LMX1A inhibits C-Myc expression through ANGPTL4 to exert tumor suppressive role in gastric cancer

Figures

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  Fig 1. Identification of LMX1A-regulated genes by using RNA-sequencing. (A) AGS cells stably expressing empty vector (AGS-EV) or Flag-tagged LMX1A (AGS-LMX1A) were constructed by retrovirus infection. (B) AGS-EV and AGS-LMX1A cells were subjected to RNA-Seq, and volcano plot of significantly regulated gene were shown. (C) Genes that were up-regulated by LMX1A overexpression for more than 3.5 folds compared to EV-expressed cells.

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  Fig 2. RNA-level and protein-level verification of ANGPTL4 activation by LMX1A. EV or LMX1A was overexpressed in AGS (A) and MKN45 (B) cells, and Q-PCR and western blot (C) was performed to detect the expression of ANGPTL4 and MDC1; D. ChIP assay of AGS with EV and overexpressed LMX1A, the ANGPTL4 enrichment was calculated in fold-values by normalization to the negative control IgG and input control. Student’s t test. *, P<0.05; **, P<0.01.

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  Fig 3. LMX1A and ANGPTL4 are downregulated and positively correlated in gastric cancer samples. (A, B) Box plots of the expression of LMX1A (A) and ANGPTL4 (B) in gastric cancer samples compared to that in normal samples in TCGA Stomach Adenocarcinoma data collection. Data were analyzed using Mann-Whitney U test. (C) Pearson’s correlation of LMX1A and ANGPTL4 in cancer samples of TCGA Stomach Adenocarcinoma data collection. (D) Pearson’s correlation of LMX1A and ANGPTL1-7 in cancer samples of TCGA Stomach Adenocarcinoma data collection.

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  Fig 4. Knockdown of ANGPTL4 rescued the suppressive effect of LMX1A on tumorigenesis. (A) shRNAs specifically against ANGPTL4 (shANGPTL4#1 and shANGPTL4#2), MDC1 (shMDC1#1, shMDC1#2) and a scramble shRNA as negative control (shNC) were designed and the knockdown efficiencies of each shRNA were verified in AGS cells. Student’s t test. **, P<0.01. (B) AGS-EV cells infected with shNC virus (AGS-EV-shNC), and AGS-LMX1A cells infected with shNC (AGS-LMX1A-shNC) or shANGPTL4#1 (AGS-LMX1A-shANGPTL4#1) virus were subcutaneously injected into nude mice. Tumors were harvested 3 weeks later and weighed. Student’s t test. **, P<0.01.

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  Fig 5. LMX1A inhibits the expression of oncogene C-Myc through ANGPTL4. (A, B) Immunoblotting of indicated proteins in AGS (A) and MKN45 (B) cells with or without LMX1A overexpression. (C) shRNAs against ANGPTL4 and MDC1 together with shNC were expressed in AGS-EV or AGS-LMX1A cells, and the indicated proteins were detected by immunoblotting. (D) shNC and shANGPTL4#1 were expressed in MKN45-EV or MKN45-LMX1A cells, and the indicated proteins were detected by immunoblotting.