Fig 2: The validation of obviously differentially expressed RNAs in the ceRNA network. (A) The ceRNA network of KLRD1, BRAF, and NFATC4 (rectangles, DEmiRNAs; triangles, DEcircRNAs; diamonds, DElncRNAs; Circles, DEmRNAs). (B) The fold changes of KLRD1, BRAF, and NFATC4 were involved in ceRNA regulatory network in the ICP group compared with the control group. (C) The validation on RNA expression in the ICP group (n=3) compared with the control group (n=3) by qRT-PCR. Each dot represents the average value of one sample in three experimental replicates. (D) The protein level of NFATC4 and BRAF in the ICP group compared with the control group. The letters represented the different placenta tissues. (E) Comparison of cytokines levels in placenta tissue between ICP and healthy controls. Cytokines concentrations of TNF-a, IFN-? and IL-10 were compared between ICP patients (n=7) and healthy controls (n=5). Data were shown as mean ± SD. Differences were analyzed by unpaired t test. All P-values are two-tailed and significantly different when P-value is <0.05. *P<0.05, **P<0.01, ***P<0.001.

Fig 3: CRISPR-RSPO4 knockout reduce adhesion potential in PTC cell lines. (A) Box plot showing the gene expression data from PTC patient’s samples considering genes from focal adhesion network, (B) RSPO and BRAF signaling and (C) Cell lysates from control, CRISPR-RSPO4, and CRISPR-BRAF of metastatic cell line were immunoblotted with antibodies against Rspo4, Braf, E-cadherin, B-catenin, aN-catenin, Y69-phospho-cMyc, PTK2 (FAK), Y397-phospho-PTK2, Paxilin, 118Y-phospho-paxillin, Src, 416Y-phospho-Src, and Gapdh as an internal control. Error Bars represent SD from two experiments.

Fig 4: RSPO4 and BRAF genes and proteins were highly expressed in our metastatic cohort (A). Using Integrative Genome Viewer (IGV) all the 10 metastatic samples harboured an A to T substitution at position 140,453,136 on chromosome 7, that is harbouring a BRAF V600E classical mutation (B). Protein expression levels reveled that BRAF and RSPO4 are overexpressed in PTC samples with locoregional invasiveness (C).

Fig 5: Survival rates in PTC patients from The Cancer Genome Atlas (TCGA) dataset considering BRAF, PTK2 (FAK), and RSPO4 gene expression. BRAF, PTK2 and RSPO4 expression levels discriminate between non-metastatic and metastatic PTC tumors with survival rates lower in PTC where these genes are overexpressed.