| Background:As one of the most common gynaecological malignant tumours,cervical cancer has become an important public health issue.The incidence rate of cervical cancer has been reported to rank the 2nd in the world among female malignant tumours,and its mortality rate ranks the 1 st among female malignant tumours of the reproductive system,rendering it a disease that seriously threatens female health.At present,surgery,chemotherapy and radiotherapy are the predominant therapeutic schemes for cervical cancer,but most cervical cancer cells are resistant to chemotherapeutic drugs,resulting in a poor therapeutic effect.There is a lack of an effective therapeutic method for advanced and recurrent cervical cancer with poor prognosis.Therefore,it is urgently necessary to investigate new RNA with more than 200 nucleotides,and it possesses similar structural features to mRNA.Most of lncRNAs are produced via RNA polymerase ? transcription.Although IncRNA does not encode a protein,it can affect the expression levels of a variety of genes at the transcriptional and post-transcriptional levels.According to recent studies,the expression of lncRNA is closely related to various tumours,such as colon cancer,breast cancer and liver cancer.However,the mechanism of IncRNA in cervical cancer still remains largely unexplored.Methods:In the present study,differentially expressed lncRNAs were identified in three pairs of cervical cancer tissues and corresponding para-carcinoma tissues using The Cancer Genome Atlas(TCGA)database.Five treatments of cervical cancer.Long non-coding RNA(lncRNA)is a type of non-coding pairs of lncRNAs that were up-regulated and down-regulated were verified via quantitative real-time reverse transcription PCR(qRT-PCR).Finally,lncRNA C5orf66-AS1 was selected as the object of our current study.Up-and down-regulation of lncRNA C5orf66-AS1 in vitro and in vivo affected the biological behaviour of cervical cancer.Therefore,it could be used to explore the target genes of lncRNA C5orf66-AS1 in the proliferation of cervical cancer.Taken together,our findings provided a new theoretical basis for the effective prevention and treatment of cervical cancer.Results:1.LncRNAC5orf66-AS1 was significantly up-regulated in cervical cancer tissues and cells.2.After the suppression of C5orf66-AS1 in SiHa and C-4 I cells,cell proliferation was significantly decreased.However,cell proliferation was significantly increased after the up-regulation of C5orf66-AS1 in SiHa and C-4 I cells.Moreover,the cell-cycle assay showed that the number of cells in the G1/G0 phase was increased with suppression of C5orf66-AS 1,while that in the G2/S phase was reduced.The number of cells in the G1/G0 phase was decreased obviously with up-regulation of C5orf66-AS1,while that in the G2/S phase was significantly increased.In addition,down-regulated C5orf66-AS1 expression promoted the apoptosis of cervical cancer cells.3.Down-regulation of C5orf66-AS1 significantly promoted the expression of miR-637 compared with the control treatment,and up-regulation of C5orf66-AS1 significantly decreased the expression of miR-637.We next constructed the fluorescent reporter enzyme plasmids C5orf66-AS1-WT and C5orf66-AS1-MUT with a miR-637 binding site.Up-regulation of miR-637 significantly reduced the luciferase activity in SiHa cells co-transfected with C5orf66-AS1-WT,while up-regulation of miR-637 had no effect on luciferase activity when the cells were co-transfected with C5orf66-AS1-MUT,suggesting that C5orf66-AS1 bound directly to miR-637.The RIP assay was used to validate the potentially endogenous interaction between C5orf66-AS1 and miR-637.The results showed that C5orf66-AS1 and miR-637 were preferentially enriched in the anti-Ago2 group compared with the IgG control group.Moreover,the expression of miR-637 was examined in 20 cases of cervical cancer and para-carcinoma tissues via qRT-PCR,and the miR-637 expression was low in cervical cancer.The expression of miR-637 in cervical cancer cell lines was lower than that in normal cervical epithelial cell lines.4.The expression of miR-637 in SiHa and C-4 I cells was increased or decreased,and the expression of RING1 at the mRNA and protein levels was changed.Next,we constructed the fluorescent reporter enzyme plasmids RING1 3'UTR-WT and RING13'UTR-MUT with the miR-637 binding site.The results of the immunofluorescence reporter assay revealed that SiHa cells co-transfected with the RING 1 3'UTR-MUT plasmid and miR-637 showed significantly less luciferase activity compared with the control group,and the up-regulation of miR-637 exhibited did not affect the luciferase activity of cells that were co-transfected with the RING 1 3'UTR-MUT plasmid,suggesting that RING1 could bind directly to miR-637.5.After up-or down-regulation of C5orf66-AS1 in SiHa and C-4 I cell lines,the expression of RING1 at the mRNA and protein levels was significantly increased or decreased,respectively.6.The over-expression of miR-637 alone significantly inhibited the proliferation of SiHa and C-4 I cells,and miR-637 partially reversed such alterations caused by C5orf66-ASl via over-expression of C5orf66-AS1 or up-regulation of miR-637.7.The over-expression of RING 1 alone significantly enhanced the proliferation of SiHa andC-4 I cells,and RING1 completely reversed these changes caused by miR-637 via over-expression of RING 1 or up-regulation of miR-637.8.Down-regulation of lncRNA C5orf66-AS1 inhibits tumour growth in vivoConclusion:LncRNA C5orf66-AS1,as a competitive endogenous RNA(ceRNA),regulated the effect of RING1 on the proliferation,apoptosis and cell cycle of cervical cancer cells through adsorbing miR-637.Taken together,our findings provided a new theoretical and experimental basis for investigating the pathogenesis and exploring effective therapeutic targets for cervical cancer. |
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