MYO1B enhances colorectal cancer metastasis by promoting the F-actin rearrangement and focal adhesion assembly via RhoA/ROCK/FAK signaling

Figures

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  Figure 1. A high expression of MYO1B in colorectal cancer increases the risk of tumor metastasis and poor prognosis for patients. (A) Quantitative real-time PCR detection of the transcriptional level of MYO1B in fresh clinical tissues. Bars represent the ratio of MYO1B expression in CRC tissues (CTcancer, CRC tissues) to that in matched adjacent normal tissues (CTNormal, adjacent normal tissues). (B) The paired Student’s t-test analysis of the difference between MYO1B transcription in fresh CRC and matched adjacent normal tissues. (C) Pearson’s chi-squared (χ2) test analysis of the relationship between MYO1B transcription and tumor metastasis, where “nmCRC” indicates non-metastatic CRC, and “mCRC” indicates metastatic CRC. (D) Pearson’s chi-squared (χ2) test analysis of the relationship between MYO1B transcription and tumor stage, where “S1”, “S2”, “S3”, and “S4” indicate stage I, stage II, stage III, and stage IV, respectively. (E) Representative figures of the MYO1B protein expression in CRC and adjacent normal tissues by immunohistochemical staining. Shown at ×100 original magnification. (F) Representative figures of the different levels of MYO1B expression in CRC and normal mucosa…

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  Figure 2. MYO1B promotes the migration, invasion, and motility of CRC cells in vitro. (A) Representative figures of the western blots for MYO1B expression in normal epithelial and colorectal cancer cell lines. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. (B) Representative figures of the western blots for MYO1B expression in cells transfected with lentivirus-MYO1B or lentivirus-shMYO1B. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. (C,D) Transwell assay without matrigel to investigate the migration property of CRC cells. Representative figures are in the left panel, and bars in the right panel are expressed as mean ± SEM. Cells were stained with crystal violet and counted. Scale bar: 200 µm. (E,F) Transwell assay with matrigel to investigate the invasion property of CRC cells. Representative figures are in the left panel, and bars in the right panel are expressed as mean ± SEM. Cells were stained with crystal violet and counted. Scale bar: 200 µm. (G,H) Wound-healing assay for investigating the motility of CRC cells. Representative figures…

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  Figure 3. MYO1B promotes the risk of tumor metastasis and poor prognosis for CRC in vivo. (A) The rate of tumor formation and metastasis in the orthotopic xenograft mouse model. (B) The Kaplan-Meier survival analysis of the effect of MYO1B on CRC prognosis in vivo. (C,D) Representative figures of in vivo imaging in the orthotopic xenograft colorectal cancer mouse model. The square region of dotted lines indicates the CRC metastasis nodule in the liver; “w” indicates week. (E,F) HE staining of the CRC metastasis nodule in the liver. Representative figures are in the left panel, and bars in the right panel are expressed as mean ± SEM. Shown at ×400 original magnification. *P<0.05, **P<0.01, vs. control. CRC, colorectal cancer; Ctrl, control; Veh, vehicle.

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  Figure 4. MYO1B promotes F-actin rearrangement and the activation of the ROCK2/LIMK/Cofilin axis in CRC cells. (A,B) Immunofluorescence assay of the rearrangement of F-actin. Representative figures are in the left panel, red arrows indicate the polymerization of F-actin, while white arrows indicate the depolymerization of F-actin. Bars in the right panel are expressed as mean ± SEM. (C) Representative figures of the western blots for the expression of the ROCK2/LIMK/Cofilin axis signatures in cells transfected with lentivirus-MYO1B or lentivirus-shMYO1B. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. (D,E) Representative figures of the immunofluorescence assay for the expression of p-LIMK. (F,G) Representative figures of the immunofluorescence assay for the expression of p-Cofilin. Shown at ×10,000 original magnification (A,B,D-G). **P<0.01 vs. control. CRC, colorectal cancer; Ctrl, control; Veh, vehicle.

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  Figure 5. MYO1B promotes the activation of RhoA and the formation of migratory focal adhesions in CRC cells. (A) Representative figures of the co-immunoprecipitation assay on MYO1B to CDC42, RAC1, and RhoA. (B) Representative figures of the co-immunoprecipitation assay on RhoA to MYO1B. (C) Representative figures of the immunofluorescence assay on the co-localization of MYO1B and RhoA in CRC cells. (D) Representative figures of the western blots for RhoA expression in cells transfected with lentivirus-MYO1B or lentivirus-shMYO1B. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. (E,F) Representative figures of the co-immunoprecipitation assay for the expression of RhoA and GTP-binding RhoA (RhoAGTP). (G) Representative figures of the western blots for the expression of the FAK/paxillin axis signatures in cells transfected with lentivirus-MYO1B or lentivirus-shMYO1B. The values under the membrane represent the expression of genes normalized to the expression of the reference gene GAPDH. Shown at ×10,000 original magnification. (H) Representative figures of the immunofluorescence assay on the formation of migratory focal…

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  Figure 6. MYO1B promotes the migration, invasion, and motility of CRC cells by targeting RhoA. (A) Representative figures of the immunoprecipitation assay for the expression of RhoA and GTP-binding RhoA (RhoAGTP) in cells treated with exoenzyme-C3, where “E-C3” indicates exoenzyme-C3. (B) Representative figures of the transwell assay with and without matrigel. Cells were stained with crystal violet and counted. Scale bar: 200µm. (C) Bars are expressed as mean ± SEM, indicating the number of migrating cells in the transwell assay without matrigel. (D) Bars are expressed as mean ± SEM, indicating the number of invading cells in the transwell assay with matrigel. (E) Bars are expressed as mean ± SEM, indicating the migration rate of cells in the wound-healing assay. (F) The fibronectin adhesive assay of the adhesive property of CRC cells. Cells were fixed and stained with 1% crystal violet. Scale bar: 100 µm. Representative figures are in the left panel, and bars in the right panel are expressed as mean ± SEM. (G) Representative figures of the immunofluorescence assay on the formation of migratory focal adhesions. Shown at ×10,000 original magnification. (H) The immunofluorescence assay of…