Knockdown of FAM83A to Verify Its Role in Cervical Cancer Cell Growth and Cisplatin Sensitivity

Summary

Here, we show the procedures for FAM83A knockdown; the assays to detect its effects on proliferation, migration, and invasion of cervical cancer cells; and the sensitization of these cells to cisplatin. This study provides a promising target gene for cervical cancer and a reference for further drug research.

Abstract

The exploration of tumor target genes holds paramount importance for the prevention and treatment of cervical cancer. In this study, we outline the steps involved in the identification of a tumor target gene FAM83A in cervical cancer. First, the Cancer Genome Atlas dataset was employed to validate the expression and prognostic significance of FAM83A in women. A small interfering RNA (siRNA) was used for knockdown of the FAM83A gene in HeLa and C33a cells. Next, 5-ethynyl-2'-deoxyuridine (EdU) staining was conducted to determine the effects on the proliferation capabilities of the tumor cells. Wound healing and porous membrane insert assays were performed to evaluate tumor cell migration and invasion abilities.

Western blotting was used to quantify apoptosis-related protein levels. JC-1 staining was employed to evaluate mitochondrial function alterations. Furthermore, cisplatin (diaminedichloroplatinum, DDP) intervention was used to assess the therapeutic potential of the target gene. Flow cytometry and colony formation assays were conducted to further validate the anticancer characteristics of the gene. As a result, FAM83A knockdown was shown to inhibit the proliferation, migration, and invasion of cervical cancer cells and sensitize these cells to cisplatin. These comprehensive methodologies collectively validate FAM83A as a tumor-associated target gene, holding promise as a potential therapeutic target in the prevention and treatment of cervical cancer.

Introduction

Cervical cancer is a global concern as it is one of the leading types of gynecological malignancy worldwide and is the major cause of cancer-related mortality in women¹. Radical surgery and chemoradiotherapy are associated with high cure rates at the primary stage. However, treatment outcomes for patients at the advanced stage of cervical cancer who develop metastatic disease are very unfavorable². Therefore, it is crucial to further understand the biological mechanisms underlying the migration and invasion of cervical cancer cells and identify potential therapeutic targets for the prevention and treatment of this disease.

Identifying target genes involved in cancer progression and finding ways to inhibit their expression or action present promising treatment options. In this study, we identified FAM83 as a cancer-causing gene and further investigated its inhibitory effects on C33a and HeLa cells. FAM83 family oncogenes (FAM83A-H) are widely reported in human cancers^3,4. Recently, FAM83A was reported to be upregulated in lung⁵, breast⁶, ovarian⁷, and pancreatic⁸ cancers,indicating that FAM83A plays an important role in cancer progression via promoting the proliferation, invasion, stem-cell-like traits, and drug resistance in the tumor cells. Importantly, FAM83A was identified as one of the novel candidate genes associated with cervical lesion progression and carcinogenesis⁹. Despite the confirmation of elevated FAM83A expression in human cervical cancer cells, the specific impact and underlying mechanisms of FAM83A in cervical cancer remain unclear.

In this study, we outline the protocols involved in the identification of FAM83A as a tumor target gene in cervical cancer and use a small interfering RNA (siRNA) for the knockdown of the FAM83A gene in HeLa and C33a cells. 5-Ethynyl-2'-deoxyuridine (EdU) staining was performed to determine the effects on tumor cell proliferation, while wound healing and porous membrane insert assays helped evaluate tumor cell migration and invasion abilities.

Western blotting was performed to determine the levels of apoptosis-related proteins, and JC-1 staining was employed to evaluate mitochondrial function alterations. Thus, we reported that FAM83A plays a critical role in cell proliferation, metastasis, and invasion in cervical cancer. Through PI3K/AKT-pathway-associated mitochondrial dysfunction and apoptosis, FAM83A knockdown sensitized cervical cancer cells to cisplatin (diaminedichloroplatinum, DDP). This study provides a new target for cervical cancer and possibly other cancers and a reference for the development of strategies to overcome the resistance of cancer cells to certain chemotherapeutic drugs.

Protocol

The study was completely in conformity with the publication guidelines provided by TCGA (https://cancergenome.nih.gov/publications/publicationguidelines). See the Table of Materials for details related to all materials, reagents, and instruments used in this protocol.

1. Data source and bioinformatics analysis

1. Obtain RNA sequencing data from the Cancer Genome Atlas (TCGA) database (https://cancergenome.nih.gov) for cluster analysis. Use the GEPIA (http://gepia.cancer-pku.cn/), a web-based tool to re-compute raw RNA-Seq data of samples from TCGA projects, to screen for candidate cancer drug targets with a standard processing pipeline.
   NOTE: The GEPIA website is freely available to all users.
2. Access the GEPIA website homepage and click on Cancer Type Analysis, select the Differential genes analysis option, and set the following parameters: Cancer name = CESE (defined as Cervical squamous cell carcinoma and endocervical adenocarcinoma on this webpage), |Log2FC| Cutoff = 1, q-value Cutoff = 0.01, Differential Methods = ANOVA, and Chromosomal Distribution = both. Then, click on List to obtain a gene list that shows differential expression between tumor and normal groups.
   NOTE: It requires an extensive literature review to identify differentially expressed candidate genes. In this study, considering the background from the available literature, we focus on the tumor gene of interest, FAM83A.
3. Then, examine the expression of FAM83A in cervical cancer and normal tissues using GEPIA by proceeding to the Expression DIY option on the website and select Boxplot as the chart type. Input FAM83A in the gene parameter field and set the following parameters: |Log2FC| Cutoff value = 1; q-value Cutoff = 0.01. Select CESE as the cancer name and Match TCGA normal data for the Matched Normal data field, leaving all other settings at their default. Click on Plot and allow the website to generate a boxplot representing the differential expression of the FAM83A gene; save the boxplot for future reference and analysis.
4. Finally, analyze the overall survival of patients bearing tumors with different levels of FAM83A expression in cervical cancer. Navigate to the Survival Plots option on the website, set the Gene to FAM83A, and select Overall Survival as the analysis type. Add the tumor name CESE, choose Months for the Axis Units, and leave all other parameters at their default settings. Click Plot and let the website generate a survival curve chart; save this chart for future reference and analysis.
5. Take into account both the differential expression of FAM83A in tumors and its correlation with patient survival analysis to identify FAM83A as a potential therapeutic target gene in cervical cancer.

2. Cell-based experiments

1. Cell culture
   1. Culture human tumor cell lines in the modified medium at 37 °C in a humidified 5% CO₂ atmosphere.
      NOTE: Refer to the Table of Materials for cell line information and culture medium components.
2. Cell transfection
   1. Use siRNA to knock down FAM83A expression in the cells.
      NOTE: See Table 1 for the specific siRNA sequence.
   2. Seed cells in 6-well plates and incubate with the mixture of 50 nM si-FAM83A or siRNA-NC and 8 µL of transfection agent or only 8 µL of transfection agent for 4-6 h after attaining 50%-70% confluency. Add only serum-free DMEM to the negative control.
   3. After the incubation, replace the medium containing the transfection agent with DMEM + 10% fetal calf serum. Further, 48 h after transfection, treat with 5 µM cisplatin (DDP) for 24 h as intended and harvest all the cells for total RNA extraction and qRT-PCR analysis.

3. EdU detection for cell proliferation assay

NOTE: Use the EdU Cell Proliferation Kit to assess cell proliferation in vitro according to the manufacturer's instructions.

1. Seed the cells in 6-well plates and incubate them with 10 µM EdU solution for 2 h. Fix the cells with 4% paraformaldehyde for 15 min at room temperature (RT) and permeabilize with 0.3% Triton X-100 in PBS for 10 min at RT.
2. Remove the permeabilization buffer and add 500 µL of 1x reaction solution. Then, incubate for 30 min at RT in the dark for nuclear staining.
3. Stain cells with 4',6-diamidino-2-phenylindole (DAPI) and visualize them under a fluorescence microscope at 200x magnification. Stain the nuclei of the proliferating cells with EdU and examine them for red fluorescence.
4. Finally, use software to quantify the results by counting at least 10 random fields and calculate the proliferation rates using the ratio of the fluorescent-positive cells to total cells.

4. Wound healing assay

1. Seed cells in 6-well plates at a density of 2 × 10⁵ cells per well.
2. When the cells attain 90% confluency, use a sterile 10 µL micropipette tip to gently create a linear scratch in the cell monolayer. Gently rinse the wells with sterile PBS to remove any detached cells and debris caused by scratching.
3. Further, incubate the cells in medium containing 1% FBS. Then, observe and take images with the inverted microscope for the healing of the wounded region at 12, 24, and 48 h.

5. Porous membrane insert assay

1. Prepare a cell suspension in cell culture medium at the desired cell concentration (containing 4 × 10⁴ cells), including transfected or control C33a cells and HeLa. Add the cell suspension to the top chamber of the membrane inserts, ensuring even distribution. Add complete medium to the lower chamber.
2. After incubation for 24 h, use methyl alcohol and 0.1% crystal violet for the fixation and staining of the cells migrating to the lower chambers, respectively.
3. Use an inverted microscope to take images and then analyze the number of migrating cells by counting at least 10 random fields with ImageJ.
   1. For the number analysis with ImageJ, open the image in ImageJ software, go to the Image tab in the tool menu, select Adjust, and then click on Threshold. Adjust the threshold settings, if necessary, until only the cells are visible against a white background.
   2. Click on Apply in the Threshold window to apply these settings to the image. Now, select the Wand (tracing) tool from the toolbar (located at the top of the window).
   3. Click on the area of the image where the cells are located to select all the cells in the thresholded image. After the cells are highlighted, go to Analyze in the Tool menu and then, click on Analyze Particles. In the Analyze Particles window, enter the desired size (e.g., 0 - infinity) and circularity values (e.g., 0.00 - 1.00) according to the experimental requirements to include all cells in the count.
   4. Click OK and wait for the software to count the cells and for a new window to appear with the analysis results.

6. Colony formation assay

1. Seed 2 × 10⁵ cells per well of Si-NC and Si-FAM83A groups in a 6-well plate with or without 5 µM cisplatin (DDP) treatment for 24 h.
2. Following a 24 h period of drug treatment, detach the cells using 0.25% trypsin and resuspend them in Dulbecco's modified Eagle medium (DMEM) containing 10% FBS. Seed the resuspended cells in a 6-well plate at a density of 1 × 10³ cells per well, and then incubate in a 5% CO₂ incubator at a temperature of 37 °C.
3. Following the incubation for ~7-10 days when colonies are visible, fix the cells with 4% Polyoxymethylene and stain them with Giemsa staining solution for 20 min.
4. Wash the cells slightly and let the plates air dry. Take images of the colony clusters by microscope and count the number of colonies that contain more than 50 cells.

7. Mitochondrial membrane depolarization (MMP) analysis using JC-1 dye

1. Seed 1.2 × 10⁶ tumor cells from Si-NC and Si-FAM83A groups into a six-well plate and culture for 24 h with or without 5 µM cisplatin (DDP) treatment.
2. Incubate with 1x JC-1 staining solution for 15-20 min under 5% CO₂ at 37 °C using a Mitochondrial membrane potential assay kit according to the manufacturer's instructions.
3. Wash the cells and examine them under a fluorescent microscope at 200x magnification using excitation and emission wavelengths for JC-1 at ~490 nm and 590 nm, respectively. To visualize the JC-1 fluorescence, use appropriate filters, such as a blue excitation filter (450-490 nm) and a red emission filter (long-pass > 590 nm) to selectively capture the emitted fluorescence signal.

8. Flow cytometric analysis of mPTP

1. Seed 1.2 × 10⁶ tumor cells from Si-NC and Si-FAM83A groups into a six-well plate and culture for 24 h with or without 5 µM cisplatin (DDP) treatment.
2. Remove the culture medium from the cells, resuspend the cells in PBS, and add 300 µL each (1x volume) of Calcein AM staining solution and fluorescence quenching working solution to cover the cells evenly. Incubate the cells at 37 °C in a light-protected environment for 30-45 min, and replace the medium with fresh preheated culture medium at 37 °C. Incubate the cells at 37 °C in a light-protected environment for an additional 30 min to ensure complete hydrolysis of Calcein AM by intracellular esterases, resulting in the generation of intracellular green-fluorescent Calcein.
3. Remove the culture medium, wash the cells 2-3x with PBS, and add detection buffer. Detect cell mPTP using flow cytometry and an excitation wavelength of 488 nm.

9. Flow cytometry

1. Seed 1.2 × 10⁶ tumor cells from Si-NC and Si-FAM83A groups into a six-well plate and culture for 24 h with or without 5 µM cisplatin (DDP) treatment.
2. Following 24 h of drug treatment, re-suspend the cells in 200 µL of binding buffer solution and gently mix 10 µL of Annexin V-FITC and 5 µL of propidium iodide (PI) into the cell suspension. Incubate the mixture for 15 min while avoiding light and add 300 µL of binding buffer solution to the cells. Detect cell apoptosis using flow cytometry at an excitation wavelength of 488 nm.

10. RT-PCR analysis

1. Seed 1.2 × 10⁶ tumor cells from Si-NC and Si-FAM83A groups into a six-well plate and culture for 24 h with or without 5 µM cisplatin (DDP) treatment.
2. Extract total RNA from the tumor cells using the RNA extraction reagent and convert the extracted RNA into complementary DNA (cDNA) with the cDNA Synthesis Mix by incubating at 42 °C for 45 min and then at 95 °C for 5 min.
3. Prepare the PCR reaction mixture containing DNA polymerase, nucleotides, buffer, and the designed gene-specific primers. Add the cDNA template to the reaction mixture for the real-time PCR reaction and perform PCR on the real-time quantitative PCR instrument with the following thermocycling conditions: denaturation at 95 °C for 30 s, followed by 40 cycles of 95 °C for 5 s and 60 °C for 30 s, and the melting curve stage at 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. Quantify the mRNA levels using the 2^−ΔΔCq method using GAPDH as an internal control.
   NOTE: The RT-PCR reaction system is shown in Table 1, and the primer sequences are shown in Table 1.
   1. To calculate the 2^−ΔΔCq value, measure the Cq values for the target gene and the reference gene (GAPDH) in the samples. Calculate the ΔCq by subtracting the reference gene's Cq value from the target gene's Cq value for each sample. Calculate the ΔΔCq by subtracting the ΔCq of a control sample from the ΔCq of each experimental sample. Finally, calculate the 2^−ΔΔCq value by raising 2 to the power of the ΔΔCq value.
      NOTE: The Cq value represents the cycle number at which the fluorescence signal reaches the set threshold.

11. Western blot analysis

1. Seed 1.2 × 10⁶ tumor cells from Si-NC and Si-FAM83A groups into a six-well plate and culture for 24 h with or without 5 µM cisplatin (DDP) treatment.
2. Collect the cultured cells and wash them with ice-cold phosphate-buffered saline (PBS) to remove any residual growth media or serum. Lyse the cells using RIPA Lysis Buffer containing phenylmethyl sulfonyl fluoride (PMSF) to release the cellular proteins. Incubate the cells on ice for a few minutes to ensure complete lysis and centrifuge at 4 °C, 12,000 × g for 15 min. Collect the supernatant to determine its protein concentration.
3. Determine the protein concentration in the cell lysate using a BCA Protein Assay Kit according to the manufacturer's instructions. Mix the cell lysate with a loading buffer and heat the mixture at 95-100 °C for 5-10 min to denature the proteins and ensure uniform separation on the gel.
4. From each sample, load 30 µg of total protein onto an SDS-PAGE gel and run the SDS-PAGE under a constant voltage of 80 V. Stop the electrophoresis when the bromophenol blue reaches the bottom of the separation gel.
5. Transfer the separated proteins from the gel onto a polyvinyldifluoride membrane using a wet transfer system on an ice bath with a current of 200 mA for 1.5 h.
6. Block the membrane with 5% nonfat milk in Tris-buffered saline with 0.05% Tween-20 buffer (TBST) for 1 h at RT and incubate overnight at 4 °C with respective antibodies given in Table of Materials.
7. Wash the membrane multiple times with TBST and incubate with the secondary antibodies (Table of Materials) for 1 h at RT, followed by washing with TBST. Visualize the protein bands using an enhanced chemiluminescence detection kit and imager system.

12. Statistical analysis

1. As all experimental data points will be independent, present the data as the mean ± SD. Perform statistical analysis.
2. Use one-way analysis of variance (ANOVA) for multiple comparisons and Dunnett's test for comparing each group with the control group. Use Student's t-test (unpaired two-tailed) for comparing two groups; consider P < 0.05 to be significant.

Results

TCGA database analysis and PCR validation

From the TCGA database analysis, we conducted a comparative analysis of mRNA expression levels in 306 cervical cancer cell samples and 13 normal cell samples to investigate the differential expression of FAM83A. FAM83A was upregulated in cervical cancer, while its expression in normal cervical tissue was negligible (Figure 1A

Discussion

The investigation of tumor target genes is of utmost importance for both the prevention and treatment of cervical cancer. Understanding the specific genes that play a significant role in cervical cancer development and progression provides valuable insight into the underlying molecular mechanisms of the disease. Furthermore, identifying these target genes can lead to the development of novel therapeutic strategies and targeted therapies. In this study, we describe the use of TCGA dataset analysis to identify FAM83A <...

Materials

Name	Company	Catalog Number	Comments
Cells and Medium Formulation
C33a	American Type Culture Collection
Hela	American Type Culture Collection
Modified medium			10% fetal bovine serum and + antibiotics (100 U/mL penicillin and 100 U/mL streptomycin)
Antibody Information
AKT	4691, Cell Signaling Technology Inc.		‘1:1,000
Bcl2	26593-1-AP, Proteintech Group, Inc		‘1:1,000
Caspase 3	19677-1-AP, Proteintech Group, Inc		‘1:2,000
cleaved-caspase3	abs132005; Absin Bioscience Inc.		‘1:1,000
Cytc	10993-1-AP; Proteintech Group		‘1:1,000
GAPDH	10494-1-AP, Proteintech Group, Inc.		‘1:8,000
mTOR	2983, Cell Signaling Technology Inc.		‘1:1,000
PI3K	4292, Cell Signaling Technology Inc		‘1:1,000
p-AKT	4060, Cell Signaling Technology Inc.		‘1:1,000
p-mTOR (Ser2448)	#5536, Cell Signaling Technology Inc.		‘1:1,000
p-PI3K p85 subunit	17366, Cell Signaling Technology Inc.		‘1:1,000
Secondary antibodies	GB23303, Servicebio		‘1:2,000
Materials
6-well plate	Corning, NPY
Alexa Fluor 555	Beyotime
BCA Protein assay kit	Beyotime, China		P0011
ChemiDoc XRS Imager System	BioRad
Enhanced chemiluminescence detection kit	Servicebio, Inc.,China		cat. no. G2014
Fluorescence microscope	Olympus Corporation, Tokyo, Japan
Hifair II 1st Strand cDNA Synthesis Super Mix	11123ES60, Yeasen Biotech o., Ltd., China
			Inverted microscope	Olympus, Tokyo, Japan;
Millicell transwell inserts	Millipore,Bedford, MA, USA
Mitochondrial membrane potential assay kit	Beyotime, China
PMSF	ST506, Beyotime Biotech, Jiangsu, China		#ST506
Real-time quantitative PCR instrument	Applied Biosystems, Thermo Fisher Scientific. China.
			RIPA Lysis Buffer	Beyotime Biotech, Jiangsu, China
TRIzol reagent	Invitrogen		15596026
TRIzol reagent	Takara Bio Inc., Otsu, Japan
Software
Image-Pro	plus 6.0