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.
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.
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 cancers3,4. 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.
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
2. Cell-based experiments
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.
4. Wound healing assay
5. Porous membrane insert assay
6. Colony formation assay
7. Mitochondrial membrane depolarization (MMP) analysis using JC-1 dye
8. Flow cytometric analysis of mPTP
9. Flow cytometry
10. RT-PCR analysis
11. Western blot analysis
12. Statistical analysis
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). To gain further insights into the prognostic implications of FAM83A expression, we performed a Kaplan-Meier curve analysis. Strikingly, patients with higher FAM83A expression displayed a markedly worse overall survival (Figure 1B). To determine the role of FAM83A in cervical cancer, we examined FAM83A expression levels in two cervical cancer cell lines, HeLa and C33a, as well as in an immortalized cervical cell line, End1/E6E7. Using qRT-PCR, we observed a significant overexpression of FAM83A in the HeLa and C33a cell lines compared with the End1/E6E7 cell line (Figure 1C). These findings underscore the importance of FAM83A in the context of cervical cancer.
Proliferation and apoptosis experiments to verify the biological function of FAM83A in cervical cancer
To investigate the functional role of FAM83A in cervical cancer, we utilized siRNA-mediated knockdown of FAM83A in C33a and HeLa cells (Figure 2A,B). We then assessed the impact of FAM83A suppression on cell proliferation. EdU staining revealed that decreased expression of FAM83A significantly inhibited cell proliferation in both C33a and HeLa cells (Figure 2C–F). Immortal malignant cells are associated with extremely low apoptosis rates10. Therefore, the levels of anti- and proapoptotic proteins were assessed in the control and si-FAM83A-treated cervical cancer cells. Western blot analyses revealed that suppressed expression of FAM83A increased the expression of Bax and cleaved caspase3 proteins (proapoptotic proteins), decreased the expression of Bcl-2 (antiapoptotic protein), and increased Cytc release (Figure 2C–E), which is consistent withthe observation that antiapoptotic proteins regulate apoptosis by blocking the mitochondrial release of Cytc (Figure 2G–J)10. Collectively, these data suggested that FAM83A plays an important role in regulating cervical cancer cell proliferation and apoptosis.
Wound-healing and porous membrane insert assays to verify the biological function of FAM83A in cervical cancer
Wound-healing and membrane insert assays were performed to explore the effects of FAM83A suppression on the migration and invasion of cervical cancer cells. The results revealed that cervical cancer cells with suppressed FAM83A expression migrated (Figure 3A–D) and invaded (Figure 3E–H) less efficiently than the control cells.
Effects on mitochondrial function and PI3K/AKT signaling pathway
Given the role of PI3K/AKT in the induction of cell apoptosis, we aimed to determine whether the suppression of FAM83A expression would inhibit the constitutive phosphorylation of the PI3K/Akt/mTOR pathway in cervical cancer. Suppression of FAM83A significantly inhibited the key phosphorylated protein levels in the PI3K/Akt/mTOR pathway including p-PI3K, p-Akt, and p-mTOR in c333a and HeLa cells (Figure 4A–D). Mitochondria are the organelles responsible for cellular metabolism and cell apoptosis induction. Accumulating evidence indicates that cancer cell apoptosis involves mitochondrial dysfunction through the intrinsic mitochondrial pathway due to enhanced mitochondrial permeability and the release of proapoptotic molecules such as Cytc into the cytoplasm10. The PI3K/AKT pathway could regulate the translocation of Bax into the mitochondria, inducing Cytc release in response to apoptotic stimuli. Therefore, it is conceivable that the oncogenic components of the PI3K-AKT signaling pathway regulate cell apoptosis directly by influencing mitochondrial behavior. Thus, a live-cell mitochondrial permeability transition pore (mPTP) assay was performed to determine the mitochondrial status. mPTP opening is associated with apoptotic and necrotic cell death11. In this assay, a green fluorescent dye (calcein AM) is retained in the cytoplasm and mitochondria when the mPTP is closed, while the dye in the cytoplasm is quenched by CoCl2, leaving only the intensive fluorescence in the mitochondria. The results revealed that cell populations with intense green fluorescence in the mitochondria decreased significantly after FAM83A suppression, indicating an opened mPTP (Figure 4E,F). Further, we examined the changes in the mitochondrial membrane potential (ΔΨm) using JC-1 staining. JC-1 forms aggregates (red fluorescence) in live cells containing intact mitochondria with high membrane potential and monomers (green fluorescence) under low MMP in apoptotic cells. FAM83A suppression gradually decreased MMP (Figure 4G).
Drug susceptibility analysis for proliferation and invasion
Cisplatin (DDP)-based chemotherapy is a standard treatment strategy for cervical cancer. However, chemoresistance remains a challenge. We investigated the role of FAM83A in the treatment of cervical cancer using cisplatin. Control, si-NC-treated, and si-FAM83A-treated HeLa cells were treated with or without 5 µM cisplatin12. Further, the effects of FAM83A suppression on cell viability were assessed. DDP induced a more significant inhibitory effect on the proliferation of HeLa cells after FAM83A suppression, compared with only DDP treatment (Figure 5A and Figure 5C). After treating cervical cancer cells with 5 µM cisplatin, FAM83A suppression also inhibited cell invasion (Figure 5B and Figure 5D).
Drug susceptibility analysis for mitochondrial function
By treating Si-NC-treated and si-FAM83A-treated HeLa cells with or without 5 µM cisplatin (DDP), we evaluated the mitochondrial membrane potential (MMP) and assessed the mitochondrial damage caused by cell death using JC-1 staining. DDP induced more mitochondrial damage as revealed by a decrease in JC-1 monomer percentage after FAM83A knockdown, compared with only DDP treatment (Figure 6A and Figure 6D). After treating cervical cancer cells with 5 µM cisplatin, FAM83A knockdown also inhibited colony formation (Figure 6B and Figure 6E) and enhanced cell apoptosis (Figure 6C and Figure 6F). In addition, FAM83A knockdown in the presence of DDP markedly increased the expression of proapoptotic proteins, Bax and cleaved caspase3, promoted Cytc release, and decreased Bcl-2 expression (Figure 6G,H). These results indicated that FAM83A knockdown sensitized cervical cancer cells to DDP via apoptosis induction.
Figure 1: FAM83A overexpression in cervical cancer cells and correlation with worse prognosis. (A) Boxplot analysis of the mRNA levels of FAM83A in cervical cancer samples. (B) Kaplan-Meier analysis of FAM83A expression for the determination of overall survival in patients with cervical cancer. (C) Relative mRNA levels of FAM83A in cervical cancer cell lines HeLa and C33a and human immortalized cervical cell line End1/E6E7. The data are presented as the mean ± SD for three independent experiments. *P < 0.05, compared with End1/E6E7 cells using one-way ANOVA. Abbreviations: CESE = Cervical squamous cell carcinoma and endocervical adenocarcinoma; HR = hazard ratio. Please click here to view a larger version of this figure.
Figure 2: Effects of suppression of FAM83A on the proliferation and apoptosis of cervical cancer. C33a and HeLa cells were transfected with FAM83A-specific siRNAs. (A,B) mRNA levels of FAM83A were assessed in cervical cancer cell lines using qRT-PCR.Cell proliferation in (C) C33a and (D) HeLa cells was determined using the EdU assay. (E,F) The EDU fluorescence images of C33a and Hela cells. (G–J) Western blot analysis for examining the levels of apoptosis-related proteins, including Cytc, Bcl-2, Bax, caspase3, and cleaved-caspase-3; GAPDH was used as the loading control. The data are presented as the mean ± SD for three independent experiments. *P < 0.05; **P < 0.01; **P < 0.001, compared with control cells using one-way ANOVA. Abbreviations: NC = negative control; EdU = 5-ethynyl-2'-deoxyuridine; CytC = cytochrome C. Please click here to view a larger version of this figure.
Figure 3: Effect of FAM83A on the migration and invasion of cervical cancer cells in vitro. (A–D) Wound-healing assay was performed to assess cell migration. Images were taken at 0, 24, and 48 h. (E–H) Transwell assays were performed to examine cell invasion. Scale bars = 100 µm (E,F). The data are presented as the mean ± SD for three independent experiments. *P < 0.05; **P < 0.01; **P < 0.001, compared with control cells using one-way ANOVA. Please click here to view a larger version of this figure.
Figure 4: Effect of FAM83A suppression on the PI3K/AKT/mTOR pathway and mitochondrial function. (A–D) Western blot analysis to determine the levels of proteins from PI3K/AKT/mTOR pathway in C33a and HeLa cells after FAM83A suppression. (E–H) Mitochondrial membrane potential analysis using JC-1 staining. JC-1 aggregates were stained in red, and JC-1 monomers were stained in green indicating a lower MMP. (I,J) Mitochondrial permeability was assessed using an mPTP kit and flow cytometry. Scale bars = 50 µm (E, F). The data are presented as the mean ± SD for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, compared with control cells using one-way ANOVA. Abbreviations: MMP = mitochondrial membrane potential; mPTP = mitochondrial permeability transition pore. Please click here to view a larger version of this figure.
Figure 5: Effects of FAM83A suppression on inhibition by DDP on cell proliferation and invasion of cervical cancer cells. HeLa cells were transfected with si-NC or si-FAM83A and treated with or without 5 µM DDP. (A,C) Effect of DDP on the cell proliferation in control, si-NC-treated, and si-FAM83-treated HeLa cells. (B,D) Effect of DDP on the cell invasion in control, si-NC-treated, and si-FAM83-treated HeLa cells. The data are presented as the mean ± SD for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the control cells. &&P < 0.01; &&&P < 0.001, compared with DDP using one-way ANOVA. Abbreviations: DDP = diaminedichloroplatinum; EdU = 5-ethynyl-2'-deoxyuridine; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Figure 6: Effects of FAM83A suppression on the effects of DDP on apoptosis in cervical cancer cells. HeLa cells were transfected with si-NC or si-FAM83A and treated with or without 5 µM DDP. (A,D) MMP analysis using JC-1 staining. (B,E) Plate cloning results. (C,F) Flow cytometry detecting apoptosis rates in different treatment groups. (G,H) Western blot analysis of mitochondria-related apoptotic proteins, including Bcl-2, Bax, Cytc, and the downstream cleaved caspase-3. GAPDH was used as the loading control. The data are presented as the mean ± SD for three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001, compared with the control cells. &P < 0.05; &&P < 0.01; &&&P < 0.001, compared with DDP using one-way ANOVA. Abbreviations: DDP = diaminedichloroplatinum; EdU = 5-ethynyl-2'-deoxyuridine; DAPI = 4',6-diamidino-2-phenylindole. Please click here to view a larger version of this figure.
Table 1: siRNA, qRTPCR primers, and the qRT-PCR reaction setup used in this study. Please click here to download this table.
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 as a target gene and investigate its mechanistic roles in cervical cancer. We observe a significantly high expression of FAM83A in cervical cancer cells, which is associated with poor prognosis of patients with cervical cancer. A series of cell-based experiments revealed high FAM83A expression in cervical cancer cells, which plays a critical role in regulating cell proliferation, migration, and invasion. Suppression of FAM83A expression inhibited proliferation and migration and promoted apoptosis in cervical cancer cells.
Constructing FAM83A-knockdown cervical cancer cell lines is the most critical step in this study. siRNAs are designed and validated according to the target gene sequences to ensure their effectiveness. Further, the cervical cancer cell lines are transfected using siRNA. The success rate of plasmid transfection is crucial for subsequent experiments, which require strict control of cell density and plasmid density. Further, the transfection efficiency is observed under a fluorescence microscope to ensure that subsequent experiments can be conducted. Western blotting and qRT-PCR results confirm that the inhibition of FAM83A suppressed the PI3K/AKT pathway and induced mitochondrial dysfunction. We hypothesize that the mechanism involves the inhibition of the translocation of Bax from the cytoplasm to the mitochondria by PI3K/AKT, which promotes cell survival13.
Advances in the treatment of cervical cancer have made it possible to develop new target molecules. The identification of a novel oncogene can be applied therapeutically to improve the clinical prognosis of cervical malignancies14,15. In this study, we successfully constructed a FAM83A knockdown system in HeLa and C33a cells and verified the effects of FAM83A knockdown on cervical cancer cells at the cellular level. We propose that the inhibition of FAM83A can be used to treat cervical cancer.
However, this study has some limitations. First, this study only performs gene screening through the database and lacks pathological data of clinical patients. Second, this study has only validated the effect in vitro, and further in vivo studies were needed to verify the results. Nevertheless, the methods of target gene identification, gene knockout, and cellular validation to identify targeted therapeutic loci elaborated in this study can provide research ideas for the discovery and validation of target genes for other diseases.
In summary, FAM83A was overexpressed and correlated with a worse prognosis in cervical cancer. FAM83A regulates the proliferation, migration, and invasion of cervical cancer cells. Suppression of FAM83A-induced mitochondrial dysfunction and apoptosis, sensitizing cervical cancer cells to cisplatin. These results indicated that FAM83A is a suitable research target for the treatment of cervical cancer. This study contributed to the advances in adjuvant chemotherapyaiming at improving the prognosis of patients with cervical cancer.
The authors have nothing to disclose.
This work was supported by the Jingzhou Science and Technology Bureau Foundation (no. 2020HC06).
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 |