Functional Role And Regulation Mechanisms Of Long Noncoding RNA LINC01137 In Oral Squamous Cell Carcinoma

Objective:Oral squamous cell carcinoma(OSCC)is the most common malignance in the oral and maxillofacial region.The 5-year mortality rate of OSCC is about 50%.Although notable improvements in the treatment of OSCC have been made in recent years,such as surgery,radiotherapy and chemotherapy,the prognosis for OSCC patients is still poor,and the survival rate has not been significantly improved.Long non coding RNA(lnc RNA)is a kind of RNA whose transcript length exceeds 200 bases and does not encode proteins.In the field of cancer research,many studys have shown that lnc RNAs could regulate the biological processes of proliferation,apoptosis,invasion and metastasis of tumor cells,and play the role of oncogene or anti-oncogene in the occurrence and development of cancer.In order to investigate the relationship between lnc RNA and OSCC,we downloaded and studied the whole genome microarray data GSE31056 from pubmed,analyzed the information of lnc RNA contained in it,screened out a number of lnc RNA differentially expressed in OSCC,and further screened out the highly expressed lnc RNA LINC01137 in OSCC for further study.Our study may provide new theoretical basis for revealing the mechanism under the occurrence and development of OSCC.Method:Reverse transcription and quantitative real-time PCR(RT-q PCR)was used to detect the expression level of LINC01137 in OSCC,adjacent normal oral mucosa.RT-q PCR was used to detect the expression of LINC01137 in OSCC cell lines HSC3,HSC4 and Tca8113,also.Two si RNA targeting LINC01137 were designed and transfected into OSCC cells and knockdown the expression of LINC01137.CCK8 assay and colony formation assay were used to analyze the effect of downregulation of LINC01137 on the proliferation of OSCC cells.Transwell assay was used to detect the effect of downregulation of LINC01137 on the migration and invasion of OSCC cells.Western Blot experiment was used to detect the expression of key proteins in the proliferation and EMT process after knockdown of LINC01137 in OSCC cells.Finally,bioinformatic software was used to find mi RNAs which could target LINC01137.It was predicted that mi R-22-3p could target LINC01137.The interaction between LINC01137 and mi R-22-3p was analyzed by Dual luciferase reporer assay and cell function assay.Results: 1.LINC01137 is upregulated in PDAC An analysis of OSCC gene profiling results(GSE31056)from pubmed GEO Data Sets showed that,among 23 matched pairs of OSCC tumor and adjacent normal oral mucosa tissues,LINC01137 was one of the most differentially expressed lnc RNA molecules in OSCC,and its expression in tumor tissues was significantly higher than that in adjacent normal oral mucosa tissues.To further validate this result,RT-q PCR was used to examine the expression level of LINC01137 in OSCC cell lines(HSC3,HSC4,Tca8113)and normal oral mucosa,and the result showed that,compared with normal oral mucosa,OSCC cells exhibited significantly higher levels of LINC01137 expression.We then detected the expression level of LINC01137 in 26 paired OSCC and adjacent normal oral tissues using RT-q PCR.The results showed that the expression of LINC01137 was significantly higher in OSCC tissues compared with normal oral mucosa.The high LINC01137 expression was significantly associated with more lymph node metastasis of OSCC patients.However,there were no statistically significant associations between LINC01137 expression level and other clinicopathological data,including age,sex,tumor size,and tumor differentiation.CPAT(CodingPotential Assessment Tool)software was used to evaluate the coding potential of LINC01137,and the results showed that the possible ORF of LINC01137 is very short,and LINC01137 does not have the coding capacity.2.Knockdown of LINC01137 inhibits OSCC cell growth,migration and invasion CCK-8 proliferation assay was used to plot the growth curve of OSCC cells transfected with si LINC01137 s.The results showed that knockdown of LINC01137 significantly inhibited the growth of HSC3 and Tca8113 cells.Colony formation assay showed that down-regulation of LINC01137 significantly inhibited the colony formation of HSC3 and Tca8113 cells,as indicated by the formation of fewer and smaller colonies in the LINC01137 knockdown group.Western Blot results showed that the expression of Cyclin D1 and Survivin in LINC01137 knockdown group was significantly lower than that in control group,suggesting that down-regulation of LINC01137 may reduce the proliferation ability of OSCC cells by inhibiting cell cycle progression and promoting apoptosis.The migration and invation of HSC3 and Tca8113 cells were detected by Transwell assay after knockdown of LINC01137 expressio.Ten visual fields were randomly selected to count the number of cells entering the lower chamber through the foramen.The results showed that the average number of cells passing through the foramen of each visual field was significantly lower in the LINC01137 knockdown group than that in the control group,indicating that the down-regulation of LINC01137 could inhibit the migrative and invasive ability of OSCC cells.Western Blot assay was used to detect the expression of EMT-related proteins after down-regulated LINC01137.The results showed that the expression of Vimentin,Twist,Snail,MMP-2 and MMP-9 significantly decreased in LINC01137 knockdown group,suggesting that down-regulated of LINC01137 reduced the migration and invasion of OSCC cells by inhibiting EMT.3.LINC01137 is negatively regulated by mi R-22-3p mi R-22-3p is predicted to bound to LINC01137 by using mi Rcode software.Overexpression of mi R-22-3p could significantly reduce the expression of endogenous LINC01137 in OSCC cells,suggesting that mi R-22-3p could negatively regulate the expression of LINC01137.Contrary to the high expression of LINC01137 in OSCC,the expression of mi R-22-3p was significantly down-regulated in the same tumor samples,resulting in a significant inverse correlation between LINC01137 and mi R-22-3p.The expression of LINC01137 and mi R-22-3p in the cytoplasm of HSC3 and Tca8113 cells was significantly higher than that in the nucleus,indicating that they were mainly located in the cytoplasm,which provided a prerequisite for their interaction.Dual luciferase reporter assay showed that the luciferase activity was significantly decreased by transfection of mi R-22-3p mimic,and the inhibitory effects of mi R-22-3p on luciferase activity were abolished by directed mutagenesis of the mi R-22-3p-binding seed region in LINC01137,which suggested that mi R-22-3p could directly target LINC01137.CCK8 and Transwell assays showed that overexpression of mi R-22-3p could enhance the inhibitory effect of si LINC01137 on the growth,migration and invation of OSCC cells HSC3 and Tca8113,while knockdown the expression of mi R-22-3p could partially abolish the effect of si LINC01137 on the growth,migration and invation of HSC3 and Tca8113 cells.Conclusion: 1.The expression of lnc RNA LINC01137 is upregulated in OSCC and is significantly associated with lymph node metastasis.2.Knockdown of LINC01137 significantly inhibits the proliferation,migration and invasion of OSCC cells.LINC01137 may play an oncogene role in OSCC.3.The low expression of mi R-22-3p in OSCC was negatively correlated with the expression of LINC01137.mi R-22-3p negatively regulated the expression and function of LINC01137 by directly targeting LINC01137.