Effects Of Long Noncoding RNA G003929 On Migration And Invasion Of Human Ovarian Cancer Cells And Its Mechanism

Objective:To screen the ovarian tissue specific Long non-coding RNAs(LncRNAs)for the clinical early diagnosis and find new targets for the bio-therapy in epithelial ovarian cancer(EOC).A high-throughput transcriptome sequencing was preformed to screen the mathed EOC tissues and contral ovarian tissues.Further,a Lnc RNA microarray was used to detect the ovarian cancer cell lines,such as SKOV3,HO8910 and A2780 and ovarian epithelial cell line HOSEpiC.Methods:(1)High-throughput transcriptome sequencing was performed between matched EOC tissue and control ovarian tisssues.Lnc RNAs microarray technology was used to detect EOC cell lines SKOV3,HO8910,A2780 and ovarian epithelial cell line HOSEpi C.Bioinformatics was used to analyze the data of transcriptome sequencing and Lnc RNAs microarray included highly conserved,tissue-specific,disease-related and process-related Lnc RNAs,etc.(2)The expression levels of Lnc RNAs were detected by q RT-PCR in SKOV3,HO8910,A2780,OVCR3,3AO and HOSEpi C cells,EOC tissues and control tissues.(3)The expression vector of Lnc RNA G003929 was construed and transfected and overexpressed in SKOV3 and HO8910 cells.(4)The cell proliferation was detected by EDU,CCK8 in SKOV3,HO8910,SKOV3-Lnc RNA G003929 and HO8910-Lnc RNA G003929.(5)Transwell migration and invasion assay and scratch assay were used to detect the migration and invasion ability of SKOV3,HO8910,SKOV3-Lnc RNA G003929 and HO8910-Lnc RNA G003929.(6)The encoding ability of LncRNA G003929 was analyzed by open reading frame(ORF)and comparative genome analysis(phylo CSF).(7)Subcellular localization of LncRNA G003929 in ovarian cancer cell SKOV3 was confirmed by fluorescence in situ hybridization(FISH)assay.(8)The Anno Lnc,lncPro and catRAPID online tools was used to predicte the binding proteins of Lnc RNA G003929.Western blot was used to detect the expression of proteins in SKOV3,HO8910,SKOV3-Lnc RNA G003929 and HO8910-Lnc RNA G003929.(9)The co-localization of Lnc RNA G003929 and ZEB1 protein in SKOV3 was detected by FISH and immunofluorescence assay(IF).RIP(RNA immunoprecipitation)experiment was used to confirm the combination of Lnc RNA G003929 with ZEB1.Results:(1)Transcriptome sequencing revealed that there were 216 up-regulated and 731 down-regulated Lnc RNAs.Microarray detection shown that there were 227 up-regulated and 2875 down-regulated Lnc RNAs.133 Lnc RNAs are highly conservative,ovarian tissue-specific and associated with the development of ovarian cancer.(2)The expression levels of seven LncRNAs were significantly different in ovarian cancer cells SKOV3,HO8910,A2780,OVCR3,3AO and ovarian epithelial cells HOSEpi C.RP3-325F22.5 and Lnc RNA G003929 were significantly different between ovarian cancer tissue and control ovarian tissue.Lnc RNA G003929 has ovarian tissue specificity and involves in the occurrence and development of ovarian cancer.Therefore,Lnc RNA G003929 was selected to study the function and mechanism.(3)LncRNA G003929 hasn’t Kozak sequence.The 22 ORFs of LncRNA G003929 were less than 200 nt.The results of BLAST showed that these short peptides had no homologous proteins in all justice chains.Phylo CSF results showed that Lnc RNA G003929 sequence was not conservative.The results indicate that Lnc RNA G003929 hasn’t the ability to encode proteins.(4)The LncRNA G003929 sequence was inserted into Plvx-Zsgreen-puro overexpression plasmid.The expression of Lnc RNA G003929 was verified by q RT-PCR in SKOV3 and HO8910.(5)The results of cell proliferation assays showed there were no significant difference between SKOV3,HO8910 and SKOV3-Lnc RNA G003929,HO8910-LncRNA G003929.(6)The migration abilities of SKOV3-Lnc RNA G003929 and HO8910-Lnc RNA G003929 were decreased but the invasion abilities of them were increased.(7)FISH results showed that Lnc RNA G003929 was localized in the nucleus of SKOV3 cell and few in the cytoplasm.(8)The predicted results of Anno Lnc,lncPro and catRAPID shown that LncRNA G003929 bind to ZEB1 protein.The prediction results of lnc Pro and Anno Lnc showed that Lnc RNA G003929 binds to MMP9 protein.Overexpression of Lnc RNA G003929 in SKOV3 and HO8910 cells resulted in decreasing the expression of ZEB1 and increasing the expression of MMP9.(9)LncRNA G003929 and ZEB1 co-localized in the nucleus of SKOV3 cell.RIP results showed that Lnc RNA G003929 binds to ZEB1.Conclusion:Lnc RNA G003929 is an ovarian tissue specific Lnc RNA and closely related to the migration and invasion of ovarian cancer and may be a new early diagnostic marker and therapeutic target of EOC.