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Long non-coding RNA for diagnosis, treatment and monitoring on bladder cancer and application of RNA

A bladder cancer and application technology, applied in the field of long non-coding RNA and its application, can solve the problems of high toxicity and weak anticancer activity

Active Publication Date: 2019-10-11
昆明医科大学第二附属医院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past 20 years, more than 3,000 platinum complexes have been synthesized, but only 28 of them have entered clinical trials as anticancer drugs, and 18 of them have undergone different stages of clinical trials due to weak anticancer activity or high toxicity. finally abandoned

Method used

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  • Long non-coding RNA for diagnosis, treatment and monitoring on bladder cancer and application of RNA
  • Long non-coding RNA for diagnosis, treatment and monitoring on bladder cancer and application of RNA
  • Long non-coding RNA for diagnosis, treatment and monitoring on bladder cancer and application of RNA

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1: Analysis of lncRNA chip expression in bladder cancer tissue

[0024] 1. Materials and methods

[0025] 1. Materials

[0026] Tissue samples were obtained from 3 pairs of inpatient surgical resection samples of bladder cancer patients, each pair containing bladder cancer tissue and paired paracancerous tissue.

[0027] 2. Method

[0028] (1) Extraction of total RNA from tumor tissue and normal tissue: Total RNA from bladder cancer tissue and paracancerous tissue was extracted according to the instructions of Qiagen's RNA extraction kit (RNeasy Micro Kit, Cat. No. 74004).

[0029] (2) Fluorescent labeling of sample RNA (ArrayStar Human LncRNA Microarray V3.0Service)

[0030] (3) Synthesis of first-strand cDNA by reverse transcription: start with Total RNA and Oligo(dT)Primer containing T7 promoter sequence as a primer, and use CbcScript enzyme to synthesize first-strand cDNA.

[0031] (4) Synthesis of second-strand cDNA: DNA polymerase uses the RNA fragment...

Embodiment 2

[0039] Example 2: In vivo experiments found that lncRNA-KMU15 was highly expressed in chemotherapy-resistant bladder cancer cell lines and its correlation with clinical stage and prognosis

[0040] 1. Experimental materials

[0041] EJ, T24, and 5637 drug-resistant bladder cancer cells were injected subcutaneously into nude mice, and the expression difference of lncRNA-KMU15 in different chemotherapy-sensitive cells was verified in vivo. Bladder cancer tissues of bladder cancer patients with different stages and grades. Bladder cancer tumor tissues resected after primary and recurrence. Tumor tissues in situ and lymph node metastases in patients with bladder cancer.

[0042] 2. Experimental methods and results

[0043] 1. Subcutaneous injection of bladder cancer cell metastasis model in nude mice: iodophor disinfected the skin of nude mice, counted drug-resistant cell lines of EJ, T24 and 5637 according to routine, digested with EDTA / trypsin, centrifuged and resuspended in ...

Embodiment 3

[0068] Example 3: construction of lncRNA-KMU15 knockdown vector

[0069] 1. Experimental method

[0070] 1. Lentiviral interference vector pSicoR-GFP digestion and recovery

[0071]The pSicoR-GFP vector was digested overnight at 37°C with Hpa I and Xho I enzymes and buffer (custmart). The enzyme digestion system:

[0072]

[0073] 2. Design lncRNA KMU15 gene lentiviral interference sequence

[0074] (1) Design the interference sequence KMU original sequence

[0075] Website for designing interference sequences: http: / / rnaidesigner.thermofisher.com / rnaiexpress / . The primer sequences were synthesized in vitro according to the requirements of the lentiviral interference vector pSicoR-GFP vector. Select the three highest-scoring interference sequences, and synthesize primers based on the designed shRNA:

[0076]

[0077]

[0078] (2) Centrifuge the primers, add water to dissolve the primers to a final concentration of 10 μmol, take 10 μl F + 10 μl R, 95° C. for 5 min...

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Abstract

The invention relates to a long non-coding RNA for diagnosis, treatment and monitoring on the bladder cancer and application of the RNA. Through a lncRNA expression chip, an expression profile of lncRNA in a sample of a patient with the bladder cancer is detected, more than ten kinds of the lncRNA closely related to generation and development of the bladder cancer are discovered through analysis,the lncRNA is subjected to knock-down inhibition in a bladder cell line T24 in sequence, the drug sensitivity of T24 cells to DDP after all lncRNA sequences are inhibited is analyzed, a lncKMU15 whichis not reported at home and abroad is discovered, after knock-down, the IC500 of the DDP is obviously reduced, and the sensibility is obviously improved; the expression amount discovered through experimental verification is in obvious positive correlation with staging and grading of the tumor and in negative correlation with the total survival rate and non-tumor survival rate of the patient withthe bladder cancer, and the novel lncRNA-KMU15 provides brand-new possibility for exploration of a novel noninvasive molecular diagnosis technology to achieve sensitive early screening on the bladdercancer and an after-operation monitoring system.

Description

technical field [0001] The invention belongs to the field of tumor molecular biology, and specifically relates to a long-chain non-coding RNA and its application. Specifically, the present invention relates to a long-chain non-coding RNA used in the preparation of bladder cancer prognosis, recurrence or chemotherapy sensitivity monitoring application. Background technique [0002] Bladder cancer is a common malignant tumor of the urinary system, ranking eighth in the incidence of tumors in my country. Even in Europe and the United States, where the medical system is developed, the mortality rate of bladder cancer ranks among the top ten in the mortality rate of various cancer patients, which seriously threatens human health and social and economic development. Therefore, bladder cancer is not only a heavy psychological and economic burden for individual patients and families, but also a severe social burden for my country's economic development. However, there are many pro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N15/11A61K31/7105A61K48/00A61P35/00
CPCC12Q1/6886A61K31/7105A61P35/00C12Q2600/106C12Q2600/178
Inventor 王海峰王剑松栾婷保欣
Owner 昆明医科大学第二附属医院
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