Myosin light chain 9 promotes the proliferation, invasion, migration and angiogenesis of colorectal cancer cells by binding to Yes-associated protein 1 and regulating Hippo signaling

Abstract

Colorectal cancer is a common type of cancer with high incidence and poor prognosis. Increased expression of myosin light chain 9 (MYL9) has been reported in early-stage and recurrent colorectal cancer tissues. This study aimed to investigate the precise role of MYL9 on the progression of colorectal cancer. MYL9 expression in several colorectal cancer cell lines was detected by Western blotting and RT-qPCR. Following MYL9 overexpression or knockdown, MYL9 expression was determined via RT-qPCR. Cell proliferation was detected with Cell Counting Kit-8 assay. Cell invasion, migration and angiogenesis were, respectively, examined with transwell, wound healing and tube formation assays. The binding between MYL9 and Yes-associated protein 1 (YAP1) was verified by a co-immunoprecipitation assay. The expression of YAP1, connective tissue growth factor and cysteine-rich angiogenic inducer 61 was examined by Western blotting. Subsequently, YAP1 silencing or Hippo antagonist was performed to clarify the regulatory mechanisms of MYL9 in colorectal cancer progression. Experimental results showed that MYL9 expression was elevated in colorectal cancer cell lines. MYL9 overexpression promoted cell proliferation, invasion, migration and angiogenesis, while silencing of MYL9 exerted the opposite effects. Results of co-immunoprecipitation assay indicated that MYL9 could bind to YAP1. Further experiments revealed that MYL9 affected the expression of YAP1 and its downstream signaling proteins. Afterward, YAP1 knockdown or the addition of Hippo antagonist inhibited the proliferation, invasion, migration and angiogenesis of colorectal cancer cells. Overall, MYL9 promotes the proliferation, invasion, migration and angiogenesis of colorectal cancer cells by binding to YAP1 and thereby activating Hippo signaling.

Keywords: Hippo; MYL9; YAP1; colorectal cancer; invasion; migration.

Figure 1.

MYL9 expression was elevated in colorectal cancer cell lines. (a-b) Detection of MYL9 protein and mRNA expression in several colorectal cancer cell lines (SW480, SW620, HT-29 and HCT116) and human normal intestinal epithelium cells (NCM460) using Western blotting and RT-qPCR. ***P < 0.001 vs. NCM460.

Figure 2.

MYL9 affected cell proliferation, migration and invasion of colorectal cancer cells after overexpression or knockdown of MYL9. (a) Detection of MYL9 mRNA expression after transfection with siRNA-MYL9-1 or siRNA-MYL9-2 in HCT116 cells by RT-qPCR assay. ***P < 0.001 vs. siRNA-NC. (b) Inspection of overexpression of MYL9 in colorectal cancer cell lines was carried out by RT-qPCR. ***P < 0.001 vs. Ov-NC. (c) CCK-8 was adopted to detect cell proliferation levels. (d-e) Wound healing was used to evaluate the ability of cell migration. (f-g) Transwell assay was performed for cell invasion. (h). Western blot assay was used to test the expression of MMP2 and MMP9. **P < 0.01, ***P < 0.001 vs. siRNA-NC; ^###P < 0.001 vs. Ov-NC.

Figure 3.

MYL9 affects the angiogenesis of colorectal cancer cells after overexpression or knockdown of MYL9. Tube formation assay was utilized to detect in vitro angiogenic capacity. *P < 0.05 vs. siRNA-NC; ^###P < 0.001 vs. Ov-NC.

Figure 4.

MYL9 bound to YAP1 and regulated the expression of YAP1 and its downstream signaling proteins after overexpression and knockdown of MYL9. (a) BioGRID database (https://thebiogrid.org/) was used to predict that MYL9 could interact with YAP1. (b) Co-IP assay was used to validate the binding between MYL9 and YAP1 in HCT116 cells. (c). Expression of YAP1 and its downstream signaling proteins CTGF and CYR61 was examined by Western blotting. ***P < 0.001 vs. siRNA-NC; ^##P < 0.01, ^###P < 0.001 vs. Ov-NC.

Figure 5.

MYL9 promoted proliferation, migration and invasion of colorectal cancer cells through YAP1-Hippo signaling. (a-b) Western blotting and RT-qPCR were adopted to test the expression level of YAP1 protein and mRNA in after YAP1 silencing. ***P < 0.001 vs. siRNA-NC. (c) CCK-8 assay was utilized to detect cell proliferation. (d-e) Wound healing was used to evaluate the ability of cell migration. (f-g) Transwell assay was performed for the evaluation of cell invasion. (h). Western blot assay was used to test the expression of MMP2 and MMP9. ***P < 0.001 vs. control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. Ov-MYL9+ siRNA-NC; ^ΔP < 0.05, ^ΔΔP < 0.01, ^ΔΔΔP < 0.001 vs. Ov-MYL9.

Figure 6.

MYL9 promoted angiogenesis in colorectal cancer cells via YAP1-Hippo signaling. Tube formation assay was implemented to detect in vitro angiogenesis. ***P < 0.001 vs. control; ^###P < 0.001 vs. Ov-MYL9+ siRNA-NC; ^ΔΔΔP < 0.001 vs. Ov-MYL9.