Renal cell carcinoma (RCC) is the most common type of kidney cancer and one of the most challenging to treat in advanced stages. While localised tumors can often be treated with surgery, metastatic RCC remains difficult to manage due to its ability to spread and resist therapy.¹ To better understand RCC progression and treatment resistance, researchers are looking at the tumor microenvironment (TME). It is the cross talk of immune cells, stromal cells, extracellular matrix and signaling molecules with tumor cells.² These interactions play a critical role in cancer progression.^3,4

A growing area of interest involves small non-coding RNAs, including PIWI-interacting RNAs (piRNAs). These molecules can be secreted in extracellular vesicles (EVs) such as exosomes, enabling communication with nearby cells. These vesicle-bound RNAs may reprogram the tumor microenvironment to promote tumor survival, cell recruitment, and metastatic potential.¹

Role of Mesenchymal stem cells in RCC and TME

Within the RCC TME, mesenchymal stem cells (MSCs) are of particular interest. Normally, MSCs are recruited to sites of injury or inflammation, where they contribute to tissue repair and immune regulation. However, in RCC, MSCs have been shown to promote tumor growth and migration, and may contribute to drug resistance. How tumors attract and influence MSCs is still being investigated.¹

While the mechanisms of MSC reprogramming are unknown, growing evidence points to tumor-derived EVs, particularly exosomes as important mediators. These vesicles carry regulatory molecules, including small non-coding RNAs such as microRNAs (miRNA) and piRNAs. Once internalized by MSCs, these RNAs can alter gene expression programs and reshape cellular behaviors in ways that support tumor progression.¹

piRNA and microRNA: Regulatory Roles in Cancer

Small non-coding RNAs (sncRNAs) don't code for proteins, but they play powerful roles in regulating gene activity. Two key types, miRNAs and piRNAs are increasingly studied for their roles in cancer progression. Among them, while miRNAs have been long studied5, piRNAs are only beginning to be explored in this context, particularly focusing on how they may influence the tumor microenvironment.

miRNAs guide Argonaute (AGO) proteins to bind messenger RNAs (mRNAs), leading to mRNA degradation or blocking translation.^6,7 piRNAs are typically 21-35 nucleotides in length and associate with PIWI proteins, a subfamily of the AGO family.^1,8 They can act in the nucleus, where they direct epigenetic silencing (eg, through DNA methylation), or post-transcriptional regulation in the cytoplasm1.

Although still an emerging area of study, evidence suggests that cancer cells may use piRNAs to modulate their surrounding environment, potentially by influencing the stromal cell behaviour or possibly modulating immune responses.

The Challenge: Isolating Small RNAs from Complex Media

Exosomes carry valuable molecular signals, including small non-coding RNAs like piRNAs but extracting these RNAs from conditioned media poses challenges. Cell culture supernatants often contain proteins, free nucleases, and other contaminants that can interfere with downstream applications like qPCR and small RNA sequencing. Reliable extraction is essential for capturing low-abundance targets and maintaining RNA integrity.

The Solution: Norgen Biotek's Exosome and RNA Purification Workflow

To overcome these challenges, the researchers implemented a two-step purification protocol using kits from Norgen Biotek.

• Researchers isolated exosomes and their RNA contents using Norgen Biotek's Cell Culture Media Exosome Purification and RNA Isolation Mini kit. This kit enabled purification of exosomes and isolation of RNA.
• Followed by RNA enrichment with RNA Clean-Up and Concentration MicroElute Kit, the resulting RNA was then concentrated to a final elution volume of 20 µL.¹

piRNA Alters Mesenchymal Stem Cell Behavior

The study demonstrated that exosomal piR_004153 is readily taken up by MSCs. Following internalization, piR_004153 localised to the nucleus and induced functional changes in MSCs, notably enhancing their migration and viability, without affecting proliferation. These behavioral shifts were accompanied by modulation of gene expression, including downregulation of FGF2, WISP1, and SLC7A5. FGF2 functions as a key mediator of cellular growth and differentiation across both normal physiology and pathological states.⁹ WISP1 is a well known regulator of MSCs proliferation and differentiation and also influences macrophage behaviour and immune activation by modulating integrin signaling and inducing macrophage migration inhibitory factor via EGFR and Src kinase pathways.^1,10 SLC7A5 (solute carrier family 7 member 5) is a regulator of MSC osteogenic differentiation, and its expression was found to be reduced by piR_004153.¹

Together, these changes suggest that piR_004153 may reprogram MSCs into a more tumor-supportive state, enhancing their ability to contribute to cancer progression.¹

Conclusion: piRNA as a Renal Cancer Biomarker and Diagnostic Tool

The study highlights the potential of piRNAs as biomarkers in renal cell carcinoma, especially when detected in exosomes isolated from biological fluids. This approach could enable non-invasive monitoring of disease progression or response to therapy. Although still in early stages, piRNA-based diagnostics may complement existing microRNA panels and offer insights into molecular dynamics of RCC.

Read the Full Study on piR_004153 in RCC

To explore the experimental details, data visualizations, and complete results, we invite you to read the full publication here.