Research Of Targeting Human PRLR Gene Inhibition In Breast Cancer Cell By RNA Interference

Now the etiopathogenisis of breast cancer is not very clear; perhaps, the interaction of heredity, hormones, immunity and all kinds of environmental factors play an important role in genesis of breast cancer. Meanwhile, to be different from other tumors, mammary gland is the organ responsing for major hormones and many cytokines, which indicates that breast cancer is hormone dependent. Hormone reacts by the combination with its receptor. With the research of function of hormone receptor family in breast tissue, and the development of molecular mechanism in breast cancer genesis and progression, the physiological and pathological regulation of prolactin and prolactin receptor in breast tissue, now is reempasized by many labortaries. Recent studies of human breast cance show that neoplastic tissues express higher level of PRLR than normal adjacent tissues. Furthermore, Y Li and his collegues demonstrated that both PRLR degradation and PRLR phosphorylation on Ser349 were impaired in breast tumor cells and tissues, and observation that directly correlated with enhanced expression of PRLR in malignant breast epithelium. Therefore, inhibition or blocking of PRLR expression may be a new method for breast cancer biotherapy.In this study, we used the newly developed technology siRNA to down regulate or block the expression of PRLR mRNA in breast cancer cell line MCF-7 specifically, then detected the inhibition of cell proliferation. Three siRNAs targeting human PRLR were designed on line and synthesized chemically, siRNA-GAPDH was used as a positive control and non-silencing siRNA was used as a negative control. After transfecting siRNA into MCF-7 cells ( express high level of PRLR) with lipofectin, expression of gene was quantified by real-time PCR. 24 hours after transfection of siRNA (terminal concertation 100 nM), compared with the negative control group, siRNA-GAPDH could inhibit expreesion of GAPDH by 68%; and among the three siRNA-PRLRs, only siRNA-PRLR1 could inhibit the expression of PRLR by 63%, while the other two siRNA-PRLRs had no siginificant effect on inhibition of PRLR expression. So we successfully selected an effective siRNA sequence targeting PRLR, which could specifically inhibit the expression of PRLR.In the following experiments, we used siRNA-PRLR1 as the treatment; cell photographing under microscope , MTT assay and flow cytometric analysis were applied to investigate change of MCF-7 cells in growth, proliferation and cell cycle after transfection of siRNA-PRLR. Meanwhile, we detected cyclin D1 expression by reverse transcription PCR(RT-PCR) and gradation analysis to study the probable mechanism of proliferation inhibition caused by siRNA-PRLR. Results of experiments revealed that: 24 hours after siRNA-PRLR ( 100 nM ) transfection, compared with the negative control group, cell growth turned worth and proliferation was inhibited obviously, cells in S phase were reduced, with some of the cells arrested in G1 phase, and expression of CCND1 mRNA was reduced.To conclude, in human breast cancer cell line MCF-7, the expression of PRLR mRNA can be inhibited by transfection of siRNA-PRLR. After down regulation of PRLR mRNA, the extent of signaling triggered by PRL is limited, which leads inhibition of breast cancer cell proliferation and part reversion of malignant phenotype.