Effect And Mechanism Of CXCL5on The Migration And Invision Of Liver Cancer Cells

Background and AimThe chemokines involve in tumor cell genotypic and phenotypic changes, as well as the formation of the tumor microenvironment, therefore promote tumor growth, invasion and metastasis through autocrine and paracrine. This research aimed to discover chemokines related to liver cancer metastasis, to identify the effect of the key chemokine CXCL5on migration and invasion of liver cancer cells, and to further explore its underlying molecular mechanisms, providing new clues for the diagnosis and treatment of liver cancer.MethodsqRT-PCR arrays was used to screen chemokines expressed differently in liver cancer cells (HCCLM3and HepG2) whit high and low metastatic potential; by RT-PCR and ELISA, the levers of mRNA and protein of key chemokines were detected; using immunocytochemistry, we observed the lever of CXCR2expression in liver cancer cells; small interfering RNA (siRNA) was transfered into liver cancer cells by Lipofectamin2000, leading to lower CXCL5expression; Wound healing assay and the Transwell chamber were used to observe the migration and invasion of liver cancer cells; key proteins of MAPK signaling pathway was analyzed by Western blot; CXCR2inhibitor SB225002and the MEK inhibitor U0126were used, respectively, to block the activation of CXCR2and ERK/MAPK signaling pathway.Results(1) qRT-PCR arrays showed that many chemokines were expressed differentially between HCCLM3(with high metastatic potential) and HepG2(with low metastatic potential).14of them were down-regulated, including CXCL1, CXCL3, IL-1A, CCL2and14were up-regulated, including CXCL5, CXCL6, IL-8; furthermore, the lever of CXCL5in HCCLM3was1000times more than that in HepG2.(2) Based on the qRT-PCR arrays and articles reported in the literature, mRNA levers of six selected chemokines (CXCL1, CXCL3, CXCL5, CXCL6, of CCL2and IL-1A) were validated by RT-PCR in four liver cancer cell lines with different metastatic potential (HepG2, SMMC7721, MHCC97L and MHCC97H, in an ascending order). The results indicated that five of them were consistent with the results of qRT-PCR arrays except for CXCL3. The differences of CXCL5remained the most significant. (3) CXCL5protein levels of cell culture supernatant from HepG2, SMMC7721, MHCC97L and MHCC97H were22.21±0.48,25.74±1.40,82.78±3.23,98.85±1.73(F=393.48, P=0.000) detected by ELISA, indicating that the CXCL5protein levels also increased in the highly metastatic liver cancer cell lines (P <0.001).(4) Immunocytochemistry results showed that the liver cancer cell expressed CXCR2on the cell membrane and in the cytoplasm, with no specific staining in nucleus and PBS control group.(5) MHCC97H was selected in interference experiment. A mixture of Lipofectame2000and siRNA with the ratio of1:20transfected into MHCC97H could achieved a transfection efficiency up to70%or more. RT-PCR and ELISA compared the interference efficiency of three CXCL5-siRNAs, of which siRNA-313was the most effective. The culture supernatant collected72hours after interference was used in cell migration and invasion assays. The results showed that the numbers of migrating cells in control group, siRNA-NC group and siRNA-313group were21.73±0.26,20.47±0.38,11.07±0.52, respectively (F=68.90, P=0.000) and he number of invasive cells were8.90±0.46,8.1±0.38,3.73±0.18, respectively (F=16.40, P=0.007). siRNA-313group with low level of CXCL5, compared with the remaining two groups could significantly decrease the capacity of migration and invasion (P<0.001).(6) HepG2was selected to be stimulated by exogenous CXCL5. The results showed that CXCL5significantly increased HepG2migration and invasion in vitro. This effect was concentration-dependent, which could be neutralized by CXCR2inhibitor SB225002, indicating that CXCL5played a key role in migration and invision of liver cancer cells through combination with specific receptor CXCR2.(7) Western blot showed that the phosphorylated proteins of three MAPK signaling pathways, p-ERK, p-of p38, p-JNK were elevated after CXCL5stimulus, and the most significant changes happened in p-ERK; ERK/MAPK pathway was blocked by U0126, resulting in decreased numbers of cell migration and invasion even after CXCL5stimulus; all of which illustrated that CXCL5/CXCR2promoted the migration and invasion of liver cancer cells mainly through activation of the ERK/MAPK signaling pathway.ConclusionThis research discovered tumor metastasis-related chemokines, and further revealed that CXCL5and its receptor CXCR2might promote the migration and invasion of liver cancer cells in vitro mainly through activation of the ERK/MAPK signaling pathway, suggesting that CXCL5might be a key molecule in liver cancer metastasis and could be expected to become an effective molecular target for liver cancer diagnosis and therapy.