High Expression Of HIFI Is Associated With Poorer Prognosis Via Regulating TβR And CDK In Breast Cancer

Objective: Recent studies showed that abnormal PIWI may play a crucial role in tumorigenesis. In this research, we studied the expression level of HIWI gene which is one of the PIWI human homologous genes in breast cancer patients. Moreover, HIWI promotes the proliferation of breast cancer cells and inhibits their apoptosis. Furthermore, to explore HIWI whether as a kind of potential biochemical marker for clinical evaluation, treatment and prognosis of breast cancer in the future.Methods: In our study, the real-time polymerase chain reaction were performed among following organizations: 25 cases of adjacent normal breast tissues; 19 cases of cancer tissues from patients(Ⅰ-Ⅱ stages and axillary lymph node-negative in breast cancer); 8 cases of cancer tissues coming from patients( Ⅲ-Ⅳ stages with metastases in breast cancer). And in each group the tissues were detected by Western blotting process. MCF-7 breast cancer cell lines were cultured to obtain the progeny cells. We constructed the interference and over-expression vector(HIWI-sh RNA and HIWI-pc DNA3.1). After the plasmids were conducted, the transfection was performed using Lipofectamine TM 2000. The cells were divided into five groups, including control group, HIWI interference group, HIWI over-expression group, HIWI interference rescue group and HIWI over-expression rescue group. The expression level of HIWI both in m RNA and protein was detected by PCR and Western blot while the rate of apoptosis was estimated by flow cytometric analysis. Finally, to test the expression of CDKs and TβR to identify if it is closely related to the expression of HIWI.Results: PCR and Western blot analysis showed that m RNA expression of MAEL, HIWI and HILI significantly increased in the tissues of breast cancer, while no significant changes detected in PIWIL3 and HIWI2. The results indicated that the highest expression of HIWI was found in over-expression group and the lowest m RNA expression was found in interference group. Meanwhile, the control group, over-expression rescue group and interference rescue group showed median expression. The expression level of the over-expression group and the interference group showed statistically significant differences compared with the rest of the three groups(P<0.05). Moreover, the protein expression showed that the over-expression group had the strongest signals compared to other groups, whereas the interference group had the lowest protein expression of HIWI among all the groups. The gene expression of m RNA and protein of Cyclin D and CDK were assayed, in which CDK4, CDK6 and CDK8 showed significant increases in over-expression group compared with other four groups(P<0.05), while the other groups had no significant differences. Therefore, the expression level of HIWI showed close correlation with the expression level of TβRⅠ, TβRⅡ, CDK4, CDK6 and CDK8(P<0.05). Furthermore, the result indicated that the lowest apoptosis rate in over-expression group in contrary with the highest apoptosis rate in interference group by flow cytometry. The over-expression group and interference group compared with the rest three groups showed significant differences(P<0.05). Moreover, the interference group showed a significant higher G2/M arrest rate compared with other groups. In addition, our study showed a significant negative correlation between the HIWI expression level and the survival rate(P<0.05).Conclusion: 1. The expression levels of HIWI and HILI, which are closely related to the tumor stages, significantly increase in the tissues of breast cancer.2. High expression of HIWI is associated with poorer prognosis, whereas HILI shows no correlation in patients of breast cancer. Accordingly, HIWI is one kind of suitable biomarker for breast cancer instead of HILI in evaluation of both progression and prognosis.3.Over-expression of HIWI promotes cell activities and inhibits the apoptosis of cells while interference of HIWI increases the apoptosis rate significantly.4. The expression levels of HIWI indicate negative correlation with the expression of TβRⅠand TβRⅡ, while cell cycle regulatory genes including CDK4, CDK6 and CDK8 are positively correlated with HIWI. Therefore, HIWI can regulate progression of breast cancer in micro level.