The Mechanism Of LncRNA DANCR In Invasion And Metastasis Of Tongue Squamous Cell Carcinoma

Objective:The incidence rate of squamous cell carcinoma of the head and neck is in the sixth place in cancer and tends increasing with each passing year in developing countries.Head and neck squamous cell carcinoma most commonly occurs in Tongue squamous cell carcinoma(TSCC),which prominent features are high proliferation rate,high lymph node metastasis rate,increasing annual incidence,and an increasing trend of younger and more feminine cases.In retrospect,although the treatment of TSCC,such as surgery,radiation or chemotherapy,is developing rapidly,the prognosis of patients with advanced tongue squamous cell carcinoma is still not gratifying.Main fatal causes are local recurrence and distant metastasis.In recent years,molecular targeting has become a research focus of comprehensive tumor management,providing a new modus for oncotherapy.Therefore,it is of great clinical significance to find effective and specific molecular targets with low toxicity and side effects as potential tumor theraputics.Long non-coding RNA(LncRNA)differentiation antagonizing non-coding RNA(DANCR)was first reported for its ability to maintain epidermal cell stemness.Studies have demonstrated that LncRNA DANCR can promote the invasion and metastasis of prostate cancer,gastric cancer,colon cancer and other malignant tumors.However,the biological role of DANCR and the underlying molecular mechanism in the progression of TSCC have not been well elucidated.This study aims to study the role of DANCR in TSCC and its molecular mechanism,so as to provide new ideas and strategies for the treatment of TSCC and the suppression of malignant biological characteristics of tumor,by conducting in vivo together with vitro experiments.Methods: 1.After knocking down or overexpressing DANCR in TSCC cell lines(SCC9,TSCCA,TCa-8113 and CAL-27),MTT,scratch and Transwell assays were executed in order to study the effects of DANCR on the proliferation,migration and invasion of TSCC cells.2.The targeting regulatory relationship between DANCR and miR-135a-5p was verified by the dual-luciferase report assay.The functional role of miR-135a-5p was verified by MTT,scratch as well as Transwell assays in TSCC cells.Western blot and qRT-PCR were used to detect the variation of Kr�ppel like transcription factor 8(KLF8)in TSCC cells with the change in DANCR or miR-135a-5p expression.3.Focusing on the miR-135a-5p/KLF8 axis,the molecular mechanism underlying DANCR-mediated regulation of the malignant phenotypes of TSCC cells was further explored.Functional rescue experiments were performed on miR-135a-5p and KLF8: to observe whether miR-135a-5p inhibitors could reverse the inhibition of cell proliferation,migration and invasion mediated by DANCR silencing in TSCC cells,and to observe whether silencing of KLF8 could reverse miR-135a-5p inhibitorinduced enhancement of cell proliferation,migration and invasion.In addition,we constructed LncRNA DANCR interference plasmid,and then transfected it into TCa-8113 and CAL-27 cells,followed by screening of stable cell lines.The stably transfected TSCC cells(DANCR shRNA group)and the control cells(NC shRNA group)were subcutaneously injected into the right armpit of nude mice,and the tumors were measured since they grew into visible sizes,and weighed 25 days after tumor cell inoculation.The expression of MMP-2,MMP-9 and KLF8 in the tumor tissues of each group were detected by Western blot.The expression of KLF8 in the tumor tissues was detected by immunofluorescence staining.Results: 1.LncRNA DANCR played a significant role in the regulation of proliferation,metastasis and invasion of TSCC cells.In TCa-8113 and CAL-27 cells,when LncRNA DANCR was knocked down,the abilities to proliferation,metastasis and invasion were weakened in evidence.By contrast,overexpression of LncRNA DANCR in SCC9 and TSCCA cell lines significantly enhanced the proliferation,metastasis and invasion ability.2.DANCR directly targeted miR-135a-5p.DANCR negatively regulated miR-135a-5p expression,while positively regulated KLF8 expression.Inhibition of miR-135a-5p induced the expression of KLF8 in TSCC cell lines and accelerated tumor cell progression.MiR-135a-5p inhibitor significantly enhanced the proliferation,migration and invasion of TSCC cells.Moreover,miR-135a-5p inhibitors can partially reverse the inhibition of cell proliferation,decreased migration capacity,and decreased invasion capacity caused by DANCR knockdown.Silencing of KLF8 partially reversed miR-135a-5p inhibitor-mediated increase in tumor malignancy.3.The in vivo experiments showed that silencing of DANCR significantly reduced tumor size and weight,and decreased MMP-2/9 and KLF8 expression in tumor tissues,and thus inhibiting tumor malignancy.Conclusions: 1.DANCR plays an important role in the regulation of proliferation,metastasis and invasion in TSCC cells.2.DANCR plays an oncogenic role in TSCC by targeting the miR-135a-5p /KLF8 axis.3.Experiments show that down-regulating DANCR can reduce TSCC tumor growth,inhibit utility protein associated with cell migration and invasion in vivo.