| Background:Tongue Squamous Cell carcinoma(TSCC)which is a serious threat to human health and life is one of the most common head and neck malignant tumors,with high morbidity and fatality rate.At present,chemotherapy is one of the main treatments for TSCC.However,due to the existence of Multidrug resistance(MDR)of tumors,many patients have poor chemotherapy effect.Therefore,how to reduce or reverse the Multidrug resistance of TSCC cancer cells has become an urgent problem to be solved in the treatment of TSCC.The expression of Long non-coding RNA Taurine up-regulated Gene 1(lncRNA TUG1)is elevated in a variety of tumors.However,its role in drug resistance in TSCC has not been studied.We will further study the expression and biological function of TUG-1 in cisplatin-resistant TSCC tissues and cells,explore the target miRNA of TUG-1 and the signaling pathway,explore the markers and related therapeutic targets in cisplatin-resistant TSCC tissues,and seek new ideas and methods for the evaluation,diagnosis and treatment of cisplatin-resistant TSCC.1 Expression and mechanism of long non-coding RNA TUG1 in patients with cisplatin-resistant tongue squamous cell carcinomaObjective:The expression of TUG 1 was detected in TSCC tissues sensitive or resistant to cisplatin chemotherapy,and the effect of TUG1 on the biological function of TSCC cells was analyzed by silencing it,so as to study the role of TUG 1 in the mechanism of cisplatin resistance in TSCC,and to explore the feasibility of TUG 1 as a molecular biomarker for the evaluation and diagnosis of patients with cisplatin resistance in tongue squamous cell carcinoma.Materials and methods:1.46 patients who have been diagnosed with tongue squamous cell carcinoma that could not be operated on directly were recruited from the Department of Oral Surgery of the First Affiliated Hospital of Zhengzhou University from May 2018 to April 2019,and they were divided into sensitive group and drug-resistant group according to the efficacy of chemotherapy;Human tongue squamous cell cell lines SCC25 and CAL27 were cultured and cisplatin-resistant cell lines SCC25/CDDP and CAL27/CDDP were established.qRT-PCR was used to detect the expression level of TUG1 in tissues and cells of tongue squamous cell carcinoma in sensitive and drug-resistant groups.2.The IC50 of cisplatin resistant cell lines was detected by CCK-8.3.Cisplatin-resistant TSCC cells were knockdowned.The abundance of TUG1,IC50 of cisplatin,viability,invasion and apoptosis were measured by qRT-PCR,CCK-8,transwell or flow cytometry,respectively.Results:1.The expression of TUG1 in cisplatin-resistant TSCC tissues and cells was higher than that in sensitive group(p<0.05).2.The IC50 of cisplatin-resistant human tongue squamous cell carcinoma cell lines SCC25/CDDP and CAL27/CDDP was increased(p<0.05).3.The expression of TUG 1,IC50 of cisplatin,viability,invasion were reduced by knockdown of TUG 1 in the two cisplatin-resistant cells(p<0.05).Flow cytometry showed that the apoptotic ability of TUG1 knockdown group was promoted(p<0.05).Conclusions:TUG1 is an oncogene of cisplatin-resistant human tongue squamous cell carcinoma cells,and knockdown of TUG1 significantly inhibited cell proliferation and invasion ability,and promoted apoptosis of cisplatin-resistant cells.2 The mechanism of TUG1 regulating miR-133b expression in cisplatin-resistant tongue squamous cell carcinoma cellsObjective:To verify whether miR-133b expression down-regulates TUG1 gene expression in SCC25/CDDP and CAL27/CDDP cisplatin-resistant tongue squamous cell cells by in vitro cell experiments,and to verify whether the expression of miR-133b affected cell activity,invasion and apoptosis,so as to provide theoretical basis for the pathogenesis of cisplatin-resistant human tongue squamous cell carcinoma cells and gene targeted therapy.Materials and methods:1.Prediction and verification of target genes:bioinformatics analyzed mitarbase or Diana Tools database to predict candidate targeted miRNAs of TUG1.The luciferase report verifies whether TUG1 and target miRNA interact each other.2.The expression of miR-133b in human tongue squamous cell carcinoma cells of the cisplatin-sensitive and drug-resistant groups was detected by qRT-PCR.3.Transfected with TUG1 overexprssion vector,the expression of TUG 1 in the two cisplatin-resistant cells was detected by qRT-PCR.In the two drug-resistant cells,the expression of miR-133b in the overexpressed or knockdown TUG1 group was detected by qRT-PCR.4.The expression of miR-133b,IC50 of cisplatin,cell viability,invasion,and apoptosis were detected in cisplatin-resistant with transfected with miR-133b mimic or miR-133b mimic+TUG1 overexpression vector by qRT-PCR,CCK-8,Transwell or flow cytometry,respectively.Results:1.Bioinformatics analysis predicted that the target miRNA of TUG1 was miR-133b.Luciferase reported that TUG1 combined miR-133b.2.qRT-PCR detection showed that the expression of miR-133b in cisplatin-resistant group was lower than that in cisplatin-sensitive group(p<0.05).3 In SCC25/CDDP and CAL27/CDDP cells,the expression of TUG1 in TUG1 overexpression group was higher than that in the control group(p<0.05),the expression of miR-133b in TUG1 overexpression group was lower than that in the control group(p<0.05),the expression of miR-133b in TUG1 knockdown group was higher than that in the control group(p<0.05).4.In cisplatin-resistant cells,the expression of miR-133b and apoptosis in the overexpressed miR-13 3b group were higher than those in the control group,respectively;IC50 of cisplatin,cell viability and cell invasion were inhibited(p<0.05);The expression of miR-133b and apoptosis in TUG1 and miR-133b overexpression groups were suppressed;IC50 of cisplatin,cell viability and cell invasion were promoted than those in the control group(p<0.05),respectively.Conclusions:TUG1 targets miR-133b;MiR-133b specifically binds to TUG1,and TUG1 negatively regulate the expression of miR-133b,promote cell viability and cell invasion,inhibit cell apoptosis,and participate in the development of cisplatin-resistant tongue squamous cell carcinoma.3 The regulatory mechanism of lncRNA TUG1-miR 133b-CXCR4 in cisplatin-resistant tongue squamous cell carcinoma cellsObjective:To predicte the target mRNA of miR-133b,To research the expression of CXCR4 in cisplatin-resistant human tongue squamous cell carcinoma cells,to explore the relationship between miR-133b and CXCR4,to find the regulatory mechanism of miR-133b on CXCR4,and to explore the mechanism of TUG1-miR133b-CXCR4 signaling pathway in cisplatin-resistant human tongue squamous cell carcinoma cells..Materials and methods:1.Prediction and validation of target genes:Bioinformatics was used to analyze the miRtarbase or Diana Tools database to predict the candidate target genes of miR-133b.The luciferase report verified whether CXCR4 combinds miR-133b.2.In cisplatin-sensitive and cisplatin-resistant cells,the expression level of CXCR4 was detected by Western blot.3 In cisplatin-resistant cells,the expression level of CXCR4 was detected in miR-133b overexpression or miR-133b knockdowned group by Western blot.4 The cisplatin-resistant cells devided into CXCR4 knockdown group and CXCR4+miR-133b knockdown group,the expression of CXCR4,IC50 of cisplatin,cell viability,cell invasion and cell apoptosis was detected by qRT-PCR,CCK-8,Transwell assay and flow cytometry,respectively.5 In cisplatin-resistant cells,TUG1 or both TUG1 and miR-133b were knocked down,and the expression level of CXCR4 was detected by Western blot,respectively.6 Tumor formation experiment in nude mice was conducted to observe the growth of cisplatin-resistant tongue squamous cell carcinoma cells in nude mice with knockdowned TUG1.The relative expression levels of TUG1,miR-133b and CXCR4 mRNA were detected in the knockdowned TUG1 group and the control group by qRT-PCR.In tumor tissues of nude mice with the knockdown TUG1 group or control group,The CXCR4 protein expression was detected by Western blot.3.To observe the growth of cisplatin-resistant tongue squamous cell carcinoma cells in nude mice with transfected sh-TUG.The mRNA relative expression levels of TUG 1,miR-13 3b and CXCR4 in the silenced TUG 1 group and the control group were detected by qRT-PCR.The expression of CXCR4 protein was detected in the sh-TUG1 group and the control group by Western blot.Results:1.The potential target of miR-133b is CXCR4.The interaction between miR-133b and CXCR4 in cisplatin-resistant cells was confirmed by luciferase report assay.2.The expression of CXCR4 in cisplatin-resistant cells was higher than that in sensitive cells by Western blot(p<0.05).3 The expression of CXCR4 in miR-133b overexpression group was lower than that in the control group(p<0.05),the expression of CXCR4 in miR-133b knockdown group was higher than that in the control group(p<0.05).4 In CXCR4 knockdown group,the expression of CXCR4 mRNA and protein,cell viability and invasion were inhibited and the apoptosis was promoted by qRT-PCR,Western blot,CCK-8 and Transwell,respectively;while knockdowned the miR-133b and CXCR4 resulted in the opposite effect(p<0.05).5 The expression of CXCR4 protein in knockdown TUG1 group decreased by Western blot(p<0.05),while the CXCR4 protein expression was increased when TUG1 and miR-133b were knocked down simultaneously(p<0.05).6 Knockdowned TUG1 of cisplatin-resistant tongue squamous cell carcinoma cells were statically transfected and and implanted into nude mice to establish xenograft model in vivo.After 5 weeks of cell inoculation,tumor volume and weight were reduced in the knockdown TUG1 group compared with the control group(p<0.05).Compared with the control group,the expression of TUG1,CXCR4 mRNA and protein in TUG1 knockdown group were decreased,while the expression level of miR-133b was increased(p<0.05).Conclusions:MiR-133b targeting CXCR4;MiR-133b specifically binds to CXCR4,and negatively regulates the expression of CXCR4,inhibiting cell viability and cell invasion,promoting cell apoptosis,and inhibiting the development of cisplatin-resistant tongue squamous cell carcinoma.By down-regulation of TUG1 up-regulate miR-133b and downregulate CXCR4,then increased the sensitivity of tongue squamous cell carcinoma to cisplatin,predicted that TUG1 may be a potential target to reverse the resistance of TSCC to cisplatin. |
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