Abstract
Background/purpose
Return of Ca2+ to endoplasmic reticulum is mediated by Orai/STIM-mediated store-operated Ca2+ entry (SOCE) channel. We aimed to investigate Orai1 and STIM1 expressions in human oral carcinogenesis.
Materials and methods
Sixty-six oral squamous cell carcinomas (OSCCs), 14 oral potentially malignant disorders (OPMD) with moderate-severe oral epithelial dysplasia (OED), 19 OPMD with mild OED, and 14 normal oral mucosa (NOM) samples were subjected to immunohistochemical staining. Two human oral cancer cell lines (OCCLs), an oral premalignant cell line (DOK), and a normal oral keratinocyte culture (HOK) were used for Western blot and real-time quantitative reverse transcription-polymerase chain reaction. OCCLs were evaluated for proliferation, migration, and invasion assays.
Results
Orai1 and STIM1 protein and mRNA expressions in OSCC were significantly enhanced as compared with normal samples. Protein expressions of Orai1 and STIM1 in OCCLs were significantly enhanced as compared with HOK. Significant decreases in degrees of proliferation, migration and invasion were noted in OCCLs with Orai1 and STIM1 siRNA transfection as compared with those without transfection. Significantly increased Orai1 and STIM1 protein levels were noted in OPMD with moderate-severe OED as compared with those with mild OED.
Conclusion
Our results indicate that Orai1 and STIM1 overexpression is associated with human oral carcinogenesis.
Keywords: Orai1, STIM1, Oral squamous cell carcinoma, Oral potentially malignant disorders, Calcium channel
Introduction
Oral squamous cell carcinoma (OSCC) is the 4th most frequently-occurring malignancy in Taiwan and is the 5th top cancer death in males.1 A portion of OSCC is preceded by human oral potentially malignant disorders (OPMDs), especially with oral epithelial dysplasias (OEDs) indicating early detection of human OPMDs is essential.2 Lesion with dysplastic changes confined to the lower-one-third of the oral epithelium is classified as mild OED whereas moderate OED is defined when dysplastic changes noted for up to the lower-middle of the thickness of the oral epithelium; and severe OED as dysplastic cells observed within lower-two-third of the oral epithelium.3
Intracellular Ca2+modulates a wide spectrum of cellular processes such as gene transcription, cell proliferation, cell migration, autophagy and apoptosis.4, 5, 6, 7, 8, 9 Reviewing literature, network controlling intracellular Ca2+ hemostasis essentially consists of four different Ca2+ channels or transporters:10 (1) Inositol 1,4,5-trisphosphate receptor mediating Ca2+ release from endoplasmic reticulum; (2) Ca2+-ATPase pumping the return of Ca2+ to endoplasmic reticulum from cytosol; (3) Ca2+ channels/transporters permitting Ca2+ influx via plasma membrane from an extracellular Ca2+ reservoir, which includes (i) voltage-gated Ca2+ channel; (ii) transient receptor potential channel; (iii) store-operated Ca2+ entry (SOCE) channel; (iv) Na+/Ca2+ exchange and purinergic receptors; (4) Mitochondrial Ca2+ uniporter modulating influx of mitochondrial Ca2+.
Additionally, the uptake of Ca2+ back into endoplasmic reticulum is chiefly mediated by the SOCE channel.4 In brief, the influx of Ca2+ to the endoplasmic reticulum mediated by the SOCE pathway is executed through the following steps:11 (1) Decrease of intracellular Ca2+ in the endoplasmic reticulum [via the above-mentioned channel (1)]; (2) The reduced endoplasmic reticulum Ca2+ store releases a signal to an endoplasmic reticulum-localized Ca2+ sensor [called stromal interaction molecule 1 (STIM1)], then inducing translocation of STIM1 to endoplasmic reticulum-plasma membrane junctions; (3) The clustering of STIM1 molecules at the endoplasmic reticulum-plasma membrane releases retrograde signals to the cell surface pore-forming unit called Orai1 to open the Ca2+ channels and allow Ca2+ influx.
To the best of our knowledge, Orai/STIM-mediated SOCE channel has not been studied in human oral carcinogenesis. The current study is aimed to evaluate Orai1/STIM1-mediated SOCE channel expression in human oral squamous cell carcinogenesis.
Materials and methods
Immunohistochemistry
Tissue specimens from 66 primary OSCC patients, 14 OPMD with moderate to severe OED patients, 19 OPMD with mild OED patients with the habits of drinking alcohol, chewing betel quid, and smoking cigarettes as well as normal oral mucosal tissues from 14 healthy persons without the oral risk factors for oral malignancy were obtained under the approval of the Ethics Committee for Scientific Research on Human Beings of the institution (KMUHIRB-E(II)-20180,225). The characteristics of the OSCC patients, including age, gender, differentiation, tumor size, histopathological lymph-node involvement, and stage were listed in Table 1. All the tissue specimens were fixed in 10% neutral buffered formalin solution, dehydrated in graded alcohols, cleaned in xylene, and embedded in paraffin for immunohistochemical staining for Orai1 and STIM1 proteins with the primary rabbit polyclonal anti-Orai1 (Invitrogen, IL, USA; Cat. No. MA5-15776; 1: 100) and anti-STIM1 (Invitrogen; Cat. No. MA5-19451; 1: 100) antibody respectively using the standard avidin-biotin peroxidase complex method with respect to the manufacturer's guidelines (Vector Laboratories).12 Each set of experiments contained a specimen known to express Orai1 and STIM1 respectively, which served as positive controls. Negative controls were included following the same procedures, but with omission of the primary antibody. The scores of the percentage of positive staining (P) were classified according to Table 2. Total score (S) was determined as P × I for each section.13 Quantification of the immunostained slides were performed by two certified oral pathologists independently using the image analysis software Image J Version 1.51e. When disagreement existed between the two observers, a consensus was reached by mutual discussion.
Table 1.
Characteristics of the oral squamous cell carcinoma patients.
| Patients characteristics |
Number (%) |
| Gender | |
| Male (mean age: 58.6 years) | 62 (93.9) |
| Female (mean age: 75.6 years) | 4 (6.1) |
| Age | |
| ≤55 years | 30 (45.5) |
| >55 years | 36 (54.5) |
| Differentiation | |
| Well | 53 (80.3) |
| Moderate- to poorly | 13 (19.7) |
| Tumor size | |
| ≤2 cm | 35 (53.0) |
| >2 cm | 31 (47.0) |
| Lymph-node metastasis | |
| Yes | 27 (57.5) |
| No | 20 (42.5) |
| Pathological stage | |
| I-II | 29 (43.9) |
| III-IV | 37 (56.1) |
Table 2.
Grading of immunoscores on immunochemical staining.
| Scores | Intensity | Scores | Percentage |
|---|---|---|---|
| 0 | No staining | 0 | <1% |
| 1 | Light yellow (weak staining) | 1 | 1–24% |
| 2 | Brown (Moderate-strong staining) | 2 | 25–49% |
| 3 | Dark brown (Strong staining) | 3 | 50–74% |
| 4 | 75–100% |
Cell culture
The human oral cancer cell lines (Ca9-22, and OECM-1) and the normal oral keratinocytes primary cultures (HOK) were cultured in high-glucose DMEM (Hyclone, Logan, UT, USA) with the addition of 10% fetal bovine serum (Hyclone) and 1% penicillin-streptomycin (Hyclone) at 37 °C in a humidified 5% CO2 atmosphere. The DOK was cultured in high-glucose DMEM (Hyclone, Logan, UT, USA) with the addition of 10% fetal bovine serum (Hyclone), 2 mM glutamine (Hyclone), 5 μg/ml hydrocortisone (Hyclone), and 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, USA) at 37 °C in a humidified 5% CO2 atmosphere. The culture medium was changed on every third day.
Western blot
Cells of the human oral cancer cell lines, DOK, and HOK were rinsed with phosphate buffered-saline (PBS; Sigma–Aldrich, St Louis, MO, USA) and lysed with radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma–Aldrich). The lysates were subsequently centrifuged at 4 °C, 14,000 rpm, for 15 min. Equal amounts of protein were denatured by adding sodium dodecyl sulfate (SDS) running buffer (Sigma–Aldrich) and β-mercaptoethanol (Sigma–Aldrich). The samples were then analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) (Sigma–Aldrich) on 15% gels, and the proteins were transferred onto a poly vinylidene fluoride (PVDF) membrane (Sigma–Aldrich) using Bio-Rad's transblot with the primary rabbit polyclonal primary anti-Orai1 (Invitrogen; 1: 1000) and anti-STIM1 (Invitrogen; 1: 1000) antibody respectively with species specificity for human tissues and observed molecular weight 55 kDa for Orai1 and 75 kDa for STIM1 respectively, and β-actin (Sigma–Aldrich; 1: 500), followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (Sigma–Aldrich; 1: 5000). The amount of protein was then quantified using a Fuji LAS-4000 lumino image analyzer (Fuji Photo Film Co., Tokyo, Japan). The ratio was normalized by the β-actin signal.
Real-time quantitative reverse transcription-polymerase chain reaction (real time qRT-PCR)
Total RNA samples were prepared from human oral cancer cell lines, DOK, and HOK using TRIzol Reagent (Invitrogen).14 The procedures of real-time qRT-PCR was then implemented according to our previous study15 with oligoprimers for Orai1 (forward: 5′-GCT TCG CCA TGG CAA T-3’; reverse: 3′-GCT GAT CAT GAG CGC AAA CA-5’; accession no. NM_032790.3), STIM1 (forward: 5′-AGA AAC ACA CTC TTT GGC ACC; reverse: 3′-AAT GCT GCT GTC ACC TCG; accession no. NM_001277962) and β-actin (forward: 5′-CAC CAT TGG CAA TGA GCG GTT C-3’; reverse: 5′-AGG TCT TTG CGG ATG TCC ACG T-3′), which were designed with reference to the published cDNA sequences in GenBank. After normalization to the expression level of β-actin mRNA, the relative expression levels of mRNA for each of human oral cancer cell line, and DOK, were determined as the percentage change as compared with HOK; and additionally, the relative expression levels of mRNA for each of human oral cancer cell line were determined as the percentage change as compared with DOK.
Establishment of human OSCC cell cultures with Orai1 and STIM1 genes knock-down
RNA interference using commercially-synthesized Orai1 and STIM1 siRNAs (Orai1: forward 5′-GGU GAG CAA CGU GCA CAA Utt-3'; reverse 3′-ttC CAC UCG UUG CAC GUG UUA-5'; STIM1: forward 5′-GGA AGU CAU CAG AAG UAU Att; reverse 3′-UAU ACU UCU GAU GAC UUC Cat-5′) to establish human oral cancer cell lines with Orai1 and STIM1 genes knock-down was performed with the procedures according to our previous study.15
Proliferation assay, migration assay, in vitro transwell invasion assay
The cell proliferation assay, migration assay, in vitro transwell invasion assay experiments for human oral cancer cell lines were measured for 48 h post-transfection of Orai1 and STIM1 siRNA respectively using the procedures according to our previous study.15
Statistical analyses
Statistical analyses were performed using SAS Statistical Package (Version 9.1.3, SAS Institute Inc.) with statistical significance when the P value < 0.05. Paired t-test was used to compare the immunohistochemical expressions among OSCCs, OPMDs and normal oral mucosa, and along with the differentiation, tumor size, histological lymph-node involvement, and pathological stage of the OSCC patients as well as to evaluate the differences in proliferation rate and degrees of migration and invasion between human oral cancer cells with and without siRNA transfection. Nonparametric Kruskal–Wallis tests were used to analyze the results of western blots and real-time quantitative reverse transcription-polymerase chain reactions.
Results
Immunochemistry
Immunohistochemistry for Orai1
Strong positive staining (cytoplasmic and nuclear) of Orai1 protein was observed in OSCC (Fig. 1A). On the other hand, weak positive staining of Orai1 protein was observed in normal oral mucosa samples (chiefly membrane) (Fig. 1B). The mean IS of the OSCC (T1-T2) and OSCC (T3-T4) cases was 5.6 and 8.1 respectively, whilst that of the normal oral mucosa samples was 1.84 (Table 3). A significant increase was noted respectively in the mean IS of the OSCC (T1 & T2) and OSCC (T3 & T4) cases in comparison with that of the normal oral mucosa samples (Table 3). Additionally, a significant difference in the mean IS of Orai1 protein was confirmed in tumor size as comparison of the OSCC (T3-T4) and OSCC (T1-T2) patients (Table 4). There was an increased mean IS of Orai1 protein with lymph node metastasis, and pathological stage; however, no significant correlations have been established for tumor size (Table 4).
Figure 1.
Immunohistochemistry for Orai1 (A) A representative strong immunohistochemical staining of Orai1 protein in human oral squamous cell carcinoma (× 100) (B) A representative weak staining of Orai1 protein in human normal oral mucosa (× 100; × 400 for inset) (C) A representative stronger immunohistochemical staining of Orai1 protein for a human oral potentially malignant disorder with moderate to severe oral epithelial dysplasia (× 100) (D) A representative weaker staining of Orai1 protein for a human oral potentially malignant disorder with mild oral epithelial dysplasia (× 100).
Table 3.
Statistical analyses for immunohistochemical expression of Orai1 protein amongst human oral squamous cell carcinomas (OSCCs), human oral potentially malignant disorders (OPMDs) with moderate to severe oral epithelial dysplasia (OED), OPMDs with mild OED, and normal oral mucosa (NOM).
|
P values |
|||||
|---|---|---|---|---|---|
| OSCC (T1-T2) | OSCC (T3-T4) | OPMD with moderate to severe OED | OPMD with mild OED | NOM | |
| OSCC (T1-T2) (5.57 ± 2.33)a | ∗<0.05 | >0.05 | ∗∗<0.01 | ∗∗<0.01 | |
| OSCC (T3-T4) (8.14 ± 2.96) | ∗<0.05 | >0.05 | ∗<0.01 | ∗∗<0.01 | |
| OPMD with moderate to severe OED (5.73 ± 1.27) | >0.05 | >0.05 | ∗<0.01 | ∗∗<0.01 | |
| OPMD with mild OED (2.42 ± 1.15) | ∗∗<0.01 | ∗∗<0.01 | ∗∗<0.01 | >0.05 | |
| NOM (1.84 ± 1.77) | ∗∗<0.01 | ∗∗<0.01 | ∗∗<0.01 | >0.05 | |
∗statistically significance, ∗∗statistically significance.
Values within brackets: Immunoscore (mean ± standard deviation)
Table 4.
Statistical comparison of immunohistochemical expression of Orai1 protein respectively in human oral squamous cell carcinomas with histopathological features.
| Patients characteristics | Immunoscores (mean ± standard deviation) | P values |
| Gender | ||
| Male | 6.8 ± 3.3 | >0.05 |
| Female | 10.5 ± 2.1 | |
| Age | ||
| ≤55 years | 6.9 ± 2.8 | >0.05 |
| >55 years | 7.0 ± 2.9 | |
| Differentiation | ||
| Well | 7.2 ± 3.0 | >0.05 |
| Moderate-to poorly | 6.5 ± 3.8 | |
| Tumor size | ||
| T1-T2 | 5.6 ± 2.3 | <0.05∗ |
| T3-T4 | 8.1 ± 3.0 | |
| Lymph-node metastasis | ||
| Yes | 8.2 ± 2.6 | >0.05 |
| No | 6.8 ± 3.0 | |
| Pathological stage | ||
| I-II | 5.6 ± 2.4 | >0.05 |
| III-IV | 7.8 ± 3.2 | |
∗ Statistically significance.
Positive staining of Orai1 protein was observed in cases of human OPMD with moderate to severe OED (Fig. 1C) and in cases of human OPMD with mild OED (Fig. 1D). The mean IS of the human OPMDs with moderate to severe OED and with mild OED was 5.73 and 2.42 respectively (Table 3), a significant increase being noted when comparing the mean IS of human OPMDs with moderate to severe OED with those with mild OED (Table 3). Also, a significant increase in the mean IS was noted when comparing cases of human OPMDs with moderate to severe OED with normal mucosa samples (Table 3); no significant increase of mean IS being found when comparing human OPMDs mild OED with normal mucosa samples (Table 3). On the other hand, a significant increase of mean IS being noted as comparing human OPMD with mild OED with samples of OSCC (T1-T2) and OSCC (T3-T4) respectively (Table 3); however, no significant increase of mean IS being observed when comparing human OPMDs with moderate to severe OED with samples of OSCC (T1-T2) and OSCC (T3-T4) respectively (Table 3).
Immunohistochemistry for STIM1
Strong positive staining of STIM1 protein was observed in OSCC (Fig. 2A). On the other hand, weak positive staining of STIM1 protein was observed in normal oral mucosa samples (Fig. 2B). The mean IS of the OSCC (T1-T2) and OSCC (T3-T4) cases of STIM1 protein was 5.7 and 7.5 respectively, whilst that of the normal oral mucosa samples was 2.6 (Table 5). A significant increase was noted respectively in the mean IS of the OSCC (T1-T2) and OSCC (T3-T4) cases in comparison with that of the normal oral mucosa samples (Table 5). Additionally, a significant difference in the mean IS of STIM1 protein was confirmed in a comparison of the OSCC (T3-T4) with OSCC (T1-T2) patients (Table 6); On the other hand, there were significant increases of the mean IS of STIM1 protein with tumor size, lymph node metastasis, and patholoigcal stage respectively (Table 6).
Figure 2.
Immunohistochemistry for STIM1 (A) A representative strong immunohistochemical staining of STIM1 protein in human oral squamous cell carcinoma (× 100) (B) A representative weak staining of STIM1 protein in human normal oral mucosa (× 100) (C) A representative stronger immunohistochemical staining of STIM1 protein for a human oral potentially malignant disorder with moderate to severe oral epithelial dysplasia (× 100) (D) A representative weaker staining of STIM1 protein for a human oral potentially malignant disorder with mild oral epithelial dysplasia (× 100).
Table 5.
Statistical analyses for immunohistochemical expression of STIM1 protein amongst human oral squamous cell carcinomas (OSCCs), human oral potentially malignant disorders (OPMDs) with moderate to severe oral epithelial dysplasia (OED), OPMDs with mild OED, and normal oral mucosa (NOM).
|
P values |
|||||
|---|---|---|---|---|---|
| OSCC (T1-T2) | OSCC (T3-T4) | OPMD with moderate to severe OED | OPMD with mild OED | NOM | |
|
OSCC (T1-T2) (5.7 ± 2.6)a |
∗∗<0.01 | >0.05 | ∗∗∗<0.001 | ∗∗∗<0.001 | |
|
OSCC (T3-T4) (7.5 ± 1.8) |
∗∗<0.01 | ∗∗<0.01 | ∗∗∗<0.001 | ∗∗∗<0.001 | |
| OPMD with moderate to severe OED (6.2 ± 1.1) | >0.05 | ∗∗<0.01 | ∗∗∗<0.001 | ∗∗∗<0.001 | |
| OPMD with mild OED (2.6 ± 1.5) | ∗∗∗<0.001 | ∗∗∗<0.001 | ∗∗∗<0.001 | >0.05 | |
| NOM (2.6 ± 1.0) | ∗∗∗<0.001 | ∗∗∗<0.001 | ∗∗∗<0.001 | >0.05 | |
∗∗statistically significance, ∗∗∗statistically significance.
Values within brackets: Immunoscore (mean ± standard deviation).
Table 6.
Statistical comparison of immunohistochemical expression of STIM1 protein in human oral squamous cell carcinomas with histopathological features.
| Patients characteristics | Immunoscores (mean ± standard deviation) | P values |
| Gender | ||
| Male | 6.5 ± 2.5 | >0.05 |
| Female | 7.5 ± 0.6 | |
| Age | ||
| ≤55 | 7.0 ± 2.4 | >0.05 |
| >55 | 6.3 ± 2.5 | |
| Differentiation | ||
| Well | 6.4 ± 2.5 | >0.05 |
| Moderate-to poorly | 7.5 ± 2.2 | |
| Tumor size | ||
| T1-T2 | 5.7 ± 2.6 | ∗∗<0.01 |
| T3-T4 | 7.5 ± 1.8 | |
| Lymph-node metastasis | ||
| Yes | 7.5 ± 2.0 | ∗<0.05 |
| No | 6.1 ± 2.3 | |
| Pathologic stage | ||
| I-II | 5.5 ± 2.5 | ∗∗∗<0.001 |
| III-IV | 7.4 ± 2.0 | |
∗Statistically significance.
Positive staining of STIM1 protein was observed in cases of human OPMD with moderate to severe OED (Fig. 2C) and in cases of human OPMD with mild OED (Fig. 2D). The mean IS of the human OPMD with moderate to severe OED and human OPMD with mild OED was 6.2 and 2.6 respectively (Table 5), a significant increase being noted when comparing the mean IS of human OPMD with moderate to severe OED with human OPMD with mild OED (Table 5). Also, a significant increase in the mean IS was noted when comparing cases of human OPMD with moderate to severe OED with normal mucosa samples (Table 5); no significant increase of IS was found when comparing human OPMD with mild OED with normal mucosa samples (Table 5).
Western blot
Western blot for Orai1
Western blot analysis of the human oral cancer cell lines (Ca9-22, and OECM-1) indicated overexpression of Orai1 protein as compared with primary cultures of human normal oral keratinocytes (HOK), these results being of statistical significance (Fig. 3A & B). On the other hand, Western blot analysis of human oral premalignant cell line DOK showed overexpression of Orai1 protein as compared with HOK with statistical significance (Fig. 3A & B). Additionally, a significant upregulation of Orai1 protein expression was also noted for human oral cancer cell lines (OECM-1 and Ca9-22) as compared with DOK (Fig. 3A &C).
Figure 3.
Western blot for Orai1. Upregulation of Orai1 protein expression in human oral cancer cell lines (Ca9-22, and OCEM-1) as compared with the primary culture of human normal oral keratinocytes (HOK) (A, B). Human oral premalignant cell line (DOK) showed overexpression of Orai1 protein as compared with HOK (A, B). Upregulation of Orai1 protein expression of Ca9-22, and OECM-1 was observed as compared with DOK respectively (A, C). Results were quantified using densitometric analysis, normalized by the level of β-actin, and expressed as fold change relative to the HOK. Bars represent means ± standard error of the mean (∗P < 0.05; ∗∗P < 0.01). A representative result of three independent experiments is shown.
Western blot for STIM1
Western blot analysis of the human oral cancer cell lines (Ca9-22, and OECM-1) indicated overexpression of STIM1 protein as compared with primary cultures of human normal oral keratinocytes (HOK), these results being of statistical significance (Fig. 4A & B). On the other hand, Western blot analysis of human oral premalignant cell line DOK showed overexpression of STIM1 protein as compared with HOK with statistical significance (Fig. 4A & B). Additionally, a significant upregulation of STIM1 protein expression was also noted for human oral cancer cell lines (Ca9-22, and OECM-1) as compared with DOK (Fig. 4A &C).
Figure 4.
Western blot for STIM1. Upregulation of STIM1 protein expression in human oral cancer cell lines (Ca9-22, and OCEM-1) as compared with the primary culture of human normal oral keratinocytes (HOK) (A, B). Human oral premalignant cell line (DOK) showed overexpression of STIM1 protein as compared with HOK (A, B). Upregulation of STIM1 protein expression of Ca9-22, and OECM-1 was observed as compared with DOK respectively (A, C). Results were quantified using densitometric analysis, normalized by the level of β-actin, and expressed as fold change relative to the HOK. Bars represent means ± standard error of the mean (∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001). A representative result of three independent experiments is shown.
Real time qRT-PCR
Real time qRT-PCR for Orai1
Real-time qRT-PCR analyses of the human oral cancer cell lines (Ca9-22, and OECM-1) indicated upregulation of Orai1 mRNA expression with statistical significance as compared with HOK (Fig. 5). On the other hand, real-time qRT-PCR analyses of the human oral cancer cell lines (Ca9-22, and OECM-1) indicated an upregulation of Orai1 mRNA expression as compared with DOK with statistical significance (Fig. 5). Additionally, there was an overexpression of Orai1 mRNA expression of DOK when compared with HOK but without statistical significance (Fig. 5).
Figure 5.
Real-time quantitative reverse transcription-polymerase chain reaction for Orai1. Upregulation of Orai1 mRNA expression was observed in human oral cancer cell lines (Ca9-22, and OECM-1) as compared with the primary culture of normal human oral keratinocytes (HOK) (∗P < 0.05). Upregulation of Orai1 mRNA expression was observed in human oral cancer cell lines (Ca9-22, and OECM-1) as compared with the human oral premalignant cell line (DOK) (✝P < 0.05). Upregulation of Orai1 mRNA expression without statistical significance was observed in DOK as compared with HOK. Results were normalized by the level of β-actin & expressed as fold change relative to HOK & DOK respectively. Bars represent mean ± standard error of the mean.
Real time qRT-PCR for STIM1
Real-time qRT-PCR analyses of the human oral cancer cell lines (Ca9-22, and OECM-1) indicated upregulation of STIM1 mRNA expression with statistical significance as compared with HOK (Fig. 6). On the other hand, real-time qRT-PCR analyses of OECM-1 indicated a significant upregulation of STIM1 mRNA expression as compared with DOK (Fig. 6). Additionally, there was a significant overexpression of STIM1 mRNA expression of DOK when compared with HOK (Fig. 6).
Figure 6.
Real-time quantitative reverse transcription-polymerase chain reaction for STIM1. Upregulation of STIM1 mRNA expression was observed in human oral cancer cell lines (Ca-922, and OECM-1) as compared with the primary culture of normal human oral keratinocytes (HOK) (∗P < 0.05; ∗∗∗P < 0.001). Upregulation of STIM1 mRNA expression was observed in the human oral premalignant cell line (DOK) as compared with HOK (∗∗∗P < 0.001); upregulation of STIM1 mRNA expression was observed in OECM-1 as compared with DOK (✝P < 0.05). Results were normalized by the level of β-actin and expressed as fold change relative to HOK & DOK respectively. Bars represent means ± standard error of the mean.
Proliferation assay
Proliferation assay for Orai1
The proliferation rates of the oral cancer cell lines (Ca9-22, and OECM-1) with Orai1 siRNA transfection for 48 h were significantly decreased as compared with the oral cancer cell lines without Orai1 siRNA transfection (Fig. 7).
Figure 7.
Proliferation assay for Orai1. The proliferation rates of the oral cancer cell lines (Ca9-22, and OECM-1) with Orai1 siRNA transfection for 48 h were significantly lower than those of the oral cancer cell lines without Orai1 siRNA transfection (∗P < 0.05).
Proliferation assay for STIM1
The proliferation rates of the human oral cancer cell lines (Ca9-22, and OECM-1) with STIM1 siRNA transfection for 48 h were significantly decreased as compared with the oral cancer cell lines without STIM1 siRNA transfection respectively (Fig. 8).
Figure 8.
Proliferation assay for STIM1. The proliferation rates of the oral cancer cell lines (Ca9-22, and OECM-1) with STIM1 siRNA transfection for 48 h were significantly lower than those of the oral cancer cell lines without STIM1 siRNA transfection (∗P < 0.05, ∗∗P < 0.01).
Migration assay
Migration assay for Orai1
The degrees of migration of the human oral cancer cell lines (Ca9-22, and OECM-1) with Orai1 siRNA transfection for 48 h were significantly decreased as compared with the oral cancer cell lines without Orai1 siRNA transfection (Fig. 9).
Figure 9.
Migration assay for Orai1. Degree of migration of the oral cancer cell lines (Ca9-22, and OECM-1) with Orai1 siRNA transfection for 48 h was significantly decreased as compared with the oral cancer cell lines without transfection (∗P < 0.05, ∗∗P < 0.01).
Migration assay for STIM1
The degrees of migration of the oral cancer cell lines (Ca9-22, and OECM-1) with STIM1 siRNA transfection for 48 h were significantly decreased as compared with the oral cancer cell lines without STIM1 siRNA transfection (Fig. 10).
Figure 10.
Migration assay for STIM1. Degree of migration of the oral cancer cell lines (Ca9-22, and OECM-1) with STIM1 siRNA transfection for 48 h was significantly decreased as compared with the oral cancer cell lines without transfection (∗P < 0.05).
In vitro transwell invasion assay
In vitro transwell invasion assay for Orai1
The degrees of invasion of the human oral cancer cell lines (Ca9-22 and OECM-1) with Orai1 siRNA transfection for 48 h were significantly decreased as compared with the oral cancer cell lines without Orai1 siRNA transfection (Fig. 11).
Figure 11.
Invasion assay for Orai1. Degree of invasion of the oral cancer cell lines (Ca9-22, and OECM-1) with Orai1 siRNA transfection for 48 h was significantly decreased as compared with the oral cancer cell lines without transfection (∗P < 0.05, ∗∗P < 0.01).
In vitro transwell invasion assay for STIM1
The degrees of invasion of the oral cancer cell lines (Ca9-22, and OECM-1) with STIM1 siRNA transfection for 48 h were significantly decreased as compared with the oral cancer cell lines without STIM1 siRNA transfection respectively (Fig. 12).
Figure 12.
Invasion assay for STIM1. Degree of invasion of the oral cancer cell lines (Ca9-22, and OECM-1) with STIM1 siRNA transfection for 48 h was significantly decreased as compared with the oral cancer cell lines without transfection (∗P < 0.05, ∗∗∗P < 0.001).
Discussion
Reviewing English literatures, disruption of normal intracellular Ca2+ is reported being associated to the formation of cancer in a number of studies.16, 17, 18, 19, 20, 21, 22, 23, 24 Dysregulated intracellular Ca2+ signaling in cancer cells has also been found to induce cancer angiogenesis, cancer progression, and cancer metastasis.16, 17, 18, 19, 20, 21, 22, 23, 24 Therefore, studying the particular genes such as Orai1 and STIM1 that attribute to dysregulated intracellular Ca2+ signaling in dysplastic cells is an essential area for oral cancer study for human oral mucosa.
In the current study, we have demonstrated that significantly increased Orai1 protein expression was observed in OSCCs as compared with normal oral mucosa; and Orai1 protein expression also increased significantly between T3-T4 and T1-T2 OSCC patients. An increased Orai1 protein level was noted in OPMDs with moderate to severe OED as compared to those with mild OED, and was also found in OPMDs with moderate to severe OED in comparison with normal mucosa. There is also a shift of membranous staining to cytoplasmic/nuclear staining from normal mucosa to lesions of OPMD and OSCC. Additionally, the in vivo data for Orai1 are largely compatible to the in vitro results. Orai1 protein and mRNA expressions were significantly enhanced as compared with the primary culture of normal oral keratinocytes (HOK). Significant decreases in the proliferation rate, degrees of migration, and invasion were noted in oral cancer cell lines with Orai1 siRNA transfection as compared with those without siRNA transfection.
Furthermore, our findings on Orai1 have been compatible with the other studies on various malignancies including breast carcinoma20 glioblastoma21 pancreatic adenocarcinoma25 melanoma26,27 renal carcinoma28 esophageal squamous cell carcinoma29 non-small-cell lung carcinoma30 prostate carcinoma.31
On the other hand, our study has confirmed a significantly increased STIM1 protein expression in human OSCCs when compared with normal oral mucosa; and STIM1 expression was also increased significantly between OSCC (T3-T4) and OSCC (T1-T2) patients. A significantly increased STIM1 protein level was noted in OSCC (T3-4) as compared with OPMD with moderate to severe OED; and was also noted in lesions of OPMD with moderate to severe OED in comparison with normal oral mucosa. These in vivo data for STIM1 are consistent to the in vitro results. STIM1 protein and mRNA expressions were significantly enhanced ranging from oral cancer cell lines, oral premalignant cell line (DOK) to HOK. On the other hand, significant reductions in the proliferation rate and degrees of migration, invasion was found in oral cancer cell lines with STIM1 siRNA transfection as compared with those without siRNA transfection. These in vitro findings were compatible to the in vivo data of STIM1 protein in which a significant increase was noted in tumor size, lymph-node metastasis and pathologic stage respectively. The aforementioned findings in the current study on STIM1 have been compatible with the other studies on miscellaneous types of cancers including breast carcinoma32,33 cervical cancer18,19 hepatocellular carcinoma24 melanoma27 pancreatic adenocarcinoma25 and colorectal cancer.34
Taken together for the current findings of Orai1 and STIM1, to our knowledge, the study would be the first to elucidate the expression of Orai1 and STIM1 in human oral mucosa ranging from normal mucosa, premalignant, and malignant lesions implicating that Orai1/STIM1-mediated SOCE channel expression is potentially associated to human oral squamous cell carcinogenesis.
Declaration of competing interest
The authors declare that they have no conflicts of interest.
Acknowledgement
The work was supported by a grant from the Ministry of Science and Technology, Taiwan (MOST-107-2314-B-037-045, MOST-108-2314-B-037-017), and grant from the Ministry of Health and Welfare (MOHW108-TDU-B-212-124016, Health and welfare surcharge of tobacco products) of Taiwan.
Contributor Information
Shyng-Shiou Yuan, Email: yuanssf@ms33.hinet.net.
Yuk-Kwan Chen, Email: k0285@ms22.hinet.net.
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