The level of miRNAs in tumor cells is different from that of healthy cells, and their up-regulated or down-regulated expression plays a key role in the activation of gene signaling pathways in cancer development [10]. Among them, dysregulation of miR-361-5p plays an important role in human cancers and its abnormal expression has been found in human lung, breast, colorectal, cervical, ovarian, gastric, and retinoblastoma cancers [11–17] and exists as a pro- or oncogenic factor. Our functional experiments demonstrated that miR-361-5p inhibited the proliferation and migration of LNCAP cells and promoted their apoptosis. lin, S et al [18] demonstrated that the combination of serum miR-361-5p with miR-34b-3p and miR-200c-3p assays had a good diagnostic value for the early screening of Pca, and that the miR-361-5p expression in expression level was significantly downregulated in serum samples of Pca. Wang YJ et al [19] showed that LncRNA LINC00308 is abnormally highly expressed in prostate cancer and has the function of acting as a competing endogenous RNA (ceRNA) that affects prostate cancer progression by inhibiting the expression of miR-361-5p. When LINC00308 is highly expressed, it decreases the level of miR-361-5p, which leads to the upregulation of TRIP13 expression, a protein that is closely related to the ability of cells to proliferate, invade, and migrate, and its upregulated expression further promotes the malignant behavior of prostate cancer cells. However, when the expression of miR-361-5p was artificially increased, this malignant effect could be effectively eliminated, demonstrating the potential value of miR-361-5p in prostate cancer therapy. Gu P et al [20, 21] demonstrated that lncRNA HOXD-AS1 was highly expressed in CRPC cells, and in vivo and ex vivo experiments verified that knockdown of HOXD-AS1 inhibited the proliferation and chemoresistance of CRPC cells. miR-361-5p, one of the most highly enriched mircoRNAs in PCa, showed a negative correlation between its expression level and that of the exosomal HOXD-AS1 (R = -0.21, P < 0.01), HOXD-AS1 was cells, HOXD-AS1 was directly internalized by PCa cells and acted as a competitive endogenous RNA (ceRNA) sponge adsorbed by miR-361-5p to upregulate the expression of FOXM1, which promoted distant metastasis in PCa cells. miR-361-5p also directly targeted SND1 and reduced its mRNA and protein expression, which in turn inhibited the growth-promoting effects of SND1 in prostate cancer [22, 23]. In addition, miR-361-5p can bind to a conserved structural domain within the 3’UTR of CLDN8 and inhibit its post-transcriptional level, thus hindering the proliferation and migration of prostate cancer cells [17, 24]. These results are consistent with our findings and again demonstrate that miR-361-5p acts as an oncogene to hinder prostate cancer progression.
Furthermore, our study revealed that miR-361-5p has a targeting effect on AR. The luciferase reporter gene results showed that miR-361-5p could target the 3’UTR region of AR. In PCa, AR is the most miR-targeted oncogene, and its 3’UTR is 2.6 times longer than its coding region. It has been shown that shortening of the 3’UTR region results in the absence of miRNA binding sites leading to the amplification of AR and thus facilitates PCa progression [25, 26]. Östling P et al [27] showed that miRNAs are important factors that interact with the 3’UTR of the AR to regulate AR protein levels, and 13 miRNAs were validated with this ability. In addition to this, it has been shown that the downstream target of miR-361-5p, MDM4 (mouse double minute 4), a nuclear protein homologous to MDM2, a negative regulator of p53, and co-overexpression of MDM4 and MDM2 at low MDM4 ratios reduces the ubiquitination of AR proteins to regulate AR [28, 29]. Interestingly, Stegeman S et al [30] found the rs4245739 SNP "C" allele with MDM4 genotype in the PC3 prostate cancer cell line, and further showed that miR-191-5p and miR-887 in PC3 cells have specific Further studies showed that miR-191-5p and miR-887 had a specific affinity for this allele in PC3 cells and reduced the expression of MDM4 protein, thus inhibiting the proliferation of PC3 cells. Therefore, we hypothesized that miR-361-5p might also play an inhibitory role in PC3 cells by reducing the expression of MDM4 in a certain way and then regulating the AR protein. The above studies suggest that miR-361-5p may regulate AR through multiple pathways, and its mechanism is very complex and needs to be further explored.
The expression of AR and changes in its protein structure play a crucial role in the process of PCa castration-resistant in the development of drug resistance [31]. Therefore, it is particularly crucial to investigate in depth how miRNAs affect the expression of AR, especially its role in the process of PCa castration-resistant transformation. We further clarified the expression of miR-361-5p in prostate cancer castration-resistant transformation, and found that during castration-resistant induction in LNCAP cells, there was no significant difference in the expression of AR at the mRNA level, regardless of whether miR-361-5p was transfected or not, whereas at the AR protein level, prior to transfection with miR-361-5p, the AR expression showed a transient elevation followed by a decrease, this change in AR mRNA not parallel to protein suggests that the effect of miR-361-5p on AR proteins acts at the post-transcriptional level. Östling P et al [27] validated using the LMA technique in LNCAP and 22Rv1 cells to show that the majority of miRNAs inhibit the translational level of the AR and rarely play a role at the function at the transcriptional level. In addition to this, Qu, F et al [32] showed that overexpression of miR-185 decreased the expression of AR protein in LNCAP cells but did not inhibit its expression at the mRNA level. These findings are consistent with our results.
Fletcher, C E et al [33] showed that inhibitors of miR-361-3p and miR-197 play a significant role in the regulation of AR. These inhibitors were able to significantly reduce the transcriptional activity of AR and decrease the ability of AR to regulate in the process of gene expression. At the same time, they were also effective in reducing the mRNA and protein levels of the AR, meaning that the AR was significantly inhibited in both quantity and function within the cell. Further studies also revealed that this inhibitory effect was not only limited to the AR itself, but also extended to a series of key biological processes in PCa cells. Specifically, by inhibiting AR, these miRNA inhibitors significantly suppressed the proliferation, migration, and invasive ability of PCa cells and promoted their apoptosis, thereby slowing down the progression of PCa, showing a superimposed effect of AR inhibition by miR-361-3p and miR-197. Enzalutamide-treated CRPC cells had elevated expression levels of both miR-361-3p and miR-197, suggesting that miR-361-3p and miR-197 may contribute to the progression of PCa denervation resistance by promoting or maintaining enzalutamide resistance to AR-targeted therapies. Larne O et al [34] demonstrated that miR-145 mediates the inhibition of AR and its downstream targets, including prostate-specific antigen (PSA), Kinase-related peptidase 2 peptidase 2 and TMPRSS2, suggesting that exogenous miR-145 may reduce AR expression in PCa to delay the onset of castration-resistant. Liu C et al [35] demonstrated that miR-185 could inhibit PCa by inhibiting AR expression through direct targeting of the AR-3’UTR, and these results have similarities with miR-361-5p that we studied, both revealing the important role of miRNAs in regulating AR expression and prostate cancer development. To further explore the function of miR-361-5p in prostate cancer, we further investigated the role played by miR-361-5p on the AR pathway during the induction of LNCAP into LNCAP-AI. We first transfected miR-361-5p into LNCAP cells and detected the expression level of AR on the seventh day of induction. Compared with the control group, although the trend was the same, the elevation of AR on the seventh day was significantly lower in the transfected group than in the untransfected group. This result suggests that miR-361-5p inhibited the dramatic elevation of AR in the early stage of castration-resistant transformation of LNCAP cells, then slowing down the transformation of prostate cancer cells to castration-resistant. When LNCAP cells were successfully induced to become LNCAP-AI, we observed that the expression levels of AR all returned to a lower level state, suggesting that progression to the advanced tumor stage may be accompanied by mutation of AR, which allows the cancer cells to ultimately progress to become AR-independent CRPCs. Therefore, we believe that miR-361-5p plays an important role in the early stage of castration-resistant transformation of prostate cancer which slows down the malignant transformation of prostate cancer cells by inhibiting AR expression, but for AR-independent CRPC. it is still necessary for us to further explore the related mechanisms and more effective therapeutic strategies.
In conclusion, our results suggest that miR-361-5p can act as an oncogenic factor in prostate cancer, which can target AR thereby inhibiting the proliferation, migration and promoting apoptosis of prostate cancer cells, and hindering the process of castration-resistant transformation.