Modulation Of The HepG2 Cell Migration, Invasion And Cell- ECM Adhesion By Human Monopolar Spindle-one-binder 2

Objective:Human monopolar spindle-one-binder 2 (hMOB2) is a member of the hMOB family of proteins, and it has been reported to regulate the nuclear-Dbf2-related kinase (NDR) activation and plays a role in centrosome duplication, cell proliferation and apoptotic signaling. However, the function of hMOB2 expression in tumor cell adhesion and motility has not been addressed. The aim of the study is to evaluate whether hMOB2 regulates cell migration, invasion and cell-extracellular matrix (ECM) adhesion of the HepG2 cells, and to clarify the mechanism of hMOB2 in the regulation of HepG2 cell motility and cell-ECM adhesion at the cellular level.Methods:Lentiviral-mediated overexpression and knockdown of hMOB2 in HepG2 cells was established to study the role of hMOB2 in the regulation of tumor cell migration, invasion and cell-ECM adhesion.(1) Real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays were initially used to examine the overexpression and knockdown of hMOB2 in HepG2 cells at the levels of hMOB2 mRNA from total RNA extracts, and the hMOB2 protein levels from whole cell extracts, normalized to GAPDH, respectively. Furthermore, western blot assay was also used to evaluate the effects of hMOB2 on the expression and activation of FAK, Src, and paxillin in theHepG2 cells.(2) MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide) and the Cell Counting Kit-8 (CCK-8) assays were performed to examine theeffect of hMOB2 on cell viability of theHepG2 cells.(3) Transwell cell migration and invasion assays were used to assess the effect of hMOB2 on the HepG2 cell migration and invasion.(4) Tumor cell adhesion to collagen type I matrix and spreading on it assays were performed to investigate the role of hMOB2 on cell-matrix adhesion and spreading of the HepG2 cells.(5) Immunofluorescence staining assay was used to study the effect of hMOB2 on theassembly of the focal adhesion and the actin cytoskeleton rearrangement, such as actin stress fibers assembly and the formation of filopodia and lamellipodia.Results:Results from RT-qPCR and Western blot assays showed that hMOB2 was overexpressed in LV-hMOB2 transduced HepG2 cells, and shown the most efficient inhibition of hMOB2 expression by LV-sihMOB2-4. Thus, LV-hMOB2 and LV-sihMOB2-4 (following named LV-sihMOB2) could be used in the following experiments.(1) Data from both MTT assay and CCK-8 assay demonstrated that neither overexpression of hMOB2by LV-hMOB2 infection nor knockdown of hMOB2by LV-siMOB2 infection had significant effects on HepG2 cell viability relative to the Mock, LV-CTL and LV-siCTL infection.(2) Transwell cell migration and invasion assaysdemonstrated that HepG2 cells infected with LV-hMOB2 showed dramatically inhibition of the cell migratory and invasive capacity when compared with those transduced with the empty vectors (LV-CTL and LV-siCTL) and the mock(p<0.01), whereas the knockdown of hMOB2 expression by LV-sihMOB2 also decreased the motile cells in HepG2 cells(p<0.05). No significant differences in the cell motility were shown among the mock and the empty vector-transduced cells.(3) Tumor cell adhesion to collagen type I matrix assay showed that hMOB2 overexpression markedly promoted the cell-matrix adhesion of the HepG2 cells(p<0.01), while the hMOB2 knockdown decreased the cell-matrix adhesion compared with those in the mock and the empty vector-transduced cells(p<0.05). Moreover, the findings also demonstrated that hMOB2 overexpression affected the HepG2 cells spreading on the matrix.(4) Data from immunofluorescence staining of paxillin (a marker of mature and nascent focal adhesion) showed that hMOB2 overexpression significantly increased both the number and the size of the focal adhesions(p<0.01), and exhibited diffuse distribution of the focal adhesions throughout the HepG2 cells, while the hMOB2 knockdown reduced the number of the focal adhesions producing a prominent peripheral distribution of the cells, when compared with those in the empty vector-transduced cells and the mock. In parallel, Rhodamine-conjugated phalloidin staining F-actin showed that more stress fibers and less filopodia and lamellipodia at the leading edgewere observed in the hMOB2-overexpressing HepG2 cells than those in the empty vector-transduced cells and the mock, whereas the hMOB2 knockdown resulted in the dissociation of the centrally located stress fibers and exhibited aperipheral distribution of the actin filaments.(5) Results from the western blot assay showed that hMOB2 overexpression upregulated the expression of phosporylation of FAK at Y397 (pY397FAK) and Y925 (pY925FAK), and phosporylation of paxillin at Y118 (pY118paxillin), and downregulated the phosphorylation of Src at Y527 (pY527Src), while thehMOB2 knockdown obviously downregulated the expression of pY397FAK, pY925FAK and pY118paxillin, and resulted in only a slight but a non-significant reduction of the expression of the pY527Src. Neither hMOB2 overexpression nor hMOB2 knockdown had significant effects on the expression of the total FAK, Src and paxillin in the HepG2 cells.Conclusion:(1) Data present here suggest that the decrease in the cell migration and invasion by hMOB2 overexpression may be attributed to the strong or excessive cell-matrix adhesion, while the reduction in the cell motility caused by hMOB2 knockdown is most likely the consequence of the decrease of the cell-matrix adhesion.(2) Our findings suggest that the modulation of cell motility and cell-matrix adhesion by hMOB2 overexpression and knockdown is possibly mediated through regulating the assembly of focal adhesions and the actin cytoskeleton reorganization.(3) Our results also demonstrat that the altered cell-matrix adhesion and cell motility induced by hMOB2 overexpression and knockdown probably requires functional FAK-Src-paxillin signaling molecules to regulate the focal adhesion formation and the actin cytoskeleton rearrangement.