To study the molecular mechanism by which miR-203a affects the development of CML, bioinformatics software was used to predict the upstream transcription factors and downstream target genes of miR-203a. A 5’-rapid amplification of cDNA ends assay was performed to detect gene transcription initiation sites. A chromatin immunoprecipitation assay was used to verify the binding of transcription factors and promoter regions. A double luciferase reporter gene vector was constructed to demonstrate the regulatory effect of miR-203a on target genes. Real-time PCR and western blotting were used to detect the relative expression levels of genes and proteins, respectively. The results showed that there was a binding site for the transcription factor EGR1 in the upstream promoter region of miR-203a. WT1, BMI1, and XIAP were identified as target genes regulated by miR-203a. EGR1 and miR-203a were downregulated in human peripheral blood mononuclear cells and the CML K562 cell line, while WT1, BMI1, and XIAP were upregulated. The transcription initiation site of miR-203a was identified in the upstream promoter region (G nucleotide at −339 bp), and the transcription factor EGR1 could bind to the promoter region (at −268 bp) of miR-203a and increase its expression. Over expression of miR-203a inhibited the proliferation of K562 cells. A rescue assay showed that overexpression of WT1, BMI1, and XIAP offset the antitumor effect of miR-203a. Conclusion, EGR1 positively regulated the expression of miR-203a, thus relieving the inhibition of miR-203a on the translation of its target genes (WT1, BMI1, and XIAP) and affecting the proliferation of K562 cells.
Background: The detail of mechanism involving in PP-10 anti-cancer activity remains to be elucidated, and the effect on HCC cells is unknown. Methods: MTT and colony formation assays were used to determine the effect of PP-10 on cell growth. Flow cytometry analysis and Hoechst 33258 were used to assess apoptosis. Gene set enrichment analysis (GSEA) was used to explore changes in apoptosis-associated pathways. Western blotting was used to detect protein expression levels. Results: In our study, PP-10 significantly suppressed cancer cell viability while had low toxicity to normal cells, with the HCC cell lines HepG2 and HuH7 being particularly sensitive to PP-10 treatment. PP-10 induced mitochondrial-related apoptosis in HepG2 and HuH7 cells. Moreover, GSEA showed that the MAPK signaling pathway could be correlated with PP-10-induced apoptosis. We used western blotting to confirm that PP-10 induced apoptosis in HepG2 and HuH7 cells by modulating the JNK/SPAK signaling and inhibiting the STAT3 signaling pathway. Conclusion: Collectively, our results show that PP-10 induces apoptosis via the JNK/SPAK and STAT3 signaling pathways in HepG2 and HuH7 hepatocarcinoma cells.
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