Cancer-associated fibroblasts promote cancer cell growth through a miR-7-RASSF2-PAR-4 axis in the tumor microenvironment

Abstract

Cancer-associated fibroblasts (CAFs), a major component of cancer stroma, play an important role in cancer progression but little is known about how CAFs affect tumorigenesis and development. MicroRNAs (miRNAs) are small non-coding RNAs that can negatively regulate target mRNA expression at post-transcriptional levels. In head and neck cancer (HNC), our analysis of miRNA arrays showed that miR-7, miR-196 and miR-335 were significantly up-regulated in CAFs when compared with their paired normal fibroblasts (NFs). FAP, α-SMA and FSP, specific markers of CAFs, were significantly expressed in CAFs. Functionally, exogenous expression of miR-7 in NFs induced a functional conversion of NFs into CAFs. In contrast, inhibition of miR-7 expression in CAFs could induce a functional conversion of CAFs into NFs. Our study demonstrated that overexpression of miR-7 in NFs significantly increased the migration activity and growth rates of cancer cells in co-culture experiments. Mechanistically, we confirmed that the RASSF2-PAR-4 axis was mainly responsible for miR-7 functions in CAFs using bioinformatics methods. Overexpression of miR-7 in CAFs led to down-regulation of RASSF2, which dramatically decreased the secretion of PAR-4 from CAFs and then enhanced the proliferation and migration of the co-cultured cancer cells. Thus, these results reveal that the inactivation of the RASSF2-PAR-4 axis controlled by miR-7 may be a novel strategy for gene therapy in HNCs.

Keywords: RASSF2; cancer microenvironment; cancer-associated fibroblasts; head and neck cancer; miR-7.

Figure 1. Identification and functional analysis of NFs and CAFs

A. Images of immunohistochemical staining showed the distribution of α-SMA-expressing fibroblasts. B. Identification of NFs and CAFs using western blot analysis. C. FSP and FAP, specific biomarkers of CAFs, were proved to up-regulation in CAFs using immunofluorescence analysis. D. Cell cycle analysis of CAFs and NFs. E. The migratory ability of NFs and CAFs were determined by transwell migration assay. F. Proliferation analysis of CAFs and NFs. (** p<0.01; ***p<0.001.)

Figure 2. MicroRNA array analysis of CAFs

A. Heat map of differentially expressed microRNA in CAFs compared with NFs. B. The expression levels of 6 miRNAs (miR-7, miR-365, miR-103a, miR-335, miR-196a and miR10a) in 8 pairs of NFs and CAFs. A log2-fold change more than 2 was regarded as significant up-regulation (dotted lines). C. A volcano plot showing the relationship between the P values and the magnitude of the differences in the expression values of the samples in different groups. D. A real-time PCR analysis was performed to validate the miR-7 expression in 8 paired samples. (***p<0.001.)

Figure 3. Functional analysis of miR-7 effects on the HNC cell proliferation

A. Compared with NFs, CAFs could promote the proliferation of HN13 cells more efficiently. B. The results of real-time PCR showing miR-7 expression levels in NFs after transfection of miR-7 mimics and higher expression of α-SMA was detected in NFs^miR-7 using Western blot analysis. C. Overespression of miR-7 in NFs could facilitate the growth of HN13 cells. D. The results of real-time PCR showing miR-7 expression levels in CAFs after treated with anti-miR-7 and decreased expression of α-SMA was detected in CAFs^Anti-miR-7 using Western blot analysis. E. Silencing the expression of miR-7 in CAFs inhibited the proliferation of HN13 cells. F. CAF-derived CM exhibited a more proliferative effect on HN13 cells than that of NFs. (*p<0.05; ** p<0.01; ****p<0.0001.)

Figure 4. MiR-7 targets RASSF2

A. Venn diagrams of the predicted candidate target genes of miR-7 by bioinformatic method and mRNA microarray. B, C. The putative target genes were futher identified in NFs^miR-7 and CAFs^Anti-miR-7 using real-time PCR analysis. D. Western blot analysis of RASSF2 protein expression after altering miR-7 expression in NFs and CAFs. E. The schematic diagram of putative miR-7-binding sites in the 3′-UTR of RASSF2. F. Down-regulation in NFs^miR-7 and up-regulation in CAFs^Anti-miR-7 of the reporter gene with the wild-type region from RASSF2-WT was apparent, whereas no effect on the RASSF2-MUT was detected. G, H. Overexpression of miR-7 or knock-down of RASSF2 in NFs could accelerate migration of HN13 cells, whereas decreasing the miR-7 expression or up-regulation of RASSF2 in CAFs severely inhibited the migratory ability of HN13 cells. (*p<0.05; ** p<0.01; ***p<0.001; ****p<0.0001.)

Figure 5. MiR-7-RASSF2-PAR-4 axis mediated the cross-talk of CAFs and cancer cells

A. Western blot analysis of RASSF2 protein expression in NFs and CAFs, and the secreted PAR-4 expression in the CM. B. The protein expressions of RASSF2 and secreted PAR-4 were determined in miR-7-expressing NFs, or CAFs transfected with anti-miR-7 using western blot analysis. C. The protein expression levels of RASSF2 and secreted PAR-4 in RASSF2-expressing NFs, or RASSF2-silenced CAFs. D. The expression of PAR-4 was down-regulated by the overexpression of miR-7 and this was rescued by coexpression of RASSF2. E. The increased expression of PAR-4 in CAFs driven by anti-miR-7 could be reduced by siRNA for RASSF2. F. Exogenous RASSF2 or PAR-4 facilitated the apoptosis of NFs, whereas miR-7 could partly attenuate RASSF2- or PAR-4-induced apoptosis and vice versa in CAFs. G. Exogenous RASSF2 or PAR-4 decreased the growth rate of NFs, whereas miR-7 could partly attenuate RASSF2- or PAR-4-induced proliferative inhibition and vice versa in CAFs. H. The soluble PAR-4 acts on HNC cells by binding to the surface receptor GRP78. I. Flow diagram illustrating the cross-talk of CAFs and cancer cells mediated by the miR-7-RASSF2-PAR-4 axis. (*p<0.05; ** p<0.01; ***p<0.001; ****p<0.0001.)