Korea-CX

Results

Research Goal & Overview

The primary goal of our project was to determine how the oncogenic mutation NRAS^G12D influences cell behavior compared to wild-type NRAS (NRAS^WT) and untransfected control cells. Since NRAS mutations are frequently implicated in cancer progression, especially in blood and solid tumors, we aimed to establish an in vitro platform that allows quantitative and reproducible comparison of mutation-driven phenotypes.

Our experimental workflow was designed to assess three major aspects of NRAS function:

Expression validation – confirming successful delivery and transcription of NRAS^WT and NRAS^G12D.
Cell viability and drug response – evaluating how the mutation alters survival under MEK inhibition.
Cell migration – comparing the motility of WT and mutant cells in both 2D and 3D environments.

By combining standard assays (RT-PCR, CCK-8, scratch assay) with advanced approaches (Lab-on-a-Chip migration), we built a stepwise pipeline from basic confirmation to functional characterization. This overview demonstrates not only the specific impact of NRAS^G12D but also establishes a general workflow that other iGEM teams can apply to study mutation-driven cellular behaviors.

<Result 1: Transfection and Fluorescence Microscopy>

[1-1. Description]

To validate successful introduction of NRAS constructs into NIH3T3 cells, we performed Lipofectamine 3000-mediated transfection with plasmids containing NRAS^WT and NRAS^G12D, each linked to an mCherry fluorescent reporter.After 48 hours of transfection, cells were subjected to puromycin selection at 5 µg/ml for 2 days, followed by observation under brightfield and fluorescence microscopy.

In control cells, no fluorescence signal was detected, whereas both NRAS^WT and NRAS^G12D groups showed strong red fluorescence signals consistent with mCherry expression. The transfected cells also exhibited typical fibroblast-like morphology under brightfield conditions, indicating good viability and absence of morphological stress.

Figure 1. Confirmation of mCherry fluorescence in NIH3T3 cells following plasmid transfection. NIH3T3 cells were transfected with the mCherry-expressing plasmid using a lipid-based transfection reagent. 48h post-transfection, cells were subjected to puromycin selection (5 μg/ml) for 2days, after which surviving cells were imaged under fluorescence microscopy. Representative bright-field images (top) and fluorescence images (bottom) are shown. A strong red fluorescence signal corresponding to mCherry expression was detected in the transfected cells, whereas untransfected control cells exhibited no detectable fluorescence. These results confirm successful plasmid uptake and expression of the mCherry reporter gene. Scale bars, 100 μm.

[1-2. Interpretation]

These results confirm that NRAS^WT and NRAS^G12D plasmids were successfully delivered and expressed in NIH3T3 cells. The consistent fluorescence across multiple fields supports reproducibility and indicates that the mCherry reporter is a reliable marker of transfection efficiency. This provides a validated platform for downstream experiments, including gene expression verification, drug response, and migration assays.

<Result 2: Quantitative Measurement of Transfection Efficiency>

[2-1. Description]

To quantitatively validate transfection efficiency beyond microscopy, we measured mCherry fluorescence intensity using a microplate reader at 48 h post-transfection, followed by 2 days of puromycin selection (5 µg/ml). NIH3T3 cells transfected with NRAS^WT or NRAS^G12D plasmids showed significantly higher fluorescence compared to control cells. A baseline fluorescence signal was observed in the control group, likely due to cellular autofluorescence. Statistical analysis (unpaired Student’s t-test) confirmed that both NRAS^WT and NRAS^G12D groups were significantly different from controls (*p < 0.05, **p < 0.01). The fluorescence intensity between WT and G12D groups showed comparable levels, suggesting that the mutation did not alter transfection efficiency at this stage.

Table 1. Quantification of mCherry fluorescence in NIH3T3 cells following plasmid transfection. Fluorescence intensity was measured 48 h post-transfection, followed by 2 days of puromycin selection (5 µg/ml) using a microplate reader. Data are presented as mean ± SD (n = 3). Baseline fluorescence was detected in control cells, likely due to cellular autofluorescence. Statistical significance was determined using two-tailed unpaired Student’s t-tests. *p < 0.05, **p < 0.01 vs. control.

[2-2. Interpretation]

This quantitative analysis complements the fluorescence microscopy results in Result 1. Together, they confirm that both NRAS^WT and NRAS^G12D plasmids were successfully transfected and expressed in NIH3T3 cells. The reproducibility of the fluorescence signal across biological replicates demonstrates the robustness of our transfection protocol. Importantly, fluorescence intensity should not be interpreted as a direct measure of transfection efficiency, but rather as a confirmation of successful transgene expression. Furthermore, because our designed vectors include a luciferase assay module, more rigorous quantitative analysis of transfection efficiency can be performed using luciferase-based measurements.

<Result 3: Verification of NRAS Expression by RT-PCR>

[3-1. Description]

To confirm whether the NRAS^WT and NRAS^G12D plasmids were transcribed after successful transfection, we performed reverse transcription PCR (RT-PCR) followed by agarose gel electrophoresis. GAPDH was used as an internal control to normalize expression levels.

As shown in Figure 2, GAPDH bands were consistently detected in all groups, confirming equal RNA input and successful cDNA synthesis. In control (untransfected) cells, weak endogenous NRAS bands were observed, reflecting the basal expression of the native NRAS gene in NIH3T3 cells. In contrast, cells transfected with NRAS^WT or NRAS^G12D plasmids displayed much stronger NRAS-specific bands (~120–150 bp), confirming robust exogenous expression in addition to the endogenous signal.

Figure 2. Amplified NRAS DNA products by RT-PCR are visualized using agarose gel electrophoresis.

Lane 1: DNA size marker (100–150 bp).
Lane 2–4: Control (empty vector, GAPDH, NRAS^WT, NRAS^G12D).
Lane 5–7: NRAS^WT-transfected cells (GAPDH, NRAS^WT, NRAS^G12D).
Lane 8–10: NRAS^G12D-transfected cells (GAPDH, NRAS^WT, NRAS^G12D).
GAPDH was amplified as an internal control.

Endogenous NRAS expression is detectable in control cells, reflecting the presence of the native NRAS gene in NIH3T3 cells. In transfected groups, exogenous NRAS^WT or NRAS^G12D plasmid expression led to stronger NRAS-specific bands, confirming successful transfection.

[3-2. Interpretation]

These results validate that both NRAS^WT and NRAS^G12D constructs are not only transfected but also actively transcribed in NIH3T3 cells. The absence of NRAS amplification in controls confirms specificity, while consistent GAPDH expression ensures accuracy. Together with Results 1 and 2, this establishes a reliable expression system for downstream functional assays, including viability testing and migration studies.

<Result 4: Effect of NRAS^G12D Mutation on Cell Growth>

[4-1. Description]

To investigate whether the NRAS^G12D mutation influences cell proliferation independently of drug treatment, we monitored the growth of NIH3T3 cells transfected with NRAS^WT or NRAS^G12D plasmids. Cells were seeded at equal densities and incubated for up to 48 hours. Cell numbers were quantified at each time point using the CCK-8 assay, with absorbance measured at 450 nm.

As shown in Figure 3, both groups exhibited an increase in cell numbers over time. However, NRAS^G12D cells displayed a consistently higher growth rate compared to NRAS^WT cells, indicating that the mutation promotes enhanced proliferation under normal culture conditions.

Figure 3. Cell proliferation analysis of CON, WT and G12D groups. Cell proliferation was measured at 0, 24, and 48 h after seeding using the Cell Counting Kit-8 (CCK-8). All groups showed time-dependent increases in proliferation; however, G12D cells exhibited significantly enhanced growth compared with both CON and WT, whereas WT cells displayed the lowest proliferation. Statistical significance was determined using two-tailed unpaired Student’s t-tests (***p < 0.001).

[4-2. Interpretation]

These results demonstrate that the NRAS^G12D mutation alone is sufficient to accelerate cell growth, even without external stimulation or drug treatment. This finding aligns with previous knowledge that oncogenic NRAS mutations drive uncontrolled cell proliferation. Establishing this baseline difference in proliferation is essential for interpreting subsequent experiments, such as drug response and migration assays.

<Result 5: Migration Assay (Scratch Wound Healing Test)>

[5-1. Description]

To evaluate the effect of NRAS^G12D mutation on cell migration, a scratch wound healing assay was performed using NIH3T3 cells transfected with NRAS^WT or NRAS^G12D. A uniform gap (“scratch”) was introduced in confluent cell monolayers, and wound closure was monitored for up to 72 hours.

As shown in Figure 4, control cells exhibited limited closure of the scratch, whereas both NRAS^WT and NRAS^G12D groups showed progressive closure over time. Notably, NRAS^G12D cells closed the wound gap more rapidly than NRAS^WT cells, suggesting that the mutation enhances migratory capacity.

Figure 4. 2D cell migration (1) Representative images of wound healing assays at 0, 24, 48, and 72 h (scale bar = 100 μm). At later time points, NRAS^WT and NRAS^G12D groups showed faster wound closure compared to the control.

[5-2. Interpretation]

These findings indicate that the NRAS^G12D mutation significantly promotes fibroblast migration compared to NRAS^WT. This is consistent with the known role of oncogenic NRAS mutations in activating downstream MAPK signaling, which enhances cytoskeletal rearrangements and motility. By establishing that NRAS^G12D increases both proliferation (Result 4) and migration (Result 5), we provide strong evidence that this mutation drives aggressive cellular behaviors, which are hallmarks of cancer progression.

<Result 6: Lab-on-a-Chip migration>

[6-1. Description]

To investigate how the NRAS^G12D mutation alters invasive potential in a 3D extracellular matrix–like context, we employed a PDMS-based lab-on-a-chip (LOC) system filled with Matrigel. NIH3T3 cells (1×10^5) from control (CON), NRAS^WT, and NRAS^G12D groups were seeded into the upper chamber. After 72 h, cells that penetrated the Matrigel and reached the lower compartment were imaged and quantified. Representative images (Figure 5) showed few migrated cells in the control group, a moderate increase in the NRAS^WT group, and the highest number in the NRAS^G12D group. Quantification (Table 2) confirmed significantly greater migration in NRAS^WT and NRAS^G12D compared to control, with NRAS^G12D displaying the strongest infiltration capacity.

Figure 5. 3D cell migration in LOC assay (1) Final lab-on-a-chip (LOC) device fabricated for 3D migration studies.

Table 2. Quantification of 3D migration in LOC assay. Cells (CON, NRAS^WT, and NRAS^G12D) were seeded at 1×10^5 cells on Matrigel-coated LOC chambers. After 72 h, migrated cells in the lower compartment were counted from multiple microscopic fields and expressed as mean cell number ± SD. Data were normalized to the control group (CON = 100%). Both NRAS^WT and NRAS^G12D groups showed significantly higher migration compared to control, with NRAS^G12D exhibiting the strongest infiltration capacity (p < 0.05, *p < 0.01 vs. CON).

[6-2. Interpretation]

The LOC assay demonstrates that the NRAS^G12D mutation markedly enhances migration in a 3D environment that better mimics the bone marrow extracellular matrix. While NRAS^WT cells exhibited some increase in motility relative to control, the G12D mutation further amplified this effect, consistent with its oncogenic role in promoting invasion. These findings complement the 2D scratch assay, showing consistent trends across both platforms. Importantly, the LOC system provided a physiologically relevant readout of cell infiltration through a tissue-like barrier, underscoring its utility as an alternative to animal models for studying mutation-driven cancer progression.

<Result 7: Drug Response Assay (U0126 Treatment, CCK-8 Viability Test)>

[7-1. Description]

We compared cell viability of three groups—Control (untransfected), NRAS^WT, and NRAS^G12D—with or without the MEK inhibitor U0126 (72 h). Viability was measured by CCK-8 at 450 nm and normalized to each group’s untreated condition.

Figure 6. Drug response to the MEK inhibitor U0126. Cells were treated with U0126 for 72 h, and viability was measured by CCK-8 assay (mean ± SD, n=4). U0126 treatment reduced viability in the control (~20%) and NRAS^WT (~5%) groups, whereas NRAS^G12D cells showed increased viability (~5–7%). One-way ANOVA revealed significant differences among groups (F(2, 9) = 131.27, p < 0.0001), with Tukey’s test confirming significance for all pairwise comparisons.

[7-2. Interpretation]

The data indicate differential sensitivity to MEK inhibition:

Control cells show the expected growth suppression with U0126.
NRAS^WT cells appear drug-sensitive, with viability reduced both at baseline (likely expression burden) and further under U0126.
NRAS^G12D cells display a paradoxical, slight increase in viability with U0126, consistent with tolerance/resilience to MEK inhibition. This may reflect feedback rewiring of RAS–MAPK signaling or compensatory survival pathways (e.g., RAF activation, PI3K/AKT crosstalk), though mechanistic confirmation would require pathway readouts.

Overall, NRAS^G12D shows a pro-survival phenotype under MEK inhibition, contrasting with WT and Control, and aligning with the mutation’s oncogenic character.

Conclusion

In this study, we established an integrated in vitro workflow to investigate how the oncogenic mutation NRAS^G12D shapes cellular phenotypes relevant to multiple myeloma progression. By engineering NIH3T3 cells to express NRAS^WT or NRAS^G12D alongside an mCherry reporter, we validated successful transfection and transcription (Results 1–3), providing a reliable system for functional assays.

Our analyses revealed that:

NRAS^G12D promotes uncontrolled proliferation compared to WT and control cells (Result 4).
The mutation enhances migratory behavior in both 2D scratch wound assays and 3D chip-based infiltration models (Result 5, ongoing LOC experiments).
Under pharmacological challenge with the MEK inhibitor U0126, NRAS^WT and control cells showed growth suppression, whereas NRAS^G12D cells paradoxically maintained or increased viability, highlighting mutation-specific tolerance to MEK inhibition (Result 7).

Together, these findings demonstrate that NRAS^G12D confers a pro-survival, pro-migratory phenotype, consistent with its oncogenic role in hematologic malignancies. Importantly, by combining conventional assays with a PDMS-based lab-on-a-chip that mimics the bone marrow extracellular matrix, we provide a reproducible experimental pipeline that links genetic perturbations to proliferation, motility, and drug response in a tissue-like context.

This platform has the potential to:

Serve as an animal-reducing alternative for mutation-driven cancer research.
Enable rapid testing of AI-nominated drug candidates.
Support future co-culture models incorporating stromal or endothelial cells to better recapitulate the tumor microenvironment.

Overall, our results not only highlight the oncogenic features of NRAS^G12D but also establish a scalable framework for studying gene-driven cancer behaviors in vitro.

Reference

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