The Role And Mechanistic Studies Of MLCK In Hypopharyngeal Carcinoma Fa Du Epithelial Cells And Intestinal Epithelial Cells

Objective: The aim of this study was to investigate the molecular mechanism of myosin light chain kinase(MLCK)permeability in hypopharyngeal carcinoma FaDu cells and mouse intestinal epithelial cells.Methods:(1)MLCK knockout hypopharyngeal carcinoma FaDu cell lines were constructed and divided into control,MLCK KO1 and MLCK KO2 groups;The knockout efficiency of MLCK was assessed by RT-q PCR and Western blot,and the effect of MLCK knockout on cell cycle and apoptosis was detected by flow cytometry while Tunel staining and Cleaved caspase-3 immunofluorescence to detect apoptosis;Moreover,the apoptosisrelated proteins and autophagy-related proteins were detected by Western blot while cell migration was detected by scratch assay and transwell migration assay.(2)Establishment of animal models: 7 mice were randomly assigned into two groups,3 in the control group and 4 in the experimental group.The model group was injected intraperitoneally with250 ug of Cytotoxic T-lymphocyte-associated protein 4(CTLA-4)and Programmed cell death 1 ligand 1(PD-L1)antibodies every 3 days.Mice were weighed and clinically scored daily,and the ileum was taken 12 days after administration.RT-q PCR was performed to detect changes in cytokines;immunofluorescence was performed to detect changes in CD8-positive cells;Tunel staining and Cleaved caspase-3immunofluorescence were performed to detect apoptosis;Ki67 immunofluorescence was performed to detect cell proliferation;The effect of immune checkpoint blockade(ICB)treatment on tight junction related proteins Claudin-2,ZO-1 and phosphorylated myosin light chains were detected by immunofluorescence staining.Results:(1)The number of Tunel staining and Cleaved caspase-3 positive cells were increased in MLCK knockout hypopharyngeal carcinoma FaDu cell lines;There is no significant change in cell cycle,while the proportion of apoptotic cells was increased;The detection of apoptosis-associated proteins Bax/Bcl-2 and Cleaved caspase-3/Caspase-3 ratio were increased by Western blot;Moreover,the expression of tight junction protein Occludin increased after MLCK knockout and decreased after silencing Occludin with si RNA;The expression of autophagy-related proteins ATG5,Beclin-1 and LC3B-II increased and the expression of P62 decreased;the cell migration rate was slowed down in both the scratch and transwell assays.(2)After 12 days of ICB treatment,there was no significant change in body weight and clinical score;RT-q PCR showed a decrease in IL-6 and IL-22 expression and an increase in Ifng expression;immunofluorescence showed an increase in CD8-positive cells;Tunel staining and Cleaved caspase-3 immunofluorescence-positive cells were increased while Ki67immunofluorescence-positive cells was decreased;the expression of tight junction protein Claudin-2 was increased while ZO-1 was decreased;Moreover,p-MLC and SGLT1 expression were increased.Conclutions: In hypopharyngeal carcinoma FaDu cells,MLCK knockout induced apoptosis,which may be regulated by upregulating the tight junction protein Occludin.Increased expression of Occludin increased the expression of Caspase-3,which in turn induced apoptosis,while silencing Occludin may reduce apoptosis.In addition,MLCK knockout induced cell autophagy and inhibited cell migration,the exact mechanism of which needs to be further investigated.Mice treated with ICB also underwent apoptosis in their ileal epithelial cells and developed an inflammatory response.Corresponding changes in their intestinal permeability were also observed,which may be associated with the expression of MLCK and the phosphorylated substrate p-MLC.