miRNAs can work in a variety of biological processes and participate in the tumor process including uterine cervix carcinoma progression. It has been reported that miR-24 is involved in keratinocyte differentiation and its expression is regulated by HPV(Human papilomavirus) E6 and E721, suggesting that the miR-24 family may be involved in the cervix carcinoma progression. miR-24-3p which is a member of the miR-24 family could inhibit the malignant progression of lacrimal adenoid cystic carcinoma through the p53/p21 signal pathway, but it promoted the development of varied tumors such as breast, lung, liver, and bladder cancer 22–26. However, the specific role and molecular mechanisms of miR-24-3p, which is a well-known cancer-associated miRNA, have not been elucidated in cervical cancer. In our studies, miR-24-3p was upregulated in SiHa and CaSki cervical cancer cells. Inhibition of miR-24-3p remarkably inhibits cell proliferation, migration, and invasion, but overexpression of miR-24-3p has the opposite effect. Moreover, animal experiments revealed that miR-24-3p antagomir could slow the growth of subcutaneous tumor in nude mice. The above results confirmed that miR-24-3p plays a promoting role in the cervical cancer progression.
One of the biggest challenges in formulating miRNA treatment methods is determining the best miRNA candidate or miRNA target for each disease. Compared to experimental verification, bioinformatics can provide a large variety of high-speed predictions of target genes and related pathways. Four online starbases were used to seek the optimal target gene. The bioinformatics screening results showed that miR-24-3p targets 95 genes in miRDB, mitaRbase, and Targetscan. Biological processes of target genes are enriched in protein autophosphorylation, positive regulation of autophagy, regulation of growth, negative regulation of cell proliferation, DNA damage checkpoint, etc. (Figure S1). Some of these target genes have been linked to cervical cancer; for example, NET1 can promote the angiogenesis of cervical squamous cell carcinoma 27; likewise, down-regulating ERBB3 of the miR-24-3p target will significantly reduce the proliferation and invasion of cervical cancer cells28. AMOTL2 was ultimately selected for validation because it is the intersection in all prediction databases.
AMOTL2 is a member of the Amot family of angiomotin 29. It has been proven to be a key factor for the normal movement of embryonic cells and is also involved in the migration and proliferation of endothelial cells18,19. AMOTL2 plays a role as a pro-oncogene in breast and colorectal cancer 30,31, but it participates in tumor growth and invasion by suppressing the activation of the target genes of YAP in gliomas as a tumor suppressor gene32. According to the existing researches, the function of the AMOTs in cervical cancer remains indistinct, and the precise roles and mechanisms of AMOTL2 in cervical cancer are also elusive. In this reasearch, we definited that AMOTL2 was a functional target of miR-24-3p in uterine cervix carcinoma. First, miR-24-3p negatively modulated AMOTL2 protein levels in cervical cancer cells and in vivo. Second, the activity of a luciferase reporter embedded the 3ʹ-UTR sequence of AMOTL2 was markedly reduced by ectopic miR-24-3p expression but was enhanced by inhibition of miR-24-3p. Third, the restoration of AMOTL2 reversed the antitumor effects of miR-24-3p inhibitors. These observations suggested that AMOTL2 was a direct functional target of miR-24-3p and served as a tumor suppressor in tumorigenesis and the progression of cervical cancer.
To elucidate possible molecular mechanisms modulated by miR-24-3p in cervical cancer, it was discovered that 36 candidate targets were cervical cancer pathogenic genes. Moreover, the KEGG signaling pathway enrichment analysis showed that they are mainly involved in microRNAs in cancer, leishmaniasis, the ErbB signaling pathway, apoptosis, the signaling pathway regulating pluripotency of stem cells, the phospholipase D signaling pathway, the hippo signaling pathway, and endocytosis.
Hippo pathway is cognized as a tumor-suppressor pathway negatively modulating the oncoproteins TAZ and YAP 33. In addition, the Hippo pathway is a conserved signaling cascade and modulate cell growth, homeostasis, fate decision, and regeneration34,35. Moreover, the Hippo pathway has recently been associated with the cervical cancer. Hyperactivation of the YAP1 oncogene actuating the initiation and development of cervical cancer was demonstrated by He et al.36. Accumulated evidence has highlighted the crucial regulatory function of AMOT family proteins in the Hippo signaling pathway 37–39. In our study, we further validated that miR-24-3p inactivated the Hippo pathway. Firstly, overexpression of miR-24-3p reduced the p-YAP1 level in western blotting, while downregulation of miR-24-3p exerted the opposite effect. Overexpression of miR-24-3p in CaSki cells augmented the mRNA level of CTGF and CYR61 by qRT-PCR. On the contrary, downregulation of miR-24-3p in SiHa cells depressed the mRNA levels of ANKRD1, CYR61, and CTGF. It indicates that miR-24-3p can inhibit the phosphorylation of YAP and activate transcription of target genes downstream of YAP. However, this requires further investigation of the specific mechanism of AMOTL2 on the YAP/Hippo pathway in cervical cancer.
In order to delve into the mechanism of function of miR-24-3p in cervical cancer, we screened out eight LncRNAs that may regulate miR-24-3p by means of bioinformatics. Among them, LINC00662, NEAT1, and ILF3-AS1 were underexpressed in cervical cancer, and the overall survival time of patients with high expression of ILF3-AS1 was longer than that of patients with low expression of ILF3-AS1, and that reminds us that ILF3-AS1 may be a good prognostic marker for cervical cancer, which will provide favorable laboratorial and theoretical bases to subsequent research.