Melatonin Inhibits The Growth Of Tongue Squamous Cell Carcinoma Via Endoplasmic Reticulum Stress-mediated Cell Death

Purpose:Tongue squamous cell carcinoma(TSCC)is one of the most aggressive forms of oral squamous cell carcinoma,which is prone to lymph node metastasis,easily resistant to chemotherapy and has poor prognosis.Therefore,it was necessary to have an effective therapy strategy to improve prognosis.Melatonin(MT)is a natural indole compound that has been shown to have anti-tumor effects in several cancers.This article will focus on the role and mechanism of MT in TSCC.Methods:1.1.CCK-8 to analyze the effect of MT on TSCC cells;Western blot to analyze whether the expression of Cyclin B1 and p21 was altered;quantitative PCR(q PCR)to analyze whether p21 was altered at the m RNA level.2.After treating TSCC cells with MT for 24 h,the effect of MT on the migration of TSCC was analyzed by wound healing assay and transwell assay;Western blot assay was performed to analyze the expression level of Zeb1,Wnt5A/B,β-catenin,c-myc,pc-myc and E-cadherin proteins,and the effect of MT on TSCC were analyzed at the protein level.3.After treating TSCC cells with MT for 24 h,the apoptosis of TSCC was analyzed by Annexin-FITC/PI staining and flow cytometry;the m RNA levels of HRK,TNFRSF10 A and TNFRSF10 B were analyzed by q PCR;the expression level of BAX and Bcl-2 proteins were detected by Western blot.4.The m TOR inhibitor rapamycin was used to treat TSCC,and the expression level of LC3,Beclin1,BAP31 and Bip proteins were analyzed by Western blot to analyze the effect of rapamycin on TSCC.5.The m RNA level of LC3 were detected by q PCR.The expression level of LC3,P62 and Beclin1 proteins were analyzed by Western blot to analyze the effect of MT on autophagy of TSCC.6.TSCC cells were treated with MT for 24 h.The m RNA level of ATF4,ATF6,BIP and CHOP were analyzed by q PCR;the expression level of BAP31,BIP,CHOP and IRE1α proteins were analyzed by Western blot to analyze whether MT induced endoplasmic reticulum stress in TSCC.7.The expression of Lnc RNAs associated with cell proliferation,metastasis,apoptosis,autophagy and drug resistance in head and neck tumors was analyzed using bioinformatics techniques,including Lnc RNA MALAT1,Lnc RNA H19,Lnc RNA TUG1,Lnc RNA PTTG3 P,Lnc RNA DICER1-AS1 and Lnc RNA GAS5.The expression level of these Lnc RNAs were analyzed by q PCR.8.The CRL-1623 cells was injected subcutaneously into the axilla of nude mice to construct a nude mouse xenograft tumor model,and the inhibitory effect of MT on TSCC in vivo was analyzed by intraperitoneal injection of PBS and MT.Result:In this study,the results suggested that MT could inhibit the proliferation of TSCC cells.Western blot results showed that MT could inhibit Cyclin B1 protein and promote the expression of p21 protein.Meanwhile,MT could inhibit the expression of Zeb1,Wnt5A/B and β-catenin proteins and promote the expression of E-cadherin protein,thus inhibiting the migratory ability of TSCC cells.In addition,our results showed that MT could promote the expression of Bax,LC3 and Beclin1 proteins and inhibit the expression of p62 protein.This suggests that MT can induce autophagy and apoptosis in TSCC cells.To further explore the role of MT,we screened several Lnc RNAs associated with cell proliferation,metastasis,apoptosis,autophagy and drug resistance,and analyzed the expression levels of these Lnc RNAs in TSCC cells treated with MT using q PCR.Our results showed a decrease in the expression of Lnc RNA MALAT1 and H19 and an increase in the expression of Lnc RNA DICER1-AS1 and GAS5.Furthermore,we found that MT inhibited tumor growth in nude mice inoculated with CRL-1623 in vivo.Conclusion:MT may induce endoplasmic reticulum stress,autophagy and apoptosis by regulating lnc RNA MALAT1 and Gas5 in TSCC.In addition,MT was able to inhibit the migratory ability of TSCC.