| Tongue squamous cell carcinoma(TSCC)is the most common malignant tumor in oral cavity,which mainly comes from the mucosa stratified squamous epithelium.Tongue squamous cell carcinoma has a high degree of malignancy and a wide range of operation,which seriously affects the quality of life of patients,and is prone to distant metastasis and poor prognosis.At present,the research direction of tongue squamous cell carcinoma is mainly focused on the following points: 1?tongue squamous cell carcinoma related gene differential expression and the role in its dysregulation of tumor biological characteristics including proliferation,apoptosis,cycle progression,invasion,metastasis,and angiogenesis in tumor biology.2.The expression of dysregulated genes in tongue squamous cell carcinoma,especially the role and mechanism of tumor related signaling pathways ncluding apoptosis,proliferation,differentiation and metastasis.3.The expression of dysregulated genes in tongue squamous cell carcinoma is related to the prognosis of patients with tongue squamous cell carcinoma,including the molecular target of prognosis and the early warning index of early metastasis of tongue squamous cell carcinoma.4.The relationship between the key molecular mechanism of the occurrence and development of tongue squamous cell carcinoma and the clinical treatment of patients.It mainly includes the prediction target and mechanism of chemotherapy sensitivity,the new target of biological therapy,the new method of gene therapy and so on.It is well known that a large number of non coding RNA are formed in the mammalian genome,including microRNA(mi RNA)and long chain noncoding RNA(long noncoding RNA).The mechanism of LncRNA is complicated,and it can regulate gene expression mainly from three aspects: epigenetics,transcriptional regulation and post transcriptional regulation.At present,a series of LncRNA expression disorders have been found in tumor tissues,and the expression of LncRNAs in liver cancer,lung cancer and prostate cancer has been found to play an important role.However,the expression and function of LncRNA in tongue squamous cell carcinoma is still rarely reported.In this study,we aimed to screen out the dysregulated LncRNA in tongue squamous cell carcinoma and further study its function and mechanism.The whole experiment is divided into three parts :The first part: the difference of LncRNA expression in carcinoma of the tongue,we used the LncRNA expression profile of high-throughput microArray detection technology,a comprehensive comparison of differential expression in tongue squamous cell carcinoma tissues and adjacent tissues of LncRNA,to find the deregulated LncRNA in tongue squamous cell carcinoma.281 of these LncRNA were up-regulated and the other 286 were down regulated.We selected 6 LncRNAs,which were significantly differentially expressed,to further study their expression in more squamous cell carcinoma and adjacent normal tissues.We designed the specific primers for these 6 LncRNAs and detected their expression in tumor samples by qRT-PCR.The results showed that these 6 LncRNAs were differentially expressed in more squamous cell carcinoma samples,which linc00152 in 73.3%(11/15)of tongue squamous cell carcinoma was increased,and studies have shown that linc00152 in gastric cancer and colon cancer to promote tumor proliferation,affecting the tumor development,and no similar reports in tongue squamous cell carcinoma.According to the results of the chip and the expression of LncRNA in tumor samples,linc00152 may play an important role in the development and progression of tongue squamous cell carcinoma.So in the next study,we focus on linc00152.The second part: the effect of LncRNA00152 on tumor cell biology.To explore the function of linc00152,we conducted both in vivo and in vitro experiment.The total RNA of the nucleus and cytoplasm was extracted by the kit.The specific orientation of linc00152 in the cells was identified by the specific primers.The results showed that the site of linc00152 was mainly in the cytoplasm.The sequence of linc00152 in tongue squamous cell carcinoma was determined by RACE.The expression of linc00152 in SCC-9 and CAL-27 was knocked down using specific interfering RNA(si-linc00152).The plasmid pcDNA3.1-linc00152 was constructed and transfected into SCC-9 and Cal-27 cells to increase the expression of linc00152.The effect of linc00152 on cell proliferation,apoptosis,migration,invasion,cell cycle and tumor growth was studied by knocking down and overexpression of linc00152 in SCC-9 and Cal-27 cells.The effect of linc00152 on the proliferation of tongue squamous cell carcinoma was conducted by CCK-8.Transwell assay and cell scratches assay showed that linc00152 promoted the migration of tongue squamous cell carcinoma,and the transwell experiments with matrix showed that linc00152 enhanced the invasion of tongue squamous cell carcinoma.The effect of linc00152 on cell cycle and apoptosis was investigated by flow cytometry.The results showed that linc00152 promoted the cell division of TSCC cell from the stationary phase and inhibited the apoptosis of the cells.In nude mice assay,the effect of linc00152 on tumor growth was not observed because of the lack the change of linc00152.Therefore,further study was needed.The third part: the mechanism of linc00152.In order to study the mechanism of linc00152 in tongue squamous cell carcinoma,we conducted qRT-PCR,WB and luciferase reporter assays.It was confirmed that the linc00152 was located in the cytoplasm.Studies have shown that LncRNA can act as a competitive endogenous RNA(ceRNA),binds to miRNAs,inhibits miRNA binding to mRNA,affects miRNA function,and plays a role in gene regulation.To investigate whether linc00152 plays a role in this way,we predict whether there are mi RNAs associated with linc00152 at http://starbase.sysu.edu.cn/mir LncRNA.php.The results show that linc00152 has a binding site with miR-193a-3p or mi R-193b-3p or miR-376c-3p.qRT-PCR was used to detect the relative expression of miR-193a-3p,miR-193b-3p and mi R-376c-3p in tongue squamous cell carcinoma.The expression of miR-193a-3p and miR-376c-3p are much lower than that of linc00152.The expression of miR-193b-3p is close to that of linc00152,and linc00152 is the most likely to inhibit the function of miR-193b-3p.To further demonstrate whether linc00152 binds to miR-193b-3p,we performed a dual luciferase reporter assay,mimic-NC or miR-193b-3p-mimics with pGL3-Promotor-linc00152-WT(WT)or PGL3-Promoton-linc00152-mut(MUT)was co-transfected to TSCC cells,mimic-NC acted as the negative control.The results showed that miR-193b-3p could significantly inhibit the luciferase activity of WT plasmid,and the inhibition rate was about 66%.The expression of c-kit,a miR-193b-3p target gene,after overexpression or knock down of linc00152 was detected of,which showed that overexpression of linc00152 increased c-kit mRNA expression,knock down of linc00152 decreased c-kit mRNA expression,so linc00152 acted as a competitive endogenous RNA to increase c-kit expression by binding to miR-193b-3p.Western blot analysis showed that linc00152 mainly activated PI3K-AKT signaling pathway and inhibited the activation of apoptosis-related protein caspase 3,which promoted the development of tongue squamous cell carcinoma. |
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