Expression Of Long Chain Non Coding RNA In HPV16 Positive Cervical Cell Lines

Objective:Gene chip was used to analyze the expression differences of long chain non coding RNA LncRNA)and messenger RNA mRNA)in cervical cancer cell line SiHa)and human cervical immortalized squamous cell line Ect1/E6E7)infected by HPV16,to explore the important role and possible molecular mechanism of expression changes of lncRNA in the development of cervical cancer CC)infected by High-risk human papillomavirus HR-HPV).Methods:1.The SiHa cell line and the Ect1/E6E7 cell line were cultured.RNA extraction Trizol method),purification mRNA-ONLY Eukaryotic mRNA Isolation Kit,Epicentre)and quality detection Agilent ND-1000 and standard denaturing gel electrophoresis).RNA marked Arraystar RNA Flash Labeling Kit)and chip hybridization Agilent SureHyb and Agilent DNA Microarray Scanner).Differential expression analysis Agilent GeneSpring GX v12.1)and functional analysis Analysis of GO and KEGG pathway)after data acquisition Agilent Feature Extraction v11.0.1.1))and standardization Agilent GeneSpring GX v12.1).The RT-PCR method was used to test the LncRNAs related to cytokine)expression of the SiHa cell line and the Ect1/E6E7 cell line,the HPV16 positive cervical cancer tissue,HPV16 positive normal cervix tissue and HPV16 positive cervical precancerous tissue.Results:1.The SiHa cell line is compared with the Ect1/E6E7 cell line,there were 2804up-regulated lncRNAs with 2 times difference expression,accounting for 11.9% of the total up regulation of LncRNAs,2372 down-regulated lncRNAs,accounting for about 12.1%,2405 up-regulated mRNAs,accounting for 28.6%,2505 up-regulated mRNAs,accounting for 27.4%.2.The up-regulated mRNAs expressed in SiHa cell line,the proportion of mRNAs that participates in the regulation of multi cell biological processes is the largest in the biological process classification,the proportion of mRNAs participating in part of the intracellular composition is the largest in the cell componentclassification and the proportion of the mRNAs with the ability to combine is the largest in the molecular functional classification.3.The down-regulated mRNAs expressed in SiHa cell line,the proportion of mRNAs that participate in the regulation process of a single organism is the largest in the biological process classification,the proportion of mRNAs that participate in the intracellular composition is the largest in the cell component classification and the proportion of the mRNAs with the ability to binding protein is the largest in the molecular functional classification.4.Pathway analysis showed that the differential expression mRNAs enriched56 Pathway,of which 28 were enriched by mRNAs and 28 were downregulated by mRMAs.5.ENST00000566282,NR₀38974,T287840 and ENST00000571370 were verified by RT-PCR method in cell lines,ENST00000566282,NR₀38974 and ENST00000571370 are identical with the chip results,T287840 is inconsistent.6.The ENST00000566282,NR₀38974 and ENST00000571370 were verified by RT-PCR method in cervical tissue,NR₀38974 and ENST00000571370 are identical with the chip results,ENST00000566282 is inconsistent.Conclusion:1.SiHa cell line has a large number of differentially expressed LncRNAs and mRNAs compared with Ect1/E6E7 cell line.2.Differential expression of LncRNAs may play an important role in the development of cervical cancer.They may be used as new markers for early diagnosis of cervical cancer.3.ENST00000566282,NR₀38974,T287840 and ENST00000571370 related with cytokine in cell lines and cervical tissues were verified by RT-PCR.The results of NR₀38974 and ENST00000571370 are consistent with the results of the chip.This indicates the reliability of gene chip results and lays a foundation for clarifying the role of cytokines in the CC immune microenvironment.