Colony Formation Assay Detecting the Proliferative Capacity of LncRNA-knockdown Osteosarcoma Cells

Summary

This experimental protocol provides a stable, cost-effective, and efficient method that combines lncRNA knockdown with colony-forming assays to quantify the effect of lncRNA on osteosarcoma cell proliferation.

Abstract

Aberrant long non-coding RNAs (lncRNAs) expression profoundly influences cellular proliferation of osteosarcoma cells, suggesting that they may serve as potential therapeutic targets. However, functional studies of lncRNAs remain largely theoretical and limited to a few validations. Colony formation assays, which specifically reflect the ultimate proliferative fate of single cells, are regarded as the gold standard for evaluating long-term proliferative potential. Here, this protocol established a reliable experimental protocol using colony formation assays in lncRNA-knockdown cells to evaluate the effect of lncRNA on osteosarcoma cell proliferative capacity. This experimental protocol provides a stable, cost-effective, and efficient assay to assess the regulatory effects of lncRNA on osteosarcoma cell proliferation. This approach is not only suitable for research on osteosarcoma cells but also serves as a feasible tool for lncRNA-regulated cell proliferative capacity in other tumors. This study included a detailed detection protocol of lncRNA-knockdown and colony formation assays, with an example of the small nucleolar RNA host gene 6 (SNHG6) knockdown in osteosarcoma cells (143B). Experimental results confirmed efficient suppression of SNHG6 expression, accompanied by a marked reduction in both the number and size of colonies. These findings suggest that SNHG6 is essential for sustaining the long-term proliferative potential of osteosarcoma cells (143B).

Introduction

Long non-coding RNAs (lncRNAs), defined as transcripts longer than 200 nucleotides without protein-coding potential, have emerged as important regulators of diverse biological processes in recent years^1,2. They play critical roles in the initiation and development of various cancers, including osteosarcoma, prostate cancer, and lung cancer^3,4,5. In osteosarcoma, accumulating evidence suggests that aberrant long non-coding RNA (lncRNA) expression has a profound influence on cellular proliferation. For instance, silencing lncRNA POU3F3 significantly suppressed the proliferation and promoted the apoptosis of osteosarcoma cells (MG63 and U2OS)⁶. Similarly, knockdown of lncRNA HOXA-AS3 reduced colony formation and inhibited growth in U2OS and SW1353 cells⁷. Given that uncontrolled proliferation is a hallmark of cancer biology and a major determinant of therapeutic strategies, elucidating lncRNA-regulated cell proliferation in tumors, particularly in osteosarcoma, remains of great importance and necessity.

Although several approaches exist to evaluate cell proliferation, each carries inherent limitations. Colorimetric or metabolic assays, such as CCK-8 and MTT, are convenient, rapid, and high-throughput. However, they primarily provide short-term readouts and are easily confounded by metabolic fluctuations, and have additional risks of optical or photochemical interference from the reagents used⁸. DNA synthesis-based methods, including BrdU and EdU incorporation, offer greater specificity while limiting to snapshot analyses of S-phase entry without reflecting long-term proliferative outcomes^9,10. Similarly, immunostaining for proliferation markers, such as Ki-67, only indicates instantaneous cell cycle activity and fails to predict clonogenic capacity¹⁰. Real-time monitoring systems, such as RTCA, enable continuous growth curve analysis with high reproducibility, yet their high cost and restriction to short- or intermediate-term assessment reduce their practicality¹¹. By contrast, the clonogenic assay uniquely captures the capacity of a single cell to undergo unlimited proliferation and form visible colonies, thereby directly reflecting its ultimate fate. Despite being labor-intensive and time-consuming, the clonogenic assay remains the gold standard for assessing long-term proliferative potential, offering insights that are unattainable by short-term or surrogate assays¹².

Here, this article includes a detailed detection protocol for lncRNA-knockdown and colony formation assays, with an example of SNHG6-knockdown in 143B osteosarcoma cells. This approach is not only applicable to osteosarcoma research but also serves as a feasible tool for investigating lncRNA-mediated proliferation in other tumors.

Protocol

The reagents and the equipment used are listed in the Table of Materials.

1. Plasmid transfection

1. Seed 5 × 10⁵ 143B cells per 10 cm culture dish (total two dishes), culture the cells in complete medium (DMEM + 10% FBS) at 37 °C in a 5% CO₂ incubator.
2. Prepare transfection reagents 24 h later, including plasmids (shNC and shSNHG6), PEI, and Opti-MEM.
   NOTE: Vortex and ensure all are equilibrated to room temperature before use.
3. Take out four 1.5 mL microcentrifuge tubes, label the plasmids (shNC and shSNHG6) and PEI (two tubes) using a marker pen.
4. Add 500 µL of Opti-MEM to each tube.
5. Add 10 µg of plasmids to the plasmid tube (shNC or shSNHG6) and add 30 µL of PEI (1µg/µL) to each PEI tube.
6. Mix gently up and down, incubate at room temperature for 5 min.
7. Combine the plasmid tube and PEI tube, mix gently, and incubate the mixture at room temperature for 20 min.
8. Take out 143B cells from the incubator and aspirate the culture medium gently. Add 7 mL of serum-free medium to each dish. Label the dishes to indicate the plasmids to be added (143B shNC or 143B shSNHG6).
9. Add the pre-prepared transfection mixture dropwise to the corresponding culture dish.
   NOTE: Add the mixture slowly, using a pipette in a circular motion, and evenly dispense it from the outer edge toward the center of the dish. Incubate at 37 °C in an incubator with 5% CO₂.
10. Discard the supernatant 8-12 h later, and add 10 mL of complete DMEM medium to each 10 cm culture dish. Incubate the cells at 37 °C in a 5% CO₂ incubator.
11. Observe under fluorescence microscope at 72 h after the supernatant is discarded and replaced with complete DMEM medium.

2. Colony formation assay

1. Remove the medium from the 10 cm culture dish (shNC and shSNHG6), add 1.5 mL trypsin-EDTA to digest the cells, gently pipette to detach them, and then add 5mL of complete medium to terminate the digestion.
2. Transfer the cell suspension into a 15 mL microcentrifuge tube.
3. Take out 20 µL of cell suspension and count cell numbers.
   NOTE: The cell count must be performed multiple times to ensure that the cell numbers in the shNC group and the shSNHG6 group are consistent. Here, we collect 3 × 10⁵ cells for RT-PCR verification of SNHG6 knockdown efficiency.
4. Seed 2 × 10³ cells per well in a 6-well plate. Add complete DMEM medium to a final volume of 2 mL and culture the cells at 37 °C in a 5% CO₂ incubator.
   NOTE: Replace the medium with fresh complete DMEM medium every other day.
5. Monitor colony formation daily.
   NOTE: Terminate the culture when most colonies in the control (shNC) group contain ≥50 cells, or after 10-14 days of incubation, whichever comes first.
6. Take out cells from the incubator and discard the supernatant.
7. Wash cells twice with 1 mL of PBS per well, and discard the PBS after each wash.
8. Fix cells with 1 mL of 10% formalin solution for 15 min, then discard the 10% formalin solution.
9. Wash cells twice with 1 mL of PBS per well, and discard the PBS after each wash.
10. Stain cells with 1 mL of 0.5% crystal violet staining solution for 10-30 min, then discard the staining solution.
11. Wash cells gently with slowly running tap water.
12. Air-dry cells at room temperature.
13. Capture images using a camera.

Results

To evaluate the effect of lncRNA SNHG6 on the proliferative capacity of osteosarcoma cells, we successfully established a plasmid-mediated knockdown system and performed colony formation assays. The experimental workflow included plasmid transfection of 143B cells and colony formation assay (Figure 1).

Transfection efficiency of 143B cells was confirmed by fluorescence microscopy 72 h after transfection (Figure 2). The results showed ...

Discussion

Therapeutically targeting cancer-associated lncRNAs, in addition to leveraging their diagnostic utility as biomarkers, represents an emerging approach in cancer management. Defining the roles of lncRNAs in osteosarcoma pathogenesis is crucial for advancing its diagnosis, therapy, and prognosis.

Critical steps must be followed to ensure the success of this assay. First, establishing a single-cell suspension is an absolute prerequisite for reliable clonal assays, as the presence of cell aggregat...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1.5 mL MICROTUBES	Merck	AXYMCT150C
10% Neutral Buffered Formalin	VICTREX	5205000
100 - 1000 μL Extended Graduated Blue Tips	Shanghai BioScience Co., Ltd	BS10401ML
100mm Cell Culture Dishes laboratory	Wuxi NEST Biotechnology Co.,Ltd	704001
143B	ATCC	ATCC CRL-8303TM
15 mL Centrifuge Tube	Shanghai BioScience Co., Ltd	BL2002150
6 Well Cell Culture Plate	Wuxi NEST Biotechnology Co.,Ltd	703001
CO2 incubator	Thermo Fisher Technology (China)Co., Ltd.	300583057
Crystal Violet	TCI Shanghai	C0428
DMEM medium	WISENT CORPORATION	319-005-CL
Eppendorf Research plus	Eppendorf	3123000233
FBS - Superior quality	WISENT CORPORATION	086-150
Mark pen	Zebra Trading (Shenzhen) Co.,Ltd.	YYST5
Opti-MEM	Gibco	31985070
PASTEUR PIPETTE	Labgic	BS-XG-O3L
PBS buffer (dry powder)	Labgic	BL601A
PEI	Polysciences	24765
Pipette tips	Merck	AXYT200Y
TrypLE Express Enzyme	Gibco	12604021
Trypsin/EDTA	Gibco	R001100