The Role And Mechanism Of LncRNA ENST00000512916 In Ameloblastoma Proliferation And Invasion

Objective: Ameloblastoma(AB)is a common tooth source tumor.It is common in oral and maxillofacial features and has a high recurrence rate,accounting for about 60%.The majority of AB is benign tumor,but there are local invasion characteristics,and the postoperative recurrence rate is higher,rarely shows distal invasion.A small number of AB shows the characteristics of malignant histology,which is called abeloblast carcinoma.AB show the clinical biological features attracted many scholars research interests。Currently,the treatment of AB is limited to surgical treatment,and there is a high recurrence rate.The malignant transformation of tumor not only increases the difficulty of clinical treatment,but also increases the patient’s pain.Therefore,it is very important to accurately reveal the pathogenesis of AB,which can provide theoretical guidance for the prevention of ameloblastoma and the exploration of new therapeutic approaches.According to genomics,less than 2 percent of all human genomes encode proteins,while the rest are transcribed into non-coding RNA(non-coding RNAs,nc RNAs).Nc RNAs play a key role in the process of regulating multiple biological processes.Ribosomal analysis of the whole genome indicates that the gene translation activity is more than expected,indicating that non-coding transcripts also have the potential of translation.More and more studies have shown that nc RNAs play a key role in maintaining gene assembly,chromatin kinetics and gene regulation.Nc RNAs can be divided into three main groups according to their length,and the length is 20-50 nt,which is referred to as short-chain non-coding RNA(short nc RNA)including micro RNAs and siRNAs.The length is50-200 nt,which is called medium nc RNA,such as sn RNAs,sno RNAs,PROMPTs,and TSSa-RNAs.Long ncrnas,which are longer than 200 nt,are called long ncrnas,such as HOTAIR,MALAT1,H19,and XIST.With the development of the research,we found that the number of LncRNAs is more than the number of encoding genes,and it can regulate gene expression on multiple levels.Lnc RNA(lnc RNAs)is a functional RNA molecule with a length of over 200 nucleotides,which can be widely transcribed in eukaryotic cells.Currently,there are nearly10,000 lncrnas.According to the literature,lnc RNA has a variety of regulatory modes.Itmainly interacts with proteins,DNA or RNA to regulate the expression of genes in epigenetic levels,transcriptional levels and post-transcriptional levels.It is also involved in many important biological processes,such as X chromosome silence,genomic imprinting,chromatin modification,transcriptional activation,transcriptional interference,and nuclear transport.Through bioinformatics analysis,we screened long chain non-coding RNAENST00000512916.Then,we explore the role of ENST00000512916 in the process of AB proliferation and invasion,and finally reveal its mechanism of regulating AB proliferation and invasion.The results of this study will provide a scientific basis for further exploring the role of ENST00000512916 in the biological behavior of AB proliferation and the molecular mechanism.Methods: 1.Through gene chip(v3.0)technology,the differential expression of LncRNA ENST00000512916 was selected.2.The expression of ENST00000512916 in 26 patients with ameloblastoma was detected by q RT-PCR and RNAscope,and the expression of HOXC13 in the normal tissue of ameloblastoma tissue was detected.3.AM-1 cell line was selected as the research object through literature review and analysis.We synthesized the si RNA of ENST00000512916 and constructed its overexpression plasmid.Then,we transfected the si RNA-512916 and pc DNA3.1-512916 to AB cells,using CCK8,plate cloning,Transwell pore experiment and flow cytometry to detect the effect of ENST00000512916 on AB proliferation,invasion and apoptosis.4.The regulation of the expression of the potential target gene HOXC13 was detected by q RT-PCR and Western-blot.Results:1.Analysis the expression of Lnc RNA changes in AB tissues and adjacent tissues by high-throughput sequencing.The expression level was more than 2 times,and the Lnc RNA with statistical significance(p <0.05)was the difference expression of Lnc RNA.It was found that there were 30 lncrnas with more than 10 times of expression.2.Through the analysis of differential multiple,statistical analysis,m RNA chip expression analysis,bioinformatics analysis,and literature report,we finally identified Lnc RNA ENST00000512916 as the focus of the study.Through q RT-PCR and RNAscope,the expression of Lnc RNA in AB tissue was significantly higher than that of normal tissue,which was consistent with the results of chip detection.3.Through CCK8,clone formation and transwell experiment,the proliferation and invasion of AB cells were inhibited after the expression of silence ENST00000512916.On the contrary,after transfection of pc DNA-512916 in AB cells,the proliferation of cells and the expression of cell cycle proteins were promoted.4.The AB cells transfected with si RNA were injected into the right armpit of the nude mouse,and the tumor was taken out after 30 days.The results showed that the volume of tumor volume in the experimental group was slower than that in the control group,indicating that Lnc RNA ENST00000512916 could inhibit the proliferation of AB in vivo.5.The expression of HOXC13 was inhibited after over-expressed ENST00000512916 in AB cells by q RT-PCR,Western blot and CCK8 analysis.In contrast,silence ENST00000512916 promotes the expression of HOXC13.We also constructed the overexpression plasmid of HOXC13 and designed the corresponding si RNA.The results showed that overexpression of HOXC13 inhibited the proliferation of AB cells and the expression of cell cycle protein(CDK2 inhibition effect was significant).After the HOXC13 gene was silenced,the proliferation of AB cells and the expression of cell cycle protein proteins were promoted.Conclusions: 1.A new Lnc RNA ENST00000512916 was first detected through gene chip(v3.0)expression and bioinformatics analysis.The expression of Lnc RNA ENST00000512916 was closely related to the development of ameloblastoma.2.Silence ENST00000512916 inhibits the proliferation of am-1 cells and the expression of cell cycle proteins.Overexpression of ENST00000512916 promotes cell proliferation and cell cycle protein expression.Whether inhibit ENST00000512916 expression or overexpress ENST00000512916 has no obvious effect on apoptosis.3.Lnc RNA ENST00000512916 regulates the proliferation of AB in the nude mice.4.Lnc RNA ENST00000512916 may play a role in the antisense chain adjacent gene HOXC13.