Fig 1: miR-204-5p restricts cell proliferation by targeting RGS20. (A) Detection of miR-204-5p using RT-qPCR. (B) RGS20 mRNA was detected by using RT-qPCR. (C) RGS20 protein was detected using Western blot. (D) miR-204-5p inhibited cell viability. (E) Representative images of the EdU experiment. (F) miR-204-5p inhibited cell replication. (G) Univariate histograms of the flow cytometry detection. (H) miR-204-5p transfection causes G0/G1 phase arrest. (I) Representative Western blot image of cell cycle proteins. (J) miR-204-5p inhibits CDK2 and cyclin E1. *P < 0.05, **P < 0.01, *** and ****P < 0.001, vs mimic NC + vector; ns, not significant; #P < 0.05, ##P < 0.01, ### and ####P < 0.001, vs miR-204-5p mimic + vector.
Fig 2: miR-204-5p is frequently downregulated in patients with HCC. (A) Venn diagram showing miRNA targeting RGS20 expression. (B) The mRNA expression between RGS20 and miR-204-5p manifested a slight negative correlation. (C) miR-204-5p were found with huge gaps in normols (NT, n = 50) and tumors (n = 370). (D) miR-204-5p levels is lower for AFP-positive HCC tissues than for AFP-negative HCC tissues. (E) miR-204-5p expression was independent of Child–Pugh grade. (F) miR-204-5p expression was less in macro vascular invasion compared with that in no or micro vascular invasion. (G) The expression of miR-204-5p was higher in G1 than in G2, G3, and G4. (H) miR-204-5p was entirely unrelated to TNM stage. (I) Higher presentation of miR-204–5p predicted better livability. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 3: miR-204-5p targets RGS20 and downregulates its expression. (A) Figure displaying the combination of miR-204-5p with RGS20 3′-untranslated region (UTR). (B) dual-luciferase reporter assay. (C) Detection of miR-204-5p using RT-qPCR. (D) Transfection of miR-204-5p downregulated RGS20 mRNA. (E) Transfection of miR-204-5p downregulated RGS20 protein expression. (F) miR-204–5p and RGS20 showed a weak negative correlation according to the cDNA microarray data set analysis. (G) RGS20 is sharply higher in tumors than in healthy individuals/adjacent liver tissue (ANT). (H) miR-204-5p was enormously less expressed in tumors than in healthy individuals/adjacent liver tissues. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.
Fig 4: (A) miR-204-5p promotes apoptosis by targeting RGS20 expression. (A) Representative images of apoptosis detected using flow cytometry. Transfection of miR-204-5p mimic increased the proportion of apoptotic cells. (B) Representative Western blot images of apoptosis-related proteins. Transfection of miR-204-5p mimic increased Bax, cleaved caspase-3, and cleaved caspase-8, but decreased Bcl-2. *P < 0.05, **P < 0.01, ***P < 0.001, vs. mimic NC + vector; ##P < 0.01, ### and ####P < 0.001, vs miR-204-5p mimic + vector.
Fig 5: miR-204-5p prevents cells from being migrated and invaded by targeting RGS20 expression. (A) Representative images of crystal violet-stained cells migrating from the upper to the lower compartment. Scale: 50 μm. Transfection of miR-204-5p mimic reduced the number of migrating cells. (B) Representative images of crystal violet-stained cells invading from the upper into the lower chamber. Scale: 50 μm. Transfection of miR-204-5p mimic reduced the number of invading cells. (C) Western blot technology for verifying proteins related to cell migration and invasion. *P < 0.05, **P < 0.01, ***P < 0.001 vs mimic NC + vector; ##P < 0.01, ### and ####P < 0.001 vs miR-204-5p mimic + vector group.
Supplier Page from Abcam for Anti-RGS20 antibody