| AIM:To explore the biological functions and mechanisms of LINC00858 in cervical cancer(CC)cells.METHODS:Firstly,human normal cervical epithelial cell line(Ectl/E6E7)and four cervical cancer cell lines(CaSki,SiHa,ME 180 and MS751)were cultured.RT-qPCR analysis was used to examine the expression of LINC00858 in five cell lines.According to the results,ME180 and MS751,the highest expression cells of LINC00858,were selected.Potential target genes(miR-3064-5p,VMA21)and corresponding binding sites downstream of LINC00858 were screened by starBase,miRmap,PicTar,TargetScan and microT databases and RT-qPCR assay.In order to further study the effects and the interaction of LINC00858,miR-3064-5p and VMA21 on CC cells,sh-RNAs and control sh-RNAs specifically targeting LINC00858 and VMA21,as well as the overexpression plasmids of miR-3064-5p simulant,miR-3064-5p inhibitor and VMA21 were constructed and transfected into cervical cancer cell lines ME180 and MS751,respectively.Then RT-qPCR was used to detect the expression of LINC00858,VMA21 and miR-3064-5p in transfected cervical cancer cells;The proliferation of cervical cancer cells knockdown or overexpressing LINC00858,miR-3064-5p and VMA21 was evaluated by EDU and colony formation assay,TUNEL assay and flow cytometry assay were conducted to assess the ability of cell apoptosis.The localization of LINC00858 in cells was analyzed by subcellular isolation assay and fluorescence in situ hybridization(FISH).The interaction of LINC00858,miR-3064-5p and VMA21 in cervical cancer cells was investigated by RNA pull-down assay,luciferase reporter assay and RNA immunoprecipitation reaction(RIP)assay.Finally,a series of rescue experiments were conducted to further verify the interaction among LINC00858,miR-3064-5p and VMA21.RESULTS:Compared with normal cervical epithelial cells,LINC00858 was highly expressed in four cervical cancer cells.In the cervical cancer cells transfected with sh-LINC00858,the expression of LINC00858 was significantly decreased,which effectively inhibited the proliferation and promoted apoptosis of cervical cancer cells.Cell localization technique confirmed that LINC00858 was mainly located in the cytoplasm of cervical cancer.It was speculated that LINC00858 mainly played a role in post-transcriptional regulation.Database query,RT-qPCR assay and bioinformatics analysis showed that miR-3064-5p expression was low in cervical cancer cells,and miR-3064-5p could be selected as a candidate target of LINC00858.VMA21 was the highest expression in cervical cancer cells among all candidate mRNAs,so VMA21 was selected as the candidate target of miR-3064-5p.miR-3064-5p,as a potential miRNA,may simultaneously bind to LINC00858 and VMA21.Luciferase reporting assay and RIP assay confirmed the interaction between miR-3064-5p and LINC00858 in cervical cancer cells.RNA pull-down assay and luciferase reporter assay showed that VMA21 was a direct target of miR-3064-5p.Compared with the control group,the expression of VMA21 was significantly increased in cervical cancer cells transfected with VMA21 overexpressed plasmid.On the contrary,the expression of VMA21 was significantly inhibited in sh-VMA21 transfected cervical cancer cells.Knockdown of VMA21 reduced the proliferation ability of ME180 and MS751 cervical cancer cells and promoted the apoptosis rate,suggesting that VMA21 promoted the proliferation and inhibited the apoptosis of cervical cancer cells.In rescue assays,miR-3604-5p silence and VMA21 up-regulation were able to counteract the effect caused by LINC00858 silence on cell proliferation and apoptosis of CC.To sum up,miR-3604-5p/VMA21 formed a regulating axis with LINC00858.CONCLUSION:1.LINC00858 inhibits the expression of miR-3064-5p and overexpresses VMA21 through competitive adsorption of miR-3064-5p,thus enhances proliferation of cervical cancer cells.2.LINC00858 promotes cell proliferation and restrains cell apoptosis of cervical cancer cells through miR-3604-5p/VMA21 axis,implying that LINC00858 may serve as a promising therapeutic target for cervical cancer. |
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