Downstream genes and pathways of ASH1-miR-375-YWHAZ axis in hepatocellular carcinoma

To explore the mechanism and downstream pathways of ASH1-miR-375-YWHAZ axis in hepatocellular carcinoma (HCC). We performed microarray assay to identify the downstream genes of YWHAZ. As a result, we obtained 507 (245 upregulation and 262 downregulation) differentially expressed genes significantly affected by YWHAZ knockdown. Function enrichment analysis showed that these genes were enriched in the “Regulation of actin cytoskeleton” pathway which is related with cell migration and tumor metastasis. We also find that most of the autophagy-associated genes, such as ATG3, ATG5 and ATG7 and EMT related gene E-cadherin were highly correlated with YWHAZ and changed by silencing YWHAZ. Real-time quantitative RT-PCR was performed to verify the microarray results. In conclusion, the expression profile of many genes were significantly affected by YWHAZ knockdown, which helps to further explore the mechanism of the effects of ASH1-miR-375-YWHAZ axis in HCC. SMMC-7721 cells transfected with si-YWHAZ_1 or siRNA-NC were used to synthesize double-stranded complementary DNA (cDNA), which was labeled and hybridized on the SurePrint G3 Human Gene Expression Microarray 8×60K v2 (Agilent Technologies, Santa Clara, CA, USA). Processed slides were scanned with the Agilent G2565CA Microarray Scanner (Agilent Technologies) after hybridization and washing. Data were extracted with Feature Extraction software 10.7 (Agilent technologies, Santa Clara, CA, US). Quantile normalization and subsequent data processing were performed using the R software limma package. Differentially expressed lncRNAs and mRNAs between two samples were identified through Fold Change filtering and Student’s t-test. The normalized intensity for each lncRNA and mRNA was presented as a log2-transformed pattern, fold changes >2 and p-values <0.05 were selected as significantly differentially expressed.