| Objective Long non-coding RNA(lncRNA)is an RNA molecule with a transcript of more than200 nucleotides that cannot encode protein,and its expression disorder plays a crucial role in a variety of physiological and pathological processes.LncRNA/miRNA/mRNA regulatory network plays an important role in the occurrence and development of prostate cancer.Long non-coding RNA small nucleolar RNA host gene 11(SNHG11)has been shown to play an important role in the development and progression of many cancers.However,the role of lncRNA SNHG11 in the development of prostate cancer is still unclear.Therefore,this study intends to focus on lncRNA SNHG11 to preliminarily explore its functional role and possible molecular mechanism in the progression of prostate cancer,so as to provide a new target for the early diagnosis and precise treatment of prostate cancer.Methods 1.Data were downloaded from TCGA database to analyze the differential expression of SNHG11 gene in prostate cancer tissues and adjacent tissues,and qRT-PCR was used to detect whether there was any difference in the expression of SNHG11 gene in prostate cancer cell lines LNCaP,DU145,PC3 and normal prostate epithelial cell lines RWPE-1.2.For prostate cancer cells DU145 and PC3 with significantly high expression of SNHG11,the localization of SNHG11 in prostate cancer cells was detected by nuclear cytoplasmic separation experiment.3.For prostate cancer cells DU145 and PC3 with significantly high expression of SNHG11,sh-SNHG11#1 and SH-SNHG11#2 were transfected with lentivirus packaging technology to construct stably low knockdown SNHG11 cell lines,and the knockdown efficiency of SNHG11 was detected by qRT-PCR.4.The effects of SNHG11 knockdown in prostate cancer cells DU145 and PC3 on cell proliferation were detected by CCK-8 assay,clonogenesis assay and EdU proliferation assay.5.Through bioinformatics analysis,it is predicted that miR-211-3p may be the downstream targeted binding miRNA of SNHG11.Changes in miR-211-3p expression levels and differences in miR-211-3p expression levels between prostate cancer cells and normal prostate epithelial cells before and after SNHG11 knockdown were detected by qRT-PCR.The interaction between SNHG11 and miR-211-3p was verified by double luciferase reporter gene assay.6.In order to explore the biological function of lncRNA SNHG11/ miR-211-3p axis in prostate cancer cells,CCK-8 assay,clonogenesis assay and EdU proliferation assay were used to detect changes in the proliferation ability of prostate cancer cells after inhibition of mir R-211-3p in stably transfected prostate cancer cell lines by sh-SNHG11.7.Through literature review and bioinformatics analysis,the downstream mrnas possibly regulated by lncRNA SNHG11/ miR-211-3p axis were predicted.qRT-PCR and Western blot were used to detect the changes in the expression levels of target genes in prostate cancer cells after SNHG11 knockdown and the differences in their expression levels between prostate cancer cells and normal prostate epithelial cells.8.An animal model was constructed to dynamically detect the growth of subcutaneous implanted tumor in nude mice to verify the effect of SNHG11 gene on the proliferation ability of prostate cancer cells in vivo.The expression changes of Ki67,a proliferation marker of implanted tumor,were detected by immunohistochemistry.Results 1.According to the analysis of data downloaded from TCGA database,the expression of SNHG11 in prostate cancer tissues was significantly higher than that in para-cancer tissues(P<0.001).qRT-PCR results showed that compared with normal prostate epithelial cells RWPE-1,the expression levels of SNHG11 in prostate cancer cell lines DU145 and PC3 were significantly increased,while there was no significant change in LNCaP cells.Therefore,PC3 and DU145 were selected as the objects of subsequent experimental studies.2.Nucleoplasmic separation of prostate cancer cells showed that SNHG11 was mainly distributed in the cytoplasm of prostate cancer cells PC3 and DU145.3.Lentivirus plasmid was used to construct SNHG11 stable knockdown cell lines of PC3 and DU145,and the knockdown efficiency of SNHG11 gene was detected by QRT-PCR.The results showed that the expression of SNHG11 was significantly reduced after transfection of corresponding SH-SNHG11 into prostate cancer cell lines(P<0.001),in which sh-SNHG11#2knockdown efficiency is better,reaching more than 70%.4.CCK-8 assay,clonogenesis assay and EdU proliferation assay showed that after SNHG11 knockdown in prostate cancer cells,the proliferation ability of prostate cancer cell lines PC3 and DU145 was significantly inhibited.5.Bioinformation analysis predicted that miR-211-3p might be the downstream targeted binding miRNA of SNHG11.qRT-PCR results showed that mir-211-3p expression was significantly upregulated after SNHG11 gene knockdown in prostate cancer cell line PC3 and DU145(P<0.001),and was negatively correlated with SNHG11 expression.Meanwhile,compared with normal prostate epithelial cells RWPE-1,the expression level of miR-211-3p in prostate cancer cell lines DU145 and PC3 was decreased.Dual luciferase reporter gene assay showed that SNHG11 could directly adsorb the sequence site of miR-211-3p and regulate its expression activity.6.CCK-8 assay,clonogenesis assay and EdU proliferation assay were performed after transfection of sh-SNHG11#2 stable knockdown cell lines with higher knockdown efficiency into miR-211-3p NC and miR-211-3p inhibitor respectively.The results showed that inhibition of miR-211-3p expression reversed the reduction of prostate cancer cell proliferation caused by SNHG11 induced knockdown in stably knocked down cell lines of prostate cancer.7.Through literature review and bioinformatics analysis,it was predicted that ESM1 might be the downstream mRNA regulated by miR-211-3p.qRT-PCR results showed that the expression level of ESM1 was significantly down-regulated after SNHG11 knockdown in prostate cancer cell line PC3 and DU145.The knockdown level was positively correlated with the knockdown efficiency of SNHG11.Western blot results also showed that ESM1 protein expression was decreased in prostate cancer cell lines PC3 and DU145 after SNHG11 knockdown.In addition,ESM1 was significantly overexpressed in prostate cancer cell lines DU145 and PC3.8.The results of subcutaneous tumorigenesis experiment in nude mice showed that knockdown SNHG11 inhibited the growth of implanted tumor in vivo.Immunohistochemical results showed that the expression level of Ki67 was decreased after SNHG11 knockdown.Conclusion LncRNA SNHG11 is abnormally elevated in prostate cancer tissues and cells,and the up-regulation of LncRNA SNHG11 expression promotes the proliferation of prostate cancer cells,which is achieved by targeting Mir-211-3p.Further studies have found that ESM1 may be a downstream target of lncRNA SNHG11/ Mir-211-3p axis.LncRNA SNHG11 competitively binds to miR-211-3p to regulate ESM1 expression.LncRNA SNHG11/ miR-211-3p /ESM1 regulatory network plays an important role in the proliferation of prostate cancer,which not only enriches the lncRNA/miRNA/mRNA regulatory network in prostate cancer,but also provides new potential targets for early screening and precision treatment of prostate cancer. |
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