CN102816862A - Application of miRNA (Micro Ribonucleic Acid) 222 to prostatic cancer serological diagnosis kit - Google Patents

Abstract

The invention relates to application of a miRNA (miR-222 shown as SEQ ID NO: 1) to a prostatic cancer serological diagnosis kit, and in particular relates to application of a reagent for detecting miR-222 in a blood sample to preparation of a kit for diagnosing object prostatic cancers. The reagent has specificity to the micro RNA (Ribonucleic Acid) miR-222 and a precursor thereof, and is concretely a probe and a PCR (Polymerase Chain Reaction) primer for detecting the specificity of the microRNAmiR-222 and the precursor thereof. The invention further relates to a prostatic cancer diagnosis kit comprising a polymerase chain reaction reagent, a miR-222 specific primer, a nematode miR-39 reference oligonucleotide sequence, a nematode miR-39 specific primer and a fluorochrome SYBR-GREEN1 which are shown in the specification. When a detection result displays that the level of the miR-222 or the precursor thereof in an object blood is higher than that in a reference serum, the object is prompted to suffer from the prostatic cancers. The kit can be effectively used for auxiliary diagnosis of clinical prostatic cancers.

Description

Application of miRNA 222 in prostate cancer serological diagnostic kit

technical field

The invention relates to the application of a microRNA (micro RNA, miRNA, miR) 222 in a prostate cancer serological diagnostic kit. It belongs to the technical field of biomedical materials. the

Background technique

Prostate cancer (PCa) is a common malignant tumor and one of the leading causes of death among men in European and American countries. the

With the extension of life expectancy and the westernization of lifestyle in my country, the incidence of PCa is increasing year by year, and it is listed as one of the fastest growing tumors in China in the 21st century. Based on the 19 up-regulated miRNAs sequences screened in the previous work, this project will further detect their content in clinical serum samples to find serological biomarkers in patients with human prostate cancer metastasis. the

At present, the research work on the diagnostic indicators of prostate cancer mainly focuses on the following aspects: (I) PSA subtypes and other kallikrein (such as serum KLK2); (II) DNA biomarkers, including some epigenetic biological markers. Markers (such as GSTPi methylation), gene fusions (such as TMPRSS2:ERG fusion) and loss of heterozygosity; (III) RNA biomarkers, such as the non-coding prostate-specific RNA PCA3 (formerly known as DD3) and α-methylacyl CoA racemase (Alpha-methylacyl CoA racemase, AMACR) mRNA expression in prostate cancer; (IV) protein molecular markers, such as PSA, prostate-specific membrane antigen (prostate-specific membrane antigen, PSMA), prostate stem cell antigen (prostate stem cell antigen, PSCA) [14] and so on. Although many candidate biomarkers are being discovered continuously, no new indicators and methods have been applied to clinical detection. the

miRNAs are a new kind of endogenous small RNAs that do not encode proteins. They play an important regulatory role in biological functions such as cell proliferation, apoptosis, and differentiation by inhibiting protein translation. Through miRNAs microarray, Northern blot and real-time PCR, some miRNAs have been found to be up-regulated and down-regulated in various types of human tumors. Studies have reported that miRNA plays an important role in the metastasis of breast cancer and liver cancer. Recent studies have found that there are other tissue-derived miRNAs in human plasma and serum, which can exist in a form that can resist endogenous RNase activity. Determining the content of tumor-derived miRNAs in plasma or serum can be used as one of the means of tumor detection. In recent years, researchers have reported that some early diseases can be diagnosed from the level of miRNA in serum and other body fluids. In 2008, Mitchell reported that miR-141 was 46 times higher in the serum of prostate cancer patients than in the control group, with a sensitivity of 60% and a specificity of 100%. The in-depth study of miRNAs in serum can provide a new and unique perspective for finding disease biomarkers. the

Studies have reported that miR-222 is frequently highly expressed in most human tumors such as liver cancer, pancreatic cancer, bladder cancer, glioblastoma, thyroid papillary carcinoma, and melanoma. By targeting tumor suppressor genes such as p27, p57, PTEN, pro-apoptotic factors Bim, Bmf, etc., it inhibits cell apoptosis, promotes cell growth and migration, and participates in the occurrence and development of tumors. The current research results suggest that miRNA-222 may be an oncogenic miRNA, which plays a crucial role in the occurrence and development of various human tumors. the

Contents of the invention

The purpose of the present invention is to provide the application of the reagent for detecting miR-222 in blood in the preparation of a kit for the serological diagnosis of prostate cancer. the

The present invention uses our invented xenograft model and next-generation sequencing technology to conduct in-depth research on the transcriptomics of miRNAs in metastatic and non-metastatic prostate cancer xenograft tissue lines, and initially finds that miR-222 is related to the malignancy of prostate cancer (Watahiki et al., PLOS One 2011). the

We optimized the method of extracting miRNA from serum and the method of detecting miRNA by real-time quantitative PCR; collated and collected the pathological data and serum samples of more than 100 prostate cancer patients; The detection of 19 miRNA sequences up-regulated in clinical serum samples revealed that the levels of miR-222 in the serum and tissues of prostate cancer patients were significantly increased. The invention has an important application prospect in the development of a serological clinical diagnosis scheme for prostate cancer in the Chinese population. the

The present invention firstly provides the application of a reagent for detecting microRNA miR-222 and its precursor in a blood sample in the preparation of a test kit for diagnosing and selecting a treatment plan for prostate cancer. 222 and its precursor specific reagents, specifically: PCR primers and probes specific for miR-222 and its precursor detection and the gene chip prepared by using the probe. the

The sequence of the miR-222 is shown in SEQ ID NO: 1;

In a preferred example, the specific primer is selected from the sequence shown in SEQ ID NO: 2;

In a preferred example, the specific probe is selected from the sequence shown in SEQ ID NO:5. the

The present invention diagnoses whether a subject suffers from prostate cancer by detecting the content of miR-222 in blood. the

In a preferred example, the determination of miR-222 by real-time quantitative PCR method includes: (1) extracting RNA in the blood sample of the subject, and adding artificially synthesized nematode miR-39 oligonucleotide as a reference; ( 2) cDNA was synthesized by reverse transcription method; (3) the content of miR-222 in the sample was detected by real-time quantitative PCR method; (4) the content of nematode miR-39 was used as a reference for normalization. If the content of miR-222 in the serum of the subject is significantly higher than that of the normal control group, it is diagnosed that the subject is suffering from prostate cancer. the

When the detection result shows that the level of the microRNA miR-222 or its precursor in the subject's blood is higher than the level in the control serum, it is suggested that the subject suffers from prostate cancer. the

Secondly, the present invention also provides a diagnostic kit for prostate cancer, the target nucleic acid detected by the kit is miR-222 having the polynucleotide sequence shown in SEQ ID NO: 1, specifically comprising:

(1) Polymerase chain reaction reagents; including hot start Taq DAN polymerase (2.5U/ul) and its reaction buffer, dNTP (10mM);

(2) miR-222-specific primers; in a preferred example, the miR-222-specific primers are selected from the sequence shown in SEQ ID NO: 2;

(3) Nematode miR-39 reference oligonucleotide sequence: 5'-UCACCGGGUGUAAAUCAGCUUG-3' (SEQ ID NO: 3);

(4) C. elegans miR-39 specific primer: 5'-CCGGGTGTAAATCAGCTTGAAA-3' (SEQ ID NO: 4);

(5) Fluorescent dye SYBR-GREEN 1;

(6) Instructions for use. the

Advantages and beneficial effects of the present invention:

Using the reagents provided in this kit, combined with RNA extraction reagents and general miRNA reverse transcription reagents well-known to researchers in the field, the present invention can specifically detect the content of miR-222 in serum, and is effectively used as an auxiliary treatment for clinical prostate cancer. diagnosis. the

Description of drawings

Figure 1 is the real-time PCR amplification curve and melting curve of internal reference miRNA and target miRNA, it can be seen that the melting curves are all single peaks, and no non-specific peaks appear;

Figure 2 is the expression level of miR-222 detected by qRT-PCR in the serum of normal people and prostate cancer patients. Column height=x ^- +SE;

Figure 3 is the ROC curve analysis of the relative expression level of miR-222 in the serum of prostate cancer patients. the

Figure 4 is the miR-222 FISH photo of prostate tissue (200×) A: Normal prostate B: PCa-1 (gleason score: 6 points) C: PCa-2 (gleason score: 9 points). the

Detailed ways

Example 1

The present invention firstly provides the application of a reagent for detecting microRNA miR-222 and its precursor in a blood sample in the preparation of a test kit for diagnosing and selecting a treatment plan for prostate cancer. 222 (as shown in SEQ ID NO: 1) and its precursor specific reagents, specifically: PCR primers and probes specific for miR-222 and its precursor detection and the gene prepared using the probe chip. the

Described specific primer is selected from the sequence shown in SEQ ID NO:2;

Described specificity probe is selected from the sequence shown in SEQ ID NO:5. the

Embodiment 2, prostate cancer diagnostic kit

The target nucleic acid detected by the kit of the present invention is miR-222 having a polynucleotide sequence shown in SEQ ID NO: 1, specifically comprising:

(1) Polymerase chain reaction reagents; including hot start Taq DAN polymerase (2.5U/ul) and its reaction buffer, dNTP (10mM);

(2) miR-222-specific primers; in a preferred example, the miR-222-specific primers are selected from the sequence shown in SEQ ID NO: 2;

(3) Nematode miR-39 reference oligonucleotide sequence: 5'-UCACCGGGUGUAAAUCAGCUUG-3' (SEQ ID NO: 3);

(4) C. elegans miR-39 specific primer: 5'-CCGGGTGTAAATCAGCTTGAAA-3' (SEQ ID NO: 4);

(5) Fluorescent dye SYBR-GREEN 1;

(6) Instructions for use. the

Embodiment 3, diagnosis effect experiment

1. Using our invented xenograft model (i.e. transplanting prostate cancer tissue into the renal capsule of immunodeficient mice, the obtained tissue line is highly correlated with the pathological characteristics of clinical tumors) and next-generation sequencing technology, the metastatic and The transcriptomics of miRNAs in non-metastatic prostate cancer xenograft tissue lines (surgical specimens from the same prostate cancer patient) has been intensively studied. By comparison, we identified 104 miRNAs with differential expression, in addition to some known miRNAs and their isoforms (isomiRs), there are some new miRNAs that have not been discovered yet. Among them, 21 miRNAs have been found to be related to the occurrence of prostate cancer, and 5 miRNAs have been reported to play a role in prostate cancer metastasis, which also proves the effectiveness of our research methods from the side. In addition to those miRNAs that have been proven to be associated with prostate cancer metastasis, we also found 36 miRNAs that have not been reported so far (Watahiki et al., PLOS One 2011), which may have potential as a role for prostate cancer in Chinese population. Serological biomarkers of miRNAs in cancer patients. the

2. Extraction of trace miRNA in serum

Since the content of miRNA in serum is very small, in the early stage of the experiment, we consulted a large number of literatures and tried the purification column kit extraction method and liquid-phase Trizol extraction method to extract miRNA. It is found that the quantity and purity of miRNA extracted by Trizol method are better (less protein content and salt content of miRNA). In the Trizol method, we have optimized two aspects: (1) Serum contains a lot of protein, and the content of Trizol can be increased to fully denature the contained protein; and use phenol/chloroform/isoamyl alcohol Perform multiple extractions to fully remove the contained protein; (2) Because the molecular weight of miRNA is very small, it is not easy to precipitate in the step of adding isopropanol precipitation, and the precipitation time can be extended appropriately to make Sufficient precipitation of miRNA increases the yield of miRNA. the

3. Detection method of trace miRNA in serum

cDNA was synthesized by reverse transcription using the All-in-One ^TM miRNA First-Strand cDNA Synthesis Kit.

According to the characteristics of miRNA and the principle of PCR amplification, the content of miR-222 and nematode miR-39 was detected on MJ Opticon II quantitative PCR instrument with the invented kit. The relative expression of miRNAs was calculated using the 2- ^ΔΔCt method.

4. Detect the content of miRNAs that are up-regulated in prostate cancer tissue in the serum of prostate cancer patients

(1) We sorted out more than 100 clinical data and patient medical records provided by the Urology Institute of the Second Affiliated Hospital of Tianjin Medical University, and screened out 28 cases of normal people and 49 cases of high grade (7-9 points) Medical records of prostate cancer patients, medical records of 13 low-grade prostate patients, and blood samples were obtained. the

(2) miRNA extraction was performed on these clinical samples, and the following results were obtained by QRT-PCR detection:

RNA was extracted from the collected serum samples, and the expression level of miRNA was detected by reverse transcription-real-time quantitative PCR using PCR primers specific to miR-222. The PCR primers used can specifically and efficiently amplify the sequence to be detected (Figure 1). The obtained data were analyzed by independent sample T test with SPSS software, and the expression of miR-222 in the serum of prostate cancer patients was increased (Figure 2). the

Table 1 The relative expression multiples of miR-222 in the serum of the normal group and the prostate cancer group

(3) Sensitivity and specificity analysis

SPSS software was used to plot the ROC curve of the qRT-PCR data of miR-222, and analyze its sensitivity and specificity in detecting the serum of the prostate cancer group: when the threshold value of miR-222 was 4.06, the sensitivity and specificity were 40.3% and 88.6%, respectively. %(image 3). the

5. The expression level of miR-222 in prostate tissue

In this experiment, using a digoxin-labeled oligonucleotide probe selected from the sequence shown in SEQ ID NO: 5, the fluorescence in situ hybridization technique well known to those skilled in the art was used to detect the expression of miR-222 in prostate cancer tissue. in the expression. First, hybridize the specific probe with the mature miRNA in the paraffin tissue section, then incubate with rhodamine-labeled anti-digoxigenin antibody, and stain the nucleus with DAPI, so that the hybrid is labeled with a fluorescent label and detected under a fluorescent microscope It can be located or quantitatively analyzed. the

The hybridization detection of this probe confirmed that the signal of miR-222 in prostate cancer tissue was more and stronger than that of the fluorescent probe in normal tissue, confirming that the expression of miR-222 in prostate cancer tissue was increased (Figure 4). the

Claims (3)

1. The reagent for detecting microRNA miR-222 and its precursor in the blood sample is used in the preparation of the test kit for object prostate cancer diagnosis and treatment scheme selection, and described reagent is to microRNA miR-222 and its precursor Specific reagents for miR-222 and its precursors, specifically: PCR primers and probes specific for miR-222 and its precursors, and gene chips prepared using the probes. 2. The application according to claim 1, wherein the sequence of the miR-222 is shown in SEQ ID NO:1. 3. A diagnostic kit for prostate cancer, characterized in that the target nucleic acid detected by the kit is miR-222 with a polynucleotide sequence shown in SEQ ID NO: 1, specifically comprising: (1) Polymerase chain reaction reagents; (2) miR-222-specific primers, as shown in SEQ ID NO:2; (3) The nematode miR-39 reference oligonucleotide sequence, as shown in SEQ ID NO: 3; (4) nematode miR-39 specific primer, as shown in SEQ ID NO:4; (5) Fluorescent dye SYBR-GREEN 1; (6) Instructions for use.

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