Method for preventing and treating tumors by using microRNA sponge technology

A sponge and tumor technology, applied in the field of biomedicine, can solve problems such as the connection between NF-κB and cancer and the unclear mechanism

Active Publication Date: 2014-02-19
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are relatively few studies on the role of non-canonical NF-κB pathway in tumors, but the activation of non-canonical NF-κB pathway in pancreatic cancer promotes the proliferation of tumor cells; blocking non-canonical NF-κB pathway in prostate cancer Can inhibit tumor growth by reducing IL8 production
[0006] However, the specific connection and mechanism between NF-κB and cancer, especially colon cancer, are not yet clear in this field, so the research and development of this invention are urgently needed in this field

Method used

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  • Method for preventing and treating tumors by using microRNA sponge technology
  • Method for preventing and treating tumors by using microRNA sponge technology
  • Method for preventing and treating tumors by using microRNA sponge technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0162] Canonical and non-canonical NF-κB pathways are constitutively activated in colorectal cancer

[0163] Test materials: Clinical tissue samples were obtained from Zhongshan Hospital Affiliated to Fudan University, and the shRelA vector was constructed with pLVX-shRNA1.

[0164] Test method: use Clontech's RNAi Designer tool to design the target shRNA. First use RNAi Target Sequence Selector to select the target sequence of shRNA, and then use shRNA Sequence Designer to design the corresponding shRNA sequence according to the target sequence. In order to facilitate the connection of shRNA to the vector, restriction enzyme sites were added at both ends of the shRNA sequence when designing the sequence. The commercially available plasmid vector pLVX-shRNA1 contains restriction sites for BamH1 and EcoR1, and restriction sites for these two enzymes were added to both ends of the designed shRNA sequence.

[0165] Targeting sequence:

[0166] 5'-GGTGCAGAAAGAGGACATT-3' (SEQ ID...

Embodiment 2

[0174] miR-221 / 222 affects the expression of RelA and affects the activation of canonical and non-canonical NF-κB pathways

[0175] Test materials: miR-221 / 222 mimics and antisense were purchased from Gemma. Real-time PCR kit was purchased from Takara Company. RNA inversion kit was purchased from Transgene. Dual fluorescent reporter gene detection kit was purchased from Promega.

[0176]Test method: 100nM miR-221 / 222 mimics or antisense were transfected into HCT116 and RKO cells, and 3 days after transfection, western blotting and real-time PCR were used to detect the expression of related genes. 100nM miR-221 / 222 mimics or antisense, 200ng NF-κB reporter gene vector and 50ngpRL-TK vector were co-transfected into HCT116 cell line, and 2 days after transfection, the activity of NF-κB reporter gene was detected. 1ng / ml CD40L stimulated HCT116 cells, collected samples after 12 hours, and detected the expression of miR-221 / 222. 100 nM of miR-221 / 222 was transfected into HCT116...

Embodiment 3

[0180] miR-221 / 222 affects the expression of RelA by regulating the coding region of RelA

[0181] Test materials: RelA expression vector was constructed with commercially available pcDNA3.1(+). The mutation kit was purchased from Takara Company. Actinomycin D was purchased from Shanghai Qianchen Biological Company.

[0182] Upper chain primer: 5'-ATA AAGCTT ATGGACGAACTGTTCCCCCTC-3' (SEQ ID NO.:12), lower strand primer: 5'-ATA GGATCC TTAGGAGCTGATCTGACTCAG-3' (SEQ ID NO.: 13), enzyme cutting sites: HindIII and BamHI.

[0183] Since miR-221 / 222 positively regulates the expression of RelA, the inventors tried to verify whether it promotes its expression by binding to the 5'UTR of RelA. Test method: 100nM miR-221 / 222 antisense and 2μg RelA expression vector were co-transfected into HCT116 or 293T cells, and 3 days after transfection, western blotting and real-time PCR were used to detect the expression of related genes. 100nM miR-221 / 222 mimics or antisense, and 200ng psiCH...

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Abstract

The invention relates to a method for preventing and treating tumors by using a microRNA sponge technology. Specifically, activation of canonical and non-canonical NFkappaB pathways can be regulated through a RelA factor, thereby regulating and controlling tumor growth. According to the invention, microRNA sponge miR-221/222 is a RelA gene inhibitor which can substantially reduce colorectal cancer cell proliferation and in vivo tumorigenesis; miR-221/222 is positively correlated to colorectal cancer cell proliferation and in vivo tumorigenesis, so activation of the canonical and non-canonical NFkappaB pathways can be substantially blocked by inhibiting miR-221/222 highly expressed in the colorectal cancer through the microRNA sponge technology; and the RelA factor or a coding sequence thereof can be used as a target point during tumor inhibition, and the RelA factor inhibitor is used for preparing drugs for tumor prevention or treatment.

Description

technical field [0001] The invention belongs to the field of biomedicine, in particular, the invention relates to a method for preventing and treating tumors by using microRNA sponge technology. Background technique [0002] Colon cancer is one of the common malignant tumors in the gastrointestinal system. In recent years, the incidence of colon cancer in my country has shown an obvious upward trend, from 1 / 10,000 in the 1970s to 3 / 10,000 now, ranking fourth in the incidence of malignant tumors. [0003] The occurrence of colon cancer is very complicated, and its pathogenesis is still not clear. But more and more evidences show that there is a close relationship between chronic inflammation and the occurrence of tumors. Inflammatory factors and other harmful compounds released during chronic inflammation can cause damage to cellular DNA, thereby changing cell proliferation and viability, and promoting tumorigenesis. For example: Chronic inflammatory bowel disease can grea...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K45/00A61K38/16A61K39/395A61K48/00C12N15/113A61P35/00
Inventor 张笑人刘三宏
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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