Fig 2: Analysis of cell proliferation, miR-143 and HK2 expression in Zn-deficient preneoplastic and neoplastic rat esophageal tissuesA. qPCR analysis of miR-143 (U87 as normalizer, n = 8 rats/group; results are shown as means, error bars represent standard deviation). B. qPCR analysis of mRNA expression of HK2 (Psmb6 as normalizer, n = 8 rats/group; results are shown as means, error bars represent standard deviation). Analysis of miR-143 and HK2 expression by in situ hybridization (ISH) and immunohistochemistry (IHC) in formalin fixed paraffin embedded esophageal samples in - C. ZD versus ZS esophagus after a 6-week dietary regimen (n = 8 rats/group) D. Archived samples of ESCC-bearing ZD esophagus versus tumor-free control ZS esophagus [28] (n = 10 rats/group). Representative hematoxylin and eosin [H&E]-stained and PCNA-stained sections are shown. miR-143 ISH signal (blue, 4-nitro-blue tetrazolium and 5-brom-4-chloro-3'-indolylphosphate; counterstain, nuclear fast red). HK2 expression (brown, 3,3'-diaminobenzidine tetrahydrochloride, counterstain, Harris modified hematoxylin). Scale bars: 50 µm (x200 magnification), 100 µm (x100 magnification, and 25 µm (x400 magnification, inset).

Fig 3: HK2 protein levels positively correlated with B7-H3 expression in CRC patient specimens.a Images of IHC analysis of B7-H3 and HK2 protein expression and hematoxylin and eosin (H&E) staining of CRC (n = 126) tissue sections. One representative image is shown. b, c B7-H3 (b) and HK2 (c) protein expression based on their staining index in nonmalignant adjacent tissues (NAT) and CRC specimens. d Correlation analysis of the staining index of expression levels of B7-H3 and HK2 protein in human CRC specimens (n = 126). e, f B7-H3 (e) and HK2 (f) protein expression based on their staining index in CRC specimens at different clinical stages. Values are expressed as means (SEMs). *P < 0.05

Fig 4: The effect of B7-H3 on the chemoresistance of CRC cells was HK2 dependent in vitro.a, b Colony formation assay of B7-H3-overexpressing HCT116 or RKO cells exposed to L-OHP. The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. c, d Cell viability was analyzed using the CCK-8 kit. B7-H3-overexpressing HCT116 or RKO cells showed resistance to L-OHP (c) or 5-FU (d). The chemoresistance of B7-H3 was abolished by HK2 siRNA or 2-DG. e B7-H3-overexpressing HCT116 or RKO cells showed less cell apoptosis than control cells (EV) after L-OHP treatment. The effect of B7-H3 on cell apoptosis was abolished by HK2 siRNA or 2-DG. f The protein levels of Bcl-2 and Bax were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. The effect of B7-H3 on Bcl-2 and Bax expression was abolished by HK2 siRNA. ß-actin served as a loading control. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. *P < 0.05

Fig 5: B7-H3 promoted the expression of HK2 in CRC cells.a, b The expression of glycolysis-related genes was detected by RT-qPCR in both B7-H3-overexpressing HCT116 (a) and RKO (b) cells. c, d The mRNA level of HK2 was detected by RT-qPCR in both HCT116 (c) and RKO (d) cells after transfection with siRNA NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. e HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both HCT116 and RKO cells after transfection with NC, B7-H3 siRNA-1 or B7-H3 siRNA-2. ß-actin served as a loading control. f HK2 protein level and STAT3 activation (examined by the p-STAT3 expression level) were detected by western blot in both B7-H3-overexpressing HCT116 and RKO cells. ß-actin served as a loading control. g Schematic representation of the proposed B7-H3/STAT3/HK2 axis. Values are expressed as means (SEMs). Five samples were analyzed per condition, and the experiments were performed in triplicate. *P < 0.05