Abstract
Background/Aim: Human dermal fibroblasts (HDFs) are widely used as a skin model in cosmetic and pharmaceutical industry due their advantages for the cosmetic industry and medical aspects. Telomeres are key players in controlling cellular aging, in which telomeres and the telomerase enzyme (hTERT) can maintain proliferative capacity and prolong cellular senescence. The primary aim of the study was to elucidate the underlying mechanisms of hTERT/SV40 immortalization of human dermal fibroblasts. Materials and Methods: Transgenic expression of hTERT and SV40 large antigen, as well as co-transfection of both factors was performed and their significance evaluated in terms of HDF immortalization efficiency. Results: The results showed that the immortalized fibroblasts of all conditions can be cultured in over 60 passages and maintain their telomere length. Further, key markers of skin cells, such as COL1A1, KRT18 and ELASTIN, were up-regulated in immortalized cells. In addition, p53 expression was enhanced in all immortalized cells, in accordance with activation of the SIRT1 gene upon transgenic immortalization. The significant role of SIRT1 in fibroblast proliferation was assessed by shRNA-knockdown, and it was found that SIRT1 silencing led to loss of Ki67, a proliferation marker. Moreover, miR-93, a SIRT1-targeted miRNA, also had a significantly reduced expression in the co-transfected immortalized cells, highlighting the linkage of the miRNA and SIRT1 pathway in the immortalization of human dermal fibroblasts. Conclusion: This evidence from this study could benefit the efficient development of human skin cell lines for use in the cosmetic industry in the future.
- Human dermal fibroblasts
- immortalization
- SIRT1
- micro-RNA
The expansion of somatic cells in vitro for research and medical applications is limited by their short proliferative lifespan (1). Cellular senescence is a state of irreversible arrest of cell proliferation, exhibiting enlarged morphological changes and senescence-associated heterochromatin foci formation. Senescence, originating from the Latin word ‘SENAX’, refers to a physiological program towards permanent cell-cycle arrest. Cellular senescence can be stimulated via a variety of factors, such as tumour suppressor proteins, oncogene activation, telomere shortening, genomic DNA damage, and activation of the p53/p21 pathway. Oncogene-induced senescence is a cellular response found in pre-malignant tumours. Senescent cells exert a pleotropic effect on development, tissue aging and regeneration, inflammation, wound healing and tumour suppression (2). Currently, cosmeceutical research is developed to correct skin problems, including skin aging, skin inflammation, skin disorders, and acne. In cosmetics, human skin cells are used as a standard model for demonstrating the therapeutic potential of cosmetic products. The use of in vitro tests is a critical step in the development of new products for treating skin problems. The study of human skin cell biology has increased the hope for a successful cell therapy in aesthetic medicine and cosmetics. Various types of skin cells are responsible for synthesizing and organizing the skin with three layers, including (i) epidermis containing keratinocytes, melanocytes and Langerhans cells; (ii) dermis that is populated with fibroblasts, vessels, and dendritic cells and (iii) subcutaneous tissue in the deepest layers (3). HDFs exist in several locations, including the dermal papilla, the hair follicle, the blood capillary and the collagen fibre. HDFs are used to explore wound-healing and aging skin phenomena, which play a pivotal role in maintaining skin function. However, a major bottleneck of using human dermal fibroblasts is their limited proliferative capacity ex vivo, and the tendency to undergo senescence (4). In some cases, the cells may overcome this replication block by virus-suppressed p53 and pRb function. However, continued proliferation results in further shortening of telomeres, extensive chromosome damage and genomic crisis leading to widespread apoptosis (5).
Cellular immortalization is a technique aiming to solve the limits of cellular senescence. Several pathways are implicated in regulating spontaneous cell immortalization and anti-senescence, including loss of tumour suppressors, in particular p53, which is necessary but not sufficient to induce immortalization (6). Telomere shortening has a definitive role in the loss of chromosomal integrity and controls aging of cells during cell division. The telomere is a protein/DNA complex at the end cap of the chromosome, and the maintenance of their length is essential for genomic stability and cell viability (7). Human telomerase reverse transcriptase (hTERT) functions to create immortalized cells by maintaining the length of the telomere. The catalytic protein subunit of telomerase plays a role in controlling the mammalian cell lifespan. Telomerase activation is highly increased in human cancer but reduced in most human somatic cells (8). Besides hTERT, DNA tumour viruses such as simian virus 40 (SV40) large T antigen could directly suppress tumour retinoblastoma protein (pRB) and activate the cell cycle of host cells. The generation of immortalised cell lines by using SV40 large T antigen provides a vital tool to examine the mechanisms of cellular immortalization (8). The extension of the cell lifespan is largely attributed to large T antigen forming complexes with pRB and p53 (9).
A recent study showed that cell proliferation and cellular rejuvenation are also regulated by sirtuin (SIRT) activation (10). SIRT1 plays multiple roles in mammalian cells, since SIRT1-deficient mice suffer multiple abnormalities, including defects in spermatogenesis and in heart and retina development (11, 12). However, the involvement of SIRT1 in cell immortalization process is not well understood. The aim of this study was to establish immortalized human dermal fibroblasts and explore the possible mechanisms underlying cellular immortalization by hTERT and SV40 large T antigen.
Materials and Methods
Chemicals and reagents. The hTERT (pBABE-neo-hTERT) and SV40 large T antigen (pBABE-neo largeTcDNA) plasmid were purchased from Addgene, Cambridge. MA.3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), 4,6-diamidino-2-phenylindole (DAPI) and dimethyl sulfoxide (DMSO) were purchased from SIGMA (Roedermark, German). Dulbecco’s Modified Eagle’s Medium (DMEM) and fetal bovine serum (FBS) were purchased from HyClone (Hyclone, Logan, UT, USA). Trypsin-EDTA (0.05%), L-glutamax and non-essential amino acids were obtained from Gibco (Gibco, Carlsbad, CA, USA). Antibodies against SIRT1, Collagen1 type I and Vimentin were purchased from Merck (Merck KGaA, Darmstadt, Germany).
Cell culture. Human dermal fibroblasts (HDFs) were obtained from cell-based assay and innovation (CBAI), biotechnology, SUT. SHSY-5Y cells were purchased from ATCC (CRL-2266; www.atcc.com). HDFs were stored in liquid nitrogen prior to experimentation. Cells were thawed, washed twice with 1× Phosphate Buffered Saline (1× PBS, pH7.4) and seeded into T25 flask (NEST, Jiangsu, PR China). The cells were cultured in high-glucose Dulbecco’s Modified Eagle Medium (DMEM/HG; Hyclone), supplemented with 10% (v/v) fetal bovine serum (FBS; Gibco BRL, Grand Island, NY, USA), 1 mM L-glutamine, 1 mM Minimal Essential Medium (MEM; Sigma-Aldrich, St. Louis, MO, USA), 100 U/ml Penicillin and 100 g/ml Streptomycin (Sigma-Aldrich) and incubated at 37°C in 5% CO2 (Thermo Scientific, Waltham, MA, USA).
Plasmid transfection. To immortalize HDFs and increase their cell proliferative capacity, the cells were transfected by plasmid hTERT (pBABE-neo-hTERT; Addgene) and SV40 large T antigen (pBABE-neo largeTcDNA; Addgene). These cells were seeded on 6-well plates at a density of 2.0×105 cells per well. When the cells reached 70% confluence, a reagent containing plasmid and Lipofectamine 2000 (Promega, Madison, WI, USA) was added and the plate incubated at 37°C in 5% CO2 for 24 h, as mentioned in the manufacturer’s protocol (13). After 48 h in culture, the transfected cells were subjected to antibiotic selection with either neomycin or geneticin by adding 300 μg/ml of G418 sulphate (Sigma-Aldrich) into the serum-free medium. G418-resistance cells were isolated and subcultured for expansion.
Cell proliferation. Cell viability and cell proliferation were measured by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay. Approximately 3×103 cells were seeded onto 96-well microplates (NEST) and cultured in 10% DMEM for 9 days. Cells were counted every 48 h until day 9. At each time point, the medium was discarded; cells were washed twice with 1× PBS, and 100 μl of 0.5 mg/μl MTT was added and incubated for 2 h at 37°C in 5% CO2. After 2 h, the MTT solution was discarded, and 100 μl dimethyl sulfoxide (Sigma-Aldrich) was added to solubilize the formazan crystals. The optical density was measured at 570 nm by a microplate reader (SPECTROstar Nano, BMG Labtech, Offenburg, Germany).
RNA isolation and cDNA synthesis. Total RNA from the cells was extracted by using the NucleoSpin RNA kit (Macherey-Nagel, Dueren, Germany) in accordance with the manufacturer’s protocol. On the other hand, miRNAs were extracted from dermal fibroblasts with the miRNeasy mini kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s protocol. The OD260/280 ratio of the RNA samples was measured, and samples with a ratio above 2.0 were used for reverse transcription. RNA (0.5 mg) was reverse transcribed by using ReverTra Ace®qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan). The 4× DNA master mix reactions were performed at 65°C for 5 min, and then 5× RT master mixes were added, and the reactions were performed at 37°C for 15 min and 98°C for 5 min.
Reverse-transcription PCR and qPCR. The expression of fibroblast and cell cycle genes was assayed by PCR (Bio-Rad, Hercules, CA, USA) using the primers shown in Table I. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the internal control gene. PCR products were separated by gel electrophoresis (1.5% agarose gel, Vivantis, Selangor Darul Ehsan, Malaysia), and visualized by EtBr staining. The stained gels were detected with a GelDoc system (Gel Doc EZ Imager, Bio-Rad) and the relative gene expression levels measured. The expression levels of Tel, 36B4, Ki67, β-actin, U6 and 5 MiRNAs were examined by qPCR using the primers shown in Table I. The QuantStudio 5 Real-Time PCR System (Thermo Scientific) was used to carry out real-time PCR experiments which were conducted in 96-well plates. To monitor cDNA amplification, qPCR BioSyGreen Mix Low-Rox (PCR BIOSYSTEMS, London, UK) was used in a 20 μl reaction volume. Cycling conditions according to the specific SYBR Green protocol were as follows: 95°C for 30 s, 60°C for 30 s and 72°C for 45 s (40 cycles). Melting curve analysis of the PCR products was performed by heating at 60°C for 60 s, 95°C for 15 s and continuous measurement of the fluorescence to verify the PCR products. The relative expression was determined by the 2^ (-delta delta C(T)) method with the expression of β-actin as the housekeeping reference gene.
Telomere measurement. DNA was isolated from fibroblasts with the genomic RNA extraction kit (Macherey-Nagel) for telomere measurement. Telomere length was measured as previously described (14) by qPCR amplification with oligonucleotide primers designed to hybridize to the TTAGGG and CCCTAA repeats. For all qPCR reactions, qPCR BioSyGreen Mix Low-Rox (PCR BIOSYSTEMS) was used for both T and S qPCR. The final telomere primer concentrations were as follows: tel 1, 270 nM and tel 2, 900 nM. The final 36B4 (single copy gene) primer concentrations were 36B4u, 300 nM and 36B4d, 500 nM. Primer sequences (5’→3’) were as follows (15):
tel 1, GGTTTTTGAGGGTGAGGGTGAGGGTGAGGGTGAGGGT;
tel 2, TCCCGACTATCCCTATCCCTATCCCTATCCCTATCCCTA;
36B4u, CAGCAAGTGGGAAGGTGTAATCC;
36B4d, CCCATTCTATCATCAACGGGTACAA.
All PCRs were performed using the QuantStudio 5 Real-Time PCR System (Thermo Scientific). For telomere PCR, 40 cycles of 95°C for 15 s and 54°C for 2 min, and for 36B4 PCR, 40 cycles of 95°C for 15 s and 58°C for 1 min were set. For telomere length, the LightCycler® 480 by QuantStudio 5 thermocycler was used to generate the standard curve for each run and to determine the dilution factors of standards corresponding to the T and S amounts in each sample. Absolute quantification results for both T and S were obtained using the Second Derivative Maximum method. The relative telomere length value was expressed as the average duplicate telomere over the average duplicate single copy gene (T/S) ratio. All presented telomere lengths were displayed as a relative level, compared to the non-transfected control HDFs. Standard deviation (SD) was obtained from three replicate values of each HDF sample, and statistical analysis was performed by Student’s t-test.
Immunofluorescence staining. Cells were cultured on coverslips and fixed with 4% paraformaldehyde (PFA) in 1× PBS for 20 min at room temperature. The fixed cells were washed twice with 1× PBS and non-specific binding blocked with 1% bovine serum albumin (EMD Millipore, Billerica, MA, USA) and 0.2% Triton X-100 in 1× PBS. After incubation for 30 min in blocking solution at 4°C, the cells were incubated in blocking buffer containing the primary antibody [anti-Collagen1 Type I (1:500; MERCK), anti-SIRT1 (1:200; DSHB, Iowa, IA, USA) and anti-Vimentin (1:500; DSHB)] overnight at 4°C. The cells were washed in washing solution, supplemented with 0.2% Triton X-100 in 1× PBS. Then, the cells were incubated in the secondary antibody either anti-mouse (1:500; Thermo Scientific) or anti-rabbit (1:500; Thermo Scientific) for 2 h at room temperature. Next, the cells were washed with 1× PBS 5 times for 5 min each, the cell nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and the coverslips were sealed with nail polish on the on the slides. Slides were observed using a fluorescence microscope (ZOE™ Fluorescent Cell Imager, Bio-Rad Laboratories).
Colony formation. The immortalized HDFs were investigated for tumour formation after plasmid transfection. The soft agar assay could detect colony formation in malignant and tumour cells. The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% FBS, 1% penicillin, and 1% NEA and incubated at 37°C in 5% CO2. The agar base was prepared as a 1% agarose gel and melted in a microwave. The 2× DMEM medium was prepared, containing 20% FBS, 1% L-glutamine, 1% penicillin and 1% NEA. The mix was blended with 1% agarose gel and placed into a 35-mm petri dish. Top agar was prepared from 0.3% agarose gel mixed with 2X DMEM and 20% FBS. The immortalized cells were counted and ~5000 cells/plate blended into the 2X DMEM solution. The cells were then transferred onto the base agar and incubated at 37°C 5% CO2 for 10-30 days. During the incubation, the cells were seeded on the top agar every 5 days. When the incubation was completed, the soft agar was stained with 0.005% crystal violet for 1 h at room temperature and the number of colonies counted using a microscope (Nikon, Japan).
shRNA preparation and vector amplification. The oligonucleotide was designed by using the basic local alignment search tool (NCBI database, https://blast.ncbi.nlm.nih.gov/Blast.cgi) as shown in Table II. The pSUPERIOR.neo vector (OligoEngine, Seattle, WA, USA) was used for inserting oligonucleotides (shRNA1 and shRNA2), of which the 5’ ends corresponded to the BglII site, and the 3’ ends contained the HindIII corresponding nucleotides. The oligonucleotides were annealed in annealing buffer at 90°C for 4 min, then at 70°C for 10 min, prior to cooling the annealed oligonucleotide to 10°C. Then, the vector was linearized with the BglII and HindIII restriction enzymes (NEW ENGLAND BioLabs; NEB, MA, USA) at 37°C for 3 h and heat inactivated at 80°C for 20 min. To ligate the vector, the oligonucleotide to pSUPRIOR vector ratio was 3:1, which was incubated in T4 DNA ligase buffer (NEB) at 37°C for 10 min and 65°C for 10 min. The ligation products were amplified by transformation into DH5α competent cells and incubated on ice for 30 min, then heat shocked at 42°C for 50 s and recovered on ice for 2 min. The mixture was added to LB broth and shaken at 200 rpm and 37°C for 60 min, prior to spreading onto LB agar plates containing ampicillin and incubating at 37°C for 16 h. The colonies on agar were selected and picked up into LB broth containing ampicillin for further selection. The plasmid was extracted using a plasmid extraction kit (GF-1 plasmid DNA extraction kit, Vivantis, Malaysia).
Statistical analysis. All experiments were performed in triplicate, and data were expressed as mean±standard deviation (16). Statistical analysis was performed using SPSS (version 16.0, SPSS Inc., Chicago, IL, USA). Significant differences between treatment effects were determined by one-way ANOVA, followed by Student’s t-tests, and *p<0.05, **p<0.01 and ***p<0.001 were considered as statistically significant.
Results
We first examined proliferative capacity and telomere length of the immortalized HDFs. HDFs were successfully immortalized by plasmid transfection of hTERT plasmid and SV40 large T antigen plasmid (SV40) and by co-transfection of both plasmids (CoTF). The cells could be cultured for over 60 passages after transfection. The non-transfected HDFs presented a spindle-shaped morphology with an approximate size of 100-150 μm, while all the transfected cells appeared clumped and were reduced in cytoplasmic volume (Figure 1A). The proliferative capacity of the immortalized cells was demonstrated by the MTT assay and the expression of the proliferation marker Ki67. Notably, all groups of immortalized cells increased their proliferation at days 2 and 4, and the highest rate was found in hTERT-HDFs at days 6 and 8 (Figure 1B). This primary observation suggested that the transfection of hTERT and SV40 could enhance HDF proliferative capacity. The level of Ki67 gene expression was used to validate the cell proliferative capacity. Ki67 is a nuclear protein, expressed in all proliferating vertebrate cells, and is widely used as a predictive marker of tumour cell proliferation and growth (17). Ki67 was significantly up-regulated in hTERT, SV40 and CoTF, compared with the control HDFs (Figure 1C). The high expression of Ki67 gene in SV40- and Co-transfected cells indicated a pivotal function of SV40 in activating human dermal fibroblast proliferation. The correlation of declining cell proliferation and telomere erosion led to the assessment of the T/S ratio, which is measured as telomere length relative to the standard reference DNA. The relative length of the telomere/single (T/S) copy gene of hTERT, SV40 and CoTF HDFs was significantly improved over the control HDFs (Figure 1D). This suggested that ectopic expression of hTERT and SV40 could elongate and maintain the telomere length of HDFs. Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface and is a hallmark of carcinogenesis. The soft agar colony formation assay was herein used to evaluate cellular transformation in vitro of the immortalized HDFs (18). The anchorage-independent growth of the transfected HDFs was compared with human neuroblastoma SH-SY5Y cells, a cancerous cell line used as a positive control (Figure 1E, F). After SH-SY5Y cells were seeded for over 20 days, the colonies grew on agar to an approximate size of 60-100 μm, while the non-transfected control HDFs formed colonies less than 10 μm in diameter. All transfected cells appeared as colonies approximately 10-20 μm in diameter, and their colony formation efficiencies were significantly less than that of SH-SY5Y cells. However, the colony formation efficiency of the transfected HDFs was clearly higher than that of control HDFs, implying the existence of cellular transformation in the immortalized HDFs to a certain degree.
Next, gene and protein expression of the control and the transfected HDFs were assessed by reverse transcriptase PCR and immunofluorescence, respectively. The key fibroblastic makers, including ELASTIN, Vimentin, COL3A1, COL1A1 and KRT18, were determined (19). The expression of Vimentin, COL3A1 and COL1A1 was at a similar level in the control and transfected HDFs; however, KRT18 and ELASTIN were radically up-regulated in all of the transfected HDFs. This suggested the maintenance and improvement of typical dermal fibroblast property of the immortalized HDFs (Figure 2A and B). Protein levels of vimentin and COL1A1 confirmed the fibroblastic property of the immortalized HDFs (Figure 2C and D). Moreover, the expression of tumour suppressor and anti-aging genes (RB, p21, p53 and SIRT1) was also evaluated to monitor the state of cellular senescence (16, 20). The direct target of SV40 large T antigen is tumour suppressor RB (pRb). Nevertheless, the expression of RB was not detected (not shown in results), but p53 was significantly increased in all immortalized HDFs. However, the high expression of p53 is not in accordance with the level of p21. SIRT1, an anti-aging gene, was investigated in this study to connect its importance to the immortalization process. SIRT1 is important for various cellular processes, including regulation of p53, cell-cycle regulation, and life span extension (21). High expression of SIRT1 was found in the immortalized HDFs, but not in control cells (Figure 2A and B). The corelation of p53 and SIRT1 was prompting further study of the relevance of SIRT1 in cellular immortalization by hTERT/SV40 transgenesis.
Importantly, SIRT1 was found abundantly expressed in all immortalized cells, compared to control HDFs (Figure 3A and B). The relative fluorescence intensity of SIRT1-positive cells was significantly high in hTERT/SV40 transgenic cells. The significance of SIRT1 in cell proliferative capacity was examined by small hairpin RNA (shRNA). SIRT1 was independently knocked-down in HDFs by two shRNAs. Immunofluorescence of SIRT1 was used to confirm the success of SIRT1 gene silencing. The relative fluorescence intensity of both SIRT1-shRNA was significantly reduced, compared to that of the normal control HDFs (Figure 3C and D). Importantly, Ki67 was found to decrease the expression in the SIRT1-knockdown HDFs (Figure 3E). The influence of SIRT1 on cell proliferation was further verified by using small molecules, resveratrol (SIRT1 activator) and sirtinol (SIRT1 inhibitor). Resveratrol and sirtinol at 1 and 5 μM were applied to the control HDFs for 24 h prior to examining SIRT1 expression. SIRT1 was significantly up-regulated by 1 μM resveratrol and down-regulated by 5 μM resveratrol sirtinol, respectively (Figure 3F). The adjustment of SIRT1 expression by small molecules directly reflected the level of Ki67 expression (Figure 3H). This evidence indicated the involvement of SIRT1 in the hTERT/SV40 immortalization process.
It is noted that miRNAs are involved in cell proliferation and senescence. Five miRNAs, which targeted SIRT1 activity, were selected and assessed regarding their involvement in the immortalization of HDFs (Table III). Four miRNAs were later selected for further analysis by qPCR due to their association with cellular senescence. It was reported that miR-22, miR-449, miR-93 and miR-34a were highly expressed during cellular senescence and aging (22-24). miR-22 and miR-449 were significantly up-regulated in the SV40-transfected HDFs, but not hTERT and CoTF HDFs (Figure 4A and B). On the other hand, the expression level of miR-93 was significantly enhanced in the hTERT-immortalized HDFs, while the level of miR-34a was reduced in both hTERT- and SV40-immortalized HDFs (Figure 4C and D). The down-regulation of miR-34a might associate with the increase in cell proliferation and cell-cycle progression by targeting the p53 gene (25). Interestingly, the hTERT/SV40 co-transfected HDFs maintained a low level of all studied miRNAs. The possible pathway of the predicted miRNAs and SIRT1 in hTERT/SV40 immortalization was illustrated in Figure 4E.
Discussion
The definition of cellular immortalization is to overcome two growth barriers in culture, senescence and crisis. Senescence is a poorly understood phenomenon exhibited by non-immortal cells in culture, characterized by an exit from the cell cycle and shortening of telomere length (26). In this study, human dermal fibroblasts (HDFs) were immortalized by using hTERT and SV40 large T antigen, in which both factors could immortalize the cells by controlling the cell cycle and preventing telomere erosion (27-29). hTERT and hTERC protein direct the elongation and metabolism of telomere, and they are widely implemented to immortalize various types of human cell lines (27, 30, 31). hTERT enabled immortalization not only of somatic cells, but also of stem cells. The hTERT-immortalized stem cells were able to maintain their differentiation capacity as well as the expression of stem cell-related cell surface markers (32). SV40 large T antigen is a tumour virus and acts as a suppressor of pRB and p53 to activate cell-cycle arrest (13, 33). The generation of an immortalised cell line by using SV40 large T antigen provides a vital tool to examine the mechanism of cell immortalization (34). The immortalized HDFs in all conditions increased their cell proliferation and maintained telomere length. Ki67, a marker of cell proliferation, was up-regulated in hTERT/SV40-transfected cells. Moreover, the anchorage-independent growth assay suggested that the immortalized HDFs by hTERT/SV40 were not transformed into cancer cells (18). The lack of transformation in the transfected HDFs could result by the function of hTERT in circumventing telomere-controlled senescence and SV40 in cell-cycle regulation without inducing global chromosomal instability.
Key dermal fibroblast markers, including COL1A1, COL3A1, Elastin and Vimentin, were constantly expressed in the immortalized HDFs, indicating the maintenance of the typical fibroblast property, and the potential application in studying skin biology (35). On the other hand, SIRT1 and p53 were highly expressed in transfected HDFs, which suggests their involvement in cell immortalization. SIRT1 regulates many physiological functions, including cell senescence, gene transcription, energy balance and oxidative stress (36), while p53 plays a role in the cell cycle and suppresses tumour formation (37). The SIRT1 level was enhanced in immortalized cells, and the silencing of SIRT1 led to cellular senescence, supporting the hypothesis that SIRT1 might mediate the process of cellular immortalization by hTERT/SV40 in this study. In some other cells, SIRT1 inhibits cellular apoptosis in response to genotoxic stress and may accomplish this by several mechanisms. For instance, SIRT1 deacetylated the p53 tumour suppressor protein, which then down-regulates p53 via effects on stability and activity (38, 39). Similarly, the overexpression of SIRT1 inhibited oncogene-induced premature senescence in mouse embryonic fibroblasts (39).
miRNAs are small non-coding RNAs of approximately 22 nucleotides, which play important roles in controlling gene activity (40). Several miRNAs could directly control SIRT1 activity and were predicted to be involved in the hTERT/SV40 immortalization process. miR-93, in particular, was found to directly regulate SIRT1 transcripts and be involved in cell growth and survival (41, 42). miR-93 expression was significantly decreased in the hTERT/SV40 transfected HDFs, while SIRT1 was upsurged in this cell type. Likewise, the expression of SIRT1 was previously found to be reduced inversely to upregulation miR-93 during aging (23). Besides SIRT1, miR-93 also targeted a number of genes that were involved in cell proliferation, for instance TGF-β1, c-Ski, E2F1 and FOXA1 (43-45). The changes in expression of specific miRNAs might be satisfied for the natural and spontaneous immortalization of human cells, and the role of miRNAs in cellular immortalization still needs further investigation (Figure 4E). This study offered the prospect of creating immortalized human dermal fibroblasts exclusively through the modification of the miRNA/SIRT1 pathway. The activation of the endogenous immortalization pathway could accelerate a spontaneous immortalization process, avoiding cellular transformation. These cells, as they are immortalized and not malignant, could dissect the important issues related to the biology of human skin. The complete image of the immortalization process for human dermal fibroblasts might accelerate the development of an advanced in vitro model for skin therapy and cosmetic product production, for instance construction of artificial skin.
In conclusion, cellular immortalization could benefit the efficient development of human cell lines, and accelerate the development of cosmeceutical products. hTERT/SV40 immortalized human dermal fibroblasts were comparable to the primary cells, regarding biological relevance, but without the limitation of proliferation. Using the well-characterized hTERT/SV40-immortalized cells, this study created a reliable cell model for toxicological studies on the skin, which could substitute to animal testing. The immortalized human dermal fibroblasts by transgenic expression of hTERT/SV40 preserved the typical skin cell properties with telomere maintenance, and the immortalization process was connected with the miRNA/SIRT1 pathway (Figure 5).
Acknowledgements
This work was supported by Suranaree University of Technology (SUT) Research and Development Fund.
Footnotes
Authors’ Contributions
Wilasinee Promjantuek provided the concept and design of the study, performed the experiments, analysed and interpreted data and prepared the article for publication. Nipha Chaicharoenaudomrung analysed and interpreted the data and prepared the article for publication. Ruchee Phonchai performed experiments, provided resources and prepared the article for publication. Phongsakorn Kunhorm analysed and interpreted data and prepared the article for publication. Parinya Noisa provided the concept and design of the study, collected samples, analysed and interpreted data, wrote the manuscript, prepared the article for publication and gave the final publication approval of the manuscript.
Conflicts of Interest
The Authors declare that they have no conflicts of interest.
- Received August 18, 2021.
- Revision received October 4, 2021.
- Accepted October 5, 2021.
- Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved