The Role And Mechanism Of LncRNA LINC01303 In Oral Squamous Cell Carcinoma

BackgroundOral squamous cell carcinoma(OSCC)is the most common head and neck malignant tumor,accounting for more than 90% of head and neck tumors worldwide.It is a tumor with high morbidity and mortality worldwide.OSCC occurs in the lips or oral cavity.Due to its special anatomical location and biological characteristics,early diagnosis of OSCC is difficult,so it may be mistaken for other common diseases.Most oral squamous cell carcinoma is caused by oral mucosa precancerous lesions,and it is easy to infiltrate surrounding tissues such as muscle,nerve,and prone to lymph node metastasis in early,so the effect of treatment is difficult,prognosis is poorer,and although in the past few decades,has been in OSCC oncogenes and suppressor genes identified,However,there is still a lack of independent biomarkers that can be routinely applied to clinical treatment,which brings great challenges to early clinical diagnosis.At present,the treatment of OSCC is mainly surgical resection in the early stage,and assisted by radiotherapy,chemotherapy and targeted therapy in the middle and late stage.Although the clinical treatment has been greatly improved,postoperative metastasis and recurrence are easy to occur,and the 5-year survival rate of most OSCC patients who are already late at the time of diagnosis and treatment is only 50%.Therefore,it is of practical significance to explore the potential molecular mechanism and new therapeutic targets of OSCC to improve the survival rate and prognosis of OSCC patients.Long non-coding RNA(LncRNA)is widely found in human genome,often located in cytoplasm or nucleus,and lacks an Open Reading Frame(ORF).Its length is more than 200 nucleotides.It’s a molecule that doesn’t have the ability to encode proteins.LncRNA are usually located in intergene sequences,intron sequences or overlapping regions.With the development of genome sequencing engineering,it has been found that LncRNA can regulate eukaryotic activities and encode and regulate genes at the transcriptional and post-transcriptional levels.More and more studies have found that LncRNA can regulate the malignant biological behavior of tumor cells from the gene and protein levels,and participate in the occurrence,development and metastasis of a variety of malignant tumors.LncRNA growth-specific inhibitor5(LncRNA of Growth crime-specific 5,LncRNA GAS5)is down-regulated in OSCC and inhibits tumor cell proliferation and invasion by activating the PTEN/AKT axis.LncRNA PDIA3P1 was significantly overexpressed in OSCC and was a prognostic biomolecule of patients,which could up-regulate the expression of Cyclin D2 and promote the proliferation of OSCC cells by targeting miR-185-5P.In addition,multiple LncRNA may act as oncogenes or tumor suppressor genes in OSCC.Previous studies have found that LINC01303 is located on chromosome 14Q23.1 It is highly expressed in gastric cancer and inhibits tumorigenesis by mediating miR-101-3p.LINC01303 is overexpressed in laryngeal squamous cell carcinoma(LARYNgeal squamous cell carcinoma)and promotes laryngeal squamous cell carcinoma by regulating miR-200 c /TIMP2 axis.Therefore,LINC01303 is a promising tumor biomarker.In this study,We observed that the expression of LINC01303 was significantly increased in tumor tissues of OSCC patients,but the role and specific biological mechanism of LINC01303 are still unclear.Therefore,exploring the role and related molecular mechanism of LINC01303 in OSCC is of great clinical significance for early diagnosis and precise treatment of OSCC.ObjectivesThe purpose of this study is to further clarify the distribution of LINC01303,explore its role and molecular mechanism in the growth,invasion and metastasis of oral squamous cell carcinoma,in order to explore a potential molecular marker for the occurrence and development of OSCC,and provide new ideas and targets for the treatment of OSCC by regulating LINC01303.Materials and MethodsClinical samples were collected to detect the difference in the expression level of LINC01303 in OSCC tissues and normal tissues.Further,the localization of LINC01303 in cells was determined by FISH assay.The effects of LINC01303 on proliferation,migration and invasiveness of OSCC cells were investigated in vitro.q RT-PCR was used to further verify the expression difference between LINC01303 and NHOK in OSCC cells.After the expression of LINC01303 in OSCC cells was decreased by siRNA or increased by transfection of overexpressed plasmid,To observe the effects of LINC01303 expression OSCC cell proliferation,migration and invasion in vitro.The effects of LINC01303 on OSCC progression and migration were investigated in vivo.OSCC cells of sh-LINC01303-knock down were injected into the tail vein to observe and compare the aggressivity and size of tumors,as well as related molecular indicators,in order to determine the effect of LINC01303 on the progression and migration of OSCC.Through star Base online database,the downstream key target miRNA of LINC01303 affecting OSCC progress--miR-429 was screened out,and the relationship between LINC01303 and miR-429 was verified by double lucifase reporting assay.Further,the downstream key target gene of miR-429--ZEB1 was screened out by Target Scan database,and the binding of miR-429 and ZEB1 was verified by dual luciferase reporting assay and miR-429 probe.The effects of LINC01303 on proliferation,migration and invasiveness of OSCC cells and progression and metastasis of OSCC were observed by in vitro and in vivo intervention of miR-429 /ZEB1 axis.ResultsThe expression of LINC01303 in OSCC tissues was significantly higher than that in normal tissues by collecting clinical samples.Further,the localization of LINC01303 in cells was confirmed as cytoplasm by FISH experiment.Further study by q RT-PCR showed that the expression of LINC01303 in OSCC cells was significantly higher than that of normal human oral epithelial keratinocyte(NHOK).After the expression of LINC01303 in OSCC cells was reduced by siRNA,the proliferation,migration and invasion abilities of OSCC cells were significantly reduced.When the expression of LINC01303 in OSCC cells was increased by transfection of overexpressed plasmid,the proliferation,migration and invasion abilities of OSCC cells were significantly increased.Further in vivo experiments were conducted to investigate the effects of LINC01303 on OSCC progression and migration.OSCC cells injected with shLINC01303-knockdown through the tail vein showed smaller tumors and significantly reduced invasiveness compared with mice injected with sh-NC-OSCC cells.Through star Base online database,the downstream key target miRNA of LINC01303 that affects OSCC progression--miR-429 was screened out,and it was confirmed that LINC01303 could directly regulate miR-429 through double luciferase reporting experiment.Further,the downstream key target gene of miR-429--ZEB1 was screened out by Target Scan database,and the binding of miR-429 and ZEB1 was verified by dual luciferase reporting assay and miR429 probe.To confirm that LINC01303 is involved in tumor development through the miR-429/ZEB1 axis,we conducted co-transfection experiments.TSCCA and SCC-25 cells were transfected with sh-NC + interactivator-NC,sh-NC+miR-429 inhibitor,sh-LINc01303+interactivator-NC and sh-LINC01303 +miR-429 inhibitor.The expression,proliferation and invasion of ZEB1 were determined experimentally.q RT-PCR results showed that the relative expression of ZEB1 in TSCCA and SCC-25 cell lines was significantly increased after transfection with sh-NC+miR-429 inhibitor.However,sh-LINc01303+miR-429 inhibitor co-trans fection inhibited miR-429 inhibitor-mediated upregulation of ZEB1.CCK-8 assay showed that OSCC cell proliferation was significantly enhanced after transfection with sh-NC+miR-429 inhibitor.Transwell assay showed that the number of invaded OSCC cells increased after transfection with sh-NC+miR-429 inhibitor.After co-transfection with SH-LINc01303 and miR-429 inhibitor,The proliferation and invasion activities of OSCC cells(TSCCAand SCC-25)were significantly reduced,suggesting that LINC01303 mediates miR-429/ZEB1 axis to promote OSCC.By inhibiting the expression of ZEB1,the enhanced proliferation and invasiveness of OSCC cells induced by LINC01303 over-expression could be significantly reduced.ConclusionsOur study confirmed that the expression of LINC01303 is significantly increased in OSCC tissues and cells,and the proliferation and invasiveness of OSCC cells can be significantly reduced by decreasing the expression of LINC01303 in vivo and in vitro experiments.Inhibition of miR-429/ZEB1 axis significantly attenuated the proliferation enhancement and invasiveness of OSCC cells induced by LINC01303 overexpression.Therefore,LINC01303 can promote the proliferation and invasion of OSCC by regulating the expression of miR-429/ZEB1 signaling pathway.LINC01303 is expected to be a biomarker for the occurrence and development of OSCC,and the regulation of LINC01303 will also provide a new idea and target for the treatment of OSCC.