The Role Of LncRNA DPP10-AS1 In Human Lung Carcinogenesis

Background and significance Long noncoding RNAs（lnc RNAs）is a novel class of non-/less-protein-coding RNAs with more than 200 nucleotides in length.Lnc RNA regulate gene expression at the transcripitional and post-transcriptional levels.Studies have shown that lnc RNA plays an important role in proliferation,apoptosis,invasion,metastasis and drug resistance of various types of cancer cells.However,the roles of dysregulated lnc RNAs in lung carcinogenesis and tumor early diagnosis and prognosis remain poorly understood.In present work,we analyzed the lnc RNA expression in lung cancer.The differentially expressed lnc RNAs were further screened and identified.Additionally,we explored the clinical significance of lnc RNA in lung cancer patients.Furthermore,we studied the function of lung cancer-specific lnc RNA in the proliferation,growth,cycle and apoptosis of lung cancer cells,and revealed the mechanism of lnc RNA in lung carcinogenesis.This study will provide a new theoretical basis for elucidating the new mechanism of lung cancer and introduce new targets for lung cancer precision medicine.Methods 1.The lnc RNA expression profile of lung cancer was analyzed by microarray: 4 pairs lung cancer and cancer-adjacent tissues were performed on arraystar V3.0 microarray by kangcheng biological technology Co,Ltd which covers 40173 lnc RNAs and 20730 m RNAs.The most significant up-regulation or down-regulation of lnc RNAs was identified by screening for the next validation.2.RT-q PCR were performed to detect the basal expression of XLOC₀12040,XLOC₀011882,RP11-536D16.2,XLOC₀07354,RP3-340N1.2,PSORS1C3,HERC2P3,FHL1,AC068039.4,C17orf76-AS1,XLOC₀09167,DPP10-AS1.3.Analysis of the initial expression of DPP10-AS1 and DPP10 in the clinical samples and cells and the correlation with the clinicopathological factors.4.MTT and cell colony formation assays were used to analyze the effect of DPP10-AS1 on cell growth and proliferation.5.The functional roles of DPP10-AS1 in cell cycle and apoptosis were measured by flow cytometry.6.Western blot to detect DPP10 protein level after up-and down-regulation of DPP10-AS1.7.We used cytoplasmic and nuclear extract isolation assays to test DPP10-AS1 nuclear cytoplasmic distribution.8.We used RNase protection assay to test the possibility of RNA duplex between DPP10-AS1 and DPP10 m RNA.9.We used methylation inhibition assay to analyze the level of DPP10-AS1 and DPP10 m RNA in lung cancer cells.Results 1.Screening of DPP10-AS1 as a candidate molecule.we used RT-q PCR to find DPP10-AS1,FHL1 and XLOC₀09167 were significant difference expression（P<0.05）.In accordance with the chip results,we chose DPP10-AS1 as the research object.2.Based on the result,the high expression level of DPP10-AS1 is related to the level of serum CYFRA21‐1,tumor size,and TNM stage。K-M survival analysis showed that the overall survival rate in patients with high expression of DPP10-AS1 was significantly lower than that in the low expression group,the recurrence survival rate in patients with high expression of DPP10-AS1 was significantly lower than that in the low expression group.3.Compared to the normal BEAS-2B cell line,the level of DPP10-AS1 in LTEP-a2 and NCI-446 cells were not statistically significant while A549,H460,SPC-A1 and NCI-H1299 cell lines were upregulated.The level of DPP10 m RNA in LTEP-a-2,NCI-H446,A549,NCI-H1299 and SPC-A1 cell lines were upregulated while DPP10 protein in A549,NCI-H1299 and SPC-A1 cell lines were upregulated.4.The expression of DPP10-AS1 and DPP10 in lung cancer tissues was significantly higher than that in adjacent tissues.In the DPP10-AS1 low expression level lung tissue samples,DPP10 also showed low expression levels;in the DPP10-AS1 high expression level lung tissue samples,DPP10 also showed high expression levels.On the other hand,it also showed that DPP10-AS1 and DPP10 were coordinated in lung cancer.5.Overexpression of DPP10-AS1 caused the upregulation of DPP10 m RNA and protein.This effect could be restored by co-transfection of si DPP10-1 and si DPP10-2.Knockdown of DPP10-AS1 caused the downregulation of DPP10 m RNA and protein while this effect could be restored by co-transfection of pc DNA3.1-DPP10.6.Knockdown of DPP10-AS1 inhibited the growth and proliferation of lung cancer cells,while overexpression DPP10-AS1 promoted cell growth and proliferation of lung cancer cells.This effect can be rescued by restoring the expression of DPP10 m RNA.7.Cell cycle progression was arrested in the G0/G1 phase and promoted the apoptosis of lung cancer cells by knockdown of DPP10-AS1.Overexpression of DPP10-AS1 promoted cell cycle progression and inhibited cell apoptosis.This effect can be restored by down-regulating the expression of DPP10 m RNA.8.RNase protection test showed that DPP10-AS1 did not form RNA duplex with DPP10 m RNA.9.Cytoplasmic and nuclear extract isolation assays showed that DPP10-AS1 was mainly located in the nucleus.10.Methylation inhibition assay showed the expression of DPP10-AS1 was increased by addition of 5-methylation inhibitor 5-azacytidine（5-Azacitidine）in SPC-A1 and NCI-H1299 cells while the expression of DPP10 m RNA was upregulated.DPP10 hypomethylation was found in squamous cell carcinoma by using the methylation database（Meth Hc）.We also found three Cp G islands in the DPP10 and one Cp G island in the DPP10-AS1 promoter,respectively.Conclusions The present study demonstrates that DPP10-AS1 is highly expressed in lung cancer tissues for the first time.Clinically,DPP10-AS1 is found to be significantly correlated to serum CYFRA21‐1,tumor size and TNM staging.Upregulation of DPP10-AS1 predicts poor prognosis and is an independent risk factor for lung cancer patients.At the cell level,DPP10-AS1 is involved in the biological effects on promoting lung cancer cell growth,proliferation and cell cycle process,as well as inhibiting cell apoptosis.Mechanically,DPP10-AS1 is coordinated with the expression of its related gene DPP10 and regulated by methylation.These results indicate that DPP10-AS1 is highly expressed in lung cancer,plays the role of oncogene,and exerts biological function by influencing DPP10 expression.This will provide novel theoretical insights into DPP10-AS1 as a prognostic maker to predict lung cancer and the development of molecular drugs targeted DPP10-AS1 in lung cancer.