Transcriptome-wide analysis reveals insight into tumor suppressor functions of 1B3, a novel synthetic miR-193a-3p mimic

Abstract

Emerging data show that microRNA 193a-3p (miR-193a-3p) has a suppressive role in many cancers and is often downregulated in tumors, as compared to surrounding normal tissues. Therefore, mimics of miR-193a-3p could be used as an attractive therapeutic approach in oncology. To better understand and document the molecular mechanism of action of 1B3, a novel synthetic miRNA-193a-3p mimic, RNA sequencing was performed after transfection of 1B3 in six different human tumor cell lines. Genes differentially expressed (DE) in at least three cell lines were mapped by Ingenuity Pathway Analysis (IPA), and interestingly, these results strongly indicated upregulation of the tumor-suppressive phosphatase and tensin homolog (PTEN) pathway, as well as downregulation of many oncogenic growth factor signaling pathways. Importantly, although unsurprisingly, IPA identified miR-193a-3p as a strong upstream regulator of DE genes in an unbiased manner. Furthermore, biological function analysis pointed to an extensive link of 1B3 with cancer, via expected effects on tumor cell survival, proliferation, migration, and cell death. Our data strongly suggest that miR-193a-3p/1B3 is a potent tumor suppressor agent that targets various key oncogenic pathways across cancer types. Therefore, the introduction of 1B3 into tumor cells may represent a promising strategy for cancer treatment.

Keywords: PTEN; RNA-sequencing; RNAi therapeutics; cancer; miR-193a; microRNA.

Graphical abstract

Figure 1

Nucleotide sequences of precursor miR-193a, miR-193a-3p, and 1B3 and transfection efficiency of 1B3 (A) Nucleotide sequences of precursor miR-193a, miR-193a-3p (according to miRBase), and 1B3. The seed sequence in the guide (anti-sense) strand is underlined. The forward qPCR primer (green) for 1B3 used in stem-loop qRT-PCR also binds to the endogenous mature miR-193a-3p sequence. (B) Six tumor cell lines were transfected with 10 nM 1B3 in the presence of RNAiMAX, and total RNA was harvested after 24 h and 72 h. RNA samples from three independent experiments were pooled. The expression levels of 1B3 in samples submitted for RNA sequencing were analyzed by stem-loop qRT-PCR. Shown are the cycle threshold (Ct) values.

Figure 2

Gene-expression regulation by 1B3 in individual cell lines (A) Numbers of differentially expressed (DE) genes that are downregulated (estimated expression 1B3/estimated expression mock < 1) and upregulated (estimated expression 1B3/estimated expression mock > 1) 24 h and 72 h after transfection with 10 nM 1B3 in each cell line are shown as compared to mock-transfection conditions. The number of downregulated genes that are predicted miR-193a-3p targets is also indicated. (B) Heatmaps showing differential gene expression induced by 1B3 per cell line at 24 h and 72 h post-transfection. All DE genes are on the y axis and on the same row across all cell lines and both time points (due to the long list, gene names are not shown). Green means downregulated, and red means upregulated genes. Gray means no observed change in gene expression.

Figure 3

Gene-expression regulation by 1B3 in multiple cell lines (A) Numbers of DE genes that are downregulated (estimated expression 1B3/estimated expression mock < 1) and upregulated (estimated expression 1B3/estimated expression mock > 1) 24 h and 72 h after transfection with 10 nM 1B3 in multiple (up to six) tumor cell lines are shown as compared to mock-transfection conditions. The number of downregulated genes that are predicted miR-193a-3p targets is also indicated. (B) Volcano plots show genes DE in at least three cell lines (blue) in the population of all DE genes in at least one cell line (black). (C) Venn diagram demonstrating DE genes common among at least three cell lines. Downward arrow indicates downregulation (average estimated expression 1B3/estimated expression mock < 1), and upward arrow indicates upregulation (average estimated expression 1B3/estimated expression mock > 1).

Figure 4

PTEN pathway activation by 1B3 (A) Canonical pathways regulated by 1B3 at 24 h (p < 0.01) according to IPA. Directionality is indicated by Z score (<−2: inhibition; >2: activation). The significance threshold for multiple testing is indicated by a black dotted line. (B) Diagram showing mRNA targets of 1B3 in the PTEN pathway. Genes highlighted in green were downregulated by 1B3 at 24 h in at least three cell lines. (C and D) Indicated human tumor cells were transfected with 10 nM of either 3A1 or 1B3 in the presence of RNAiMAX and lysed after 72 h. Clarified whole-cell lysates were immunoblotted for PTK2, RPS6KB2, PIK3R1, TGFBR3, and PDPK1 (C). TGFBR3 could not be detected in HEP3B and HUH7. Additionally, samples were immunoblotted for pSer473 AKT and AKT total protein (D). Vinculin served as a loading control. Green boxes highlight observed protein downregulation as measured by densitometry.

Figure 5

Biological functions affected by 1B3 (A) The top 100 biological functions (ranked by p value) affected by 1B3, according to IPA at 72 h, were filtered for functions relevant to tumor cells. Shown are functions with Z score < −2 and > 2. All p values are smaller than 0.00001. (B) Categories of top 100 biological functions (all p < 0.00001) regulated by 1B3 were ranked based on the number of functions in each category.