HIF1α/CCL7/KIAA1199 axis mediates hypoxia-induced gastric cancer aggravation and glycolysis alteration

Abstract

Gastric cancer is a common digestion tumor with high malignant severity and prevalence. Emerging studies reported C-C motif chemokine ligand 7 (CCL7) as a regulator of various tumor diseases. Our research explored the function and underlying mechanism of CCL7 during gastric cancer development. RT-qPCR, Western blot and other datasets were employed to evaluate CCL7 expression in tissues and cells. Kaplan-Meier and Cox regression analyses were recruited to evaluate the correlations between CCL7 expression and patients' survival or clinical features. A loss-of-function assay was performed to evaluate the function of CCL7 in gastric cancer. 1% O₂ was utilized to mimic hypoxic condition. KIAA1199 and HIF1α were included in the regulatory mechanism. The results showed that CCL7 was up-regulated and its high expression was correlated with poor survival of gastric cancer patients. Depressing CCL7 attenuated proliferation, migration, invasion, and induced apoptosis of gastric cancer cells. Meanwhile, CCL7 inhibition weakened hypoxia-induced gastric cancer aggravation. Besides, KIAA1199 and HIF1α were involved in the mechanism of CCL7-mediated gastric cancer aggravation under hypoxia. Our research identified CCL7 as a novel tumor-activator in gastric cancer pathogenesis and hypoxia-induced tumor aggravation was regulated by HIF1α/CCL7/KIAA1199 axis. The evidence may provide a novel target for gastric cancer treatment.

Keywords: CCL7; HIF1α; KIAA1199; gastric cancer; glycolysis; hypoxia.

Fig. 1.

The expression of CCL7 in GC tissues and correlation with the pathological features of GC patients. (A, D, E) RT-qPCR was performed to evaluate CCL7 expression in GC patients. (B) The image of immunohistochemical staining for CCL7 in tissues. (C) CCL7 expression was recruited from GEPIA. (F) The Cox regression analysis between CCL7 expression and clinical features of GC patients. (G, H) The overall survival of GC patients with low or high expression of CCL7.

Fig. 2.

The role of CCL7 during GC progression in vitro. MKN-45 and KATO III cells were transfected with CCL7 shRNAs for 48 h at a concentration of 50 nM. (A, C) The expression of CCL7 was measured using RT-qPCR. (B, D) Western blotting showed the protein level of CCL7. (E) Elisa assay was utilized to evaluate the BrdU positive cells. (F) Cell apoptosis was determined using a Cell Death Elisa kit. (G, H) Wound healing and Transwell assays showed cell migration and invasion. In 2A, *p<0.05 vs GES-1; ^#p<0.05 vs HGC-27; ^†p<0.05 vs KATO III. In 2C, *p<0.05 and **p<0.01 vs shRNA NC; ^#p<0.05 vs sh-CCL7a; ^†p<0.05 vs sh-CCL7b. In 2E–2H, *p<0.05 and **p<0.01 vs shRNA NC.

Fig. 3.

The function of CCL7 in GC cells under hypoxia treatment. MKN-45 and KATO III cells were transfected with CCL7 shRNAs for 48 h at a concentration of 50 nM before treated under hypoxia condition. (A, B) The expression of CCL7 was measured using RT-qPCR and Western blotting. (C) Elisa assay was utilized to evaluate the BrdU positive cells. (D) Cell apoptosis was determined using a Cell Death Elisa kit. (E, F) Wound healing and Transwell assays showed cell migration and invasion. *p<0.05 and **p<0.01 vs control; ^#p<0.05 and ^##p<0.01 vs shRNA NC.

Fig. 4.

HIF1α was important in hypoxia-induced CCL7 alteration and GC progression. GC cells were transfected with HIF1α shRNAs, with or without pc-CCL7 overexpression plasmid, for 48 h at a concentration of 50 nM before treated under hypoxia condition. (A, C) The expression of CCL7 was measured using RT-qPCR. (B, D) The protein expressions of CCL7 and HIF1α were determined by Western blotting. (E) Elisa assay was utilized to evaluate the BrdU positive cells. (F) Cell apoptosis was determined using a Cell Death Elisa kit. (G, H) Wound healing and Transwell assays showed cell migration and invasion. In 4A, *p<0.05 and **p<0.01 vs GES-1; ^#p<0.05 vs untreated; ^†p<0.05 and ^††p<0.01 vs hypoxia + shRNA NC; ^§p<0.05 vs hypoxia + sh-HIF1α-2. In 4C–4H, *p<0.05 and **p<0.01 vs shRNA NC; ^#p<0.05 vs sh-HIF1α-2; ^††p<0.01 vs sh-HIF1α-2 + 0.2 μg pc-CCL7.

Fig. 5.

HIF1α/CCL7/KIAA1199 mediates hypoxia-induced GC aggravation. GC cells were transfected with CCL7 and HIF1α shRNAs, with or without pc-KIAA1199 overexpression plasmid for 48 h at a concentration of 50 nM before treated under hypoxia condition. (A, B) The expression of CCL7 was measured using RT-qPCR and Western blotting. (C) Elisa assay was utilized to evaluate the BrdU positive cells. (D) Cell apoptosis was determined using a Cell Death Elisa kit. (E, F) Wound healing and Transwell assays showed cell migration and invasion. In 5A, *p<0.05 and **p<0.01 vs GES-1; ^#p<0.05 and ^##p<0.01 vs untreated; ^†p<0.05 and ^††p<0.01 vs hypoxia + shRNA NC; ^§§p<0.01 vs hypoxia + sh-HIF1α-2; ^¶p<0.05 vs hypoxia + sh-CCL7c. In 4C–4F, *p<0.05 and **p<0.01 vs shRNA NC; ^#p<0.05 and ^##p<0.01 vs sh-HIF1α-2; ^†p<0.05 and ^††p<0.01 vs sh-CCL7c.

Fig. 6.

HIF1α/CCL7/KIAA1199 mediates hypoxia-induced GC glycolysis. GC cells were transfected with CCL7 and HIF1α shRNAs, with or without pc-KIAA1199 overexpression plasmid, for 48 h at a concentration of 50 nM before treated under hypoxia condition. (A, C) A Glucose Assay Kit and a Lactate Assay Kit were used to measure the glucose consumption and lactate production. (B, D) Using a Lactate Dehydrogenase Activity Assay Kit and a Luciferase-based ATP Assay Kit, the LDH activity and ATP level were evaluated. (E, F) RT-qPCR and Western blotting determined the expression of HK2. *p<0.05 and **p<0.01 vs normoxia; ^#p<0.05 and ^##p<0.01 vs shRNA NC under hypoxia; ^†p<0.05 and ^††p<0.01 vs sh-HIF1α-2 under hypoxia; ^§p<0.05 and ^§§p<0.01 vs sh-CCL7c under hypoxia.