Objective To evaluate the stability of U6 and let-7a as internal reference genes of miRNAs in RTqPCR by using femoral head samples of cartilage tissue from inbred DA rats.Methods Total RNA was extracted from femoral head cartilage tissues of female DA rats at three different time points,i.e.at birth (D0),ablactation (D21) and maturation (D42).The expressions of different miRNAs (miR-1,-25,-26a,-140,-146a,-150,-181a,-195,-223 and-337) were detected by RT-qPCR using U6 or let-7a as the internal reference.The two sets of miR expression were compared with the results from Solexa sequencing in our pioneer work to evaluate the stability of the two internal references.Results The relative values of U6 (P =0.045) and let-7a (P =0.021 5) revealed significant difference in the D42 sample.Both in U6 and let-7a systems,miR-26a,-140,-223,and-337 showed a similar tendency in expression and quantification but miR-1 and-146a did not have significant differences.miR-25,-150,-181a and-195 differed significantly (P＜0.05).Comparison of absolute quantification results between the two generations' sequencing showed that let-7a is more stable than U6.Conclusion Let-7a is more suitable to be used as the internal reference gene in RT-qPCR for miRNAs in cartilage tissue.

OBJECTIVETo investigate the expression of microRNA-100(miR-100) and the relationship with cisplatin resistance in human ovarian epithelial cancer SKOV3/DDP cells.

METHODSThe SKOV3/DDP cells were transfected with the mimics or inhibitor of miR-100 or negative control RNA (NC) or inhibitor negative control RNA (inhibitor NC) by lipofectamine 2000. The experiment was divided into six groups: SKOV3 group, SKOV3/DDP group, miR-100 mimices group, NC group, miR-100 inhibitor group and inhibitor NC group. The expression of miR-100 and the cisplatin IC50 were measured by real-time PCR and CCK8 assay respectively.

RESULTS(1)The cisplatin resistance index of SKOV3/DDP was 2.23; (2)The express level of miR-100 in SKOV3/DDP cells was significantly lower than that in SKOV3 cells (P<0.001); (3)After transfected with miR-100 mimics, SKOV3/DDP cells showed that the level of miR-100 was 38.29 times higher than that in the NC group(P<0.01). The cisplatin IC50 of miR-100 mimices group was significantly lower than that in the NC group (P<0.001); (4) After transfected with miR-100 inhibitor, the level of miR-100 0f SKOV3/DDP was decreased by 97.7%. The cisplatin IC50 of miR-100 inhibitor group was significantly increased as compared with that in the inhibitor NC group (P<0.001).

CONCLUSIONThe expression of miR-100 is downregulated in SKOV3/DDP cells. Overexpressing miR-100 may effectively increase the sensitivity to cisplatin of human ovarian epithelial cancer SKOV3/DDP cells and may reverse cisplatin-resistance of EOC (epithelial ovarian cancer).

Antineoplastic Agents ; Cell Line, Tumor ; Cisplatin ; pharmacology ; Down-Regulation ; Drug Resistance, Neoplasm ; Female ; Humans ; MicroRNAs ; metabolism ; Neoplasms, Glandular and Epithelial ; metabolism ; pathology ; Ovarian Neoplasms ; metabolism ; pathology ; Real-Time Polymerase Chain Reaction ; Transfection