Utilizing miRNA Lockers in miRNA loss of function researches

Here, we demonstrated how miRNA Lockers can be used in miRNA researches as a promising substitute of current miRNA inhibitors by identifying a new RNF8-targeting miRNA and establish its regulatory relationship with EMT.

By setting RNF8 as the target downstream gene in three different computational algorithms that analyze miRNA binding sites on the 3’-UTR of a specific gene (TargetScan, miRBase and miRWalk), miR-654-5p (miR-654 for short) was predicted as a promising candidate regulator of RNF8 by directly binding with its 3’-UTR (Data not shown). However, such regulatory relationship has not been experimentally validated yet, while the relationship between miR-654 and cancer metastasis also remains poorly discussed.

Hence, we designed four Lockers for miR-654, named as Lc-1p，Lc-1np，Lc-2p and Lc-2np. Locker-1p (Lc-1p) and Locker-1np (Lc-1np) contained two miR-654 binding sites, while Locker-2p (Lc-2p) and Lc-2np (Lc-2np) contained three miR-654 binding sites. Suffix ‘p’ and ‘np’ was marked to distinguish the non-perfect complementary base pairing (np) and perfect complementary base pairing (p) between miRNA Locker and target miRNA (figure 3a). The control Locker (Lc-C) was constructed similarly as the control Locker of miR-214 by replacing the miRNA binding sites of Lc-1p with equal length of poly-C sequence to avoid unnecessary cross talks with other host miRNAs while maintain the same structure.

Figure 1. Schematic representation showing the anticipated effect of miR-654 on EMT process of human lung adenocarcinoma cells, the relation between miR-654 and RNF8 is unverified. Four miRNA Lockers, Lc-1p, Lc-1np, Lc-2p, Lc-2np are expected to promote EMT by blocking the possible repressive effect of miR-654 on RNF8.

As such, IP-PCR was performed to verify the miR-654 binding ability of the designed Lockers. Similarly, primers were designed on the supporting module amplifying one of the loop structure of the Lockers. A549 cells transfected with specific sets of nucleotides were cultured for 36 hours and harvested for IP-PCR assay. As shown in Figure 3b, ~80bp (for Lc-1p and Lc-1np) or ~100bp (for Lc-2p and Lc-2np) DNA fragments was amplified from the precipitates of the groups co-transfected with Flag-hAgo2 expressing plasmid and Lc-1p/Lc-1np/Lc-2p/Lc-2np miRNA Lockers by using anti-Flag antibody, while the negative control that did not use antibodies or adopted the normal rabbit IgG showed no amplification signal. Meanwhile, the control group transfected with Flag-hAgo2 expressing plasmid alone did not display any amplification signal. Besides, the knockdown effect of miR-654 Lockers on miR-654 in A549 cells were validated through Taqman-based RT-qPCR assay. Results showed that

Figure 2. IP-PCR assay and qPCR assay of A549 cells transfected with miR-654 Lockers.(a) IP-PCR assay determining the binding ability of miRNA Lockers with miRNA in A549 cells. Schematic representation of primer design is shown above. Each group was co-transfected corresponding Lockers/Lc-C with hAgo2 expression plasmid. Input indicates an aliquot of total DNA. Antibodies used for immunoprecipitation are indicated above the lanes. (b) RT-qPCR results demonstrating miR-654 abundance in A549 cells transfected with miR-654 Lockers Lc-C/Lc-1p/Lc-1np/Lc-2p/Lc-2np or Antagomir An-654/An-C. Relative gene expression was calculated using the 2^-ΔΔCT method, with initial normalization of genes against U6 snRNA within each group. The expression levels of each gene in the control groups (Lc-C or An-C) were arbitrarily set to 1.0. The relative expression values were averaged from the data in three parallel reactions. Error bars represent SD.

Since prediction results suggested that miR-654 might downregulate RNF8 expression by directly targeting 3’UTR of RNF8 mRNA. To validate such prediction, Western blot analysis was performed to examine the protein expression level of RNF8 under miR-654 knock-down condition. As showed in Figure 3d, western Blot results showed 2-fold to 3.5-fold increases on RNF8 protein level in Locker-supplemented groups comparing to the Lc-C group, while the RNF8 expression level in An-654 group only increased for 1.17 folds comparing to the control group. In addition, miR-654 knockdown reduced epithelial marker (E-cadherin) and increased mesenchymal marker (Snail, N-cadherin) (Figure 3e), which suggested that Locker-mediated miR-654 knockdown might be related with enhanced EMT via upregulation of RNF8. Taking together, these results demonstrated miRNA Locker-induced knockdown of miR-654 could increase expression of RNF8 and plausibly promote EMT.

Figure 3. Western blot analysis results showing RNF8 and EMT associated marker.(a) Western blot analysis results showing RNF8 expression level in A549 cells transfected with miR-654 Lockers Lc-C/Lc-1p/Lc-1np/Lc-2p/Lc-2np or Antagomir An-654/An-C. (b) Western blot analysis determining the expression level of epithelial marker E-cadherin and mesenchymal markers (N-cadherin and Snail) in A549 cells transfected with miR-654 Lockers Lc-C/Lc-1p/Lc-1np/Lc-2p/Lc-2np or Antagomir An-654/An-C.Relative protein expression levels were calculated by using β-actin expression level as initial normalization and then set the protein expression level in the control groups (Lc-C or An-C) arbitrarily as 1.0. the results were obtained from at least three independent experiments. Error bars represent SD.

With previous results, we could conclude that miR-654 participant in the regulation of RNF8 expression in A549 cells. Since it has been proved that RNF8 participated in cell proliferation and migration in non-small lung cancer cell A549. We anticipate miR-654 might be involved in migration and proliferation of lung cancer by regulating RNF8 expression. To further study the role of miR-654 in A549 proliferation and migration, Cell proliferation assay using Cell Counting Kit-8 (CCK8) was performed to detect the migration capacity of A549 cells transfected with four types of miR-654 Lockers or with Lc-C control. Results showed that the absorbance of 450nm (indicating the living cell number) in four Lockers-groups were significantly increased comparing to the Lc-C groups (figure 3f), indicating that the knockdown of miR-654 could significantly strengthen cell proliferation capacity. Subsequently, Transwell assay was carried out to test the migration capacity of Locker-mediated miR-654 knockdown groups. Compared with control, miR-654 knock-down dramatically increased the number of migrated A549 cells with 3.5~5-fold changes (Figure 3h), suggesting that knockdown of miR-654 accelerates cell migration of non-small cell lung cancer A549 cell. Transwell results of miR-654 knockdown by antagomir (~2-fold change) confirmed such conclusion. Taken together, we found that block of miR-654 using our miRNA locker promoted cell proliferation and migration of lung adenocarcinoma cells.

Figure 4. Cell proliferation assay and Transwell assay determining proliferation and migration capacity of A549 cells transfected with Lockers.(a) CCK8 Assay indicating the proliferation of A549 cells transfected with Lockers Lc-C/ Lc-1p/Lc-1np/Lc-2p/Lc-2np. (b) and (c) Tranwell assay determining of migration in A549 cells transfected with miR-654 Lockers Lc-C/Lc-1p/Lc-1np/Lc-2p/Lc-2np or Antagomir An-654/An-C. The results were obtained from at least three independent experiments. Error bars represent SD.