| Objective: To explore the effects of long non-coding RNA small nucleolar RNA host gene 16(lncRNA SNHG16)and WNT signaling pathway inhibitor Dickkopf 3(DKK3)on epithelial-mesenchymal transition(EMT)in gastric cancer(GC)cells.Methods:(1)The GC HGC-27 and AGS cells with high expression of SNHG16 were selected as the research object.These cells were divided into three groups(blank control,negative control and si-SNHG16).RNAi interference technique was used to construct the cell model of SNHG16-knockdown.Next,the expression level of SNHG16 and the level of DKK3 mRNA expression were detected by reverse transcription-quantitative polymerase chain reaction(RT-qPCR)assay.The expression levels of EMT-associated proteins(E-cadherin,Claudin-1,Vimentin,Snail,Slug,Zeb1)and DKK3 protein were observed by Western blot assay.(2)To find the best interference sequence that can effectively knock down DKK3,three si-DKK3 sequences were designed.RT-qPCR and Western blot assays were combined to verify the interference effect of DKK3.In order to construct low-expressed SNHG16 stable lines,HGC-27 and AGS cells were infected lentivirus of SNHG16 shRNA.Then these stable cells were transfection of si3-DKK3 to further silence the DKK3 expression.It was divided into 4 groups(si-NC group,sh-SNHG16 group,si-DKK3 group and sh-SNHG16 associative si-DKK3 group).The EMT-associated proteins levels were measured by western blot assay.(4)We performed RT-qPCR assay to observe the expressions of SNHG16 and DKK3 at RNA level in 20 GC tissues and their adjacent tissues.Meanwhile the correlation of two genes in GC tissues was analyzed by Spearman correlation analysis.Results:(1)After knockdown of SNHG16 transitorily,the interference efficiency of SNHG16 in HGC-27 and AGS cells were up to(69.56 ± 3.86)% and(82.14 ± 1.09)%(all P<0.001),respectively.In addition,the RNA level of DKK3 was no obvious difference(P>0.05),but the DKK3 protein abundance was significantly increased(P<0.05).(2)As detected by Western blot analysis,SNHG16 knockdown upregulated the expression of epithelial markers E-cadherin and Claudin-1,whereas,reduced mesenchymal markers N-cadherin and Vimentin in HGC-27 or AGS cells(all P<0.05).In addition,SNHG16 knockdown could reduce the level of transcription factors Zeb1(P<0.05).However,Slug,Snail were no significant difference(all P>0.05).(3)The different siRNAs of DKK3 were screened by RT-qPCR and western blot assays,and it was found that the efficiency of si3-DKK3 sequence was the best.Thus,si3-DKK3 sequence was selected for follow-up experiments.(4)The interference efficiency of HGC-27 and AGS stable lines were(76.74±2.60)%,and(67.32±2.60)%(all P<0.001),respectively.(5)SNHG16 knockdown alone could inhibit EMT,while DKK3 knockdown alone could promote EMT in GC cells,indicating that SNHG16 and DKK3 were all involve in the regulation of EMT to some extent.And the SNHG16 knockdown-induced inhibition of EMT was reversed by co-transfection of sh-SNHG16 and si3-DKK3 into HGC-27 and AGS cells,implying that SNHG16 affects EMT via regulation of DKK3 in GC cells.(6)SNHG16 was higher but DKK3 was lower expression in GC tissues than their paired adjacent tissues.Furthermore,it was found that the expression of SNHG16 and DKK3 was negatively correlated in GC tissues.Conclusion: The expression of SNHG16 and DKK3 was a negative correlation in GC in some extent,and SNHG16 may affect the EMT of GC cells by regulating the expression of DKK3.It would provide a certain experimental basis for the study of GC metastasis. |
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