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Long non-coding RNA sequence for early diagnosis of human prostate cancer and its application

A long-chain non-coding, prostate cancer technology, applied in the field of long-chain non-coding RNA expression inhibitors and cancer combined diagnostic markers, can solve the limitation of the clinical application value of PCA3, and has not been found to be able to effectively or accurately detect the early occurrence of prostate cancer. and other problems, to achieve the effect of accurate cDNA sequence results, high accuracy, and simple steps in the cloning process

Active Publication Date: 2019-09-20
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Over the years, with the goal of identifying markers beyond PCA3 for the diagnosis of prostate cancer, a variety of genes have been evaluated, but no molecular markers that can effectively or accurately detect the early occurrence of prostate cancer and disease progression have been found so far
In addition, it is reported that the diagnostic sensitivity of PCA3 in urine is only 67%, and the specificity is 83% (Han Renqiang. 2003-2007 Analysis of Prostate Cancer Incidence and Death in China. Chinese Cancer. 2012; 21(11): 805-11), The low sensitivity of PCA3 detection in urine samples limits the clinical application value of PCA3

Method used

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  • Long non-coding RNA sequence for early diagnosis of human prostate cancer and its application
  • Long non-coding RNA sequence for early diagnosis of human prostate cancer and its application
  • Long non-coding RNA sequence for early diagnosis of human prostate cancer and its application

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Experimental program
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Embodiment 1

[0061] This embodiment provides a method for cloning the precise full-length cDNA sequence of a long-chain non-coding RNA (PRCAT38), which specifically includes the following steps:

[0062] 1), design a set of gene-specific primers according to the known sequence of PRCAT38 in the Mitranscriptome database, named in turn as GSP1, GSP2, GSP3, GSP4, GSP5, GSP6, the primer set can specifically recognize PRCAT38, the primer set in The sequences of each primer are shown in Table 1.

[0063] Table 1 Primer sets specifically recognizing PRCAT38

[0064]

[0065] 2), extract the total RNA of human prostate cancer cell lines PC3, LNCaP, 22Rv1 and DU145, and use ThermoFisher's The reverse transcription kit of III First-Strand Synthesis System uses oligo dT as a reverse transcription primer to reverse transcribe total RNA into cDNA.

[0066] 3), use the cDNA in step 2) as a template, use the primer pair GSP1-F, GSP1-R and GSP2-F, GSP2-R, and GSP3 to amplify the PRCAT38 specific fra...

Embodiment 2

[0073] This embodiment provides a bioinformatics method to analyze the conservation, transcriptional activity and coding ability of the long-chain non-coding RNA (PRCAT38 precise full-length cDNA sequence), as follows:

[0074] 1. Use Roadmap to obtain the data of H3K4me3, H3k36me3 and H3K27ac in the prostate cancer cell line PC3, and use the ENCODE database (https: / / www.encodeproject.org / ) to obtain the data of H3K4me3, H3k36me3 and H3K27ac in normal tissues and prostate cancer tissues, The GEO accession numbers are GSE96019 (H3K4me3, PC3), GSE96418 (H3k36me3, PC3) and GSE96399 (H3K27ac, PC3), and the ENCODE Experiment IDs are ENCSR748RBT (H3K4me3, Normal prostate), ENCSR499FXI (H3k36me3, Normal prostate) 6ENCSR7 Normal prostate). The data obtained in the prostate cancer cell line PC3 were displayed on the UCSC website (http: / / genome.ucsc.edu / ), and the enrichment of H3K4me3, H3k36me3 and H3K27ac in the PRCAT38 gene promoter and gene region was obtained ( figure 2 A). Tran...

Embodiment 3

[0078] This embodiment provides a RT-PCR and bioinformatics detection of the expression of long-chain non-coding RNA (PRCAT38) in prostate cancer cells, normal prostate tissues and cancer tissues, and various other normal human tissues and cancer tissues. Specifically include the following steps:

[0079] 1. RT-PCR detection of the expression of PRCAT38 in different prostate adenocarcinoma cell lines:

[0080] (1) Primer design

[0081] Based on the full-length cDNA sequence of PRCAT38, the amplification primers for RT-PCR are designed, wherein the nucleotide sequence of the upstream primer PRCAT38-F is shown in SEQ ID NO.10, and the nucleotide sequence of the downstream primer PRCAT38-R is shown in SEQ ID Shown in NO.11.

[0082] (2) Extract the total RNA of human prostate cancer cells PC3, LNCaP, DU145 and 22Rv1 respectively according to the following method:

[0083] ①Wash a 6cm culture dish with any of the above-mentioned cells with PBS (pH 7.4), add 1ml Trizol, transfe...

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Abstract

The invention provides a full-length cDNA sequence of long chain non-encoding RNA for early diagnosis of human prostatic cancer and its application. An accurate sequence of the PRCAT38 is cloned for the first time, and a foundation for further studying the function of the PRCAT 38 in the generation and development processes of the prostatic cancer is laid. The specificity high expression of the PRCAT 38 in the prostatic cancer cells and tissues can influence the proliferation, clone formation and migration of the prostatic cancer cells, and the PRCAT 38 can be applied to prepare the product for the early diagnosis, prognosis evaluation and / or treatment of the prostatic cancer. The invention provides a cancer diagnosis marker, which comprises PRCAT 38, EZH2 and miR-24-2, the marker has highsensitivity and high specificity to detect prostate. The invention provides an expression inhibitor of the long chain non-encoding RNA, which comprises siRNA and / or shRNA; the expression inhibition can inhibit the expression of PRCAT38 and EZH2 at the same time, and is good for the gene treatment and clinical drug research and development of the prostatic cancer.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and in particular relates to a precise cDNA sequence of a prostate cancer-specific long-chain non-coding RNA, a preparation method of the sequence, a combined cancer diagnostic marker, and an expression inhibitor of a long-chain non-coding RNA and its uses. Background technique [0002] Prostate cancer (PCa) is a malignant tumor that occurs in the prostate epithelium. It is the most common and unique malignant tumor in the male reproductive system. The incidence of prostate cancer continues to increase with age, and 95% of it occurs in people over 60 years old. elderly men. In the past ten years, about one million prostate cancer patients are newly diagnosed each year, accounting for about 15% of new male cancer cases worldwide, ranking second in the incidence of all male malignant tumors. The incidence of prostate cancer in the United States has surpassed that of prostate cancer, becomin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12Q1/6886C12N15/11A61K48/00A61K31/713A61P35/00
Inventor 高山杨晓辉
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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