CN107674917B - A kind of UCR sequence highly expressed in B cell lymphoma tissue, kit and detection method - Google Patents

Abstract

Translated fromChinese

本发明公开了一种检测在B细胞淋巴瘤组织中高表达的UCR序列、试剂盒及检测方法。本发明在系统探索B细胞淋巴瘤的UCR表达谱后，发现与B细胞淋巴瘤侵袭转移特异性相关的一组UCRs，结果筛选到一条在B细胞淋巴瘤组织中显著高表达的UCR，即uc.189。本发明探索出与B细胞淋巴瘤发生发展特异性相关的超保守基因序列189号，作为B细胞淋巴瘤药物的应用。

The invention discloses a UCR sequence highly expressed in B-cell lymphoma tissue, a kit and a detection method. After systematically exploring the UCR expression profile of B-cell lymphoma, the present invention finds a group of UCRs specifically related to the invasion and metastasis of B-cell lymphoma, and as a result, a UCR that is significantly highly expressed in B-cell lymphoma tissue is screened, namely uc .189. The present invention explores the ultra-conservative gene sequence No. 189 specifically related to the occurrence and development of B-cell lymphoma, which is used as a B-cell lymphoma drug.

Description

UCR sequence, kit and detection method for detecting high expression in B cell lymphoma tissue

Technical Field

The invention belongs to the field of tumor molecular biology, and particularly relates to a UCR sequence, a kit and a detection method for detecting high expression in B cell lymphoma tissues.

Background

Malignant lymphoma is one of ten malignant tumors in China, and the morbidity and mortality are in the front of the tumor. The tumor is divided into Hodgkin lymphoma and non-Hodgkin lymphoma according to clinical pathological characteristics, the Hodgkin lymphoma is a single tumor cell disease and can be cured by reasonably and effectively treating most cases, while the non-Hodgkin lymphoma has high heterogeneity and consists of a plurality of subtypes, wherein the B cell lymphoma is a type of lymphoma with high invasiveness and recurrence and is a main reason for treatment failure and death of patients of the B cell lymphoma. For the research of molecular mechanism of lymphoma, researchers are constantly searching, for example, translocation occurs in chromosome 3q27 of bcl-6 gene of B cell located in germinal center, so that cell differentiation is inhibited, and proliferation capacity is strongest; the protooncogenes (pim-1, myc, RhoH/TTF and PAX5) in 50% of non-Hodgkin lymphoma cells undergo high frequency mutations, and these genes control tumor cell growth, proliferation, apoptosis and metastasis. In addition, the occurrence and development of B lymphoma cells are influenced by mutation of p53 gene, deletion of p16 gene, and amplification of rel and myc genes.

uc.189 is a member of super-conserved gene Sequences (UCRs), is a long-chain non-coding RNA (LncRNA) absolutely highly conserved in the biological evolution, and the gene sequence of the long-chain non-coding RNA maintains high homology in high-class organisms such as mice, rats and the like. Research shows that UCRs transcribed into RNA exert their specific biological functions, namely, regulate and control the expression of functional genes by regulating other RNAs, participate in the biological processes of growth and development, apoptosis, cell cycle, invasion and metastasis and the like of tumor cells, and are becoming new hotspots for diagnosis and research of tumors at present. Many UCRs have been identified to date as differentially expressed and performing important regulatory functions in diseases including bladder cancer, breast cancer, colorectal cancer, and inflammatory bowel disease. However, no literature reports exist on the research on the UCR expression in B cell lymphoma and the functionality of related indexes thereof.

Disclosure of Invention

In view of the above technical problems, the present invention provides a UCR sequence, a kit and a detection method for detecting high expression in B cell lymphoma tissue. According to the invention, after the UCR expression profile of the B cell lymphoma is systematically explored, a group of UCRs related to the invasion and metastasis specificity of the B cell lymphoma is found, and a UCR which is obviously and highly expressed in the B cell lymphoma tissue, namely uc.189, is screened as a result. The invention explores the super-conservative gene sequence No. 189 related to the transfer specificity of B cell lymphoma, and the super-conservative gene sequence is used as a B cell lymphoma medicine.

The technical scheme for solving the technical problems is as follows:

in order to systematically research new UCRs closely related to the generation and development of B cell lymphoma, 20B cell lymphomas and 15 matched normal tissue specimens are obtained, fresh specimens are obtained and are frozen and stored in a liquid nitrogen tank, and after the fresh specimens are collected, Arraystar Human T-UCR chip 2.0 of Shanghai Kangbio Limited company is adopted to detect and screen two UCRs highly expressed in B cell lymphoma tissues, wherein uc.189 is obviously highly expressed. The gene sequence is shown in a sequence table SEQ ID NO. 1. And in the later stage, the expression of the UCR in the sample is remarkably up-regulated by 106 pairs of B cell lymphomas and matched normal tissue samples qRT-PCR verification. The UCR is expected to become a marker for diagnosis and prognosis judgment of lymphoma, and provides a new target for treatment of the lymphoma.

The invention aims to provide a uc.189 sequence for detecting high expression in B cell lymphoma tissues, which has a nucleotide sequence shown as SEQ ID NO.1 in a sequence table.

The invention also provides a detection method of the uc.189 sequence, and a preparation for auxiliary diagnosis or curative effect prediction of B cell lymphoma is prepared according to the sequence. Namely exploring the application of the compound in preparing the medicine for the B cell lymphoma.

According to the invention, 3 pairs of primers are designed according to the uc.189 sequence for detecting the uc.189 sequence.

Pair 1 upstream primer: SEQ ID NO.2

Pair 1 downstream primer: SEQ ID NO.3

Pair 2 upstream primer: SEQ ID NO.4

Pair 2 downstream primer: SEQ ID NO.5

Pair 3 upstream primer: SEQ ID NO.6

Pair 3 downstream primer: SEQ ID NO.7

The invention designs and synthesizes a detection primer group for qRT-PCR according to the uc.189 sequence. The primer group is suitable for detection of SYBR Green, TaqMan probes, molecular beacons, double-hybrid probes, composite probes and the like.

The preferred primer sets for dye qRT-PCR detection are respectively:

pair 2 upstream primer: SEQ ID NO.4

Pair 2 downstream primer: SEQ ID NO.5

The invention provides a dye qRT-PCR kit for detecting UCR expression level, which comprises the following components: the kit comprises a specific upstream primer sequence, a specific downstream primer sequence, a DNA template, a fluorescent dye and qRT-PCR reaction liquid, wherein the specific upstream primer and the specific downstream primer comprise SEQ ID NO.4 and SEQ ID NO. 5.

The qRT-PCR reaction solution comprises buffer solution, Taq enzyme and Mg²⁺And dNTPs.

SYBR Green II is preferred for fluorescent dyes, and hot-start enzyme is preferred for Taq enzyme.

The invention also discloses a detection method of UCR in B cell lymphoma, which comprises the steps of extraction of sample total RNA, preparation of sample cDNA and amplification of uc.189. The specific contents are as follows:

1) extraction of total RNA of the sample: extracting total RNA of B cell lymphoma tissue or tumor according to the required reagent and procedure of TRIZOL (R) reagent (product number 15596018) of ThermoFisher Scientific company; and quantified using a NanoDropND 1000 micro uv-vis spectrophotometer (NanoDrop Technologies, Wilmington,

delaware) quantified the purity and concentration of the extracted RNA.

2) Preparation of sample cDNA: cDNA was synthesized by reverse transcription of the extracted total RNA using TaKaRa kit PrimeScript First Strand cDNA Synthesis (cat # 6110A).

The reaction system and conditions were as follows:

reagent	Amount of the composition used
		Template RNA/Primer Mixture	2.0μl
Total RNA	0.5μg
		RNaseFree dH20	Upto10μl
Total volume	10μl

The cDNA is obtained after the components are evenly mixed and then are heated to 37 ℃ for 15 minutes and then are heated to 85 ℃ for 5 seconds.

3) Amplification of uc.189: real-time fluorescent quantitative PCR was performed using the PrimeScriptTM RT Master Mix kit from Takara. And (3) carrying out qRT-PCR amplification by using the reverse transcribed cDNA as a template.

The reaction system and conditions were as follows:

qRT-PCR procedure: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles: 5s at 95 ℃ and 30-60s at 60 ℃.

Through the detection of positive samples, the detection accuracy of the dye fluorescence quantitative kit disclosed by the invention is over 82-87%, and the test result is stable after 10 times of repeated experiments.

The invention also designs and synthesizes a specific RNA interference sequence aiming at the uc.189 sequence: siRNA1(SEQ ID NO.8) and siRNA2(SEQ ID NO.9), wherein the interference sequence can remarkably knock down the expression level of uc.189 in tumor cells (FIG. 8) and can cause that the invasive capability of the tumor cells is obviously weakened compared with the prior art (FIG. 9).

The invention has the beneficial effects that:

1. in order to systematically explore UCR expression profiles of B cell lymphomas and discover a group of UCRs which are specifically related to the occurrence and development of the B cell lymphomas, the invention adopts a long-chain non-coding RNA-UCR chip technology to screen a UCR which is remarkably and highly expressed in B cell lymphomas, compares a UCR genome database to discover that uc.189 is positioned between 23027 and 23599 bases of chromosome 6-like positive strand, and the total length of the gene is about 573.

2. Compared with paired normal tissues, the UCRs are remarkably and highly expressed in B cell lymphoma tissues, and further prove that the expression of uc.189 in the B cell lymphoma tissues is remarkably higher than that of the paired normal tissues in a later-stage large sample tissue real-time qRT-PCR experiment.

3. The invention relates to a qRT-PCR kit for detecting uc.189 expression level. The qRT-PCR kit is suitable for all types of fluorescent quantitative gene amplification instruments on the market at present, and has the advantages of high sensitivity, high speed and accuracy in quantification, good stability and good application prospect.

4. The invention focuses on UCR expression spectrum of B cell lymphoma tissue, and the novel gene regulatory factor is expected to further enrich and perfect the research on tumor pathogenesis including B cell lymphoma, and also brings hope for finding markers for tumor diagnosis and prognosis judgment and new tumor treatment targets.

Drawings

FIG. 1.UCR chip diagram

It was shown that uc.189 was expressed in 20 cases of B-cell lymphoma tissues (15 cases in the control group), the fold differential expression was 2.741-fold, the light color indicated decreased expression, and the dark color indicated increased expression.

FIG. 2 is a diagram showing the screening results of specific primers

The effect of the primers was tested by agarose gel electrophoresis after PCR amplification of 3 pairs of specific primers designed against the sequence of uc.189.

FIG. 3 preliminary qRT-PCR assay results of uc.189 in first 40 clinical samples of B-cell lymphoma (2)^-△CtPlotted).

FIG. 4. second 90 clinical samples of B-cell lymphoma uc.189 revalidated the qRT-PCR assay (2)^-△CtPlotted).

FIG. 5. analysis of qRT-PCR detection system for differential expression of 130 total samples uc.189 in cancer and paracarcinoma tissues (2)^-△△CtValue comparison, p <0.05, p < 0.01).

FIG. 6 correlation analysis of qRT-PCR detection of uc.189 expression with patient clinical staging (2)^-△△CtValue comparison, p <0.05, p < 0.01).

FIG. 7.uc.189 expression correlation analysis with B-cell lymphoma lymph node metastasis (2)^-△△CtValue comparison, p <0.05, p < 0.01).

FIG. 8.qRT-PCR assay for siRNA knockdown uc.189 efficiency.

FIG. 9.Transwell assay detects the invasive ability of tumor cells after siRNA knockdown of uc.189.

FIG. 10. nude mice tumor-bearing experiment tests the tumor cell growth ability after siRNA knockdown of uc.189 (uc.189 knockdown group and control group nude mice transplanted tumor growth curve chart and nude mice transplanted tumor tissue).

Detailed Description

The present invention is further illustrated below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. It will be appreciated by those skilled in the art that various changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers.

Example 1 differential expression analysis of UCR chips for B-cell lymphoma tissues and paired Normal tissues

Materials and methods

1. Material

Tissue samples were obtained from surgical resection samples of 20 hospitalized cases of B cell lymphoma patients (numbered 1-20), of which 15 pairs were paired B cell lymphoma tissue and paraneoplastic normal tissue.

2. Method of producing a composite material

2.1 extraction of Total RNA from tumor tissue and paired Normal tissue

Total RNA for extracting B cell lymphoma tumor tissue and paired normal tissue was performed according to the procedure of the Qiagen's RNA extraction kit (RNeasy Microkit, #74004), which contains RNase-FreeDNase I (Lyophilized) and can effectively remove DNA from genome. The purity and concentration of the extracted RNA was then quantified using aNanoDrop ND 1000 nucleic acid quantifier (NanoDrop Technologies, Wilmington, Delaware).

2.2 fluorescent labelling of sample RNA: (

cRNA amplification labeling kit, catalog No.: 360060-10).

2.2.1 Synthesis of first Strand cDNA by reverse transcription

First strand cDNA was synthesized using CbcScrpt enzyme using Total RNA as a Primer and T70 oligo (dT) Primer containing T7 promoter sequence.

2.2.2 Synthesis of 2nd strand cDNA

The RNA in the hybrid strand was cut into short fragments with RNase H, DNA Polymerase was extended with the RNA short fragments as primers to synthesize 2nd strand cDNA, and double-stranded cDNA was purified.

2.2.3 in vitro transcription Synthesis of cRNA

Synthesizing cRNA by using the T7Enzyme Mix by using the cDNA as a template; then purified with RNA Clean-up K it (MN).

2.2.4 random primer reverse transcription

5ug of cRNA was reverse transcribed with CbcScipt II enzyme, Random Prime, and the reverse transcription product was purified with PCR NucleoSpin extract II Kit (MN).

2.2.5cDNA labelled with Klenow enzyme

Taking the reverse transcription product, using Random Primer as Primer to make KLENOW enzyme labeling, using PCR NucleoSp in extract II Kit (MN) to purify the labeled product, then pumping out after purification. (Cy5-dCTP or Cy 3-dCTP (GE healthcare) for the same purpose.

2.3 hybridization and washing

The labeled DNA was dissolved in hybridization solution (2 XGEx Hyb Buf er (HI-RPM), 25% formamide) and hybridized overnight at 45 ℃. After hybridization, the cells were washed in a solution containing 0.2% SDS and 2 XSSC at about 42 ℃ for 5min and then in 0.2 XSSC for 5min at room temperature. The slide can be used for scanning after being dried.

2.4 chip Scan

The chip was scanned with lncRNA-TUCR Scanner to obtain a hybridization image.

2.5 acquisition of chip images and data analysis

And (3) analyzing the chip image by using Feature Extraction image analysis software and GeneSpr ing GX software, converting the image signal into a digital signal, and screening the differential genes.

Second, result in

A diagram of the TUCR chip differential gene screening for B-cell lymphoma is shown in figure 1. Chip screening found multiple TUCRs with up-and down-regulated expression, with uc.189 showing significant up-regulation in tumor tissue, whereas in view of its possible presence of specific expression in B-cell lymphoma tissue, the invention performed repeated validation of the index using large-scale sample batches by the following examples.

Example 2 real-time fluorescent quantitation qRT-PCR preliminary validation of differential expression of uc.189 in B-cell lymphoma tissues and paired paracancerous normal tissues

First, experimental material

40 other pairs (numbered 21-60) of B cell lymphoma tissues and paired paracancerous normal tissues were selected and qRT-PCR first instance validation of uc.189 expression differences was performed.

Second, Experimental methods and results

1. Primer specificity screening and identification

Extracting uc.189 related transcript gene sequences from a UCSC Genome Brower database according to uc.189 gene loci, and designing primers for uc.189 by Primer design software according toPrimer 5;

(1) evaluating the designed primers by using Oligo7 to obtain 3 pairs of designed primer sequences;

a first pair: upstream primer SEQ ID NO.2

Downstream primer SEQ ID NO.3

The second pair: upstream primer SEQ ID NO.4

Downstream primer SEQ ID NO.5

And a third pair: upstream primer SEQ ID NO.6

Downstream primer SEQ ID NO.7

Verified by common PCR and agarose gel electrophoresis experiments, the primer sets for dye qRT-PCR detection are screened as follows (FIG. 2):

a second pair of upstream primers: SEQ ID NO.4

A downstream primer: SEQ ID NO.5

(2) Extracting total RNA of B cell lymphoma tissue or tumor from the paired paracancerous normal tissue according to the required reagent and procedure of TRIZOL (R) reagent (Cat. 15596018) of ThermoFisher Scientific company; the purity and concentration of the extracted RNA was then quantified using aNanoDrop ND 1000 micro-UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, Delaware).

(3) Preparation of sample cDNA: cDNA was synthesized by reverse transcription of the extracted total RNA using TaKaRa kit PrimeScript First Strand cDNA Synthesis (cat # 6110A).

The reaction system and conditions were as follows:

reagent	Amount of the composition used
		Template RNA/Primer Mixture	2.0μl
Total RNA	0.5μg
		RNase Free dH20	Upto10μl
Total volume	10μl

The cDNA is obtained after the components are evenly mixed and then are heated to 37 ℃ for 15 minutes and then are heated to 85 ℃ for 5 seconds.

Setting a reaction group and a negative control group without a cDNA template aiming at 3 pairs of primers by taking the synthesized cDNA as a template, and performing PCR reaction according to the primers designed in the step (2) except the possibility of primer dimer;

(4) and (4) carrying out electrophoresis detection, wherein Marker DL1000(TaKaRa) is selected. Selecting a specific qRT-PCR primer according to an electrophoresis detection result, wherein the selection standard is as follows: a. the amplified fragment size was the same as expected; b. only one amplification product is needed to clarify the specificity of the amplification product. As a result, the optimal primer pair among the three pairs of primers was the second primer pair (FIG. 2), and the upstream and downstream specific primer sequences, which are represented by SEQ ID NO.4 and SEQ ID NO.5 in the sequence Listing.

2. Extraction of total RNA from 40 samples:

grinding with liquid nitrogen, according to ThermoFisher Scientific

Reagents (cat 15596018) reagents and procedures were required to extract total RNA from B cell lymphoma tissues or tumors. The main operation steps are as follows:

(1) the sample is frozen in liquid nitrogen quickly after being separated, when RNA is extracted, the tissue is put into a precooled mortar for grinding, liquid nitrogen is added while grinding, and the liquid nitrogen is not volatilized to dry in the whole process.

(2) After the tissue sample was ground into a powder, 1.5ml of TRIZOL reagent was added to each mortar when the liquid ammonia was substantially volatilized, and after mixing, the mixture containing TRIZOL was transferred to a 2ml centrifuge tube and left at room temperature for 8 min.

(3) 200ul of chloroform was added, shaken vigorously by hand for 30 seconds, centrifuged at room temperature for 8 minutes at 4 ℃ and 12000 g.times.5 min.

(4) Transfer 600ul of supernatant to a fresh centrifuge tube, add 0.5ul of isopropanol, precipitate at room temperature for 8 minutes, centrifuge at 4 ℃ and 12000g × 5 min.

(5) Discard the supernatant, pipette excess supernatant with a small gun, add 1ml 75% ethanol to wash RNA, shake for a while,12000g 5 minutes, carefully discard the supernatant.

(6) ) standing at room temperature for 5-15min to dry the RNA precipitate, and adding 20ul DEPC water to dissolve.

(7) After using spectrophotometer RNA concentration. Partially used or stored in liquid nitrogen for a long time.

3. The total RNA of the above 40 pairs of B cell lymphoma tissues and paired paracancerous normal tissues was collected and used as per ThermoFisher Scientific

Reagents required for reagents (cat 15596018) and procedures to extract total RNA from B cell lymphoma tissues or tumors; the purity and concentration of the extracted RNA was quantified using aNanoDrop ND 1000 micro UV-Vis spectrophotometer (NanoDrop Technologies, Wilmington, Delaware).

(1) Preparation of sample cDNA: cDNA was synthesized by reverse transcription of the extracted total RNA using TaKaRa kit PrimeScript First Strand cDNA Synthesis (cat # 6110A).

The reaction system and conditions were as follows:

reagent	Amount of the composition used
		Template RNA/Primer Mixture	2.0μl
Total RNA	0.5μg
		RNase Free dH20	Upto10μl
Total volume	10μl

The cDNA is obtained after the components are evenly mixed and then are heated to 37 ℃ for 15 minutes and then are heated to 85 ℃ for 5 seconds.

(2) Amplification of uc.189: real-time fluorescent quantitative PCR was performed using the PrimeScriptTM RT Master Mix kit from Takara. And carrying out qRT-PCR amplification by using the reverse transcribed cDNA as a template.

The reaction system and conditions were as follows:

qRT-PCR procedure: pre-denaturation at 95 ℃ for 30s, followed by 40 cycles: 5s at 95 ℃ and 30-60s at 60 ℃.

According to the relative transport formula of qRT-PCR: 2^-△CtThe expression level of uc.189 in tumor tissue (T) and paired normal tissue (N) of patients with B cell lymphoma was calculated, and the comparison results are shown in FIG. 3: the qRT-PCR amplification result is stable, wherein the expression level of uc.189 in paired normal tissues is mainly concentrated between 0.000 and 0.001, while the expression level of uc.189 in tumor tissues is mainly concentrated between 0.002 and 0.150, and the expression level is obviously highIn normal tissue pairing, the above results indicate that the marker is generally highly expressed in tumor tissue. Then according to the relative expression quantity T-N>0 is defined as the index expression up-regulation; t N<0 is defined as the down-regulation of the expression of the marker. The experimental result shows that: uc.189 is up-regulated in 40 cases of B cell lymphoma and paired normal tissues in 33 cases, according to the formula: the positive detection rate of the index is defined by the up-regulation expression example number/total detection example number x 100%, and then the positive rate of the index is 82.5%.

Example 3qRT-PCR to further verify the differential expression of uc.189 in tumor tissue and paired normal tissue of B cell lymphoma

1. qRT-PCR kit composition

1.1 dye uc.189PCR kit composition:

(1) an upstream primer: SEQ ID NO.4

(2) A downstream primer: SEQ ID No. 5;

other reagents were referred to as SYBR Premix Ex Taq^TMII (Tli RNaseH plus) fluorescent quantitation kit (Code No. RR820A).

Detection of uc.189

2.1 preparation of Total RNA

Another 90 pairs of tumor tissues and paired normal tissues were selected for B-cell lymphomatosis (numbers 61-130) according to ThermoFisher Scientific Inc

Reagents (cat 15596018) reagents and procedures required for total RNA extraction, see the description. The purity and concentration of the extracted RNA was quantified using aNanoDrop ND 1000 micro-UV-Vis spectrophotometer (NanoDrop Techno regions, Wilmington, Delaware).

2.2cDNA Synthesis

The total RNA extracted as described above was subjected to reverse transcription reaction using TaKaRa kit PrimeScript First Strand cDNA Synthesis (cat. 6110A).

2.3qRT-PCR detection

The apparatus for qRT-PCR used an Applied Biosystems 7500Real-Time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.); qRT-PCR reaction procedure the expression level of uc.189 was determined as in example II using qRT-PCR with fluorescent dyes.

3 results of detection

The results show that: the tumor tissues of 90 pairs of B cell lymphoma diseases (numbered 61-130) and the amplification sample amount of the matched normal tissues are selected for verification, qRT-PCR detects that the index in 76 pairs of samples is up-regulated in the lymphoma tissues, and the detection positive rate reaches 84.4 percent (figure 4). The above results again demonstrate that the index is generally highly expressed in tumor tissues. The qRT-PCR test is repeated for 3 times on the samples, and the result repeatability reaches 100 percent, which shows that the kit has better repeatability and stability.

Example analysis of the potential value of Tetrauc.189 in diagnosis and prognosis of B-cell lymphoma

On the basis of detecting high expression of uc.189 in B-cell lymphoma, the correlation between the expression condition of uc.189 and different pathologies and clinical stages is further analyzed by combining clinical pathological data of patients (specifically referring to American Joint Committee on Cancer criterion), and the potential value of uc.189 in diagnosis of diseases in B-cell lymphoma, including the relation between the expression and clinical/pathological stages, prognosis judgment, selection of treatment schemes and the like, is discussed.

Statistical processing is carried out on the correlation between the RNA expression of uc.189 and clinical pathological parameters of the B cell lymphoma by using SPSS 16.0software package (SPSS Inc., Chicago, IL, USA) statistical software, the correlation data is subjected to T-test orchi 2 test and data analysis, and P <0.05 considers that the differential expression of the index has statistical significance. Statistical results showed that the RNA expression level of uc.189 in 130 pairs of B-cell lymphoma samples was significantly higher than that of paired normal tissues (fig. 5, P < 0.01); the expression of uc.189 is closely related to lymph node metastasis (fig. 7): lymph node metastasis group was significantly higher than non lymph node metastasis group patients (P < 0.05); the expression of uc.189 is highly correlated with clinical staging (fig. 6): the expression of uc.189 in patients with stage III-IV B cell lymphoma is significantly higher than that in patients with stage I-II (P < 0.05).

After an interfering RNA (siRNA) sequence is designed on the basis of detecting high expression of uc.189 in B cell lymphoma tissues, interfering RNAs (siRNAs) synthesized by Life technology company, siRNA1(SEQ ID NO.8) and siRNA2(SEQ ID NO.9), can obviously knock down the expression level of the marker in B cell lymphoma cell strains A20 and DOHH2 (FIG. 9), and obviously weaken the invasive ability of tumor cells compared with the prior art (FIG. 9). In the experiment of transplantation tumor in nude mice, a B cell lymphoma cell strain DOHH2 containing uc.189-siRNA1 and a control group are injected into the nude mice to carry out subcutaneous tumor growth, and as a result, the growth of the transplantation tumor can be inhibited after the uc.189 expression is knocked down (figure 10).

In a word, the B cell lymphoma is obviously highly expressed in a B cell lymphoma tissue, has an index for regulating the growth and the transfer capacity of tumor cells, is expected to become a biomarker related to the diagnosis, treatment and prognosis evaluation of the auxiliary B cell lymphoma, and has very important clinical application value. With the later stage of further and gradually clarifying the action mechanism of uc.189 for regulating the growth and metastasis functions of tumor cells in B cell lymphoma, uc.189 can become a biomarker related to diagnosis and prognosis judgment, and is more expected to become a new B cell lymphoma treatment target to improve and improve the clinical B cell lymphoma treatment effect.

Sequence listing

<110> Yangzhou university

<120> UCR sequence, kit and detection method for detecting high expression in B cell lymphoma tissue

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agatggttgt actgatggct tgtttttcat tttttttgtg ctttttggtc catctattaa 60

taaaaatgaa ccccgttaca gagtcaccat catgtctctt ctcaccaccc tctgaatctg 120

cattagccag tcaactagcc ctttcagcgt catgtgacca gcgcgcccca ttcagcttgg 180

ctggtgtcgt ttcacatgac ccaggctggc cagtcgtcag gttgcaccgc cctttggttc 240

ccgagcatgc tgttttctct cagccttctc tccaacctta accaaatcgg cagcagccac 300

ctcgaccgcc cacacattcc tggccaatca gctcagctgt ttatttacca aatgtcttca 360

caacaactac agcagcagcc ttcggctaac aaaaaagcag gaaaaatcca caacaccccc 420

ttcgccaacc aactaaatcc aacgcaacat ctggcaaaac cttttcagca aattcttcct 480

ggccgtcagt ccggcagcct cacctcacca tttctagctt gttgaaaccc aaaactagta 540

agtttttcct gcttatacag tttactgctg gtt 573

<210> 2

<211> 18

<212> DNA

<213> Artificial sequence ()

<400> 2

gatggttgta ctgatggc 18

<210> 3

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tggtcacatg acgctgaa 18

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cacacattcc tggccaat 18

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<213> Artificial sequence ()

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ccagatgttg cgttggat 18

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ggttgtactg atggcttg 18

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aggttacccc tgtttcag 18

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aactagccct ttcagcgtca tg 22

Claims (5)

1. An application of a specific primer for detecting UCR expression level in preparation of a kit for auxiliary diagnosis or curative effect prediction of B cell lymphoma is characterized in that the nucleotide sequence of UCR is shown in a sequence table SEQ ID NO.1, the specific primer is a primer Pair Pair 1 or a primer Pair Pair 2, and the primer Pair Pair 1 or the primer Pair Pair 2 is respectively:

pair 1 upstream primer: SEQ ID number 2;

pair 1 downstream primer: SEQ ID number 3;

pair 2 upstream primer: SEQ ID number 4;

pair 2 downstream primer: SEQ ID number 5.

2. The use of claim 1, wherein the specific primers are shown in SEQ ID NO.4 and SEQ ID NO.5 of the sequence Listing.

3. The use of claim 2, wherein the kit further comprises a DNA template, a fluorescent dye, and a qRT-PCR reaction solution, wherein the qRT-PCR reaction solution comprises buffer solution, Taq enzyme, Mg^2＋And dNTPs.

4. Use according to claim 3, wherein the fluorescent dye is SYBR Green II and the Taq enzyme is a hot start enzyme.

5. The application of siRNA for knocking down uc.189 expression in preparing a medicament for treating B cell lymphoma is characterized in that the sequence of uc.189 is shown in a sequence table SEQ ID NO.1, and the sequence of siRNA is shown in sequence tables SEQ ID NO.8 and SEQ ID NO. 9.

Images