Bioinformatics Analysis Of Prognostic Role Of KIF4A And Study On The Upstream Regulation Of MiR-335

Objective:Although advances in breast cancer screening and treatment have improved the survival rate in recent years,breast cancer is still a major disease that impacts women's health with huge individual differences in treatment outcomes.The current genetic prognosis prediction tools are mainly based on gene chips,which may bring additional financial burden in the absence of specificity.Therefore,discovering new accurate and economical biomarkers for prognosis prediction has important clinical significance.The human kinesin family member 4A(KIF4A)plays a key role in a variety of cellular physiological activities,such as chromosome condensation,cell mitosis.In recent years,there is evidence to prove KIF4A plays an important role in various tumors development,such as gastric cancer,lung cancer,cervical cancer and hepatocellular carcinoma.This study explored the relationship between the expression of KIF4A as an effective prognostic biomarker and the breast cancer clinical features by bioinformatics analysis of TCGA and GEO datasets,and investigated the possible regulatory factors by bioinformatics analysis and in vitro verification.Methods:In the first part of this study,we downloaded human breast cancer gene expression datasets with corresponding clinical follow-up information from the GEO database and the TCGA database for assessing the clinical significance of KIF4A in breast cancer.The patient was divided into four subgroups(Q1,Q2,Q3,and Q4)based on mRNA expression percentile.The expression level below the median was defined as low KIF4A(Q+ + Q2),and the expression level upper the median was considered to be high KIF4A(Q3 + Q4).The relationship between the expression of KIF4A and breast cancer prognosis was evaluated using R language.The expression and clinical relevance and biological effects of KIF4A were studied using GSEA analysis.In the second part,a variety of breast cancer celllines were selected,RT-PCR and western-blot method were used to detect the expression of KIF4A in various breast cancer cells.MDA-MB-231 cells with high expression of KIF4A were selected for further function identification experiment.The shRNAs targeting KIF4A with corresponding control were constructed and transfected into breast cancer cells respectively.CCK-8 experiment was used to detect cell proliferation activity,colony formation assay was used to detect cell colony formation ability,and flow cytometry was used to detect apoptosis.At the same time,western-blot method was used to detect the expression of exogenous apoptosis-related proteins in the cells after KIF4A expression knockdown.In the third part,miRNAs associated with KIF4A expression in breast cancer were identified using the GSE22220 dataset in GEO database and TCGA database.The miRanda algorithm was used to predict miRNAs with KIF4A mRNA binding sites and the miRNAs most likely to be involved in KIF4A expression regulation.The dual luciferase reporter assay was used to predict the direct effect of miR-335 and KIF4A.Results:In the first part of this study,the expression level of KIF4A was significantly correlated with tumor size and grade,and was not significantly associated with lymphatic metastasis.In the GSE1456 dataset,KIF4A expression was significantly higher in basal-like breast cancer.Further GSEA analysis of the GSE1456 dataset indicated KIF4A high expression phenotype significantly enriched gene signatures associated with high tumor grade,suggesting that KIF4A is closely associated with breast cancer susceptibility,invasion,metastasis and recurrence.Survival analysis of the TCGA dataset and the GEO dataset revealed that high expression of KIF4A was significantly associated with low overall survival(OS)and progression-free survival(PFS).As the expression of KIF4A increased,OS and PFS decreased gradually in a dose-dependent manner.Further analysis of GEO pooled data showed that KIF4A expression levels in ER positive and ER negative subtypes were significantly associated with poor OS and PFS,and a significant dose dependency was observed in ER positive subtype.These findings suggest that KIF4A could be a powerful predictor for breast cancer prognosis.In the second part,cell function experiments were performed.RT-PCR and Western-blot showed that KIF4A was highly expressed in breast cancer.MDA-MB-231 cells were selected for knock-down assay.The expression of KIF4A was down-regulated and the proliferation of breast cancer cells was significantly inhibited and the number of apoptosis was significantly increased compared with the control group.In the third part,there showed a correlation between miR-335 expression and patient survival or lymph node metastasis in TCGA database analysis.The miRanda algorithm was used to predict miRNAs with KIF4A mRNA binding sites,indicating miR-335 and miR-411 might be KIF4A-associated regulatory miRNAs of KIF4A.The KIF4A and miR-335 and miR-411 expression levels were negatively correlated.By further stratification analysis,the prognosis(PFS)was associated with miR-335 expression in a dose-dependent manner,rather than miR-411 expression.Using the dual luciferase reporter assay,miR-335 inhibits KIF4A expression by targeting the 3'-UTR of KIF4A mRNA.Conclusion:In the first part of this study,KIF4A expression was significantly associated with breast cancer size and grade,and KIF4A expression was significantly elevated in basal-like breast cancer.GSEA analysis suggested that KIF4A is closely associated with breast cancer susceptibility,invasion,metastasis and recurrence.Further survival analysis found that high expression of KIF4A was significantly associated with poor prognosis,especially in ER-positive subtype breast cancer,KIF4A can be a powerful predictor of breast cancer prognosis.In the second part,we identified the expression and function of KIF4A in breast cancer cell lines by in vitro experiments.KIF4A is highly expressed in a variety of breast cancer cell lines,with high expression in MDA-MB-231.The proliferation of MDA-MB-231 cells was significantly decreased by KIF4A shRNA knockdown.Flow cytometry analysis showed a significantly increased apoptosis in MDA-MB-231 cells after KIF4A knockdown.This suggests that KIF4A affects tumor progression by regulating breast cancer cell proliferation and apoptosis.In the thrd part,miR-335 and miR-411 may be the corresponding regulatory miRNAs of KIF4A,and the KIF4A expression is negatively correlated with miR-335.Further TCGA database analysis and dual luciferase reporter assays suggest that miR-335 inhibits KIF4A expression by targeting the 3'-UTR of KIF4A mRNA,which in turn affects the prognosis of breast cancer.