Study On The Mechanism Of LINC01929 In Promoting The Development Of Oral Squamous Cell Carcinoma By Regulating FOXC1 Expression Through MiR-137-3p

BackgroundOral squamous cell carcinoma(OSCC)is a common malignant tumor in head and neck.It can occur in the skin and mucosa,mostly in the tongue,palate,and gingiva.Lymph node metastasis usually occurs,and distal metastasis may also occur in late stage.Currently,surgery is the main treatment method,combined with radiotherapy and chemotherapy,and once the timing of cancer treatment is delayed,the quality of life and survival of patients with prognosis will also be reduced,so the principle of early detection,early diagnosis and early treatment is particularly important.To better understand the carcinogenesis of OSCC,it is important to find effective biomarkers and therapeutic targets for the treatment of oral squamous cell carcinoma.Long noncodingRNA(lncRNA)is a class of non-codingRNA with more than200 nucleotides in length,which has little or no protein-coding function.However,more and more evidence now shows that lncRNA can participate in the regulation of tumor development at the epigenetic,pre-transcriptional and post-transcriptional levels.In recent years,a number of lncRNA have been found to be significantly correlated with the proliferation,migration and invasion of OSCC cells,and play an important role in promoting or inhibiting cancer in the development of OSCC.Studies have reported that LINC01929 is highly expressed in NSCLC and significantly correlated with tumor stage,and patients with low expression of LINC01929 also have a better prognosis,suggesting that LINC01929 can promote the development of NSCLC.However,other studies have found that LINC1929 plays an inhibitory role in cancer,and high expression of LINC01929 is associated with improved long-term survival in HCC patients without fibrosis.Xiong et al.demonstrated that LINC01929 was up-regulated in OSCC by high-throughput sequencing,but its function in OSCC remains unclear.In this study,the expression level of LINC01929 was detected in OSCC and adjacent tissues,and its correlation with clinical and pathological data was analyzed.The effects of abnormal LINC01929 expression on the proliferation,migration,invasion and apoptosis of OSCC cells were investigated by in vitro cell experiment and in vivo animal experiment respectively.Furthermore,the molecular mechanism of LINC01929 promoting cancer was explored through relevant experimental methods,which provided a new direction and theoretical basis for targeted therapy of OSCC.Objectives:1.The expression level of LINC01929 in OSCC tissue and adjacent tissue was detected and the correlation between LINC01929 and clinicopathological features of OSCC patients was analyzed.2.To investigate the effects of LINC01929 expression on proliferation,migration,invasion and apoptosis of OSCC cells.3.Using competitive endogenousRNA(ceRNA)as the starting point,to explore the regulation mechanism of LINC01929 promoting OSCC proliferation,migration and invasion.Methods:Part ⅠA total of 27 cases of OSCC tissue and adjacent normal tissue samples were collected.The expression level of LINC01929 in OSCC tissue and adjacent tissue samples was detected by qRT-PCR,and the correlation between LINC01929 expression level and clinicopathological characteristics of OSCC patients was analyzed.The expression of LINC01929 in OSCC cell lines(CAL-27,CAL-33,SCC-9,SCC-25 and HSC-3)and normal keratinocytes was detected by qRT-PCR.Kaplan-meier survival curve was analyzed.Part ⅡOSCC cell lines CAL-27 and SCC-9 were transfected with sh-LINC01929 and pLINC01929,respectively.The expression of LINC01929 was detected by qRT-PCR to verify the knockdown and overexpression efficiency.The effects of LINC01929knockdown/overexpression on proliferation,migration,invasion and apoptosis of OSCC cells were detected by CCK-8 proliferation assay,scratch assay,transwell assay and flow cytometry.In vivo tumorigenesis experiments were performed to verify the effects of LINC01929 knockdown on tumorigenesis and related molecular expression in nude mice.Part ⅢThe subcellular localization of LINC01929 in OSCC cells was detected by nuclear cytoplasmic separation assay.The bioinformatics database predicted that miR-137-3p was the downstream miRNA molecule of LINC01929 and the target gene of miR-137-3p was FOXC1.The targeted binding of LINC01929 to the mutual binding sites of miR-137-3p,miR-137-3p and FOXC1 was verified by dual luciferase experiment.The correlations between LINC01929 and miR-137-3p,miR-137-3p and FOXC1 were analyzed by Spearman correlation analysis.Western blot was used to detect the effect of LINC01929/ miR-137-3p/ FOXC1 expression on FOXC1 protein expression.Recovery experiments demonstrated that LINC01929 regulates FOXC1 through miR-137-3p and affects the biological function of OSCC cells.Results:Part ⅠLINC01929 was highly expressed in 27 OSCC tissue samples and OSCC cell lines,and the expression level of LINC01929 was not related to age,gender,tumor size,TNM stage,etc.,but was related to advanced lymph node metastasis.Kaplan-meier survival curve results indicated that the survival rate of OSCC patients with high LINC01929 expression was significantly lower than that of patients with low LINC01929 expression.Part ⅡAfter transfection of CAL-27 or SCC-9 with sh-LINC01929 or p LINC01929,the expression level of LINC01929 was decreased and increased,respectively.CCK-8assay,scratch assay,transwell invasion assay and flow cytometry showed that knockdown LINC01929 significantly inhibited the proliferation,migration and invasion ability of OSCC cells,and accelerated cell apoptosis.Overexpression of LINC01929 promoted proliferation,migration and invasion of OSCC cells and inhibited apoptosis.Knockdown of LINC01929 inhibited the growth of transplanted tumor in mice in vivo.Part ⅢThe bioinformatics database predicted that miR-137-3p was the miRNA molecule targeted by LINC01929,and the luciferase reporter gene assay results confirmed that the two could combine with each other.Knockdown of LINC01929 significantly up-regulated the expression level of miR-137-3p.After co-transfection of miR-137-3p inhibitors with sh-LINC01929,the inhibition of proliferation,migration and invasion of OSCC cells caused by LINC01929 knockdown could be partially reversed.Luciferase reporter gene assay showed that FOXC1 was the target gene of miR-137-3p,and overexpression of miR-137-3p or knockdown of LINC01929 could inhibit the expression level of FOXC1.After co-transfection of pc DNA-FOXC1 and sh-LINC01929,the inhibition of proliferation,migration and invasion of OSCC cells caused by LINC01929 knockdown could be partially reversed.Conclusions:1.LINC01929 was highly expressed in OSCC and was associated with poor prognosis of OSCC.2.High expression LINC01929 can significantly promote the proliferation,invasion and migration of OSCC cells,and inhibit cell apoptosis.3.LINC01929 regulates FOXC1 mainly by targeting miR-137-3p in OSCC,which promotes cancer and provides a new target and theoretical basis for new treatment strategies for OSCC.