MLF1IP promotes cells proliferation and apoptosis by regulating CyclinD1 in breast cancer

Abstract

Breast cancer is the most frequently diagnosed cancer and the leading causes of cancer death among females in worldwide. It is urgent to develop novel biomarkers to improve risk stratification and optimize therapy choice. In our previous study, we firstly found that MLF1IP was upregulated in breast cancer tissue compared with adjacent normal tissue and patients with high MLF1IP expression had significantly lower overall survival. However, the biological function and cellular mechanisms of MLF1IP in breast cancer is still need to be elucidated. Here, we further investigated the role of MLF1IP in breast cancer by in vivo experiments. Our results showed that the expression level of MLF1IP was associated with lymph nodes metastasis and tumor size in clinical characteristic features. By biological function experiment, we found MLF1IP is correlated with cell proliferation and apoptosis and arrest cell cycle G1 through regulating Cyclin D1. Taken together, our findings suggested that MLF1IP could contribute to the oncogenic potential of breast cancer. To the best of our knowledge, it was firstly reported that MLF1IP was involved in breast cancer. This study provided a potential new marker and a target for gene therapy in breast cancer treatment.

Keywords: Breast cancer; MLF1IP; cyclinD1.

Figure 1

MLF1IP expression in TCGA and Oncomine database. A. MLF1IP expression level of breast cancer in TCGA database. B. MLF1IP expression level of breast cancer in Richardson Cohort from Oncomine database. C. MLF1IP expression level of breast cancer in Radvanyi Cohort from Oncomine database. D. MLF1IP expression level of breast cancer in Zhao Cohort from Oncomine database. ***P<0.001 in comparison with the normal breast tissues using Student’s t-test.

Figure 2

Immunohistochemical staining of MLF1IP proteins in breast cancer and adjacent normal tissues. A, B. High MLF1IP immunohistochemical staining was shown in breast cancer tissues. C. MLF1IP immunohistochemical staining was shown in breast cancer (red arrow) and adjacent normal tissues (black arrow). D. Low MLF1IP immunohistochemical staining was shown in adjacent normal tissues as negative control.

Figure 3

Target cell selection and lentivirus transfection efficiency. A. The relative expression of MLF1IP in MDA-MB-213, MCF-7, T47D. Compared to the other cell lines, MCF-7 cells exhibited the highest MLF1IP expression. B. The relative expression of MFL1IP in six groups. The expression of MLF1IP in shRNA2 group was lowest, compared with other groups. C. The relative expression of MLF1IP in protein level. Left: MLF1IP expression is knocked down after transfected shRNA2 measured by Western blot. Right: Relative quantification of MLF1IP expression. *P<0.05, **P<0.01, ***P<0.001 in comparison with the NC group using Student’s t-test.

Figure 4

Down-regulated MLF1IP expression inhibits breast cancer cell proliferation and increases apoptosis. A. The relative cell proliferation in MFC-7 cells was measured after the cells were transfected with shRNA2 or negative control (NC) using CCK-8 assays. B. Cell cycle distribution was performed by flow cytometric analysis. Representative flow cytometric histograms at 48 hrs showing the distribution of cell cycle. Knockdown of MLF1IP by shRNA2 in MCF-7 cells induced cell cycle arrest in G1 phase at 48 hours after transfection. C. Down-regulated MLF1IP induced cell apoptosis. The number of apoptosis MLF1IP-transfected MCF-7 cells was significantly higher than in the NC groups. *P<0.1, **P<0.05, ***P<0.01 in comparison with the NC group using Student’s t-test.

Figure 5

Effect of downregulation of MLF1IP on protein expression level of cell cycle in MCF-7 cells. A. Total cellular protein were extracted at 48 hours after transduction and determined by Western blot analysis using antibodies against CDK2, CDK4, CDK6, Cyclin D1, Cyclin E and β-Actin as a loading control. B. Relative quantification of MLF1IP expression. Down-regulated MLF1IP significant knocked down Cyclin D1 expression. **P<0.01 in comparison with the NC group using Student’s t-test.