Cancer-derived exosomal miR-221-3p promotes angiogenesis by targeting THBS2 in cervical squamous cell carcinoma.

AIMS: Recently, cancer-derived exosomes were shown to have pro-metastasis function in cancer, but the mechanism remains unclear. Angiogenesis is essential for tumor progression and is a great promising therapeutic target for advanced cervical cancer. Here, we investigated the role of cervical cancer cell-secreted exosomal miR-221-3p in tumor angiogenesis. METHODS AND RESULTS: miR-221-3p was found to be closely correlated with microvascular density in cervical squamous cell carcinoma (CSCC) by evaluating the microvascular density with immunohistochemistry and miR-221-3p expression with in situ hybridization in clinical specimens. Using the groups of CSCC cell lines (SiHa and C33A) with miR-221-3p overexpression and silencing, the CSCC exosomes were characterized by electron microscopy, western blotting, and fluorescence microscopy. The enrichment of miR-221-3p in CSCC exosomes and its transfer into human umbilical vein endothelial cells (HUVECs) were confirmed by qRT-PCR. CSCC exosomal miR-221-3p promoted angiogenesis in vitro in Matrigel tube formation assay, spheroid sprouting assay, migration assay, and wound healing assay. Then, exosome intratumoral injection indicated that CSCC exosomal miR-221-3p promoted tumor growth in vivo. Thrombospondin-2 (THBS2) was bioinformatically predicted to be a direct target of miR-221-3p, and this was verified by using the in vitro and in vivo experiments described above. Additionally, overexpression of THBS2 in HUVECs rescued the angiogenic function of miR-221-3p. CONCLUSIONS: Our results suggest that CSCC exosomes transport miR-221-3p from cancer cells to vessel endothelial cells and promote angiogenesis by downregulating THBS2. Therefore, CSCC-derived exosomal miR-221-3p could be a possible novel diagnostic biomarker and therapeutic target for CSCC progression.

Cervical squamous cell carcinoma-secreted exosomal miR-221-3p promotes lymphangiogenesis and lymphatic metastasis by targeting VASH1.

Cancer-secreted exosomal miRNAs are emerging mediators of cancer-stromal cross-talk in the tumor environment. Our previous miRNAs array of cervical squamous cell carcinoma (CSCC) clinical specimens identified upregulation of miR-221-3p. Here, we show that miR-221-3p is closely correlated with peritumoral lymphangiogenesis and lymph node (LN) metastasis in CSCC. More importantly, miR-221-3p is characteristically enriched in and transferred by CSCC-secreted exosomes into human lymphatic endothelial cells (HLECs) to promote HLECs migration and tube formation in vitro, and facilitate lymphangiogenesis and LN metastasis in vivo according to both gain-of-function and loss-of-function experiments. Furthermore, we identify vasohibin-1 (VASH1) as a novel direct target of miR-221-3p through bioinformatic target prediction and luciferase reporter assay. Re-expression and knockdown of VASH1 could respectively rescue and simulate the effects induced by exosomal miR-221-3p. Importantly, the miR-221-3p-VASH1 axis activates the ERK/AKT pathway in HLECs independent of VEGF-C. Finally, circulating exosomal miR-221-3p levels also have biological function in promoting HLECs sprouting in vitro and are closely associated with tumor miR-221-3p expression, lymphatic VASH1 expression, lymphangiogenesis, and LN metastasis in CSCC patients. In conclusion, CSCC-secreted exosomal miR-221-3p transfers into HLECs to promote lymphangiogenesis and lymphatic metastasis via downregulation of VASH1 and may represent a novel diagnostic biomarker and therapeutic target for metastatic CSCC patients in early stages.

Clinical Significance of CD163+ and CD68+ Tumor-associated Macrophages in High-risk HPV-related Cervical Cancer.

Objective. To explore the influence of M2-polarized tumor-associated macrophages (TAMs) on high-risk human papillomavirus (hr-HPV)-related cervical carcinogenesis and metastasis. Methods. CD68+ and CD163+ macrophages were examined immunohistochemically in a series of 130 samples, including 26 cases of normal cervical tissues, 59 cases of cervical intraepithelial neoplasia (CIN), and 45 cases of squamous cell carcinoma (SCC), and the results were statistically analyzed. The macrophage count was corrected for the epithelial and stromal compartments respectively. Clinical data were also obtained. Results. High counts of CD68+ and CD163+ macrophages were associated with hr-HPV infection (both p < 0.05) and positively correlated with cervical carcinogenesis (Spearman's rho = 0.478, p = 0.000; Spearman's rho = 0.676, p =0.000, respectively). The immunostaining pattern of CD163 exhibited clearer background than that of CD68. CD163+ macrophages showed a more obviously increasing migration into the epithelium along with the progression of CIN to invasive cancer. Notably, a high index of CD163+ macrophages was significantly associated with higher FIGO stages (p = 0.009) and lymph node metastasis (p = 0.012), but a similar finding was not found for CD68+ macrophages (p = 0.067, p = 0.079, respectively). Conclusions. Our study supported a critical role of TAMs as a prospective predictor for hr-HPV-related cervical carcinogenesis. CD163, as a promising TAMs marker, is superior to CD68 for predicting the malignant transformation and metastatic potential of cervical cancer.

Orthotopic Xenograft Mouse Model of Cervical Cancer for Studying the Role of MicroRNA-21 in Promoting Lymph Node Metastasis.

Cervical cancer is the most frequent cause of gynecologic cancer-associated death worldwide. Animal models that demonstrate metastatic patterns consistent with the clinical course of cervical cancer are urgently needed to conduct studies focused on understanding the mechanisms of the disease and identifying optimal treatments. To address this, we established an orthotopic xenograft model of cervical cancer in female NOD-SCID mice using SiHa and ME180 cell lines stably expressing green fluorescent protein to evaluate the role of microRNA-21 (miR-21) in spontaneous lymph node metastasis in vivo. In this case, SiHa and ME180 cells were transduced by lentivirus to stably express green fluorescent protein and miR-21. Overexpression of miR-21 promoted proliferation, migration, and invasion of SiHa and ME180 cells in vitro. Finally, an orthotopic xenograft model of human cervical cancer was successfully established in NOD-SCID mice. Using this model, we confirmed that overexpression of miR-21 resulted in an increase in the size of primary tumors and in the frequency of spontaneous lymph node metastasis at the time of excision. Therefore, the use of the orthotopic xenograft model should allow for the investigation of novel factors that affect metastasis of cervical cancer and presents an opportunity to evaluate potential therapeutic agents that may inhibit the spread of the disease.

Molecular characterization of HbCZF1, a Hevea brasiliensis CCCH-type zinc finger protein that regulates hmg1.

KEY MESSAGE: The HbCZF1 protein binds to the hmg1 promoter in yeast and this interaction was confirmed in vitro. The hmg1 promoter was activated in transgenic plants by HbCZF1. Biosynthesis of natural rubber is known to be based on the mevalonate pathway in Hevea brasiliensis. The final step in the mevalonate production is catalyzed by the branch point enzyme, 3-hydroxy-3-methyl-glutaryl coenzyme A reductase (HMGR), which shunts HMG-CoA into the isoprenoid pathway, leading to the synthesis of natural rubber. However, molecular regulation of HMGR expression is not known. To study the transcriptional regulation of HMGR, the yeast one-hybrid experiment was performed to screen the latex cDNA library using the hmg1 (one of the three HMGR in H. brasiliensis) promoter as bait. One cDNA that encodes the CCCH-type zinc finger protein, designated as HbCZF1, was isolated from H. brasiliensis. HbCZF1 interacted with the hmg1 promoter in yeast one-hybrid system and in vitro. HbCZF1 contains a 1110 bp open reading frame that encodes 369 amino acids. The deduced HbCZF1 protein was predicted to possess a typical C-X7-C-X5-C3-H CCCH motif and RNA recognition motif. HbCZF1 was predominant in the latex, but little expression was detected in the leaves, barks, and roots. Furthermore, in transgenic tobacco plants, over-expression of HbCZF1 highly activated the hmg1 promoter. These results suggested that HbCZF1 may participate in the regulation of natural rubber biosynthesis in H. brasiliensis.