Our preliminary study has reported that miR-542-5p played an important role in glioma progression. HOXA-AS3 also has been found in regulating tumor cells proliferation, migration and apoptosis [14]. In the present study, we discovered and assessed HOXA-AS3 as the upstream of miR-542-5p when regulating glioma cells, and manipulated HOXA1/WNT5A axis. Our results may provide the missing piece of the well-known oncogenic and tumor suppressor network puzzle.
The HOX family belongs to homeodomain-containing Transcription Factors encoding several homeobox genes, such as HOXA, HOXB, HOXC, and HOXD [15]. Hox proteins play a key role in regulating gene expression, morphogenesis, and maintenances of differentiated cell populations [16]. HOXA11-AS, HOTTIP, HOXA-AS3, and HOXA-AS2 were found to be within the HOXA gene cluster [17]. HOXA1, one of the HOXA gene cluster members, was notably found to be up-regulated in several human cancer, such as hepatocellular carcinoma [18] and breast cancer [19], and function as an oncogene.
Here, we used online tool RNA22 v2 to find that HOXA-AS3 and HOXA1 not only could bind to miR-542-5p, but also high expressed in glioma tissues compared to normal. Next, GO and pathway analysis found that both HOXA-AS3 and HOXA1 were enriched in “transcriptional misregulation in cancer” in KEGG analysis, and GO analysis showed that HOXA-AS3 and HOXA1 were enriched in similar terms. Considering the interaction among miR-542-5p, HOXA-AS3 and HOXA1, we designed three different siRNA to silence HOXA-AS3. HOXA-AS3 expression was inhibited by three siRNA transfection in U373 cells, meanwhile, HOXA1 expression wwas also inhibited. Then, silence miR-542-5p augmented HOXA-AS3 and HOXA1 expression in U373 cells. We wondered whether regulating HOXA-AS3 or miR-542-5p expression would affect apoptosis, proliferation or migration in glioma cell lines. We knocked down or overexpressed HOXA-AS3 or miR-542-5p expression using cell transfection technology in glioma cells. We found that silenced HOXA-AS3 or enhanced miR-542-5p blocked U373 cells viability and migration, and cell apoptosis was induced. If inhibited miR-542-5p expression, the effect of silencing HOXA-AS3 was antagonized. Finally, we manipulated HOXA-AS3 or miR-542-5p expression, then test miR-542-5p, HOXA-AS3, HOXA1 and WNT5A expression. In accordance with the change of cell phenotype, mRNA expression of which was downregulated when silenced HOXA-AS3, while upregulated when inhibited miR-542-5p.
As we predicted, several pathways were related to the biochemical activity of HOXA-AS3/miR-542-5p axis, and inhibition of anti-apoptotic factors, including Wnt5. Moreover, it was reported that pristimerin treatment suppressed miR-542-5p and PTPN1 expression, result in restraint of glioma progression in vitro [3]. Overexpression of miR-542-5p in patients’ plasma was correlated with worse overall survival of colorectal cancer patients, indicating a novel signature for colorectal cancer diagnosis [20]. When activated, wnt5a involved in multiple biological processes, leading to cell growth, differentiation, and tumor metastases, which might be a target for H3K23me3 in cancer-associated fibroblasts, loss of H3K23me3 leaded to increased Wnt5a in cancer-associated fibroblasts, predicted a poor prognosis in patients, and suppressed WNT5A expression in cancer-associated fibroblasts effectively inhibited cancer cell growth and migration [21]. In addition, we showed a decreased percentage of arginase-1+ F4/80+ cells in glioma cells when HOXA-AS3 and/or hsa-miR-542-5p are silenced in tumor-bearing mouse. Aiginase-1 is essential for the suppression of anti-tumor T cell proliferation and attenuation of anti-tumor immune response [22]. Indeed, our results showed that hsa-miR-542-5p silence significantly restricted tumor growth in mouse model.
Though T cells are main tumor-infiltrating immune cell types in multiple solid tumor microenvironment, a growing number of evidences shows that tumor-associated macrophages could substantially facilitate metabolism of tumor microenvironment, especially glioma[23], and are responsible for anti-tumor response and worse outcomes in patients. Up-regulated HOXA1 expression could be observed in several cancer types, including breast cancer[24], head and neck Squamous cell carcinoma[25], and glioma[26]. It was reported that lncRNA HOTAIRM1 could promote glioblastoma multiforme progression by increasing the HOXA1 expression[26]. HOXA1 restrain led to impaired tumor growth and migration in endometrial cancer[27]. However, it was still unclear if HOXA1 could impact the glioma development via tumor-associated macrophages. Here, we showed that the silence of HOXA-AS3 or enhanced hsa-miR-542-5p remarkably suppressed arginase-1 secreted by tumor-associated macrophages. And we further found a positively correlation between the HOXA1/WNT5A and tumor-associated macrophages. We also observed that the high level of HOXA1/WNT5A significantly correlated with the survival in LGG patients. It has been shown that the expression level of WNT5A increased in glioma cell lines (A172, SHG44, and LN229) and corresponding cancer tissues, and cell migration could be inhibited by targeting miR-139-3p, which could regulate the WNT5A level[28]. For the first time, we found the knockdown of HOXA-AS3/HOXA1 suppressed the migration ability of glioma cells, and inhibited the arginase-1 secreted by tumor-associated macrophages.
Our findings provided the basis for further exploring the molecular mechanisms in glioma growth and invasion, and provided potential novel targets for anticancer treatment. However, the interplay between the HOXA-AS3/hsa-miR-542-5p/HOXA and cells in tumor microenvironment still needs to be characterized.