Keratin 23 Mediates The Inhibition Effect Of Melatonin On Proliferation Of Gastric Cancer Cells

Objective:In order to elucidate the anti-tumor mechanism of melatonin,this study used keratin 23(KRT23)as the entry point,and used si RNA,Western-blot,Flow Cytometry,ECIS Ztheta cell analysis to reveal its effects on the proliferation of gastric cancer cells and further verify whether melatonin plays an anti-gastric cancer role through KRT23 by cellular functional experiments,so as to provide a theoretical basis for enriching the understanding of the mechanism of melatonin against gastric cancer,and offer possible new molecular targets for the treatment of gastric cancer.Methods:1.The expression of KRT23 in GC cell lines were detected by Western-blot.2.The highly efficient si RNA interference sequence targeting KRT23 were screened,the lentiviral expression vector of KRT23 sh RNA were constructed and stable transfected in SNU216 by lentivirus infection.The effect of KRT23RNAi-knockdown was detected by Western-blot and immunofluorescence.3.The proliferation activity of GC cells was measured by MTS Assay,Clone Formation Assay,Flow Cytometry,ECIS dynamic cell analyzer.4.The molecular mechanism of KRT23 on the proliferation of gastric cancer cells were preliminarily elucidated by Western-blot.5.KRT23 knockdown cells were treated with melatonin and MTS assay,Clone Formation Assay,Flow Cytometry and ECIS dynamic cell analyzer were implemented to explore the possible function of melatonin on the anti-gastric effects by down-regulating KRT23 expression.Results:1.The expression of KRT23 in two GC cells was significantly higher than that in normal gastric epithelial cells.2.The lentiviral expression vector of sh KRT23 was successfully constructed,and a stable KRT23 knockdown gastric cancer cell model was obtained.3.The results of MTS Assay showed that KRT23 RNAi-knockdown could significantly inhibit the proliferation of GC cells,compared with the non-sense interference group(P<0.01).In addition,knockdown of KRT23 significantly inhibited the colony formation rate,and significantly increased the proportion of cells in G1 phase,and decreased the proportion of S phase and G2/M phase.Cells dynamic analysis of ECIS revealed that KRT23 RNAi-knockdown significantly reduced the Z value of gastric cancer cells.4.We measured and analyzed changes in the activity of MAPK family-associated proteins.The results showed that knockdown of KRT23 could down-regulate the expression of p-ERK1/2 and p-p38,but had no significant effect on the total levels of ERK1/2 and p38,indicating that knockdown of KRT23 could inhibit the activation of ERK1/2 and p38.5.In GC Cells with KRT23 RNAi-knockdown,melatonin can markedly inhibit GC cell lines proliferation,colony formation and led to the G1/S block,compared with non-sense interference group.In addition,ECIS cell dynamic analysis found that the effect of melatonin on the proliferation of gastric cancer cells was more pronounced after KRT23 knockdown.Conclusion:1.This study preliminarily confirmed that KRT23 plays an important role in the proliferation of gastric cancer cells,and its effect may be related to the regulation of ERK1/2 and p38 MAPK signaling pathways.2.KRT23 mediates the inhibitory effect of melatonin on the proliferation of gastric cancer cells.