Fig 2: The workflow of entire study. First, with the aid of global microRNA expression analysis, a total of 9 prognostic miRNAs for GIC were identified. Second, after analysis of disease-specific survival and progress-free interval, miR-194 turn out to be an optimal prognostic biomarker for GIC. Third, 18 potential target genes greatly downregulated by miR-194 were screened using transcriptome sequencing. Forth, genome-wide CRISPR-CAS9 knockout screening identified 4 miR-194 target genes required for cell proliferation. Fifth, the Ago-HITS-CLIP-seq, qRT-PCR, immunoblotting and cell proliferation assays together confirmed miR-194 inhibited GIC proliferation by targeting ATP6V1F, BTF3L4, PPP1R14B and SLC7A5.

Fig 3: MiR-194 inhibited GIC cell proliferation by targeting ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4. (A-C) The expression of miR-194 was negatively associated with the expression of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 in gastric cancer (STAD), colorectal cancer (COADREAD) and liver cancer (LIHC). (D) The expression of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 were determined after overexpression of miR-194 in GIC cell lines. (E) Overexpression of miR-194 greatly downregulated the protein level of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4. (F-H) Cell proliferation assays showed that overexpression of miR-194 had similar effects on cell proliferation as siRNA pools containing siRNAs targeting ATP6V1F, PPP1R14B, SLC7A5, and BTF3L4. **P < 0.01.

Fig 4: Ago-HITS-CLIP-seq analysis identified ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4 as target genes of miR-194. (A-D) An obvious binding peak of miR-194 was present within the mRNA of ATP6V1F, PPP1R14B, SLC7A5 and BTF3L4, respectively. The statistical results were shown on the right panel. The binding site of miR-194 and PPP1R14B gene is located in in the coding region. While the binding site of miR-194 and other three genes is located in the 3’ untranslated region. (E-H) There was an obvious miR-194 seed sequence binding site inside the miR-194 binding peaks identified by Ago-HITS-CLIP-seq analysis. The binding sites of miR-194 in 4 genes identified by Ago-HITS-CLIP-seq analysis are highly consistent with the predicted results of bioinformatics analysis.