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Molecular marker for development of gastric cancer diagnostic products and application

A product, gastric adenocarcinoma technology, applied in the field of molecular markers, can solve problems such as ambiguity of molecular mechanism

Active Publication Date: 2020-07-17
XUZHOU MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, research on the mechanism of gastric cancer development has continued, but the exact molecular mechanism is still unclear

Method used

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  • Molecular marker for development of gastric cancer diagnostic products and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Screening of gene markers associated with gastric cancer

[0046] 1. Sample collection

[0047] The cancer tissues and corresponding paracancerous tissue samples of 4 cases of gastric adenocarcinoma were collected for high-throughput sequencing. All patients did not receive chemotherapy, radiotherapy or endocrine therapy before operation.

[0048] 2. RNA sample preparation and quality analysis

[0049] Total RNA was extracted using Tiangen Animal Tissue Total RNA Extraction Kit (catalogue number: DP431), and the steps are detailed in the instructions.

[0050] 1) Homogenization treatment

[0051] Add 300μl Lysis Solution RL for every 10-20mg tissue, and grind the tissue thoroughly with a grinding pestle; then add 590μl RNase-Free ddH to the homogenate 2 O and 10 μl Proteinase K, mix well and treat at 56°C for 10-20min.

[0052] 2) Centrifuge at 12,000rpm for 2-5min, and take the supernatant for the following operations.

[0053] 3) Slowly add 0.5 times th...

Embodiment 2

[0085] Example 2 QPCR sequencing verification of differential expression of RP11-199F11.2 gene

[0086] 1. The differential expression of the RP11-199F11.2 gene was verified by large-scale QPCR on the cancer tissue samples and para-cancerous tissue samples collected from 31 gastric adenocarcinoma patients according to the collection method in Example 1.

[0087] 2. RNA extraction

[0088] The total RNA was extracted using Tiangen Animal Tissue Total RNA Extraction Kit (Cat. No. DP431). See Example 1 for specific steps.

[0089] 3. QPCR

[0090] Primers were designed according to the gene sequences of RP11-199F11.2 and GADPH. The primer sequences are as follows:

[0091] RP11-199F11.2:

[0092] Forward primer: 5'-GGCTTCTGTCCTTCAATG-3' (SEQ ID NO.1)

[0093] Reverse primer: 5'-CAAATGGGAAACAACTCAAA-3' (SEQ ID NO.2)

[0094] GAPDH:

[0095] Forward primer: 5'-AATCCCATCACCATCTTCCAG-3' (SEQ ID NO.3)

[0096] Reverse primer: 5'-GAGCCCCAGCCTTTCTCAT-3' (SEQ ID NO.4)

[0097] Th...

Embodiment 3

[0105] Example 3 Silencing of RP11-199F11.2 and its effect on gastric adenocarcinoma cells

[0106] 1. Transient transfection

[0107]The siRNA interference fragment targeting RP11-199F11.2 gene was designed and synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. The negative control is general siRNA-NC, RP11-199F11.2-siRNA group: 5′-AAAGAUGACGUAAGUACGGCA-3′ (SEQ ID NO.5); 5'-CCGUACUUACGUCAUCUUUUU-3' (SEQ ID NO.6). Gastric adenocarcinoma MGC-803 cells were inoculated in six-well plates 24 hours before transfection. When the cell density reached 50%-70% confluence, the medium was replaced with serum-free medium. Mix the diluted interference fragment with Lipofectamine TM 2000 Liposomes were gently mixed and incubated at room temperature for 20 minutes to form a transfection complex; 2 , Cultivate in a 37°C incubator, and replace the complete medium after 6 to 8 hours. After 48h, the interference efficiency was detected.

[0108] 2. QPCR detection interference ...

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Abstract

The invention discloses a molecular marker for the development of gastric cancer diagnostic products and application. The expression of the molecular marker RP11-199F11.2 is up-regulated in patients with gastric adenocarcinoma. The invention discloses the application of the RP11-199F11.2 to preparation of products for diagnosis and treatment of gastric adenocarcinoma.

Description

technical field [0001] The invention belongs to the field of biomedicine, and relates to molecular markers and applications for developing gastric cancer diagnostic products. Background technique [0002] Gastric cancer (GC) is a disease with a high mortality rate, ranking second in cancer-related deaths worldwide. In 2011, more than 400,000 patients were diagnosed with gastric cancer in China, and about 300,000 patients died of gastric cancer (DiGesualdo F, Capaccioli S, Lulli M.A pathophysiological view of the long non-codingRNA world. Oncotarget. 2014; 5: 10976 -10996.). Environmental factors and lifestyle are key causes of this phenomenon. Although the treatment options for patients with gastric cancer are still being improved, surgery and systemic chemotherapy are currently the main treatment methods (Kanat O, O'Neil BH. Metastatic gastric cancer treatment: a littleslow but worthy progress. Med Oncol. 2013; 30:464. ). However, research on the mechanism of gastric ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/118C12Q2600/158C12Q2600/178
Inventor 董东郑骏年孙强
Owner XUZHOU MEDICAL UNIV
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