The Effect Of Hsa- MiR- 660 Expression Regulation To CYP3A4 Protein Expression At The In Vitro Liver Cells

Background and Objective As a/an opioid analgesics fentanyl has a good analgesic effect. Factually it performs 50 to 100 times better than morphine, thus it has been widely adopted in clinical anesthesia and analgesia of cancer patients. However clinical cases show that the analgesic effect and side effect offen fentanyl varies from patient to patient. Fentanyl becomes Norfentanyl through the CYP3A4 in liver whose enzymatic activity of different patients changes significantly, therefore gene polymorphism determines CYP3A4 enzymatic activity..In recent years, it is found that a small, single-stranded molecules RNA(miRNA), which can act on the gene After the transcription process and further affect theprotein expression, plays an important role in life’s origin and development. In liver tissues, numerous kinds of miRNA are discovered. Though some of them are nonfunctional, some are functional, which needs further trials. Some researches indicate that there may be a correlation between Hsa-mi R-660 and CYP3A4 enzymatic activity Nonetheless Validation of a cellular level hasn’t been conducted yet.Our laboratory explores the correlation between Hsa-miR-660 relative expression and CYP3A4 protein expression in liver cells. Materials and Methods All cells are chang liver cells. The stable expression chang liver cell line is cultivated in 37 ° 5% CO2 incubator, whose culture medium is include 10% fetal calf serum RPMI1640, which is changed every 2 to 3 days to ensure the cells’ normal growth.If the cells grow well, Virus transfection can be prepared. The cells are firstly paved on 6 orifice, and then continuously cultivated until the Cell alignment reaches above 80%.Four groups are divided: mi RNA overexpression(H)、miRNA Silence(L)、control group and Negative carrier group(N).The transfection viruses are slow ones with target gene. The viruses in the group H contains miRNA overexpression target gene.; The viruses in the group L contains restrain miRNA expression; for group C, equal amount of salted water is added; for group N lentivirus with empty gene is added.According to MOI=50 adding virus culture together. 24 hours later, after the culture medium with virus is replaced by culture mediumwith10% fetal calf serum, the cells are cultivated continuously.44 to 72 hours later, transfection efficiency are observed under fluorescence microscope. When transfection has completed, the total RNA is extracted from a portion of the cells. With the method of real-time fluorescence quantification, the CYP3A4 mRNA ralative expression and Hsa-miR-660 ralative expression are determined. internal reference are GAPDH and U6 respectively. The rest cells are cultivated continuously. When they relatively thrives, total protein are extracted. With the method of western blot, the western blot is determined. The internal reference is GAPDH. Results 1 The measurement of transfection efficiency Under fluorescence microscope Group fluorescence microscope transfection efficiency of transfection efficiency of 50% to 70% of group H, L, N. 2 The determination of relative expression of Hsa- miR – 660 The relative expression of Hsa- miR – 660 is determination by Real-time fluorescence quantitative PCR method. Group L is significant decreased than group N and group C. group H is significan increased than group N and group C. 3 CYP3A4 mRNA expression The CYP3A4 mRNA relative expression is determination by RT-PCR. The CYP3A4 mRNA relative expression of four groups has no significant difference. 4 CYP3A4 determination of the amount of protein expression The CYP3A4 protein relative expression is determined by western blot.There is no statistical difference between group N and group C. The CYP3A4 protein relative expression of group L was obviously increased than group N and group C,Group H was obviously decreased than group N and group C. Conclusion 1. Hsa- miR- 660 does not affect the CYP3A4 mRNA stability. 2. Hsa- miR- 660 inhibits the expression of CYP3A4 protein.