Fig 2: Identification of putative downstream genes of Loc100506691 in gastric cancer by applying the microarray approach(A) Differential expression genes were identified in HR with Loc100506691 overexpression and TSGH with Loc100506691 knockdown and compared with the corresponding control using a microarray approach. The heatmap presents putative downstream targets of Loc100506691in gastric cancer cells. (B) Expression levels of CHAC1 were examined by real-time PCR in TSGH and HR cells with Loc100506691 knockdown. (C) Expression levels of CHAC1 were assessed in HR cells with pLoc100506691-V1 or -V2 overexpression by applying the real-time PCR approach. (D) Expression levels of CHAC1 were examined by western blotting in gastric cancer cells with Loc100506691 knockdown or overexpression in TSGH and HR, respectively. Relative expression was further quantification (below panels). (E) The CHAC1 expression levels in human gastric cancer cells were examined through western blotting. Relative expression was further quantification (below panel). (F and G) After transfection for 24 h, CHAC1 expression levels were examined in TSGH with siCHAC1 using western blotting and real-time PCR. (H) Cell proliferation was examined in TSGH with CHAC1 knockdown. (I and J) The colony-formation ability of TSGH was investigated and quantified following siCHAC1 transfection. (K and L) The migration and invasion ability were assessed and quantified in TSGH with CHAC1 knockdown (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 3: Loc100506691 was involved in suppressing CHAC1 expression by modulating miR-26a-5p and miR-330-5p expression(A) Identification of downstream miRNA candidates of Loc100506691 in gastric cancer cells by using next-generation sequences. Venn diagrams reveal that miR-26a-5p and miR-330-5p were regulated by Loc100506691 and were identified using microRNA.org as having potential to target the 3' UTR of CHAC1. (B) Expression levels of miR-26a-5p and miR-330-5p were assessed in gastric cancer cells with miR-26a-5p or miR-330-5p mimic transfection, respectively. (C and D) Ectopic expression of miR-26a-5p or miR-330-5p significantly inhibited and suppressed the endogenous mRNA and protein levels of CHAC1 in TSGH cells. (E) Luciferase constructs: The miR-26a-5p (left panel) or miR-330-5p (right panel) targeting sequence in the 3' UTR sequence of CHAC1 is presented in the upper panel, and the mutant of its seed region is presented in the color red. (F and G) Relative luciferase activity of the reporter with wild-type and mutant 3' UTR of CHAC1 was determined in TSGH cells cotransfected with miR-26a-5p (left panels), miR-330-5p mimic (right panels), and the scramble control, respectively. Firefly luciferase activity was the normalization control. (H–J) TSGH cells were used to perform Ago2 protein precipitation, and the expression levels of miR-26a-5p, miR-330-5p, and CHAC1 were examined using real-time PCR (*p < 0.05, **p < 0.01, ***p < 0.001).

Fig 4: CHAC1 transcriptional activation and protein levels in I-152-treated cells. (A) RTqPCR analysis of Chac1 mRNA levels in cells treated with different concentrations of I-152 for 6 h and 24 h and in cells treated with 1 mM I-152 for different times. mRNA levels were normalized to the housekeeping genes Gapdh and Gusb and expressed as fold-change versus untreated cells (CTR). The values are the mean ± SD of two independent experiments with two technical replicates (B) Cells were incubated with different concentrations of I-152 for 2 h, 6 h and 24 h, and whole lysates corresponding to 10 µg of proteins were separated on 12% (w/v) polyacrylamide gels. The blots were probed with anti CHAC1 antibody. ß-ACTIN was stained as a control. Untreated cells receiving only fresh medium served as a control (CTR). Immunoreactive bands were quantified with the Image Lab software. CHAC1 levels, normalized to ß-ACTIN content, are reported in the graph as fold change relative to CTR. Values are the mean ± SD of at least three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001.

Fig 5: The proposed model for the mechanistic metformin suppression of gastric cancer cell growth through modulation of the Loc100506691-microRNAs-CHAC1 axis