Role of miRNAs, miR-135b-5p & miR-21-5p in metastasis of cervical cancer

2.1. Cell culture

In this study, two distinct cell lines were employed: the SiHa cell line, which originates from HPV16-positive cervical cancer, and HEK293T, a non-malignant, immortalised human embryonic kidney cell line. Both were sourced from the National Centre for Cell Science (NCCS), Pune, India. Cultures were maintained at 37°C with 5% CO₂ in a humidified incubator. Growth medium comprised Dulbecco’s Modified Eagle Medium (DMEM, Gibco, USA), enriched with 10% foetal bovine serum (FBS) and supplemented with 1% penicillin-streptomycin (Gibco, USA) to maintain cell health and sterility^20,21.

2.2. RNA extraction and quantitative real-time PCR

RNA was extracted from cultured cells using TRIzol reagent (Invitrogen, USA) as per standard protocols. Reverse transcription was conducted using M-MuLV reverse transcriptase (Promega, USA) to generate cDNA for downstream analysis. qRT-PCR was carried out using SYBR Green Master Mix (Applied Biosystems, USA), with B2M as the internal normalisation control gene¹¹. Primer details are provided in supplementary table I.

For profiling of miRNA expression, reverse transcription was performed using the miRCURY® LNA® RT Kit (Qiagen, Germany), followed by qPCR using the associated SYBR Green PCR Kit and miRNA-specific primers (listed in Supplementary Table II). SNORD44 (Qiage, Germany) served as the endogenous control²². Relative expression values were computed using the 2^−dCt method²³.

2.3. Cell transfection

SiHa cells were seeded in 6-, 24-, or 96-well plates (Corning, USA) and cultured for 24 h until reaching 50-70 per cent confluence. Cells were transfected with 50 nM of miRCURY LNA anti-miR-135b-5p (Qiagen, Germany) and/or anti-miR-21-5p (Qiagen, Germany) alone or in combination (50nM each for dual inhibition). A scrambled RNA (negative control A, Qiagen, Germany) was used as a control. Transfection was performed with Lipofectamine 3000 (Invitrogen, USA) in Opti-MEM (Gibco, UK) according to the manufacturer’s protocol^17,19. To better understand the functional roles of miR-135b-5p and miR-21-5p in cervical cancer progression, their individual and combined effects were assessed in SiHa cells using specific inhibitors targeting each miRNA. Initial optimisation of inhibitor concentration and time points was performed using two doses (25 nM and 50 nM) of anti-miR-135b-5p and anti-miR-21-5p, based on previously published studies^17,19. At first, the effect on LIMD1 and VHL mRNA expression were assessed at 6, 24, 48, and 72 h post-treatment (Supplementary Figs. 1A and B). All experiments were performed in triplicate.

2.4. Colony formation assay

To evaluate the clonogenic potential of SiHa cells, 1,000 cells per well were plated in 6-well plates. After an initial 48-h transfection period, the medium was replaced and refreshed every two days. After 14 days, colonies were fixed with 4% paraformaldehyde and stained using 0.05% crystal violet. Colony numbers were determined via ImageJ software²⁴.

2.5. Cell cycle analysis

SiHa cells (3×10⁵/well) were seeded in 6-well plates and synchronised with thymidine (2mM) for 18 h, followed by an 8-h release in prewarmed DMEM. A second thymidine treatment (2mM) was then administered for another 18 h²⁵. After 1X PBS washing, cells were transfected for 48 h, followed by 70% ice-cold ethanol fixation at 4°C for 24 h. Following RNase treatment cell cycle distribution was analyzed by adding 30µL of 0.05g/L propidium iodide (Sigma, USA) staining via flow cytometry (BD Biosciences, Germany)²⁶.

2.6. Apoptosis assay

To evaluate apoptosis, SiHa cells (3×10⁵ per well) were plated in 6-well plates and incubated for 24 h. Post 48-h transfection, apoptotic cells were quantified using the Annexin V-FITC Apoptosis Detection Kit (Sigma-Aldrich, Germany) as per supplier’s instructions²⁷. Flow cytometric analysis was conducted using BD Biosciences instruments, and data were processed with FlowJo software.

2.7. Cell viability assay

The viability of SiHa cells was determined via the MTT assay (Himedia)¹⁴. In this protocol, 1×10⁴ cells per well were plated in a 96-well format and incubated for 24 h. After 48 h of transfection, 5 mg/mL MTT reagent was introduced and incubated at 37°C for 4 h. Resulting formazan crystals were dissolved in 100 µL of DMSO, and the absorbance was recorded at 570 nm using the MULTISKAN SkyHigh microplate reader (Thermo Scientific, USA).

2.8. Wound healing assay

To study cell migration, a scratch assay was performed. SiHa cells were grown in 24-well plates until they reached ∼80% confluency. After 48-h transfection, a straight scratch was made across the cell monolayer using a sterile 200 µL pipette tip¹¹. Images of the wound were captured at 0, 24, and 48 h using an inverted microscope, and wound closure was analysed using ImageJ.

2.9. Invasion assay

The invasive capacity of SiHa cells was evaluated using the CytoSelect™ cell invasion assay kit (Cell Biolabs, USA) in a 24-well format, adhering to the manufacturer’s instructions. Absorbance was measured at 560 nm using the MULTISKAN SkyHigh reader (Thermo Scientific, USA)²⁸.

2.10. Western blot analysis

After 48 h of transfection, cells were lysed using RIPA buffer (Sigma Aldrich, Germany). The lysate was then quantified, followed by analysis of target protein expression by western blot based on the standard protocol of Roy et al²⁹, 2019. Details regarding the primary and secondary antibodies along with their respective dilutions, were provided in supplementary table III. Protein bands were quantified with ImageJ with peak densities were normalised to α-tubulin and represented relative to the control group²⁹.

2.11. Luciferase reporter assay

To investigate whether miR-135b-5p targets the LIMD1 gene, luciferase reporter vectors were engineered containing either the wild-type or mutated 3′UTR region of LIMD1. Mutations were introduced using the QuickChange mutagenesis kit (Agilent, USA), and constructs were cloned into the psiCHECK2 vector. Co-transfection was performed with either anti-miR-135b-5p or scrambled RNA in SiHa cells using lipofectamine 3000. After 48 h, luciferase activity was assessed using the dual-luciferase reporter assay kit (Promega, USA), and normalised Renilla to Firefly ratios were calculated to quantify miRNA-mediated repression¹⁷.

2.12. Statistical analysis

Data were analysed by comparing experimental groups (anti-miR-135b-5p, anti-miR-21-5p, and dual inhibition) to the scrambled RNA control. Statistical significance was evaluated using a two-tailed unpaired t-test. A P<0.05 was considered statistically significant. All data are presented as mean ± standard deviation (SD). All assays were performed in triplicate to ensure reproducibility.

3.1. miR-135b-5p and miR-21-5p dual upregulation perturbs LIMD1 and VHL function in SiHa cells

A significant upregulation of miR-135b-5p and miR-21-5p was observed in SiHa cells compared to HEK293T cells (P=0.002 and 0.001, respectively) (Figs. 1A and B). In contrast, the expression levels of their respective target genes, LIMD1 and VHL, were significantly downregulated in SiHa cells (P=0.012 and 0.003) (Figs. 1C and D). These findings indicate that LIMD1 and VHL expression might be regulated by their respective targeting miRNAs, miR-135b-5p and miR-21-5p. When luciferase activity was measured, cells transfected with the WT LIMD1 3' UTR exhibited a significant increase in relative luciferase activity following miR-135b-5p inhibition (P=0.012), compared to control cells (Fig. 1E). In contrast, mutated LIMD1 3’-UTR abolished this effect, indicating direct and specific binding of miR-135b-5p to the LIMD1 3′-UTR, thereby regulating LIMD1 mRNA expression. Both miRNA inhibitors demonstrated maximal efficacy at 50 nM after 48 h, as evidenced by a significant restoration of LIMD1 and VHL mRNA levels. (P=0.004 and P=0.003 for LIMD1; P=0.003 and P=0.002 for VHL in individual and dual inhibition settings, respectively) (Fig. 1F). These mRNA changes were corroborated at the protein level (Supplementary Figs. 1C and D). Since, LIMD1 and VHL are known to regulate HIF-1α stability, we hypothesised that inhibition of miR-135b-5p and miR-21-5p would affect HIF-1α expression. Indeed, immunoblot analysis revealed a significant reduction in HIF-1α levels following individual inhibition of miR-135b-5p (P=0.004) and miR-21-5p (P=0.003). Notably, a further decrease in HIF-1α expression was observed with dual inhibition (P=0.001), compared to scrambled RNA treated control cells (Fig. 1G).

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Fig. 1.

Transfection efficiency of anti-miR-135b-5p and anti-miR-21-5p was evaluated by analysing the expression alteration of their respective targets, LIMD1 and VHL in SiHa cells: (A-D) The mRNA expression of LIMD1 and VHL and the corresponding miRNAs (miR-135b-5p and miR-21-5p) were analysed in SiHa and HEK293T cell lines by real-time PCR, using B2M and SNORD44 as endogenous controls for mRNA and miRNA, respectively. (E) The LIMD1 3′UTR containing the predicted miR-135b-5p binding site was cloned into luciferase reporter constructs (wild-type and mutant) to validate direct targeting. SiHa cells were co-transfected with anti-miR-135b-5p or anti-miR-NC for 48 h prior to luciferase assay. (F) SiHa cells were transfected with anti-miR-135b-5p and anti-miR-21-5p, individually and in combination, followed by real-time PCR analysis of LIMD1 and VHL mRNA expression using B2M as control. (G) HIF-1α protein levels were assessed by Western blot after individual and combined inhibition of miR-135b-5p and miR-21-5p. (All data are presented as mean with standard deviation (SD), where Error bars represent the SD; P ^*<0.05, ^**<0.01, ^***<0.001, respectively).

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3.2. The dual upregulation of miR-135b-5p and miR-21-5p promotes proliferation of SiHa cells

Transfection of anti-miR-135b-5p and anti-miR-21-5p into SiHa cells resulted in a significant reduction in colony numbers compared to scrambled-RNA-treated control cells (P=0.0002 and P=0.0003, respectively) (Fig. 2A and Supplementary Fig. 2A). Dual inhibition of both miRNAs led to a more pronounced decrease in colony number (P<0.0001), suggesting a synergistic effect on suppressing cell proliferation. Given these findings, it was hypothesized that miR-135b-5p and miR-21-5p may contribute to cell cycle dysregulation through suppression of LIMD1 and VHL. To test this hypothesis, double thymidine treated G0/G1 synchronized cells were transfected separately with anti-miR-135b-5p and anti-miR-21-5p.Interestingly, a significant increase (P=0.024 and P=0.029, respectively) in the number of cells in the G1 phase was observed, indicating G1-S cell cycle arrest (Fig. 2B and Supplementary Fig. 2B). This cell cycle arrest was further corroborated by a marked decrease in Cyclin D1 protein expression, a key regulator of G1-S progression, in cells treated with anti-miR-135b-5p and anti-miR-21-5p (P=0.002 and P=0.012, respectively) (Fig. 2C). This data indicates that miR-135b-5p and miR-21-5p promote cellular proliferation.

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Fig. 2.

Effect of miR-135b-5p and miR-21-5p inhibition on proliferation of SiHa cells. Cells were transfected with anti-miR-135b-5p, anti-miR-21-5p, both anti-miRs, or anti-miR negative control (anti-miR-NC). (A) Colony formation assay was performed to assess proliferative potential following individual or combined anti-miR transfection. Colonies were quantified using ImageJ software. (B) Cell cycle distribution was analysed by flow cytometry after 48 h of transfection. Percentage of cells in different phases of the cell cycle was calculated from flow cytometry data. (C) Cyclin D1 protein expression was evaluated by Western blot using α-tubulin as loading control. Band intensities were quantified using ImageJ software (All data are presented as mean with standard deviation (SD), where Error bars represent the SD; P ^*<0.05, ^**<0.01, ^***<0.001, respectively).

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3.3. Hyper proliferative SiHa cell escapes apoptosis

To further assess the impact of miR-135b-5p and miR-21-5p on apoptosis in SiHa cells, the data showed a significant increase (P=0.017 and P=0.03, respectively) in the total apoptotic cell populations (both early and late apoptotic) compared to the scrambled-RNA treated cells (Fig. 3A and Supplementary Fig. 2C). Notably, dual inhibition of both miRNAs showed a further significant increase (P=0.007) in cellular apoptosis, compared to the control cells (Fig. 3A and Supplementary Fig. 2C). These observations indicate that upregulation of either miR-135b-5p or miR-21-5p allows SiHa cells to evade apoptosis, with a more pronounced anti-apoptotic phenotype observed when both miRNAs are co-expressed. Conversely, their inhibition significantly reduced SiHa cell viability, as assessed by MTT assay. Anti-miR-135b-5p and anti-miR-21-5p treatments individually caused marked reductions in cell viability (P=0.002 and P=0.001, respectively), while dual inhibition produced an even greater effect (P=0.0002), aligning with the apoptotic data (Fig. 3B). Molecular analysis further supported these findings. Inhibition of miR-135b-5p and miR-21-5p significantly upregulated the expression of proapoptotic Bax (P=0.008 and P=0.002. respectively), downregulated the expression of anti-apoptotic Bcl-2(P=0.007 and P=0.004, respectively) and elevated the Bax/Bcl-2 ratio (P=0.008 and P=0.003, respectively) (Figs. 3C-F). Moreover, dual inhibition of these two miRNAs resulted in a more pronounced increase in Bax expression (P=0.0008), a greater reduction in Bcl-2 expression (P=0.0003) and a significantly higher Bax/Bcl-2 ratio (P=0.002) relative to control cells (Figs. 3C-F). Thus, our findings suggest that SiHa cells with upregulated miR-135b-5p and miR-21-5p effectively evade cellular apoptosis.

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Fig. 3.

Effect of miR-135b-5p and miR-21-5p inhibition on apoptosis of SiHa cells. Cells were transfected with anti-miR-135b-5p, anti-miR-21-5p, a combination of both, or anti-miR negative control (anti-miR-NC). (A) Apoptosis was evaluated by flow cytometry to determine early and late apoptotic populations. The total percentage of apoptotic cells (early+late) was analysed using FlowJo software. (B) Cell viability was assessed by MTT assay. (C) Protein expression of Bax and Bcl-2 was examined by Western blot using α-tubulin as a loading control. Band intensities for (D) Bax, (E) Bcl-2, and (F) Bax/Bcl-2 ratio were quantified using ImageJ software (All data are presented as mean with standard deviation (SD), where Error bars represent the SD; P ^*<0.05, ^**<0.01, ^***<0.001, respectively).

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3.4. Dual miRNA upregulation modifies metastatic potential of SiHa cells

Knockdown of either miR-135b-5p or miR-21-5p, significantly impaired the wound healing ability of SiHa cells, as indicated by reduced wound closure percentages at 48 h (P=0.004 and P=0.002, respectively), compared to the control group (Fig. 4A and Supplementary Fig. 3A). Notably, co-inhibition of both miRNAs led to a more pronounced impairment in wound healing, with significant reductions observed at both 24 h (P=0.003) and 48 h (P=0.0004) (Fig. 4A and Supplementary Fig. 3A). Invasion assays showed that anti-miR-135b-5p and anti-miR-21-5p each significantly reduced the number of invading SiHa cells (P=0.016 and P=0.021), while dual inhibition produced an even greater suppression (P=0.004), indicating a synergistic role of these miRNAs in promoting cervical cancer cell motility and invasiveness (Fig. 4B and Supplementary Fig. 3B).

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Fig. 4.

Effect of miR-135b-5p and miR-21-5p inhibition on EMT of SiHa cells. Cells were transfected with anti-miR-135b-5p, anti-miR-21-5p, both anti-miRs, or anti-miR negative control (anti-miR-NC). (A) Cell migration was evaluated using a wound-healing assay at 0, 24, and 48 h post-transfection. Wound closure rate was quantified using ImageJ software. (B) Cell invasion was analysed using the CytoSelect™ 24-well cell Invasion Assay Kit. The number of invasive cells was quantified and plotted. (C) Protein expression of E-cadherin and N-cadherin was assessed by Western blot with α-tubulin as a loading control. (C-E) Band intensities for (D) E-cadherin and (E) N-cadherin were quantified using ImageJ software. (All data are presented as mean with standard deviation (SD), where Error bars represent the SD; P ^*<0.05, ^**<0.01, ^***<0.001, respectively).

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3.5. Enhanced metastatic potential of SiHa cell is rendered by Epithelial-Mesenchymal transition

To elucidate the role of miR-135b-5p and miR-21-5p in regulating epithelial-to-mesenchymal transition (EMT) in SiHa cells, the data showed a significant increase in E-cadherin, a hallmark epithelial protein, following individual or combined inhibition of these miRNAs (P=0.042, P=0.03, and P=0.021, respectively). Concurrently, there was a significant decrease in the mesenchymal marker N-cadherin (P=0.036, P=0.04, and P=0.018, respectively), suggesting a reversal of EMT (Figs. 4C-E).

These observations indicate that blocking miR-135b-5p and miR-21-5p suppresses EMT-associated changes, highlighting their pro-EMT roles in cervical cancer progression through regulation of epithelial and mesenchymal marker expression.

3.6. HIF-1α stabilisation induces metastasis via its downstream target gene expression

Several HIF-1α-regulated genes—many of which serve as potential tumour biomarkers—were significantly downregulated after treatment with anti-miR-135b-5p or anti-miR-21-5p. These include the growth factor, TGF-α (P=0.003 and P=0.003, respectively), proteolysis markers of extracellular matrix remodelling during wound healing, MMP9 (P=0.004 and P=0.005, respectively); and the G1-S phase cell cycle regulator, Cyclin D1 (P=0.001 and P=0.003, respectively) (Figs. 5A-C). Notably, dual inhibition produced an even more pronounced suppression of TGF-α (P=0.002), MMP9 (P=0.002), and Cyclin D1 (P=0.0004) expression at the mRNA level (Figs. 5A-C). The expression of the angiogenic factor VEGF was also significantly reduced (P=0.002) upon dual inhibition of both miRNAs compared to control cells (Supplementary Fig. 4A). The mRNA expression was further validated at the protein level, revealing a statistically significant reduction in the expression of MMP9 (P=0.0003) and Cyclin-D1 (P=0.003) in the dual inhibitor-treated group (Supplementary Figs. 4B and C). A significant reduction of VEGF protein (P=0.012, P=0.008, and P=0.002, respectively) were also found following treatment with anti-miR-135b-5p, anti-miR-21-5p, or the dual inhibition of both miRNAs, when compared to control cells (Fig. 5D). HIF-1α functions as an upstream activator of Snail and Slug, both of which are key transcription factors involved in regulating genes responsible for EMT, such as Vimentin. Our data demonstrated a significant reduction in Snail, Slug, and Vimentin expression (P=0.018, P=0.003, and P=0.002, respectively) following dual inhibition compared to control cells (Supplementary Figs. 4D-F). These findings suggest that miR-135b-5p and miR-21-5p may contribute to cervical cancer progression by promoting the expression of HIF-1α target genes.

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Fig. 5.

Effect of miR-135b-5p and miR-21-5p inhibition on HIF-1α target gene expression. Cells were transfected with anti-miR-135b-5p, anti-miR-21-5p, both anti-miRs, or anti-miR negative control (anti-miR-NC). The mRNA expression of HIF-1α downstream target genes (A) TGF-α, (B) MMP9, and (C) Cyclin D1—was analysed by real-time PCR using B2M as the endogenous reference control. (D) VEGF protein expression was evaluated by Western blot with α-tubulin as a loading control, and band intensities were quantified using ImageJ software. (All data are presented as mean with standard deviation (SD), where Error bars represent the SD; P ^*<0.05, ^**<0.01, ^***<0.001, respectively).

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