubated at 42 ℃ for 1 h.

3. QPCR reaction

A25. mu.l reaction system was used, with 3 parallel channels per sample, and all amplification reactions were repeated three more times to ensure the reliability of the results. The reaction system of QPCR is: SYBR Green Realtime PCR Master Mix 12.5. mu.l, forward primer (5. mu.M/. mu.l) 1. mu.l, reverse primer (5. mu.M/. mu.l), template cDNA 2.0. mu.l, 8.5. mu.l without enzyme water. The amplification procedure for QPCR was: 10min at 95 ℃; 95 ℃ for 15s, 60 ℃ for 60s, 45 cycles. SYBR Green is used as a fluorescent marker, and PCR reaction is carried out on a LightCycler fluorescent real-time quantitative PCR instrument. The sequence of a forward primer for detecting miR-30d is shown as SEQ ID NO.1, and the sequence of a reverse primer is shown as SEQ ID NO. 2; the sequence of a forward primer for detecting miR-10 is shown as SEQ ID NO.3, and the sequence of a reverse primer is shown as SEQ ID NO. 4. Taking snRNA U6 as an internal reference gene, and detecting that the forward primer sequence of the internal reference gene snRNA U6 is shown as SEQ ID NO. 6; the sequence of the reverse primer is shown as SEQ ID NO. 7. The band of interest was determined by melting curve analysis and electrophoresis, and the Δ Δ CT method was used for relative quantification, the results of which are shown in fig. 1. As can be seen from FIG. 1, the content of miR-30d and miR-10 in the blood of healthy control patients is lower than that of the esophageal cancer patient group. And the detection result is consistent with that of the miRNA chip.

Example three: analysis of diagnostic value of miRNA on esophageal cancer

According to the QPCR result in example II, the expression levels of miR-30d and miR-10 in 2 groups of plasma samples (an esophageal cancer patient group and a healthy control group) are analyzed, a single-side 95% reference value range of the expression amounts of miR-30d and miR-10 in the healthy control group is taken as a standard, the SPSS 21.0 software is used for drawing a ROC curve to evaluate the sensitivity and specificity of prediction, and further the judgment capability of the 2 miRNAs on the esophageal cancer onset is evaluated, and the result is shown in FIG. 2. As can be seen from FIG. 2, when the esophageal cancer patient group and the healthy control group were distinguished by using miRNA-30d alone as an indicator, the AUC value was 0.676, the sensitivity at the optimal cut-off point was 71% and the specificity was 74%. When the miRNA-10 is used alone to distinguish an esophageal cancer patient group from a healthy control group, the AUC value is 0.724, the sensitivity of the optimal critical point is 56%, and the specificity is 70%.

Based on the poor specificity of a single miRNA, the two miRNAs are combined to be used as a miRNA pane, risk prediction is carried out on esophageal cancer patients and healthy control groups again according to cut-off values of a single index to obtain a composite index after the two miRNAs are combined, and an ROC curve graph is manufactured by using sps software (see figure 2). As can be seen from FIG. 2, when the miRNA panle is used for distinguishing the esophageal cancer group from the normal group, the AUC value is 0.804, namely the miR-30d and miR-10 are combined to be used as one miRNA panle, the esophageal cancer patient group and the healthy control group can be separated by 80.4% of AUC, the sensitivity of the optimal critical point is 90%, and the specificity is 81%, so that the combination of miR-30d and miR-10 can well distinguish the healthy control from the esophageal cancer patient, and the sensitivity and the specificity for diagnosing esophageal cancer are higher than those of the miRNA alone.

Example four: preparation of esophagus cancer auxiliary diagnosis kit

The preparation and operation process of the esophageal cancer auxiliary diagnosis kit is based on miRNA chip detection and QPCR quantitative detection technology. The esophageal cancer auxiliary diagnosis kit is used for detecting miR-30d and miR-10 in blood; the kit comprises a detection reagent for detecting miR-30d and miR-10; the detection reagent comprises an RNA reverse transcription primer and a DNA amplification primer used in a QPCR experiment, wherein the RNA reverse transcription primer is an Oligo (dt) primer; the nucleotide sequences of the DNA amplification primers for detecting miR-30d are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the DNA amplification primers for detecting miR-10 are shown as SEQ ID NO.3 and SEQ ID NO. 4. Further, the kit also comprises a detection reagent for detecting the reference gene SnRNA U6, wherein the detection reagent for detecting the reference gene SnRNA U6 comprises an RNA reverse transcription primer and a DNA amplification primer used in a QPCR experiment, the nucleotide sequence of the RNA reverse transcription primer is shown as SEQ ID NO.5, and the nucleotide sequence of the DNA amplification primer for detecting the SnRNA U6 is shown as SEQ ID NO.5 and SEQ ID NO. 6. Further, the kit may further comprise conventional reverse transcription reagents and PCR amplification reagents, such as reverse transcriptase, dNTPs, MgCl₂Double distilled water, Taq enzyme, etc., which are well known to those skilled in the art, and may further contain a standard and a control.

The detection method of the kit comprises the following steps: (1) extracting total RNA of the sample; (2) reverse transcribing the extracted RNA into cDNA; (3) performing amplification detection on miRNA and a reference gene on a fluorescent real-time quantitative PCR instrument; (4) determining a target band through melting curve analysis and electrophoresis, and carrying out relative quantification by a delta Delta CT method; see example three for the specific operation of each step.

The kit has the value that only peripheral blood is needed, other tissue samples are not needed, the expression of miR-30d and miR-10 is detected through the simplest and most specific primers to assist in judging the esophageal cancer, the kit is stable, convenient to detect and accurate in quantification, and sensitivity and specificity of disease diagnosis are greatly improved, so that the kit is put into practice and can help to guide clinical accurate diagnosis.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the present invention, but rather as the following description is intended to cover all modifications, equivalents and improvements falling within the spirit and scope of the present invention.

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Claims (7)

1. The application of the blood miRNA marker in preparing an esophageal cancer auxiliary diagnosis kit or reagent is characterized in that the blood miRNA marker is a combination of miR-30d and miR-10.

2. The use of claim 1, wherein the kit comprises detection reagents for detecting miR-30d and miR-10.

3. The use of claim 2, wherein the detection reagent comprises an RNA reverse transcription primer and/or a DNA amplification primer used in QPCR assay, wherein the RNA reverse transcription primer is an Oligo (dt) primer; the nucleotide sequences of the DNA amplification primers for detecting miR-30d are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the DNA amplification primers for detecting miR-10 are shown as SEQ ID NO.3 and SEQ ID NO. 4.

4. The use of claim 2, wherein the kit further comprises a detection reagent for detecting an internal reference gene; the internal reference gene is SnRNA U6, the detection reagent of the internal reference gene comprises RNA reverse transcription primer and/or DNA amplification primer used in QPCR experiment, the nucleotide sequence of the RNA reverse transcription primer is shown as SEQ ID NO.5, and the nucleotide sequence of the DNA amplification primer used for detecting SnRNA U6 is shown as SEQ ID NO.6 and SEQ ID NO. 7.

5. Application of detection reagents of miR-30d and miR-10 in preparation of auxiliary diagnosis reagents or kits for esophageal cancer.

6. The use of claim 5, wherein the detection reagents comprise RNA reverse transcription primers and/or DNA amplification primers.

7. The use of claim 6, wherein the RNA reverse transcription primer is an Oligo (dt) primer; the nucleotide sequences of the DNA amplification primers for detecting miR-30d are shown as SEQ ID NO.1 and SEQ ID NO.2, and the nucleotide sequences of the DNA amplification primers for detecting miR-10 are shown as SEQ ID NO.3 and SEQ ID NO. 4.

Images