Effects Of Hippo Signaling Pathway On Morin Regulated Biological Behavior Of Oral Squamous Cell Carcinoma Cells

Backgroud and ObjectivesOral cancer is a common malignant tumor in the world,especially in developing countries.Among oral malignant tumors,oral squamous cell carcinoma(OSCC)accounts for the largest proportion,about 80%.Even though medical technology is constantly evolving,the mortality rate of OSCC is still relatively high.Statistics show that the diseased population tends to be younger,and the overall prognosis of cancer is poor,with a survival rate of about 55%to 65%.The treatment of OSCC emphasizes the mode of comprehensive treatment,mainly based on surgery,supplemented by radiotherapy and chemotherapy.However,chemotherapeutic drugs have effects on both normal cells and tumor cells,and there is a dose-limiting toxic effect,and it is possible to induce drug resistance of tumor cells.Therefore,it is very important to screen and develop highly effective and low-toxic anti-tumor drugs.Morin(3,5,7,2’,4’-pentahydroxyflavone)is a pale-yellow flavonoid extracted from the mulberry plants such as yellow mulberry and mulberry orange.A variety of flavonoids have been demonstrated in research and have important potential medicinal properties.Morin can exert its important physiological functions such as anti-oxidation,anti-diabetes,anti-inflammatory,anti-hypertensive,anti-bacterial and neuroprotective by participating in various signaling pathways regulating cells.Morin has been shown to exert its anticancer activity in a variety of cancers.For example,Morin inhibits cancer cell proliferation by interfering with cell cycle and exerts anticancer activity in human colon cancer cells;Morin can induce apoptosis in prostate cancer,human leukemia HL-60 cells,and multiple myeloma cells.Studies have confirmed that Morin is a biologically active compound with broad pharmacological activity and very low cytotoxicity.Therefore,exploring the regulation of Morin on OSCC cell biology and its molecular mechanism are important foundations for guiding us to screen and develop anti-tumor drugs for OSCC.The Hippo signaling pathway is a highly conserved pathway that can play a role in tumor suppression in a variety of tumors.Hippo signaling pathway is a kinase cascade reaction chain composed of four important core kinases,MST1,SAV1,LATS1/2 and MOB1a/b.Hippo signaling pathway regulates the subcellular localization of YAP by phosphorylation.Activation of the Hippo signaling pathway can restrict the downstream effector protein YAP to the cytoplasm by phosphorylating,where degrades YAP by ubiquitination;inactivation of the Hippo signaling pathway leads to decreased YAP phosphorylation levels and nuclear enrichment,thereby affecting the transcriptional activity of a variety of downstream related factors plays a regulatory role in the biological behavior of cells.As a transcriptional coactivator,YAP protein is involved in the regulation of biological signals related to tumorigenesis.The increase of YAP protein level and nuclearlocalization can induce the development of various tumors.It has been reported that Morin can inhibit the nuclear localization of YAP by activating the Hippo signaling pathway,and induce apoptosis of human hepatoma cells,thereby exerting the anti-cancer effect of Morin.It has also been confirmed that Morin can induce cell cycle arrest in human oral squamous cell carcinoma cells,thereby exerting a tumor suppressor effect.However,to date,studies on the anti-cancer effect of Morin in OSCC have been very limited,and the molecular mechanism of its anti-cancer effect in OSCC is still unclear and remains to be clarified.Therefore,our study aimed to explore Morin’s anti-cancer effect on OSCC in a more comprehensive and in-depth manner,and to verify whether Hippo signaling pathway get involve in the anti-cancer role of Morin in OSCC.Materials and MethodsOral squamous cell carcinoma cell lines Cal27 and SCC15 were selected as subjects to investigate the regulation of Morin on the biological behavior of OSCC and its potential mechanism.1.Comparison of cytotoxic effects of Morin on normal oral epithelial cells(HOEC)and OSCC cellsHOEC and OSGC cells were treated with Morin,respectively,and cells treated with DMSO(dimethyl sulfoxide)were used as blank control.The cytotoxicity of Morin to HOEC and OSCC was detected by CCK-8,and the IC50 of Morin in HOEC and OSCC cells was calculated.The effect of Morin on the expression of phospho-ERK in HOEC and OSCC cells was detected by Western blot.2.The regulation of Morin on the biological behavior of OSCC cellsThe Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control:(1)The Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control:the effect of Morin on the colony forming ability and EdU positive rate of Cal27 and SCC15 were detected.(2)The Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control:cell cycle distribution and apoptosis of OSCC cells were detected by flow cytometry.The expression level of cyclin-dependent kinase inhibitors P53,P21;apoptosis-related proteins CASPASE-3,CASSASE-9,BCL-2 of Cal27 and SCC15 were detected by Western blot;thus to explore the regulation of Morin on OSCC cell cycle and apoptosis levels.(3)The Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control,and the migration ability of OSCC cells was detected by scratch test.The expression levels of E-CADHERIN and VIMENTIN which were the markers related to epithelial-mesenchymal transition in OSCC cells were detected by Western blot.(4)Tumor formation capacity in nude mice:20 nude mice with good growth status were randomly divided into A and B groups,with 10 rats in each group.The tumor models of nude mice were constructed with Cal27 and SCC15 cells respectively.The nude mice in group A and B were randomly divided into A1,A2,B1 and B2 groups,with 5 rats in each group:nude mice in group A1 and group B1 were injected with 50 mg/kg Morin via tail vein once a day;nude mice in group A2 and group B2 were injected with the same dose of DMSO via the tail vein once a day.After 25 days of normal feeding,the nude mice were sacrificed,the tumor was removed,and the tumor was weighed and photographed.Thus,the effect of Morin on the tumor formation capacity of OSCC cells was verified.3.The effects of Hippo signaling pathway in Morin regulated biological behavior of OSCC cells(1)To investigate whether Morin has a regulatory effect on the activation level of Hippo pathway in OSCC cells,Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and DMSO-treated cells were used as blankcontrol:①Western blot was used to detect the key kinases MST1,MOB1 total’protein level and phosphorylation level,②Western blot was used to detect the phosphorylation level of the key effector YAP,and its expression level in the cytoplasm and nucleus,③immunofluorescence staining was used to detect thesubcellular localization of YAP protein,④RT-PCR was used to detect the mRNAexpression levels of Ctgf,Cyr61,Ankrd;(2)To explore whether the key effector factor YAP of Hippo pathway mediates the regulation of the biological behavior of OSCC cells,and to indirectly speculate whether the Hippo pathway mediates the regulation of Morin on the biological behavior of OSCC cells,OSCC cells with silencing and overexpressing YAP were constructed:①EdU staining was used to detect cell proliferation ability,②flow cytometry was used to detect cell cycle distribution and apoptosis level,③scratch test was used to detect cell migration ability.(3)To directly confirm the mediated role of the Hippo pathway in the regulation of Morin on the biological behavior of OSCC cells,Mstl-silenced OSCC cells were constructed.① Western blot was used to detect the expression level of phospho-MST1 phospho-MOB1 phospho-YAP,cytoplasmic-YAP and nuclear-YAP;so as to explore the effect of Mstl silencing on the activation level of Hippo pathway.②Infected OSCC cells were treated with Morin,and cells were divided into OSCC+DMSO group,OSCC +Morin group,LV3-ctrl-OSCC+Morin group,LV3-shMstl-OSCC+Morin group:EdU staining was used to detect cell proliferation ability;Cell cycle detection and apoptosis level were detected by flow cytometry;cell migration ability was detected by scratch assay;Western blot was used to detected the expression level of P53,P21,CASPASE-3,CASSASE-9,BCL-2,E-CADHERIN,VIMENTIN.Results1.Comparison of the cytotoxicity of Morin on HOEC and OSCC(1)Compared with the control group,Morin inhibited the proliferation of HOEC,Cal27 and SCC15 cells in a dose-dependent manner,and the half inhibitory concentration of Morin was much higher in HOEC than in the OSCC cells.(2)Compared with the control group,Morin inhibited the expression of phospho-ERK in HOEC,Cal27 and SCC15 cells;and the concentration of Morin produced significantly inhibition in HOEC was much higher than in the OSCC cells.2.The regulation of Morin on the biological behavior of OSCC cells(1)The Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control,with the increase of Morin concentration:cell clone formation ability and EdU positive rate decrease,(2)The Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control,with the increase of Morin concentration:cell cycle arrested in the G1 phase,and the protein expression level of P53 and P21 was significantly up-regulated.Cell apoptosis rate increased,and the protein expression level of CASPASE-3 and CASPASE-9 was significantly up-regulated,and the protein expression level of BCL-2 was significantly down-regulated.(3)The Cal27 and SCC15 cell lines were treated with different concentrations of Morin,and the cells treated with DMSO were used as blank control,with the increase of Morin concentration:the migration ability of OSCC cells decreased,the protein expression level of E-CADHERIN was significantly up-regulated,and the protein expression level of VIMENTIN was significantly down-regulated.(4)After subcutaneous transplantation of OSCC cells in nude mice,the weight of the tumor in Morin group was significantly lower than that of in the DMSO group.3.Hippo pathway-mediated the regulation of Morin on the biological behavior of OSCC cells(1)Cal27 and SCC15 cells were treated with different concentrations of Morin,the cells treated with DMSO were used as blank control.As the concentration of Morin increased:the results from Western blot showed that the total protein levels of MST1 and MOB1 were unchanged,and the phosphorylation levels of MST1,MOB1 and YAP were increased.The level of cytoplasmic-YAP protein was increased,and the level of nuclear-YAP protein was decreased.Immunofluorescence staining showed that the cytoplasmic localization of YAP protein was increased.RT-PCR results showed that the transcription levels of CTGF,CYR61 and ANKRD were down-regulated.(2)Silencing of YAP in Cal27 and SCC15 cells,compared with control cells:decreased proliferation ability,arrested cell cycle,up-regulated the apoptosis level,decreased cell migration ability;overexpressing of YAP in Cal27 and SCC15 cells,compared with control cells:proliferative capacity was up-regulated,cell cycle was accelerated,apoptosis level was down-regulated,cell migration ability was enhanced.(3)①Compared with control cells,the Mstl-silenced OSCC cells had increased phosphorylation levels of MST1,MOB1,and YAP,increased levels of nuclear-YAP protein,and decreased levels of cytoplasmic-YAP protein;②OSCC cells infected with virus were treated with Morin,and cells were divided into OSCC+DMSO group,OSCC+Morin group,LV3-ctrl-OSCC+Morin group,LV3-shMstl-OSCC+Morin group:LV3-shMstl-OSCC+Morin group compared with OSCC+Morin group and LV3-ctrl-OSCC+Morin group partially reversed the changes of EdU staining positive rate,cell cycle distribution,apoptosis level and cell migration ability after Morin treatment;but LV3-shMstl-OSCC+Morin group compared with the OSCC+ DMSO group,the cells did not return to the state before the Morin treatmen;③OSCC cells infected with virus were treated with Morin,and cells were divided into OSCC+DMSO group,OSCC+Morin group,LV3-ctrl-OSCC+Morin group,LV3-shMstl-OSCC+Morin group:the results from Western blot showed that compared with OSCC+Morin group and LV3-ctrl-OSCC+Morin group,the LV3-shMstl-OSCC+Morin group reversed the changes of related protein expression levels caused by Morin-treatment;however,LV3-shMstl-OSCC+Morin group Compared with the OSCC+DMSO group,the expression level of the relevant protein did not return to the state before the Morin treatmen.Conclusions1.Morin has cytotoxic effects on HOEC and OSCC cells,and has a stronger cytotoxic effect in OSCC cells.2.Morin has a regulatory effect on the biological behavior of OSCC cells,which is specifically to inhibit cell proliferation,arrest cell cycle,promote apoptosis,and inhibit cell migration.3.(1)Morin up-regulates the activation level of Hippo pathway in OSCC cells(2)The key effector of the Hippo pathway,YAP,does have a regulatory effect on the biological behavior of OSCC cells.To some extent,indicated that the regulation of Morin on OSCC cells may be mediated by the Hippo pathway.(3)By silencing Mstl in OSCC cells,inhibiting the activation level of Hippo pathway:partially reversing the regulation of Morin on OSCC cells,further directly confirming that the regulation of Morin on OSCC cells is mediated by Hippo pathway.