Study On Mechanism Of HSDL2 Protein On Cell Proliferation And Prognosis Evaluation Of Breast Cancer

Objectives:(1)To explore the expression of HSDL2 protein in breast cancer and normal breast tissues,and analyze the correlation between HSDL2 expression and clinicopathological features.(2)To investigate the role of HSDL2 protein in breast cancer cell proliferation and cell cycle distribution,and clarify the molecular mechanisms.Finally,provide potent prognostic biomarkers and molecular targets for the clinical trials of breast cancer.Materials and methods:1.Database and breast cancer clinnical specimens analysis:Oncomine database was used to compare the expression level of HSDL2 mRNA between breast cancer and normal breast tissues;The expression level of HSDL2 in 119 breast cancer tissues and 40 normal breast tissues were evaluated by immunohistochemical(IHC)staining.Then,the correlations between HSDL2 expression and clinicopathological features were evaluated by using the chi-square test.The relations between HSDL2 and the survival rates were calculated by the Kaplan-Meier,Cox proportional hazards model and also confirmed by Protein Atlas database.2.Experiments in vitro:The expression of HSDL2 in different breast cancer cell lines was confirmed by Western Blot.Knockdown of HSDL2 in MDA-MB-231 and MDA-MB-468 cells were constructed with short interfering RNA transduction.The greatest effective sequence of HSDL2 siRNA was selected by Western Blot analysis.HSDL2 protein localization in cells was observed by immunofluorescence(IF)assay.The effect of HSDL2 on cell proliferation and cell cycle progression were observed by MTT and colony formation assay and flow cytometry.The expression of cell cycle related proteins and PI3K/AKT signaling pathway-related proteins were detected by Western Blot.Results:1.HSDL2 was overexpressed in breast cancer tissues and significantly associated with the differentiation and clinical stage of breast cancer:Oncomine database analysis showed that the expression level of HSDL2 mRNA was upregulated in breast cancer tissues compared with normal breast tissues(p=0.01);IF staining showed that HSDL2 protein was mainly located in the cytoplasm;The IHC staining showed that the positive rate of HSDL2 protein was significantly higher in breast cancer tissues(87.4%,104/119)than in adjacent normal breast tissues(25%,10/40)(p<0.01).Similarly,the strongly positive rate of HSDL2 protein was also significantly higher in breast cancer tissues(48.7%,58/119)than in normal breast tissues(12.5%,5/40)(p<0.01).Moreover,HSDL2 overexpression was significantly correlated with differentiation and clinical stage(p<0.05).However,the positive rate of HSDL2 expression was not associated with age,lymph node metastasis,ER,PR or HER2 expression status.2.High expression of HSDL2 was an independent biomarker for poor prognosis of breast cancer patients:The correlation between the mRNA expression of HSDL2 and survival time of breast cancer patients were analysed by using KM plotter and the Protein Atlas database.A high expression of HSDL2 mRNA was correlated with short survival time in breast cancer patients(p<0.05).The correlation between HSDL2 expression and the overall survival(OS)rates of 119 breast cancer patients were determined by using the Kaplan-Meier method.The results showed that patients with high HSDL2 expression had lower OS rates than those with low HSDL2 expression(p<0.05).Remarkably,for PR(-)breast cancer patients,OS rates were significantly higher in patients with low HSDL2 protein expression than in those with high HSDL2 expression(p<0.05).However,the expression status of HSDL2 protein was not correlated with OS in PR(+)breast cancer patients.Univariate analysis using the Cox proportional hazards model demonstrated that clinical stage,ER status,PR status,and HSDL2 expression status were significantly associated in patients with breast cancer(p<0.05).Multivariate analysis revealed that HSDL2 overexpression was a significant independent prognostic factor for survival along with clinical stage and PR status(p<0.05).3.HSDL2 knockdown suppressed breast cancer cell proliferation by inducing cell cycle arrest:MTT assay showed that knockdown of HSDL2 expression significantly decreased cell viability in vitro(p<0.01).Colony formation assay showed that knockdown of HSDL2 expression significantly decreased the colony formation ability of the breast cancer cells.PI staining showed that HSDL2 silencing increased the percentage of cells in G0/G1 phase arrest,which was accompanied by a decrease in the G2/M phase.Western Blot showed that cyclin-dependent kinase 1(CDK1),cyclin B1,and cyclin D1 expression were decreased after HSDL2 silencing.In contrast,cyclin-dependent kinase inhibitors p21,known as a tumor suppressor,was increased in the si-HSDL2 group,while the expression of p53 was not altered.4.HSDL2 knockdown suppressed the activation of PI3K/AKT signaling pathway:Western Blot showed that knockdown of HSDL2 down-regulated the.expression of PI3K/AKT signaling pathway-related proteins(p-AktSer473,p-S6KThr389)(p<0.01),but the expression of AKT and S6 was not change.Conclusions:1.HSDL2 was overexpressed in breast cancer tissues and significantly associated with the differentiation,clinical stage and survival time of breast cancer;2.High expression of HSDL2 is an independent biomarker for poor prognosis of breast cancer patients;3.HSDL2 knockdown suppressed breast cancer cell proliferation through the induction of cell cycle arrest,the mechanisms maybe associated with the activation of PI3K/AKT signaling pathway.