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Application of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha

A technology of enzyme activity and medicine, applied in the field of disease treatment, to achieve the effect of good specificity, less side effects and less toxicity

Active Publication Date: 2009-10-21
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far there is no direct evidence and molecular mechanism research on the relationship between PI4K and tumor growth

Method used

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  • Application of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha
  • Application of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha
  • Application of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Materials and methods

[0069] cell culture

[0070]Human cervical cancer HeLa cells (ATCC: CCL2), human breast cancer MCF-7 cells (ATCC: HTB-22) and human renal epithelial HEK-293 (ATCC: CRL-1573) cells were obtained from American Type Cell Culture Center (ATCC) . The cells were cultured in DMEM nutrient mixture (Kaighn's modified), which contained 10% fetal calf serum (Hycloe), streptomycin sulfate (100 μg / ml), and ampicillin (100 units / ml), and the cells were placed in Incubator 37°C, 5% CO 2 cultured under conditions. According to the manufacturer's instructions, the cells were transfected (30%-60% confluent) in 6-well or 6-cm plates by using a cationic lipid: Lipofectamine 2000 (invitrogen) (see invitrogen Lipofectamine for specific methods) 2000 Product Manual). After transfection, cells were cultured for 6 hours, and the medium was changed to DMEM medium containing 10% fetal bovine serum and double antibodies to continue culturing.

[0071] semi-...

Embodiment 2

[0080] Example 2: Study on the Regulatory Function of Overexpression of PI4KIIα on HIF-1α, a Hypoxia Inducible Factor Closely Related to Tumor Growth in Tumor Cells

[0081] Human renal epithelial HEK293 cells were transiently transfected with the plasmid PEGFP-PI4KIIα or PEGFP-C1 according to the method described above (PEGFP-C1 was purchased from Promage, and PEGFP-PI4KIIα was constructed according to the "Molecular Biology Experiment Guide"). After 48 hours, the cells were lysed, and the PI4KIIα in the cells was detected by the above immunoblotting method to determine the transfection efficiency. On the basis of detecting the expression of transfected GFP-PI4KIIα, the protein content of HIF-1α in the overexpressed cells was detected, and the housekeeping protein Actin (Actin) in the cells was used as an internal reference for comparison. In order to ensure the reliability of the experiment, we designed a positive control experiment, we used 0.2mM cobalt chloride to treat th...

Embodiment 3

[0083] Example 3 Interfering with the regulatory function of PI4KIIα on hypoxia-inducible factor HIF-1α

[0084]Synthesize a small RNA sequence that specifically interferes with PI4KIIα (sense strand: UGAAGCAGAACCUCUUCCUCATT (SEQ ID NO: 1); antisense strand: UCAGGAAGAGGUUCUGCUUCATT (SEQ ID NO: 2)) and a non-specific control small RNA sequence (sense strand: UUCUCCGAACGUGUCACGUTT (SEQ ID NO: 3); antisense strand: ACGUGACACGUUCGGAGAATT (SEQ ID NO: 4)). According to the instructions in the above method, HeLa cells and MCF-7 cells were transfected and cultured for 72-96 hours, the cells were lysed and the protein content of PI4KIIα was detected by the above Western blot to determine the interference effect of the interference sequence. On the basis of proving that the interference efficiency of the small interference RNA is very high, the protein content of HIF-1α in the interference cell and the control cell is detected and compared, and the housekeeping protein Actin (Actin) in ...

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Abstract

The invention discloses a new biological function of Phosphatidylinositol4-kinase type II alpha PI4KIIalpha and an application based on the new function. PI4KIIalpha plays a key role in regulating andcontrolling a hypoxia-inducible factor HIF-1alpha and a downstream vascular endothecial growth factor VEGF. The selective reduction of PI4KIIalpha expression can obviously inhibit tumor growth and tumor angiogenesis. The PI4KIIalpha can be used as a target molecule for intervening in diseases which are relevant to a gene regulated and controlled by the HIF-1alpha and the diseases which are relevant to angiogenesis. In addition, the target molecule can be used for screening medicines for the relevant diseases.

Description

Background technique [0001] The inositol phospholipid (PI) signaling pathway plays a central role in transmembrane signal transduction and signaling pathways for the regulation of cell growth by various growth factors and hormones. Inositol phospholipid 4-position kinase PI4K is the first kinase that plays an important role in the PI signaling pathway. During the inositol phospholipid cycle, PI4-K first catalyzes the phosphorylation of D4 on the inositol phospholipid (PI) ring to generate 4-phosphate Phosphatidylinositol (4-phosphatidyl-inositide, PIP), and then further catalyzed by PIP5-K kinase to synthesize (4,5-phosphatidyl-inosididediphosphate, PIP2); PIP2 is hydrolyzed under the action of phospholipase C (Phospholipase C, PLC) Inositol triphosphate (1,4,5-trisphosphoinositol, IP3) and diacylglycerol (DAG), as the second messenger, trigger intracellular calcium release and activate protein kinase C (Protein kinase C, PKC), start Intracellular two-messenger system. In ad...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48A61K31/713A61K38/00A61P35/00A61P19/02A61P3/10A61P9/00A61P27/00A61P17/02
Inventor 陈畅李江美
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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