A new target for inhibiting metastasis of bladder cancer and its application

A technology for bladder cancer and bladder cancer cells, applied in the field of new targets for inhibiting bladder cancer metastasis, can solve the problems of unclear molecular mechanism of bladder cancer metastasis, and achieve the effect of preventing or/and treating bladder cancer metastasis

Active Publication Date: 2020-03-27
WENZHOU MEDICAL UNIV
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However, the roles and molecular mechanisms of most o

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  • A new target for inhibiting metastasis of bladder cancer and its application
  • A new target for inhibiting metastasis of bladder cancer and its application
  • A new target for inhibiting metastasis of bladder cancer and its application

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Experimental program
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experiment Embodiment 1

[0023] Bioinformatics analysis of miR-3648 in TCGA database was up-regulated in bladder cancer patients.

[0024] 1) Bioinformatics Download and analyze the level 3 data of RNA-seq V2 and miRNA-seq in the TCGA database.

[0025] 2) Collect the barcode information of the samples, select Primary solid Tumor (sampe ID ends with -01) and Solid Tissue Normal (samples ID ends with 11) samples for analysis, a total of 19 pairs and 38 samples. The R package edgeR was used to analyze the differential expression of miRNAs in paired samples of cancer (tumor) and para-cancer (normal), which adopted a weighted trimmed mean of M values ​​(TMM) normalization method of the logarithmic expression ratio . In order to reduce false positives (false discovery rate, fdr), p-value was corrected by Benjamini–Hochberg (BH) method. The screening threshold for the significance of the difference was log2fold-change (logFC) not less than 3, fdrfigure 1 As shown, among the 19 patients, the expression lev...

experiment Embodiment 2

[0027] Expression of miR-3648 in clinical bladder cancer samples and human bladder cancer cells

[0028] 1) Tissue samples

[0029] Tissue samples from 33 patients who underwent bladder cancer surgery in the Department of Urology, The First Affiliated Hospital of Wenzhou Medical University between March 2014 and December 2016 were collected. Collect patient name, gender, age, pathology number, pathology and other information. Most of the selected patients were male and all underwent total cystectomy. Preoperative biopsy and postoperative pathological examination confirmed bladder urothelial carcinoma. Tumor tissue was collected from each sample, and normal tissue was cut about 3cm away from the tumor as a control. Each sample was frozen in liquid nitrogen after tissue isolation. The experimental operation was approved by ethical review.

[0030] 2) Tissue / cell total RNA extraction

[0031] a. Take out the bladder cancer clinical samples from the -80°C refrigerator, put ab...

experiment Embodiment 3

[0071] Inhibition of miR-3648 expression significantly reduces the invasive ability of bladder cancer cells

[0072] 1) T24T and UMUC3 cells were selected as the research object. LV3-pGLV-h1-GFP-puro-miR-3648-inhibitor-sponge plasmid was used to inhibit miR-3648 in T24T and UMUC3 cells, and stable transfection cells T24T (miR- 3648-inhibitor), UMUC3 (miR-3648-inhibitor) and its control T24T-Vector, UMUC3-Vector, using U6 as an internal reference, using 2-△△ct to calculate the gene expression of miR-3648 in stably transfected cells fold change. The result is as image 3 A, 3B show that a stable transfection cell line inhibited by miR-3648 was successfully constructed, and the difference was statistically significant.

[0073] 2) Using Transwell experiment, T24T (miR-3648-inhibitor), UMUC3 (miR-3648-inhibitor) and control cells were resuspended with 0.1% corresponding medium and planted in the upper chamber, and the lower chamber was cultured in full culture After 24 hours, f...

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Abstract

The present invention belongs to the field of biotechnology, and relates to a new target for inhibiting the metastasis of bladder cancers, and applications thereof. According to the invention, bladder cancer T24T cells and bladder cancer UMUC3 cells are used as models, and the metastasis of bladder cancer cells can be reduced in vitro and in vivo by inhibiting the expression of miR-3648, such that the miR-3648 expression inhibitor can inhibit the metastasis of bladder cancers; by using a real-time fluorescence quantitative PCR technology, a bioinformatics analysis TCGA database and other methods, the detection results show that the miR-3648 expression in most of the clinical high-grade invasive bladder cancer tissues is significantly up-regulated, such that the miR-3648 expression detection results show that the miR-3648 expression is closely related to the high-grade invasive bladder cancers; and the new target and the corresponding measures can be expected to be provided for the inhibition of the metastasis of bladder cancers.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a new target for inhibiting bladder cancer metastasis and application thereof. Background technique [0002] Bladder cancer is the most common malignant tumor of the urinary system. In recent years, statistics show that the incidence of bladder cancer is gradually increasing. 70-80% of bladder cancers are diagnosed as non-muscle-invasive tumors. The 5-year survival rate of patients with non-muscle-invasive bladder cancer is close to 90%. It is difficult to cure due to the lack of relatively complete molecular targets and corresponding measures. This makes the recurrence rate as high as 50-70% in this population. In addition, 15% of recurrent bladder cancer will develop into muscle-invasive disease, and the 5-year survival rate of patients with muscle-invasive bladder cancer is only close to 60%, and nearly 80% of patients with lymph node metastasis die in the first disease after diag...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12N15/11A61K45/00A61P35/00
CPCA61K45/00C12Q1/6886C12Q2600/158C12Q2600/178
Inventor 黄海山金红蕾孙文瑞古佳艳王帅邓文海
Owner WENZHOU MEDICAL UNIV
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