Application of ILF3 detection reagent in preparation of colorectal cancer diagnosis reagent

A technology for detection reagents and detection kits, which is applied in the field of colorectal cancer prognostic diagnostic reagents/kits, and can solve problems such as unsatisfactory treatment of CRC

Active Publication Date: 2020-04-17
THE SIXTH AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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  • Application of ILF3 detection reagent in preparation of colorectal cancer diagnosis reagent
  • Application of ILF3 detection reagent in preparation of colorectal cancer diagnosis reagent
  • Application of ILF3 detection reagent in preparation of colorectal cancer diagnosis reagent

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Embodiment 1

[0083] Example 1 Patient and tissue samples

[0084] Fresh paired frozen samples of primary colorectal cancer and adjacent normal colon tissue were extracted from the Department of Surgery of the Sixth Affiliated Hospital of Sun Yat-sen University. All patients had stage II or III disease at the time of sample collection.

[0085] Additionally, paraffin-embedded samples of primary colorectal adenocarcinoma (prepared as TMA) were obtained from three independent cohorts of CRC patients: (1) 79 patients in the Sixth Affiliated Hospital of Sun Yat-sen University (test cohort); ( 2) 270 patients from the First Affiliated Hospital of Sun Yat-sen University (validation cohort 1); (3) 134 patients from the 150th Central Hospital of the Chinese People's Liberation Army (validation cohort 2). Raw immunohistochemistry slides were scanned by Aperio Versa (Leica Biosystems), which captured digital images of immunostained slides. Genie calculates H-scores for regions selected by pathologi...

Embodiment 2

[0086] Example 2 Cell Culture, Reagents and Transfection

[0087] All cells were obtained from ATCC and stored at 37 °C and 5% CO 2 condition. Among them, DLD-1 and HCT-8 cells were kept in RPMI 1640 medium (RPMI) containing 10% (v / v) fetal bovine serum (FBS). 293T, RKO, HT29, WiDR and SW620 cells were cultured in DMEM medium (containing 10% FBS). All transient transfections of plasmid and siRNA-introduced cell lines followed the instructions of Lipofectamine 2000 Transfection Reagent (Thermo Fisher, #11668019).

Embodiment 3

[0088] The shRNA knockdown of embodiment 3ILF3

[0089] The present invention screens four hairpin-type shRNAs against human ILF3 transcripts, and finds two independent sequences that can reduce mRNA levels by >70%. The shRNAs are in the pLKO.1 vector (#31 and #32). #35 was designed to target the 3'UTR of ILF3.

[0090] The steps for preparing lentiviral particles are as follows: the 55cm 2 1 x 10 in dish 7 HEK293T cells were co-transfected with 10 μg pLKO.1 shRNA construct, 5 μg psPAX2 and 5 μg pMD2.G. 48 and 72 hours after transfection, the supernatant containing virus particles was collected and filtered through Millex-GP filters (0.45 μm pore size, Millipore). To infect cancer cells with lentivirus, cells were infected twice with a medium containing 2 mL lentivirus, 200 μL FBS, and 5 mg / mL polybrene (Sigma) at 37° C. for 24 hours and 48 hours. To increase knockdown efficiency, puromycin selection was performed on infected cells for several days.

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Abstract

The invention, which belongs to the field of biological medicines, particularly relates to application of an ILF3 detection reagent in preparation of a colorectal cancer diagnosis reagent and a colorectal cancer prognosis diagnosis reagent/kit. The research finds that ILF3 is overexpressed in a specimen of a primary CRC patient and is related to poor prognosis; and the gene is suggested to be possibly related to the malignant phenotype of CRC at the clinical level. With the molecular biological means, interference of ILF3 can significantly inhibit CRC proliferation, so that the ILF3 is shown to have an important function in colorectal cancer occurrence and development. In addition, the invention also discovers that the ILF3 promotes tumor growth by directly adjusting the mRNA stability ofthe SGOC gene and increasing the expression of the SGOC gene for the first time; that is, the ILF3 influences tumors by adjusting the SGOC pathway. Researches show that the SGOC inhibitor can be usedfor treating CRC patients with ILF3 overexpression and has important clinical significance.

Description

technical field [0001] The invention belongs to the field of biomedicine, more specifically, the application of an ILF3 detection reagent in the preparation of a colorectal cancer diagnostic reagent, and a colorectal cancer prognosis diagnostic reagent / kit. Background technique [0002] Colorectal cancer (CRC) is one of the most common tumors of the digestive system. According to the latest global cancer survey report, colorectal cancer is the third most common tumor in the world after breast cancer and lung cancer in both incidence and mortality. With the rapid development of China's social economy and the changes in residents' living habits and dietary structure, the incidence and mortality of colorectal cancer in my country are still increasing. Rich historical research has precipitated many key genes and important pathways related to the malignant progression of CRC, including WNT, RAS-MAPK, PI3K, TGF-β, P53 and DNA mismatch repair pathway. However, the treatment of CR...

Claims

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Application Information

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IPC IPC(8): A61K45/00A61K31/496A61P35/00C12Q1/6886G01N33/574
CPCA61K45/00A61K31/496A61P35/00C12Q1/6886G01N33/57419G01N33/57446G01N33/57484C12Q2600/118C12Q2600/158G01N2800/52
Inventor 方乐堃李孟鸿李凯蒙曼琦
Owner THE SIXTH AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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