阅读说明：本技术 一种piRNA NU9作为检测靶点的儿童胚胎性恶性肿瘤诊断试剂 (Child embryonic malignant tumor diagnostic reagent taking piRNA NU9 as detection target ) 是由 何大维 王璋 于 2020-03-20 设计创作，主要内容包括：本发明公开了一种未知的新piRNA NU9作为检测靶点的儿童胚胎性恶性肿瘤诊断试剂。未知piRNA NU9作为检测靶点在制备儿童肾母细胞瘤、神经母细胞瘤、肝母细胞瘤或髓母细胞瘤诊断试剂中的应用。检测ND5表达量的试剂在制备儿童肾母细胞瘤、神经母细胞瘤、肝母细胞瘤或髓母细胞瘤诊断试剂中的应用。本发明通过研究发现piRNA NU9在肾母细胞瘤、神经母细胞瘤、肝母细胞瘤或髓母细胞瘤的肿瘤组织与正常组织中的表达差异显著,可以做为区分胚胎性恶性肿瘤与正常组织的潜在标志物。(The invention discloses an unknown new piRNA NU9 diagnostic reagent for children embryonic malignant tumor as a detection target. Unknown piRNA NU9 is used as a detection target for preparing a diagnostic reagent for children nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma. Application of a reagent for detecting expression level of ND5 in preparation of a diagnostic reagent for children nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma. According to the invention, researches show that the expression difference of the piRNA NU9 in tumor tissues and normal tissues of nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma is obvious, and the piRNA NU9 can be used as a potential marker for distinguishing embryonic malignant tumors from normal tissues.)

Application of piRNA NU9 as a detection target in preparation of a diagnostic reagent for children nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma.

2. Application of a reagent for detecting expression level of ND5 in preparation of a diagnostic reagent for children nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma.

3. A diagnostic reagent for childhood embryonic malignant tumor using ND5 as detection target point is characterized by comprising a reagent for detecting the expression level of piRNA NU 9.

Technical Field

The invention belongs to the field of biological detection, and relates to a child embryonic malignancy diagnostic reagent taking piRNA NU9 as a detection target, in particular to a child embryonic malignancy diagnostic reagent taking piRNA NU9(TCTCGTGATGAAAACTCTGTCCAGT) as a detection target.

Background

The theory of tumor stem cells (CSCs) suggests that CSCs are the root cause of tumor recurrence and metastasis and resistance to chemoradiotherapy. Understanding the origin and the occurrence process of tumor stem cells helps us to better judge the prognosis of tumor and to perform targeted therapy more accurately. Subject group early stage reprogramming normal FB cells with Piwil2 to establish stable expression Piwil2-GFP labeled tumor stem cells (Piwil2-iCSC), and cell biological behavior tests and tumor stem cell marker tests prove that reprogramming is successful, so that the in vitro CSCs cell model can be used as an ideal in vitro CSCs cell model.

piRNA is a sncRNA of 23-31nt in length, characterized by a uracil ribonucleic acid with a common monophosphate at the 5 'end and a methylation modification at the 3' end^[8]. The action mechanism is mainly combined with PIWI protein (PiWIl2/PiWIl4), the function of inhibiting transposon through the ping-pong effect plays a role in stabilizing gene, and piRNA can also participate in gene regulation through DNA methylation and histone modification. Through the reprogramming of fibroblasts by Piwil2, an in vitro CSCs model is formed, and the fact that the changes of the expression quantity and the function of related piRNA caused by Piwil2 gene overexpression are probably important in the tumorigenesis process is presumed, and the piRNA with differential expression is probably of great value for the tumorigenesis, the tumor diagnosis and the prognosis of the tumor.

Disclosure of Invention

The invention aims to provide a reagent for diagnosing the embryonic malignant tumor of children by taking piRNA NU9 as a detection target against the defects in the prior art.

Another objective of the invention is to provide a diagnostic reagent for child embryonic malignant tumors by using ND5 as a detection target.

The purpose of the invention can be realized by the following technical scheme:

the ND5 is used as a detection target for preparing a diagnostic reagent for children nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma.

Application of a reagent for detecting expression level of ND5 in preparation of a diagnostic reagent for children nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma.

A diagnostic reagent for child embryonic malignant tumors by taking ND5 as a detection target point is characterized by comprising a reagent for detecting the expression level of ND 5.

Has the advantages that:

according to the invention, the unknown piRNA NU9 is found to have significant expression difference between tumor tissues and normal tissues of nephroblastoma, neuroblastoma, hepatoblastoma or medulloblastoma through research, and can be used as a potential marker for distinguishing embryonic malignant tumors from normal tissues, and the reagent for detecting the piRNA can be used for preparing a diagnostic reagent for children nephroblastoma neuroblastoma, hepatoblastoma or medulloblastoma.

Drawings

Figure 1 GO analysis of predicted target genes expressing significantly different pirnas.

FIG. 2 protein interaction analysis of predicted target genes for differential piRNA (a: protein interaction network; b: protein interaction composite score; c: number of protein interactions).

FIG. 3 shows the expression of piRNA NU9, NU13, ND5, ND7, ND9 and their target genes NOP56, RPS8, MTRNR2L1 in nephroblastoma tissue.

Detailed Description

The invention is further described below with reference to the accompanying drawings.

NU9 sequence TCTCGTGATGAAAACTCTGTCCAGT (SEQ ID NO.1)

NU13 sequence TGGAGGTGATGAACTGTCTGAGCCTGACCTT (SEQ ID NO.2)

ND5 sequence ATGTTGGATCAGGACATCCCAATGGTGCAGC (SEQ ID NO.3)

ND7 sequence ATGTTGGATCAGGACATCCCAATGGTGCAGCC (SEQ ID NO.4)

ND9 sequence TTGGATCAGGACATCCCAATGGTGCAGCC (SEQ ID NO.5)

1 specimen and Material

1.1 major reagents and instruments CO2 incubator (Thermo corporation, USA), thermostatic water bath (Shanghai medical instruments factory) cell culture dish (Hyclone corporation, USA), pipette (Eppendorf corporation, Germany), ultracentrifuge (Thermo corporation, USA), fluorescent microscope (Nikon corporation, Japan), fetal bovine serum (Hyclone corporation, USA), F-12 culture medium (Gibico corporation, USA), puromycin (Sigma corporation, USA), Trizol (Thermo corporation, USA), miRNA reverse transcription first strand kit (TIANGGEN corporation, China), miRNA fluorescent quantitative PCR kit (TIANGGEN, China), mRNA reverse transcription first strand kit (Takara corporation, Germany), mRNA fluorescent quantitative PCR kit (Qiagen corporation, Germany)

1.2 cell task group prophase, respectively transfecting primary culture foreskin Fibroblast (FB) with Piwil2-GFP marked lentivirus and no-load GFP marked lentivirus of target genes, and successfully constructing Piwil2 induced tumor stem cells through cytobehavioral and in vitro cell karyotype analysis, wherein the Piwil2 induced tumor stem cells are stored in liquid nitrogen for a long time.

1.3 clinical specimens surgical specimens of patients with nephroblastoma who were admitted to the department of Chongqing medical university affiliated Children hospital urology surgery in 2015 6 to 2019 6 were collected, and were first-line tumor resection and confirmed as nephroblastoma tissue by 2 physicians in the pathology department. The specimens were obtained from 34 children, 15 men and 19 women, with the average age of 29.9 (4.0-104.0. ANG.) months, and the tissue well-differentiated type (FH)27, poorly-differentiated type (uFH)7, stage I7, stage II 9, stage III 11, stage IV 1 and stage V6 according to the American national nephroblastoma study group (NWTS-5) criteria. The tumor tissue of each patient is taken as a tumor group, normal kidney tissue with the distance between the paracarcinoma tissue and the tumor being more than or equal to 3cm is taken as a control tissue, and normal kidney tissue on the same side of the tumor is taken as the bilateral tumor in stage V.

2 method of experiment

2.1 cell culture Piwil2-FB and GFP-FB cells were taken out from a-80 ℃ refrigerator, rapidly rewarming in a 37 ℃ water bath, transferring the cell suspension to a 10ml EP tube, adding 2ml F12 medium, centrifuging, washing, culturing in 10% fetal bovine serum F12 medium (10cm dish), culturing in a 37 ℃ 5% CO2 incubator, changing the medium every 48 hours, and extracting total RNA by trizol digestion when the cells grow to 80% or more fusion degree.

2.2 Total RNA extraction, reverse transcription and qPCR trizol method for extracting total RNA of cells and tissues, digesting the cells in a 10cm culture dish with 1ml trizol for 10min, sucking the cells into a 1.5ml enzyme-removed EP tube (an electric homogenizer fully homogenizes the cells after adding 100 mug/ml trizol to the tissues for 1-2 min), adding 200 mug chloroform, shaking and mixing uniformly, standing at room temperature for 15 min, centrifuging at 4 ℃ for 15 min, sucking the supernatant, adding 500 mug isopropanol, mixing uniformly, standing at room temperature for 10min, centrifuging at 4 ℃ for 10min, discarding the supernatant, adding 1ml 75% ethanol, gently shaking, centrifuging at 4 ℃ for 5 min, discarding the supernatant, vacuum drying for 5-10 min, and adding 50 mug DEPC for dissolving. And performing a NanoDrop ND-1000 spectrophotometer and agarose gel electrophoresis for quality inspection, wherein the standard A260/A280 is between 1.8 and 2.0, and two bright bands of 28s and 18s can be seen in electrophoresis. Total RNA was reverse transcribed into cDNA using miRNA reverse transcription kit (TIANGEN) and mRNA reverse transcription kit (Takara). qPCR primer design was from shanghai life worker (table 1), and qPCR experimental procedures were found in miRNA fluorescence quantification kit (TIANGEN) and mRNA fluorescence quantification kit (Qiagen).

TABLE 1

2.3 second generation sequencing and target Gene prediction the second generation sequencing was performed on the Shanghai boon research organism using 2 sets of samples (Piwil2-FB, GFP-FB). Samples of model species humans were sequenced at high throughput by the single-ended 50bp sequencing mode of the illumina Hiseq2000 sequencing platform with Piwil2-FB (tumor stem cells of Piwil2 transfected FB by lentivirus) as experimental group and GFP-FB (empty GFP-labeled lentivirus transfected FB) as control group. The original data is removed by a primer and an adaptor sequence and subjected to quality inspection on the 3' end of a sequencing fragment, and a fragment with the quality reliable length range of 24-30nt is selected for predicting the piRNA. The prediction of the piRNA adopts a k-mer statistical method to establish a mathematical model for identifying and distinguishing sequences with piRNA characteristics and non-piRNA sequence characteristics, establishes a piRNA sequence characteristic classifier through a machine training learning algorithm, and is used for predicting the piRNA. The screening standard of the differential genes is that P is less than or equal to 0.05 and | logFC | ≧ 1, target gene prediction is carried out on the differential genes through Miranda algorithm, and function analysis is carried out on the target genes.

3 statistical analysis

Statistical analysis and mapping are carried out by using SPSS and Graphpad, data description of abnormal distribution is expressed by using median (quartile interval), expression difference of target genes in matched samples is compared by using wilcoxon test, and Mann-whitney test is used for comparing difference of target genes in tumor tissues of different ages, sexes, stages and types. Differences of P <0.05 were statistically significant.

4 results

4.1 PiRNA expression profile and differential piRNA in Piwll 2-iCSC: the number of significantly different piRNAs selected from Piwll2-iCSC was 230 compared to The piRNA database, with 180 found and reported genes (46 reported in human genes and 134 reported in non-human organisms) and 50 unreported new piRNAs (unknown up-regulation represented by NU and unknown down-regulation represented by ND). Of these, 106 were significantly up-regulated in tumor stem cells (22 of these were unknown), and 124 were significantly down-regulated (28 of these were unknown).

4.2 prediction and enrichment analysis of piRNA target genes: and performing target gene prediction on 230 differentially expressed genes by using a miRanda algorithm, wherein the target genes are predicted by 61 piRNAs and the predicted target genes are 43. GO analysis is carried out on the 43 target genes (P is less than or equal to 0.05), and the biological processes involved in the GO analysis are mainly used for regulating the calcium ion concentration of cells, and the molecular functions are mainly involved in the activity of ATPase and poly-A tail RNA combination (figure 2). The protein interaction network analysis was performed by String database (interaction score ≧ 0.15), in which 8 interacting proteins were present in myosin IXA, 7 interacting proteins were present in profilin 2, 6 interacting proteins were present in protein tyrosine phosphatase D-type receptor (PTPRD), 4 interacting proteins were present in NOP56 ribonucleoprotein (NOP56), 3 interacting proteins were present in 40S ribosomal protein S8(RPS8), and the combination with the highest combined interaction score was NOP56 and RPS8 (0.994). Although the target gene MT-RNR 2-like protein 1(MTRNR2L1) is not in the protein interaction network, 3 piRNAs target the unknown piRNAs in the previous ten downregulations of differential expression levels (FIG. 3).

4.3 expression of range piRNA and its target gene in 34 tissues of patients with nephroblastoma: the piRNA NU13 (position 13 of unknown up-regulated differential piRNA) and its target gene NOP56, the piRNA NU9 (position 9 of unknown up-regulated differential piRNA) and its target gene RPS8, and the piRNA ND5, ND7, ND9 (positions 5, 7, and 9 of unknown up-regulated differential piRNA) and its target gene MTRNR2L1 were selected for qPCR detection in 34 patients with renal cell tumors. The results show that the expression level of NU13 is significantly reduced (P <0.01) in the tumor tissues, and the expression of the target gene NOP56 is not significantly different (P ═ 0.58); the expression level of NU9 is obviously reduced (P <0.01), and the expression of a target gene RPS8 is not obviously different (P ═ 0.29); while the expression levels of ND5, ND7, ND9 and its target gene MTRNR2L1 were all significantly reduced (P <0.01) (FIG. 3). Among them, the expression levels of NOP56 and RPS8 in tumor tissues were correlated with age and sex, and the remaining genes were not correlated with the stage, age and sex of wilms (P > 0.05) (Table 2, 3).

TABLE 2 relationship between expression of piRNA NU9, NU13, ND5, ND7 and ND9 in 34 patients with nephroblastoma and clinical pathological parameters

TABLE 3 relationship between the expression of the target genes MTRNR2L1, NOP56 and RPS8 in 34 patients with nephroblastoma and the clinical pathological parameters

Thus, the expression difference of the piRNA NU9, NU13, ND5, ND7 and ND9 between the nephroblastoma tissue and the normal tissue is large, so the piRNA can be used as a new diagnosis target of embryonic malignant tumor.

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