MiR-192 and miR-204 modulate expression of multiple HOTTIP downstream genes.

<p>(A) Cellular localization of lncRNA HOTTIP in HCC cells. (B)miR-192and miR-204 inhibit HOXA13 expression. 20 nmol/L miR-192 mimics, miR-204 mimics, siHOTTIP-1, siHOTTIP-2 or NC RNA were transfected into SMMC7721 and HepG2 cells. HOXA13 mRNA expression was detected using qRT-PCR. All data of HOXA13expression were normalized to <i>β-actin</i> or <i>GAPDH</i> mRNA expression levels. All results of the mean of triplicate assays with standard deviation of the mean are presented. *<i>P</i>< 0.05, **<i>P</i>< 0.01. (C) Deregulated genes in HepG2 cells after delivery of miR-192, miR-204 or siHOTTIP-1 using gene profiling arrays. (D) Cluster analysis of mRNAs expression profiles of the miR-192 group, the miR-204 group or the siHOTTIP-1 group versus the NC RNA group. Overexpression is coded in red, whereas under expression is indicated in green. (E) Candidate gene expression was validated using qRT-PCR. All data of gene expression were normalized to <i>β-actin</i> or <i>GAPDH</i> mRNA expression levels.(F) Inhibition ofGLS1 expression inhibits SMMC7721 cell growth. Up left panel, different doses of NC RNA or GLS1 siRNAs (20–60 nM) was transfected into SMMC7721 cells. Cell number was counted at 48 h after transfection. Up right panel, 30 nM NC RNA or GLS1 siRNA was transfected into SMMC7721 cells. Cell number was counted at 24, 48 or 72 h after transfection. Relative GLS1 gene expression was detected using qRT-PCR (low). (G) Colony formation assays. 30 nM NC RNA or GLS1 siRNA was transfected into SMMC7721 cells. After 10 days, colony number in each well was counted. *<i>P</i>< 0.05, **<i>P</i>< 0.01.