An Alternatively Spliced Variant Of CXCR3 Mediates The Metastasis Of CD133+ Liver Cancer Cells Induced By CXCL9

Background and aimHepatocellular carcinoma(HCC)is a common malignant tumor,prone to early metastasis.HCC mortality ranks third in China’s malignant tumor.Hepatitis B(HBV)is the main cause of HCC,and compared to non-HBV liver cancer,HBV-related liver cancer is more prone to early hematogenous metastasis.Cancer stem cell hypothesis postulates that not all cancer cells can resist the killing role of drugs and immune system,and cancer stem cells can resist them and are the root of recurrence.CD133 is an important marker of hepatocellular carcinoma stem cells.Previous studies have shown that HBX upregulates chemokine CXCL9 expression in HCC.Chemokines and their receptors in tumor microenvironment play an important role in the regulation of tumor stem cell metastasis.The aim of this study was to investigate the mechanism of CXCR3 variable splicing in the metastasis of CD133 + hepatocarcinoma cells induced by CXCL9,in order to find potential targets for early detection and treatment of HCC.Methods1)Collect the cancer tissue and adjacent tissue in clinical HBV-related liver cancer patients.At the same time,the clinical data of patients with liver cancer were collected and the patients are followed up regularly(once every 3 months).The expression of CXCR3 in hepatocellular carcinoma(HCC)was detected by immunohistochemistry(IHC),quantitative polymerase chain reaction(q-PCR)and Western blotting(WB).Analyze the association between CXCR3 expression and clinicopathological features.2)CD133+ liver cancer cells were sorted by flow cytometry,and then the expression of CXCR3 receptor in CD133+ liver cancer cells were detected by immunocytochemistry and immunofluorescence.The invasion and migration of CD 133+ liver cells were tested by Transwell assays and wound healing tests.The CXCR3-A and CXCR3-B receptor were expressed highly and lowly respectively using overexpression plasmids and siRNAs.Then the CXCR3 receptor isoform is confirmed by Transwell assays and WB assays.And CCK8 reagent was used to detect cell adhesion ablilties.The effect of CXCR3 receptor splice on CD133+ liver cancer cells was detected in nude mice.3)CD133+ liver cancer cells were co-cultured with human umbilical vein endothelial cells(HUVEC),including direct contact co-culture,indirect co-culture and CD 13 3+ liver cancer cells culture system.Transwell assay was used to detect the chemotaxis of CD 133+ hepatocarcinoma cells by culture supernatants.The expression of CXCL9 in culture supernatant was tested by enzyme-linked immunosorbent assay(ELISA).Flow cytometry was used to sort out the direct co-cultured CD 133+ liver cells and HUVEC cells,and then the mechanism of up-regulated CXCL9 expression in culture supernatant was verified by WB experiment.Result:In the cancer tissues and corresponding adjacent noncancerous tissues of the patients with HBV-related hepatocellular carcinoma,the results of IHC showed that CXCR3 was highly expressed in the cancer tissues compared to the corresponding adjacent noncancerous tissues.CXCR3 was mainly expressed in the cytoplasm of hepatocellular carcinoma cells and focused on the perivascular areas.Q-PCR and WB showed that CXCR3 mRNA expression and protein expression were significantly higher in cancer tissues than in adjacent noncancerous tissues.The high expression of CXCR3 was closely related to tumor size,tumor differentiation,portal invasion and metastasis.2)The expression of CXCR3 receptor in CD133+ liver cancer cells(including CD133 + PLC/PRF/5 cells,CD133 +Hep-3B cells and CD 133 + Huh7 cells)was shown by ICC and IFC assays,chemokine CXCL9 at different concentrations was added into the lower transwell chamber,ranging from 0 ng/ml to 200 ng/ml,and CD133+ liver cancer cells was added into the upper chamber.The portion of CD133+ Huh7,Hep-3B and PLC/PRF/5 cells migrate to the lower chamber in the experimental group were larger than those in the control group,which 100 ng/ml group reached the highest.The western blot results showed that chemokine CXCL9 at 100ng/ml could cause MMP2 and MMP9 to obvious increase in the CD133+ PLC/PRF/5,Hep-3B and Huh7 cells at 12h.The CD133+ Huh7 and Hep-3B with stable expression of CXCR3-A exhibited enhanced invasion and migration compared to the controls and the CXCR3-B groups in transwell assays and wound healing tests.The RNA interference(RNAi)was used to knockdown the CXCR3-A or CXCR3-B expression of CD133+PLC/PRF/5 cells,the tests showed the cells in which CXCR3-A expression were knockdown exhibited lower invasion and migration ability.the CD 133+ Huh7,Hep-3B and PLC/PRF/5 cells were each cultured with different concentration of CXCL9 for 24h,then added to the 96-well plates,The CXCL9 100ng/ml groups showed the strongest inhibition of the cell adhesion for CD133+ PLC/PRF/5,Hep-3B,but for CD133+ Huh7 cells,the CXCL9 50 ng/ml and 100 ng/ml groups were both suppressed the most.Subsequently,CXCR3-A splicing was confirmed in nude mice to promote the formation of metastatic foci of CD133+ liver cells.3)CD133 + PLC/PRF/5 cells and CD133 + Hep-3B cells were co-cultured with HUVEC cells.The results showed that the direct co-cultured supernatants promoted the migration and invasion of CD 133+ liver cancer cells.It was further investigated that the expression level of chemokine CXCL9 was significantly elevated in the culture supernatants of direct co-culture system by activating the NF-kB,rather than in the indirect co-culture system or mono-culture system.High expression of CXCL9 in the direct co-cultured supernatants played a significant role in enhancing the migration and invasion of CD133+ liver cancer cells.The chemotaxis of CD133+ liver cancer cells was significantly decreased by culture supernatants after direct addition of NF-kB inhibitor or CXCL9 neutralizing antibody.Conclusion:CXCR3 is highly expressed in HBV-related hepatocellular carcinoma and is closely related to tumor size,tumor differentiation,portal invasion and metastasis.CXCR3-A splicing,induced by CXCL9 can lead to upregulated the ERK1/2 phosphorylation levels of the MAPK signaling pathway,followed by up-regulated the MMP2 and MMP9 expression,so promoting CD 133 + liver cancer cell invasion and metastasis.CXCR3-A also promotes tumor metastasis in nude mice.In addition,overexpression of CXCR3-A inhibited the adhesion of CD 133+ live cancer cells induced by CXCL9 after 24 hours.In the culture supernatants of the CD133+ liver cancer cells and HUVEC direct co-cultured group(to some extent it can simulate the contact between the endothelial cells and the tumor cells during the transendothelial migration of the tumor cells)CXCL9 showed high expression,thereby promoting CD 133 + liver cancer cell metastasis.In summary,CXCR3-A mediates the metastasis and adhesion of CXCL9-induced tumor cells in the extracellular matrix,and CXCL9 may have an important potential role in tumor cell transendothelial barrier Intervention of CXCL9/CXCR3-A is expected to be a therapeutic target for metastatic liver cancer.