miR-4770 promotes colorectal cancer progression by targeting PRKAA2 via m6A modification

Figure 1 The regulation of METTL3 on miR-4770 expression and N6-methyladenosine modification level. A: The expression of METTL3 in colorectal cancer (CRC) tissues and normal tissues was detected by qRT-PCR; B: The expression of miR-4770 was positively correlated with METTL3 in CRC tissues; C-E: The expression of METTL3 (C), miR-4770 (D) and pri-miR-4770 (E) was detected in siNC, si-METTL3, NC and METTL3-oe cells (qRT-PCR); F: RIP assay was used to detect the binding level of pri-miR-4770 and DGCR8 in the above four groups of cells; G: MeRIP assay was used to detect the N6-methyladenosine modification level of pri-miR-4770 in the above four groups of cells. Data were expressed as mean ± SD. ^aP < 0.01 vs normal group; ^bP < 0.05 and ^cP < 0.01 vs siNC group; ^dP < 0.01 vs NC group. Clinical tissue analyses were based on 10 paired CRC and adjacent normal tissues, whereas in vitro experiments were performed with three independent biological replicates. CRC: Colorectal cancer.

Figure 2 The expression of miR-4770 in colorectal cancer and its effect on cell viability, migration and invasion. A: The expression of miR-4770 in colorectal cancer (CRC) tissues and adjacent normal tissues was detected by qRT-PCR; B: The expression of miR-4770 in FHC and HCT116 cells was detected by qRT-PCR; C: The expression of miR-4770 in the NC inhibitor, miR inhibitor, NC mimic, and miR mimic groups was detected by qRT-PCR; D: CCK-8 method was used to detect the proliferation activity of the above four groups of cells at 24 hours, 48 hours, and 72 hours; E and F: Transwell assay was used to evaluate the migration (E) and invasion (F) abilities of the above four groups of cells. Scale: 100 μm. The data were expressed as mean ± SD. ^aP < 0.01 vs normal group; ^bP < 0.01 vs FHC group; ^cP < 0.05 and ^dP < 0.01 vs NC inhibitor group; ^eP < 0.05 and ^fP < 0.001 vs NC mimic group. Clinical tissue analysis included 10 paired CRC and adjacent normal tissues, and in vitro experiments were performed with three independent biological replicates. CRC: Colorectal cancer.

Figure 3 Verification of the targeting relationship between PRKAA2 and miR-4770. A: The mRNA levels of PRKAA2 in colorectal cancer (CRC) tissues and normal tissues were determined by qRT-PCR; B: The expression of the two in CRC tissues was negatively correlated; C: The expression of PRKAA2 was detected by qRT-PCR in four groups of cells: NC inhibitor, miR inhibitor, NC mimic and miR mimic; D: Western blot was used to detect the level of PRKAA2 protein in the above four groups of cells; E: Predicting the target binding sequence of miR-4770 and PRKAA2; F: Dual luciferase reporter assay verified the direct targeting relationship between the two. Data were expressed as mean ± SD. ^aP < 0.01 vs normal group; ^bP < 0.01 vs NC inhibitor group; ^cP < 0.05 vs NC mimic group; ^dP < 0.01 vs miR-4770 NC group. Clinical tissue analyses included 10 paired CRC and adjacent normal tissues, and in vitro experiments were performed with three independent biological replicates. CRC: Colorectal cancer.

Figure 4 miR-4770 regulates the viability, migration and invasion of HCT116 cells through PRKAA2. A: QRT-PCR was used to detect the mRNA expression of PRKAA2 in siNC group and si-PRKAA2 group; B: CCK-8 method was used to detect the proliferation activity of four groups of cells at 24 hours, 48 hours and 72 hours: NC inhibitor + siNC, miR inhibitor + siNC, NC inhibitor + si-PRKAA2, miR inhibitor + si-PRKAA2; C and D: Transwell assay was used to evaluate the migration (C) and invasion (D) abilities of the above four groups of cells. Scale: 100 μm. The data are expressed as mean ± SD and are derived from three independent biological replicates. ^aP < 0.01 vs siNC group; ^bP < 0.05 and ^cP < 0.01 vs NC inhibitor + siNC group; ^dP < 0.05 and ^eP < 0.01 vs miR inhibitor + siNC group; ^fP < 0.05 and ^gP < 0.01 vs NC inhibitor + si-PRKAA2 group.

Figure 5 Expression of miR-4770 in colorectal cancer cells and its regulation of PRKAA2. A: The mRNA levels of miR-4770, METTL3 and PRKAA2 in FHC, HCT116 and SW480 cells were detected by qRT-PCR; B and C: In SW480 cells, qRT-PCR was used to detect the expression of miR-4770 (B) and PRKAA2 (C) in each group, which were divided into NC inhibitor, miR inhibitor, NC mimic and miR mimic; D: Western blot was used to detect the level of PRKAA2 protein in the above four groups of SW480 cells. Data are expressed as mean ± SD and are derived from three independent biological replicates. Statistical difference: ^aP < 0.01 vs FHC group; ^bP < 0.05 and ^cP < 0.01 vs NC inhibitor group; ^dP < 0.01 vs NC mimic group.