In this study, we demonstrated that intraperitoneal administration of miR-214/5AE can induce apoptosis and growth suppression in an intraperitoneal dissemination mouse model of HSA. We also found that the administration of miR-214/5AE did not result in any adverse event.
Mature miRNAs are hydrolyzed immediately by nuclease in vivo. Thus, for effective therapeutic application, it is necessary to confer nuclease resistance to miRNAs. To enable the administration of miR-214 in vivo, a previous study developed synthetic miR-214/5AE, which showed higher cytotoxicity, nuclease resistance, and transfection rate than mature miR-214 in vitro (Yoshikawa et al. 2020). In the present study, we found that the administration of miR-214/5AE induced tumor suppression in mice intraperitoneal dissemination lesions. Previous reports have suggested that intraperitoneal administration of cisplatin or paclitaxel can have beneficial effects for progressive ovarian cancer (Moore et al.1991; Olsen et al. 1994; Armstrong et al. 2006). Intraperitoneal administration of cisplatin has been reported to facilitate exposure of the intraperitoneal tumor to a higher concentration of the anticancer drug than intravenous systemic administration (Pretorius et al. 1981). Herein, we expected that intraperitoneally administered miRNA would be delivered into the tumor via two routes: endocytosis due to direct exposure to the tumor and hematogenous distribution through the capillaries. It has been demonstrated that the residual amount of miR-214/5AE decreases over time with prolonged exposure to RNase (Yoshikawa et al. 2020). Therefore, it is desirable that miRNAs are introduced into tumor cells immediately after administration. Since intraperitoneal administration can expose miR-214/5AE directly to tumor cells, the miRNAs are likely to be less susceptible to immediate degradation and can be introduced into the tumor more efficiently using this method.
To determine the cytotoxic mechanism of miR-214/5AE, we analyzed the expression of cleaved caspase-3, p53, COP1, and Ki-67. In the 5AE group, p53 and cleaved caspase-3 expression levels tended to be higher than those in the ns miR group, and the proportion of Ki-67-positive cells in the 5AE group was significantly lower than that in the ns miR group. The p53 pathway is known to be involved in both apoptosis induction and cell proliferation suppression (Heishima et al. 2015). A previous study reported that p53 was downregulated in HSA cells, and its expression was recovered by transfection of miR-214 into cells (Heishima et al. 2015). In this study, activation of p53 was observed, but the detailed mechanism has not been elucidated and needs to be investigated in the future. We hypothesized that the p53 pathway was activated by the same mechanism, inducing an antitumor effect. Moreover, cleaved caspase-3 protein expression was higher in the 5AE group than in the ns miR group. It was previously revealed that miR-214/5AE induces apoptosis in HSA cells (Yoshikawa et al. 2020). In our study, we found that miR-214/5AE induced apoptosis in vivo. Additionally, there were fewer Ki-67-positive cells in the 5AE group than in the ns miR group. Ki-67 is widely used as a marker of cell proliferation (Gerdes et al. 1984); hence, we considered that the administration of miR-214/5AE also suppressed tumor cell proliferation in our mouse model. Taken together, these results indicate that miR-214/5AE has an antitumor effect on HSA by inducing apoptosis and suppressing cell proliferation.
In this study, we developed a mouse model for intraperitoneal dissemination of HSA. Similar intraperitoneal dissemination mouse models have been reported for stomach and ovarian cancers (Prantner et al. 2018; Miwa et al. 2019); however, it was difficult to evaluate the lesions in vivo in these models because they presented intraperitoneally. Herein, we succeeded in evaluating the intraperitoneal lesions using MRI. According to previous reports, HSA masses are observed as regions of high signal intensity on T2-weighted images and regions of low signal intensity on T1-weighted images when compared to the signal intensity of normal liver and spleen (Kim et al. 2015; Clifford et al. 2004; Kippenes et al. 1999). Our results are consistent with the findings of these previous reports; the fat-suppressed T2 images showed high signal enhancement of the tumor. Furthermore, the in vivo imaging findings and the macroscopic findings after dissection were in agreement, and the results strongly supported our hypotheses.
Adverse events due to the miR-214/5AE administration were evaluated using the body weight and blood test results. Our findings show no difference between the ns miR and 5AE groups. The blood test results showed an elevation in the white blood cell count and a reduction in the hematocrit value in the ns miR group compared to the non-treat group. These findings were consistent with the clinicopathological findings of HSA and may represent a tumor effect. In a prior study, Lipofectamine RNAiMAX (a cationic carrier) exhibited toxicity to the liver and kidney by non-specific binding with blood components (Hongtao et al. 2006; Hatanaka et al. 2010). Intraperitoneal administration is considered to have a risk of hepatic injury because miRNA absorbed from capillaries is transferred into the liver via the portal vein. However, no obvious hepatic injury was observed in this study. Although longer-term evaluation is required in the future, intraperitoneal administration of miR-214/5AE according to the protocol of this study is considered to be well tolerated without any significant side effects.
In contrast to previous in vitro reports, COP1 expression did not significantly differ between the 5AE and ns miR groups in vivo. Unfortunately, we were not able to clarify the cause of this problem in this study. Further investigation into the mechanism of the anti-tumor effect is necessary.
In conclusion, we showed that intraperitoneal administration of miR-214/5AE exerts an antitumor effect in an intraperitoneal dissemination mouse model of HSA by inducing apoptosis and suppressing cell proliferation. In addition, no significant side effects were observed. These results could form the basis for the development of a new treatment method using miR-214/5AE for spontaneous HSA cases. Further studies regarding its clinical application are needed to support these claims.