MicroRNA-708 emerges as a potential candidate to target undruggable NRAS

doi:10.21203/rs.3.rs-1954766/v1

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MicroRNA-708 emerges as a potential candidate to target undruggable NRAS

https://doi.org/10.21203/rs.3.rs-1954766/v1

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RAS (KRAS, NRAS, HRAS), the most frequently mutated oncogene in cancers, drives tumorigenesis by promoting cell proliferation, survival, and motility, was perceived undruggable for the past three decades. Intense research mainly focused on KRAS mutation, however, targeted therapy for NRAS mutation, which is frequently observed in several cancer types, including melanoma (15–20%), leukemia (10%), and occasionally other cancer types, remained an unmet medical need. Here we report using miRNA-708 that targets the distinct 3’ untranslated region (3’UTR) of NRAS to develop a miRNA-based precision medicine to treat NRAS mutation-driven cancers. We identify NRAS as a direct target of miRNA-708. Overexpression of miRNA-708 successfully reduced NRAS protein levels in NRAS-mutated melanoma, leukemia, and lung cancer cell line, resulting in suppressed cell proliferation, anchorage-independent growth, and promotion of reactive oxygen species-induced apoptosis. Consistent with the functional data, the activities of NRAS-downstream effectors, PI3K-AKT-mTOR or RAF-MEK-ERK signaling pathway, were impaired in miR-708 overexpressing cells. On the other hand, cell proliferation was not disturbed by miRNA-708 in cells carrying wildtype NRAS. Collectively, our data unveil the therapeutic potential of using miRNA-708 in NRAS mutation-driven cancer through direct depletion of constitutively active NRAS and thus inhibit its downstream effectors to decelerate cancer progression. Harnessing the beneficial effects of miR-708 may therefore offer a potential avenue for small RNA-mediated precision medicine in cancer treatment.

MicroRNA-708

NRAS

Targeted therapy

Melanoma

Leukemia

Precision Medicine

RAS gene (KRAS, HRAS, and NRAS) encodes a small GTPase which acts as a binary switch, toggling between GDP-bound inactive state (OFF) and GTP-bound active state (ON) that communicate the environmental growth factor cues and the downstream signaling network to control cell cycle, differentiation, and survival ¹. Up to 30% of all human cancers carry oncogenic mutations in one of these three RAS isoforms ². Codon 12, 13, and 61 are the hotspot for amino acid substituting mutation, trapping RAS protein in GTP-bound active state ³. The inhibition of GTP hydrolysis results in the constitutively active RAS, leading to uncontrolled cell growth and malignant transformation of cells ⁴.

RAS is one of the earliest identified oncogenes ⁵ and the whole world has spurred intense resources in developing RAS inhibitors for over 40 years. However, no effective RAS inhibitor was approved in clinical practice until 2021. RAS was even perceived as an “undruggable” target for many years ⁶. This traditional perception has been revolutionized with the pioneering research done by Shokat and colleagues, who paved a way for the design of some promising allele-specific covalent inhibitors (e.g. AMG510 and MRTX849) to target the KRAS^G12C mutant ^{7, 8}. Still, this discovery is restricted only to KRAS^G12C-mutant and cannot be translated into drugs targeting other RAS isoforms-mediated oncogenesis.

Given that KRAS is predominantly mutated in cancers harboring RAS mutation, drug development targeting NRAS mutation is lagged far behind and remained an unmet medical need. NRAS is mutated in cutaneous melanoma and acute myeloid leukemia (AML) at 20% and 15% frequency respectively. Some other cancers including lung cancer, colon cancer, and neuroblastoma are also reported to carry NRAS mutation at a lower frequency ². Melanoma cells with NRAS mutation present elevated mitotic activity causing thicker lesions and a higher rate of metastasis as a consequence of persistent NRAS signaling ⁹. Melanoma patients harboring this mutation were found to have shorter survival as compared to patients harboring other mutations ¹⁰. NRAS mutant-mediated aggressiveness is not only limited to melanoma, its correlation with the higher mortality rate in pediatric AML is also observed in a meta-analysis ¹¹. Nonetheless, no clinical trial was conducted specifically in treating NRAS mutant cancer since direct inhibition of the NRAS activity is extremely challenging. The lack of deep hydrophobic pockets on NRAS catalytic G domain, the high binding affinity to GTP, and identical G domains among different RAS isoforms all posed obstacles in developing efficacious NRAS-selective drugs ¹. Current management of NRAS mutant melanoma mainly relies on MEK inhibitors (MEKi) and immune checkpoint blockade (ICB) therapy ¹². However, third-generation MEKi, such as trametinib and binimetinib, only showed a modest response rate when solely used ^{13, 14}. MEKi in combination with CDK4/6 or PI3K/AKT inhibitors showed enhanced antitumor activity but also caused undesirable toxicities in clinical trials ^{15, 16}. Instead, ICB therapy shows promising result for advanced metastatic melanoma with NRAS mutation ^{17, 18, 19, 20}. However, some patients who receiving ICB therapy developed resistance ²¹ or recurrence after treatment²². Therefore, developing drug selectively targeting NRAS mutant is urgently required.

Since pan-RAS inhibitors may lead to substantial toxicity as the RAS signaling pathway accounts for many critical cellular events in non-cancerous cells ²³, the isoform-specific inhibitor should be taken into consideration when drugging NRAS. Here, we offer a workable solution to circumvent the off-target toxicities and deplete the NRAS protein effectively by using a microRNA-based approach. MicroRNA (miRNA) is a short non-coding RNA that can negatively regulate gene expression post-transcriptionally ^{24, 25}. It binds generally to the 3’-untranslated regions (3’UTR) of target mRNA and induces translational repression, degradation, or cleavage ^{26, 27}. To date, dysregulated miRNAs are shown to be associated with tumor development, progression, metastasis, and response to therapy, suggesting their potential use as diagnostic, prognostic, and predictive biomarkers ^{28, 29}. Meanwhile, in the light of the first siRNA drug approved by the FDA in 2018 ³⁰, the number of miRNA drugs entering or getting closer to clinical trials has increased ³¹. Harnessing the power of miRNA may help us to develop a therapeutic strategy to suppress those “undruggable proteins” through epigenetic inhibition ³². In the case of RAS, different isoforms share more than 90% homology on their protein sequences, hence targeting sequences on the 3’untranslated region (3’UTR) of RAS isoforms by miRNA can result in unique inhibition on each individual isoform of RAS and thus provide a great opportunity to specifically deplete NRAS protein expression.

MicroRNA-708 (miR-708), a known tumor suppressor ³³, is located within the first intron of the ODZ4 gene ³⁴. Mature miR-708 formed after pre-miR-708 spliced out from the host mRNA and processed by several nucleases. The discovery of miR-708 has drawn oncologists’ attention on its role in suppressing cancer progression. Studies have reported that downregulation of miR-708 correlates with the occurrence, advanced stage and poor survival in a variety of different malignancies while restoring miR-708 expression in different cancer cells results in decreased cell migration and invasion in vitro, as well as reduced tumor growth and metastasis in vivo ^{35, 36, 37, 38, 39, 40}. Few targets of miR-708 were proved for participating in cell motility including Rap1B in ovarian cancer ³⁵, KPNA4 in prostate cancer ³⁹, and NNAT in breast cancer ⁴⁰. MiR-708 restoration in those cancer cells significantly inhibited those targeted genes and eventually attenuated distant metastasis. Besides acting as an anti-metastatic factor in cancer, miR-708 also exerts a profounded anti-proliferative effect by suppressing pro-survival protein (e.g. survivin ³⁶, Bcl-2 ³⁸), DNA repair mechanism (e.g. BMI1 ³⁶, PARP1 ³⁸), as well as proteins related to cell cycle, for example, cyclin D1 ³⁸ to suppress tumor growth. However, the role of miR-708 in melanoma and acute myeloid leukemia (AML) is yet to be characterized, not to mention that NRAS, one of the most important oncogenes in these cancers is a predicted target of miR-708.

In this study, we identified and confirmed that miR-708 directly targets NRAS to deplete its protein levels in different cancer cell lines driven by NRAS mutation, resulting in the decreased signaling on effector pathways, PI3K-Akt-mTOR or RAF-MEK-ERK, and subsequently alleviate cancer cell proliferation, anchorage-independent growth, motility, and resistance to apoptosis. On the other hand, cell proliferation was not suppressed by miRNA-708 in non-NRAS mutation cacner or normal lung epithelial cells. Overall, our data provide a potential new approach that miR-708 can be used as a promising precision medicine for NRAS mutant cancers in the near future.

NRAS is the direct target of miR-708

To identify miRNAs that potentially deplete NRAS expression and subsequently suppress tumorigenesis driven by NRAS mutation, we applied TargetScan databases to retrieve miRNAs capable of binding 3’ UTR of NRAS. TargetScan is an online portal that is used to predict the potential alignments between miRNAs and mRNAs by using a computational quantitative model ⁴¹. Among all miRNAs with canonical binding sites on NRAS 3’UTR (Figure S1), we chose miR-708 as our candidate to perform further molecular and functional analysis since miR-708 is a well-known tumor-suppressive miRNA in various cancer types ³³. To investigate whether miR-708 is a suitable drug for cancers driven by NRAS mutation, we collected three cell lines carrying NRAS mutation from different cancer types, which are SK-MEL-2, THP-1, and H1299 that belong to malignant melanoma, AML, and non-small cell lung cancer (NSCLC) respectively (Fig. 1A).

To examine whether miR-708 can deplete NRAS, we measured NRAS protein levels in these three cell lines transfected with miR-708 (Figure S2). Significant downregulation of NRAS protein level was observed in all cell lines with miR-708 overexpression through Western Blot analysis (Fig. 1B), as well as mRNA level of NRAS in SK-MEL-2 (Fig. 1C). 3’UTR luciferase reporter assay was employed to confirm whether NRAS is directly regulated by miR-708. Wild type (WT) or mutated putative binding sequence of miR-708 on NRAS 3’UTR was inserted into a luciferase reporter construct (Fig. 1D). Expression of miR-708 caused a significant decrease in luciferase activity in cells transfected with a luciferase reporter construct inserted with the WT NRAS 3’UTR, while this effect was rescued in cells expressing mutant form (Fig. 1D). These data suggest that NRAS is the direct downstream target of miR-708 and lead us to determine how miR-708 modulates cellular activities through NRAS depletion in NRAS mutated cells.

MicroRNA-708 exerts anti-tumor activities in NRAS-mutated cancer cells

To evaluate the function of miR-708 in different cancers carrying NRAS mutation, we transiently transfected SK-MEL-2, H1299, and THP-1 cells with miR-708 to evaluate the impact of miR-708 on uncontrolled cell proliferation driven by NRAS mutation. Expression of miR-708 impaired the proliferation rate of SK-MEL-2 and H1299 cells in 2D clonogenic assay (Fig. 2A, S3A). To assess the role of miR-708 on anchorage-independent cell growth, we seeded these three cell lines on soft agar and incubated those cells for 7–14 days. At the end of the experiment, we measured the number and area of colonies as the indicators for survival and anchorage-independent cell division. The number and the size of colonies in SK-MEL-2 cells transfected with miR-708 were significantly reduced compared to the control group (Fig. 2B). Intriguingly, the results were different in THP-1 and H1299 cells in which miR-708 overexpression only led to dramatical inhibition in the size of the colony but not in the colony number (Figure S3B, C). To further explore the function of miR-708, we checked whether miR-708 modulates cell motility. We used the Boyden chamber with or without Matrigel to assay the invasiveness through the extracellular matrix and cell migration towards the chemoattractant concentration gradient. Three cell lines exhibited different metastatic behavior when transiently overexpressed miR-708. We found that miR-708 effectively suppressed both migration and invasion of SK-MEL-2 cells (Fig. 2C). Similar to SK-MEL-2 cells, the motility of THP-1 cells was decreased with miR-708 overexpression (Figure S3D). We only assayed the chemotaxis of THP-1 cells transfected miR-708 because invasion is not frequently discussed in non-solid cancer. On the other hand, H1299 behaved differently when transiently overexpressed miR-708. To be specific, invasion of H1299 cells was significantly inhibited by miR-708 while migration was only slightly inhibited in the presence of miR-708 (Figure S3E).

Constitutively active NRAS is known to protect cells from apoptosis ⁴², thereby we also examined apoptosis of these three cell lines with miR-708 overexpression in the presence of reactive oxygen species (ROS) donor, hydrogen peroxide (H₂O₂). Flow cytometry analysis of early apoptosis marker, Annexin V, and late apoptosis marker, 7-AAD, co-staining showed that miR-708 transfection enhanced H₂O₂-triggered apoptosis (Annexin V positive) in SK-MEL-2 (Fig. 2D) and H1299 (Figure S3F) cells, but not in THP-1 cells (Figure S3F). Taken together, these findings indicate miR-708 exerts profound but different anticancer effects on NRAS-driven melanoma, AML, and NSCLC cell lines. To verify the relationship between NRAS depletion (Fig. 1) and miR-708-mediated anticancer activities (Fig. 2 and S3), we next investigated whether cells with NRAS knockdown resulted in similar phenotypes as presented in cells with miR-708 overexpression.

MicroRNA-708 impairs cell proliferation and survival through NRAS-mediated pathway in NRAS-mutated cancer cells

Melanoma is the major cancer type caused by NRAS mutation, therefore, we carried out NRAS depletion experiments in SK-MEL-2 cells. Owing to the inhibition of cell proliferation, anchorage-independent growth, migration, and invasion by miR-708 in NRAS mutated cells (Fig. 2 and S3), we are interested in finding out whether these miR-708-regulated cellular activities are majorly through NRAS depletion. We used small interfering RNA (siRNA) to deplete NRAS expression and the silencing effect was validated by Western blot analysis (Fig. 3A). SK-MEL-2 cells transiently transfected with siNRAS presented lower proliferation rate and reduced anchorage-independent growth compared to control groups indicated by clonogenic assay (Fig. 3B) and soft agar colony formation assay (Fig. 3C). Surprisingly, in contrast to miR-708 transfection, migration was not suppressed in cells with NRAS knockdown (Fig. 3D). Meanwhile, invasion of SK-MEL-2 cells transfected with siNRAS was reduced to a lesser extent compared to miR-708 overexpression (Fig. 3D). This result suggests that NRAS depletion by miR-708 may not play the central role to inhibit cell migration and invasion. It is possibly dominated by other downstream targets mediated by miR-708, for example, Rap1B and NNAT as reported previously in ovarian and breast cancer respectively ^{35, 40}. Moreover, siNRAS transfection was also unable to fully recapitulate the effect of miR-708-enhanced ROS-induced apoptosis in SK-MEL-2 (Fig. 3E), implying that miR-708 possibly induced cell apoptosis through additional targets, such as survivin ³⁶. To further elucidate the underlying mechanism of the miR-708-NRAS signaling pathway, we analyzed the activities of two dominant NRAS downstream effectors, RAF-MEK-ERK cascade and PI3K-AKT-mTOR pathway. Phosphorylation of AKT was significantly downregulated in miR-708-transfected SK-MEL-2 and THP-1 while modestly decreased in H1299 cells (Fig. 3F). In contrast, phosphorylation of ERK protein in H1299 was significantly suppressed while SK-MEL-2 was barely affected. Unexpectedly, ERK phosphorylation was even 3-fold higher in THP-1 cells transfected with miR-708, showing three different responses of ERK activation in three different cell types (Fig. 3F). Taken together, these findings demonstrate that miR-708 negatively regulates NRAS expression and subsequently suppresses either one of the main effector pathways, to decelerate cancer cell proliferation, in the three cell lines we examined. On the other hand, it also indicates that the activation of these pathways in these cells may be not only dependent on NRAS activation but also rely on a much more complex mechanism.

Cell proliferation is not affected by microRNA-708 in cells carrying wildtype NRAS

To further support the idea of miR-708 as a precision medicine for NRAS mutation-driven cancers, we overexpressed miR-708 in breast cancer cells carrying KRAS mutation, MDA-MB-231, and normal lung epithelial cells, BEAS-2B. In MDA-MB-231 cells, NRAS expression was greatly reduced (Fig. 4A), whereas clonogenic cell proliferation was not suppressed by miR-708 overexpression (Fig. 4B). As the proliferation of MDA-MB-231 relies on aberrant KRAS signaling, NRAS knockdown only results in minimal effects on the cells and thereby showing no growing defects. Similar with the data from MDA-MB-231 cells, expression of miR-708 reduced expression of NRAS protein (Fig. 4C), but cell proliferation was not significantly suppressed in BEAS-2B cells overexpressing miR-708 (Fig. 4D). Overall, these results indicate miR-708 will exclusively exert its tumor suppressive function on NRAS mutation-driven cancers. Collectively, our hypothesis in which miRNAs can act as a good candidate for RAS isoform-specific targeted therapy is feasible.

MicroRNA-708 presents moderate survival benefits in NRAS-mutated cancer patients

To investigate the prognostic value of miR-708 in NRAS mutated cancer, we conducted a correlation analysis between miR-708 expression and patients’ survival with TCGA database. Selected information from patients with NRAS single somatic mutation (#SSM) in Skin Cutaneous Melanoma (SKCM), Acute Myeloid Leukemia (LAML), or Lung Adenocarcinoma (LUAD) dataset was applied for further analysis. Compared with the survival plot of patients among all SKCM, LAML, LUAD, high expression of miR-708 presented a moderately better survival rate in patients with NRAS mutation from three different cancer types (Fig. 5), especially in patients from LAML (Fig. 5C) and LUAD (Fig. 5E). Although the sample sizes for patients with NRAS mutation in LAML and LUAD are too small (n = 12 or 10, respectively) and that could lead to statistical bias, it is still encouraging to see the survival difference, supporting our hypothesis that miR-708 can serve as a therapeutic option in treating NRAS mutation-driven cancers.

In this work, miR-708 emerges as a novel therapeutic option for NRAS mutation-driven cancer which has long been considered a tough nut to crack in drug design. We showed that miR-708 directly suppressed NRAS expression and this NRAS depletion leads to the reduced proliferation of cancer cells through subsequent oncogenic signaling inhibition. MicroRNA-708 restoration also weakened cancer cell motility in the NRAS-independent manner. Our findings provide convincing evidence for developing miR-708 as the NRAS specific targeted therapy and we hope by this way, patients who suffer from cancers driven by NRAS mutation will get benefit from our research (Fig. 6).

It is well-documented that oncogenic NRAS signaling facilitates cells to degrade the surrounding extracellular matrix by enhancing its proteolytic activity and thus increases the invasiveness of NRAS-mutated melanoma cells ⁴³. Our result echoes the previous studies in which NRAS knockdown could moderately suppress the invasion of melanoma cells while the migration is not affected (Fig. 3D). By taking the advantage of miR-708 as a powerful tumor suppressor through targeting numerous cancer-initiating or facilitating genes ³³, we could use miR-708 to combat cancers from multiple aspects besides NRAS inhibition and results in a net positive outcome. This benefit is confirmed by strongly abrogated migration and invasion in miR-708-transfected cells (Fig. 2C and S3D, E) and promotion of ROS-induced apoptosis (Fig. 2D, S3F), instead of only mildly reduced invasiveness and mildly induced apoptosis in cells with NRAS knockdown, respectively (Fig. 3D, E). Taken together, the fact that we found miR-708 can effectively reduce the metastatic potential of NRAS-mutated cancer suggests a potential therapeutic strategy to treat NRAS mutation-driven tumorigenesis.

In this study, we have tried to uncover the underlying signaling transduction regulated by miR-708/NRAS axis. Given that ERK and AKT are both downstream effectors of RAS, our results were intriguing as their activation was differently regulated when NRAS was suppressed by miR-708 in different cancer cell lines, suggesting the complexity of the NRAS signaling network. Earlier lines of work have shown that RAF/MEK/ERK and PI3K/AKT/mTOR pathways are not functioning independently, crosstalk between these two pathways occurs in the forms of cross-inhibition or -activation ⁴⁴. Here our data showed that NRAS suppression led to single pathway inhibition in three cell lines while the other pathway was barely affected or even activated in THP-1 cases (Fig. 3F), in consistency with what several reports revealed before ^{45, 46}. By inference, we speculated that the compensatory mechanism was triggered to sustain growth, proliferation, and survival signals in NRAS driven malignancies when NRAS was blocked. Although the exact regulation of miR-708 on the NRAS signaling pathway remains inconclusive, we believe that there is an opportunity for miR-708 to cooperate with ERKi and AKTi to tackle NRAS mutant cancer effectively.

Several microRNAs have been shown to act as a tumor suppressor via targeting NRAS in various cancer types, including breast cancer ⁴⁷, lung cancer ⁴⁸, colorectal cancer ⁴⁹, prostate cancer ⁵⁰ et cetera. However, all these works were restricted to a single type of cancer. Moreover, the current concept for precision cancer treatment is based on the genetic profile of a cancer patient and provides the most accurate gene-specific but not organ-specific treatment ⁵¹. Therefore, we tested our hypothesis of using miR-708 to inhibit NRAS mutation-driven cancer in three types of cancer cell lines: melanoma, AML, and NSCLC. Our study presents a proof of concept that using one microRNA to combat multiple cancers harboring the same mutation. In our case, it is NRAS mutation.

Previous studies on NRAS knockout mice showed that NRAS is dispensable during normal development ⁵², revealing that there might be some functional redundancy between NRAS and HRAS isoforms ⁵³. Moreover, when nanoparticle encapsulated miR-708 distributed in several major organs, no toxicity was observed, indicating that elevated miR-708 level has no impact on normal tissues ⁵⁴. Here our data further confirmed that depletion of NRAS protein level in normal lung epithelial cells did not disturb cell proliferation (Fig. 4C, D), echoing previous study that miR-708 has no impact on proliferation of normal lung cell line, WI-38 ⁵⁵. Overall, it is suggested that using using miR-708 to suppress NRAS mutation-driven cancer will be a safe and less toxic approach for a standard NRAS-targeted therapy.

The success of miR-708 suppresses NRAS is a proof of concept of using microRNA to antagonize the “undruggable” cancer target. Similar to NRAS, many oncogenes in cancer are still deemed intractable. These targets are difficult to drug with conventional pharmacological inhibitors because of the lack of deep pockets ¹, large protein-protein interactions ⁵⁰, or the druggable domain was spliced out ⁵⁶. Some notorious examples of undruggable protein include KRAS, MYC, and androgen receptor variant 7 (AR-V7) ⁵⁷. Therefore, applying our strategy extensively to all these “yet to be drugged” targets by using microRNA to deplete their mRNA transcripts or block their translation is an alternative option to target cancers driven by these undruggable oncogene mutations.

Cell culture

Human melanoma cell line SK-MEL-2 and human breast cancer cell line MDA-MB-231 were maintained in DMEM (Invitrogen) while acute myeloid leukemia cell line THP-1. Non-small cell lung cancer cell line H1299 and normal lung epithelial cell line BEAS-2B were cultured in RPMI (Invitrogen). All cancer cell lines were obtained from Bioresource Collection and Research Center (BCRC), Taiwan. BEAS-2B was a gift from Dr. I-Ching Wang of National Tsing Hua University (Taiwan). All culture media were supplemented with 10% fetal bovine serum (FBS; Biological Industries, Israel or Hyclone, USA) and 1% of penicillin-streptomycin (GIBCO), cultured at 37°c with 5% CO₂.

Nucleotides and reagents

The precursor miR-708 was purchased from Applied Biosystem (Carlsbad, CA). The NRAS-specific siRNA and negative control siRNA (NC) were from MDBio, Inc (Qingdao, China). Detailed sequences of primers and small RNAs are listed in Table S1.

Transfection and transduction

For transfection of adherent cells, 20 or 50nM of miR-708 precursor, siNRAS, or negative control siRNA were delivered using TransIT-X2 (Mirus Bio) or Lipofectamine® RNAiMax (Invitrogen) into SK-MEL-2, H1299, BEAS-2B, and MDA-MB-231 cells in Opti-MEM (Gibco) for 5h, then replaced with normal culture medium. For transfection of suspension cells, THP-1 was electroporated using Neon® Transfection System at the condition recommended (1600V/10ms/3 pulses), 10⁶ cells were transfected with 200nM miRNA or siRNA respectively in Buffer T provided. Transfected cells are harvested 48–72 h post-transfection for subsequent analysis.

Quantitative real-time PCR for genes and miRNAs

RNA was extracted from cells using TRIzol (Invitrogen) according to the manufacturer’s protocol. First-strand cDNA was synthesized by ReverTraAce (Toyobo, Japan) using oligo-dT or specific microRNA RT primers designed based on a previous study ⁵⁸ from 1ug of total RNA. Real-time RT-PCR was performed on a qTower 3 real-time PCR Thermal Cycler (Analytik Jena). The KAPA SYBR FAST Universal qPCR Kit (KAPA Biosystems, MA) was used for NRAS detection. The KAPA PROBE FAST universal qPCR Kit together with Universal Probe Library no. 21 (Roche Applied Science) was used for miR-708 detection. The mRNA levels were normalized with actin, while miRNA levels were normalized with U6. All the primer sequences used in this study are provided in Table S1. Data are means ± SD from 3 independent experiments and presented as fold changes relative to the control.

Western Blot analysis

Cells were lysed in 10mM Tris buffer, pH 7.4, containing 0.15 M NaCl, 1% Triton X-100, 100mM EDTA, protease inhibitor (Roche) and phosphatase inhibitor (Cyrusbio) mixture. The cell lysates were resolved in 10% SDS-Polyacrylamide gel, transferred onto PVDF membrane, and probed with antibodies. Antibodies against NRAS (sc-519), β-actin (sc-47778) were purchased from Santa Cruz Biotechnology, while antibodies against Akt (pan) (4691S), phospho-Akt (Ser 473) (4060S), p44/42 MAPK (t-ERK 1/2) (9102S), and phospho-p44/42 MAPK (p-ERK 1/2) (Thr 202 and Tyr 204) (9101S) were purchased from Cell Signaling Technology. Protein expression levels were quantified with Image J (NIH, USA). To be precise, the raw blot was quantified with uncalibrated OD. All bands within the same lane were selected with rectangular tools and plotted. The maximum background value of the membrane on the lane was then subtracted. The area of each peak was quantified by integration using Wand tools and regarded as protein expression levels. The expression of NRAS, p-AKT, and p-ERK1/2 were then normalized with Actin, Akt (pan), and MAPK p44/42 respectively. Data are means ± SEM from 3 independent experiments and presented as fold changes relative to the control. Full uncropped Western blots are presented in Figure S4.

3’UTR luciferase reporter assay

Sequences containing the predicted miR-708 binding site (157 bps) on NRAS mRNA 3’UTR region were amplified and cloned into the pGL3-Basic Vector (Promega). The mutant form of 3’UTR was generated using site-directed mutagenesis. In 3’UTR luciferase reporter assay, SK-MEL-2 cells were transfected with 1ug of pGL3-NRAS 3’UTR (wild type, WT or mutant), and 0.1ug of GFP construct was used as the internal control. 24 h later, miR-708 was transfected into the cells. The cells were lysed with passive lysis buffer for the detection of luciferase signal with Dual-Luciferase Reporter System (Promega) or Neolite Reporter Gene Assay System (PerkinElmer, USA). The Luciferase and GFP activity were read by BioTek Synergy HTX Multi-Mode Reader. Primer sequences used are provided in Table S1. Luciferase activity was normalized with GFP activity. Data are means ± SD from 3 independent experiments and presented as fold changes relative to the control.

Cell proliferation assay

Cell proliferation was measured through the clonogenic assay. SK-MEL-2 cells ($3\times {10}^{3}$ cells), MDA-MB-231 cells ($3\times {10}^{3}$ cells), BEAS-2B ($0.5 \times {10}^{3}$ cells), and H1299 cells ($1\times {10}^{3}$ cells) were seeded into 24-well plates in triplicate, 5 hours after miR-708 transfection. Then the cells were incubated for 7 (H1299, MDA-MB-231, and BEAS-2B) or 9 days (SK-MEL-2). Colonies were fixed and stained with 0.1% crystal violet for 30 min. The dye was then washed away with tap water and let dry overnight, photos of triplicate wells were then scanned. Cell proliferation was quantified with Image J by a plugin called Colony Area developed by Manish Bagga et al ⁵⁹. The parameter used to illustrate the result is colony intensity percentage, defined by an equation:

$$\text{C}\text{o}\text{l}\text{o}\text{n}\text{y} \text{i}\text{n}\text{t}\text{e}\text{n}\text{s}\text{i}\text{t}\text{y} \text{%}= \frac{\sum pixel intensities in a region}{\sum maximum intensities possible in the same region} \times 100$$

Data are means ± SD from 3 independent experiments and presented as fold changes relative to the mock control.

Soft agar colony formation assay

To prepare for the experiment, the bottom agar layers were prepared by mixing 2X DMEM or RPMI (20% FBS and 2% penicillin/streptomycin supplemented) and 1.2% of sterile high-melting agarose (FocusBio) in the ratio of 1:1, then placed 350 µL of the mixture into a 24-well culture dishes and allowed it to be solidified in room temperature. $1\times {10}^{3}$ of SK-MEL-2 cells, H1299 cells, and THP-1 cells that transfected with miR-708 and siNRAS were harvested to perform the experiment in order to determine the anoikis and anchorage-independent growth of cells in soft agar. The harvested cells are resuspended in 100 µL of culture medium. Cells were then mixed with 100 µL of 2X DMEM or RPMI and 1.2% of sterile low-melting agarose (LONZA SeaPlaqueTM agarose) in the 1:1:1 manner. The plate was then incubated at 4°c for 5 min to solidify the middle layer of agar-containing cells. To prevent the culture from drying out, 350µL of culture medium was added to each well before transfer to the incubator. The cells were allowed to grow into colonies for one (THP-1) or two (SK-MEL-2, H1299) weeks. After that, cells were stained with iodonitrotetrazolium violet (INT) for 2 days. Images of cell colonies from 3 separate fields of 2 different vertical planes of each well were taken using a microscope with 40X and 100X magnification (IX51, Olympus). The number of colonies in each field of view was quantified using the cell counter plugin in ImageJ and summed up for each group. For area quantification, each colony was circled and the area (pixel²) was analyzed by ImageJ as well. The mean colony area was obtained from the average of the total colony area. Data are means ± SD from 3 independent experiments and presented as fold changes relative to the mock control.

Transwell cell migration and cell invasion assay

Cell migration was assayed in 8.0-mm Falcon Cell Culture Inserts (Corning), and for the cell invasion assay, the BD biocoat Matrigel invasion chamber was applied (Corning) and performed as previously described ³⁵. Briefly, 2 x 10⁴ to 1 x 10⁵ of miR-708 or siNRAS-transfected SK-MEL-2 or H1299 cells were suspended in serum-free DMEM (500 µL), and placed in the upper transwell of 0.3 cm² in area. The bottom well was filled with 500 µL DMEM with FBS. After incubation for 16 h to 24 h, cells on the upper side of the inserts were removed by cotton swabs, and cells on the underside were fixed and stained with crystal violet. Photos of three (migration) to five (invasion) regions were taken from duplicated inserts, and the numbers of cells were counted using Image J. Data are means ± SD from 3 independent experiments and presented as fold changes relative to the mock control.

Chemotaxis assay

Chemotaxis of THP-1 cells was examined by using 8.0-mm Falcon Cell Culture Inserts (Corning). 1 x 10⁵ THP-1 cells transfected miR-708 were resuspended in serum-free RPMI 1640 (500 uL), and placed in the upper transwell of 0.3 cm² in area. The bottom well was filled with 500 µL DMEM with 8% FBS. After 24 h incubation, the inserts were removed and the migrated cells in the bottom well were stained with CellTracker™ Red CMTPX Dye (Invitrogen) for 20 minutes. Then, photos of five regions were taken from the duplicated bottom wells and the number of cells was counted automatically using ImageJ. Data are means ± SD from 3 independent experiments and presented as fold changes relative to the mock control.

Apoptosis assay

Cells transfected with miR-708 or siNRAS were treated with 200 µM hydrogen peroxide (H₂O₂) (SHOWA) to induce apoptosis for 24 hr after 48 hr transfection. Cells were then collected and double stained with 80 ng/mL Annexin V (BioLegend, 640920) and 5 µg/mL 7-AAD (BioLegend, 420403) in calcium rich binding buffer (10mM pH7.8 HEPES, 140mM NaCl, 2.5mM CaCl₂) for 15 minutes. The stained cells were then subjected to flow cytometry analysis (Beckman Coulter) and the raw data was analyzed by using CytExpert 2.0.

Survival Correlation Analysis

To investigate the correlation between miR-708 expression and overall survival of patients with different types of cancer, including Skin Cutaneous Melanoma Project (TCGA-SKCM), Acute Myeloid Leukemia Project (TCGA-LAML), and Lung Adenocarcinoma Project (TCGA-LUAD), we retrieved clinical data and miRNA expression quantification from The Cancer Genome Atlas (TCGA) database (http://cancergemome.nih.gov/). Statistical analysis was carried out using the R environment for statistical computing. The survival and survminer packages were used to model patient survival data. The "ggpubr" package was used to model scatter plot data. For survival data, we divided the patients into high and low expression groups based on the median value of the miR-708 expression in the miRNA-sequencing file. The survival period of each patient was defined as the days to last follow-up. Overall survival was demonstrated with the Kaplan-Meier method. The p-value was analyzed by Pearson chi-square test. The detailed clinical information is provided in Table S2.

Statistical analyses

The statistical analyses were all assessed by GraphPad Prism using Student’s t-tests to compare two means. ANOVA followed by Tukey’s post-hoc test was used for the statistical analysis when more than two means were compared. The correlation significance between miR-708 expression and survival was tested by log-rank test.

Data availability

All data generated or analyzed during this study are included in this article.

Acknowledgements

We are grateful to National Tsing Hua University for funding support our Competitive Research Team (Project No. 110Q2729E1 and 111Q2713E1) through the Ministry of Education's Higher Education Sprout Project.

Funding

This research was funded National Science and Technology Council (NSTC) (108-2314-B-007-003-MY3 and 111-2320-B-007-005-MY3) and National Tsing Hua University (110Q2729E1 and 111Q2713E1).

Author information

These authors contributed equally: Jia Meng Pang and Po-Chen Chien.

Authors and Affiliations

Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan

Jia Meng Pang, Yu-Ling Hsu, and Kai-Ti Lin

Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan

Jia Meng Pang, Po-Chen Chien, Ming-Chien Kao, Pei-Yun Chiu, Pin-Xu Chen, Yu-Ling Hsu and Kai-Ti Lin

Interdisciplinary Program of Life Science, National Tsing Hua University, Hsinchu, Taiwan

Pei-Yun Chiu

College of Life Sciences, Wuhan University, Wuhan, Hubei, China

Chengyang Liu, and Xiaowei Liang

Author contributions

Conceptualization, JM and KT; Software, PY; Investigation, JM, PC, MC, PY, PX, YL, CY, XW and KT; Validation, JM and KT; Visualization, JM and KT; Writing – Original Draft, JM; Writing – Review & Editing, KT; Resources, KT; Supervision, KT; Funding Acquisition, KT

Corresponding author

Correspondence to Kai-Ti Lin

Ethics declarations

Competing interests

The authors declare no competing interests.

Data availability

The data presented in this manuscript are available upon reasonable request from the corresponding authors.

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MicroRNA-708 emerges as a potential candidate to target undruggable NRAS

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Abstract

Figures

Introduction

Results

NRAS is the direct target of miR-708

MicroRNA-708 exerts anti-tumor activities in NRAS-mutated cancer cells

MicroRNA-708 impairs cell proliferation and survival through NRAS-mediated pathway in NRAS-mutated cancer cells

Cell proliferation is not affected by microRNA-708 in cells carrying wildtype NRAS

MicroRNA-708 presents moderate survival benefits in NRAS-mutated cancer patients

Discussion

Material And Methods

Cell culture

Nucleotides and reagents

Transfection and transduction

Quantitative real-time PCR for genes and miRNAs

Western Blot analysis

3’UTR luciferase reporter assay

Cell proliferation assay

Soft agar colony formation assay

Transwell cell migration and cell invasion assay

Chemotaxis assay

Apoptosis assay

Survival Correlation Analysis

Statistical analyses

Declarations

References

Additional Declarations

Supplementary Files

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