Mechanisms Of Hepatic Stellate Cells In Promoting Colorectal Cancer Metastasis In Liver Microenvironment

Objective: Colorectal cancer liver metastases and liver microenvironment are inseparable.Tumor cells that metastasize to the liver need to create an environment that is more suitable for their own growth.Hepatic stellate cell(HSC)is one of the main components of hepatic matrix.Activated HSC can secrete a variety of substances,such as extracellular matrix components HGF,TGF-?,COMP and so on.Previous studies have shown that activated hepatic stellate cells can promote tumor proliferation and invasion.Until now,the mechanisms of HSC on the promotion of colorectal cancer metastasis is still limited.Therefore,on the basis of establishing the model of liver metastasis of colorectal cancer,we discussed HSC could regulated colorectal cancer liver metastasis in liver microenvironment.Methods: 1.The liver metastasis model of colorectal cancer was established by splenic injection.2.Expression level of COL6A3 gene mRNA in tumor cells was meatured by RT-qPCR technique.3.SiRNA-COL6A3 and lipofectamin 2000 and were used to knockdown gene expression in Caco2-H and RKO-H cells.4.The short-term proliferation ability of cells(0,24,48,72 hours)was determined by MTT assay.5.The ability of colorectal cancer cell migration was detected by Transwell method.6.The ability of long-term proliferation of colorectal cancer cells was detected by colony formation experiment(cell was cultured for 14 days).7.Cell-extracellular Matrix adhesion assay was used to detect the adhesion ability of cells to extracellular Matrix.8.Western blot could be used to detect the protein expression in cells.9.ELISA was used to detect the concentration of COL6A3 in the patient plasma and the concentration of HGF in the cell supernatant and.10.The protein expression level of COL6A3 was detected by immunohistochemistry in the colorectal cancer tissues and paired liver metastasis tissues(N=30),and the expression difference of COL6A3 was determined.11.Network database download and data analysis.12.Statistical analysis: All the experiments were repeated for 3 times.We use SPSS 17.0 to analyse the data by mean ± standard deviation.In orde to comparise the groups,T test was used.There is a statistical difference when p value is less than 0.05.13.Graphpad prism 7.0 software is used to make pictures.Results:1.Caco2-H and RKO-H increased liver tumorigenesis in nude mice.We established an mouse model of colorectal cancer liver metastasis and colorectal cancer metastasis liver cell lines(RKO-H,Caco2-H).Caco2-H and RKO-H can increased the number of liver metastasis significantly.2.Compared with Caco2 and RKO cells,the proliferation ability of Caco2-H and RKO-H cells were increased,and the migration and adhesion ability were significantly increased.3.EMT occurred in Caco2-H and RKO-H.Caco2-H and RKO-H cells had an elongated appearance and changed from an epithelial sheet-like structure to a spindle-like fibroblast morphology.We also examined the EMT markers.The E-cad was downregulation and the N-cad and vimentin was upregulation.The transcription factor ZEB1,snail and slug were also upregulation based on Western blot.4.HSC could be activated by the supernatant of Caco2-H and RKO-H.Immunofluorescence assay showed that the expression of ?-SMA protein increased in HSC which was cultured with Caco2-H-CM and RKO-H-CM.Western blotting also proved that the expression of ?-SMA,Fibronectin and Collagen I increased.Alpha-SMA immunohistochemistry of liver metastases in nude mice suggests stronger expression in Caco2-H and RKO-H tumor-forming groups 5.Activated HSC enhanced the ability of proliferation,migration and adhesion in the four cells.Caco2-H and RKO-H cells were enhanced more significantly.The morphology of four tumor cells began to change after cultured with HSC-CM for four hours.The migration and adhesion ability of the four cell lines were enhanced,and the colony test showed that the ability of long-term proliferation also increased.6.The expression of COL6A3 was up in Caco2-H and RKO-H cells.The four cell lines were sequenced by mRNA level transcriptome.We selected COL6A3 by overlapping the up-regulated molecules of Caco2-H cell and RKO-H cell.The expression of COL6A3 was upregulated in Caco2-H and RKO-H by qRT-PCR and Western Blot analyses.7.High expression of COL6A3 protein in nude mice liver tumors of Caco2-H and RKO-H cells.8.The migration and adhesion ability of Caco2-H and RKO-H cells were reduced after slience of COL6A3.9.COL6A3 participated in the regulation of Integrin ?v/FAK/AKT pathway.According to the results of cell mRNA sequencing,ECM-receptor interaction and Focal adhesion were enriched.On the GEPIA website,there was great correlation between COL6A3 and Integrin ?v.Western Blot analysis showed that knockdown of COL6A3 inhibited the expression of Integrin ?v,p-FAK and p-Akt,but did not alter the expression of FAK and Akt.10.COL6A3 was upregulated by HSC-CM,and activated HSC could promote the abilitiy of migration and adhesion through Integrin ?v/FAK/AKT pathway.The expression of COL6A3 was further increased by culured HSC-CM.The pathway of ECM-receptor interaction and Focal adhesion were enriched.Western blotting showed that after cultured with HSC-CM,the expression of COL6A3 protein increased after 4 hour and reached maximum level at 12 hour.The expression changes of ITG ?v,p-FAK and p-AKT were consistent with COL6A3,but FAK and AKT proteins did not change.When COL6A3 was knocked-down with si-RNA,the migration and adhesion of Caco2-H and RKO-H cells can't fully restored even after cultured with HSC-CM.At the same time,the activation of Integrin ?v/FAK/AKT pathway cannot fully restored even cultured with HSC-CM.11.HGF which driced from HSC could improve the ability of migration and adhesion via the activation of Integrin ?v/FAK/AKT signaling pathways in Caco2-H and RKO-H cells.High concentration of HGF was detected in the supernatant of activated hepatic stellate cells.After the applicationof anti-HGF antibody,up-regulation of COL6A3,Integrin ?v,p-FAK and p-AKT.were partly inhibited.The inhibitor blocked HSC-CM induced adhesion and migration significantly.12.The increase of COL6A3 in plasma indicated the presence of colorectal cancer liver metastasis.The expression of COL6A3 was 590.1±88.19(pg/ml)in 38 patients with synchronous liver metastasis and was312.7 ±54.84 in 21 patients without distant metastasis(pg/ml),p<0.05.ROC curve analysis showed that the area under the curve(AUC value)of COL6A3 was 0.6654.13.Immunohistochemical analysis of 30 patients showed that the expression level of COL6A3 was up in liver metastasic tissues.The expression of COL6A3 could be divided into 7 groups according to the expression of cytoplasm and nucleus in primary and metastatic tissues.There were 14 cases of cytoplasmic expression in both primary and metastatic tissue.The overall expression of COL6A3 protein in liver metastasis tissue was higher than in primary lesions(p< 0.05),and the expression of COL6A3 protein in the nuclear of liver metastasis tissue was higher than primary tissue(p< 0.05).But there was no significant difference in cytoplasmic expression.14.Bioinformatics analysis found that COL6A3 was associated with liver metastasis of colorectal cancer and was a poor prognostic factor of colorectal cancer.According to the TCGA-COAD transcriptome data and clinical information,the patients were divided into two groups including high COL6A3 expression group(140 cases)and lowCOL6A3 expression group(143 cases).It was found that compared with the low expression of COL6A3,the patients with high expression of COL6A3 had shorter OS and PFS,and the prognosis was poor.The results of forest map showed that high expression of COL6A3,age and tumor stage were all poor prognostic factors.GSE22834 dataset confirmed that the expression of COL6A3 in liver metastases was higher than that in colon cancer.Conclusion: 1.The liver metastasis model of colorectal cancer was established.2,Caco2-H and RKO-H cells have stronger proliferation,adhesion and migration ability and liver tumorigenesis ability in nude mice.3.Caco2-H and RKO-H have relationgship with liver microenvironment.4.COL6A3 enhances the migration and adhesion of colorectal cancer cells through ITG/FAK/AKT pathway.5.HSC secretes HGF to up-regulate the expression of COL6A3 in colorectal cancer cells which could promote the ability of adhesion and migration in colorectal cancer cells.6.COL6A3 is highly expressed in peripheral blood of patients with liver metastasis.7.The expression of COL6A3 in the metastatic focus of the liver is higher than that in the primary focus.8.COL6A3 can be used as a predictor of liver metastasis.