Evaluation of the Altered Tissue Expression of HSP60 and HSP70 Genes in Oral and Cutaneous Lichen Planus Compared to Normal Healthy Tissues.

Background: Recent highlights have investigated the possible roles of molecular chaperons like heat shock proteins (HSPs) into Lichen Planus (LP)-onset and pathogenesis. This study for the first, determine the expression of both HSP60 and HSP70 genes in cutaneous LP (CLP) and oral LP (OLP) lesions compared to normal healthy cases and between different subtypes of OLP lesions by real-time (RT)-PCR. Materials and
Methods: Paraffin blocks of LP lesions including 56 OLP and 56 CLP samples were selected from theMashhad University of Medical sciences, Mashhad, Iran. Also 56 biopsy samples of healthy normal participants were selected. The demographic and clinical characteristics were extracted from their medical records. The expression of HSP60 and HPS70 genes were evaluated using the real-time RT-PCR method.
Results: The comparison of the expression of HSP60 and HSP70 genes among the patients with CLP and OLP showed a significant overexpression of HSP60 and HSP70 genes in both groups compared to the normal participants (P = 0.001). The expression of HSP60 and HSP70 genes was high in both the groups of CLP and OLP patients, but the amount was not significantly different between the two groups. Comparing the two mucosal subgroups of OLP lesions (non-erosive and erosive) showed that the expression of the HSP60 and HSP70 in erosive subtypes of OLP was significantly higher than the non-erosive subtypes of OLP (P = 0.001).
Conclusion: Regarding the overexpression of HSP60 and HSP70 in the LP lesions compared to healthy biopsies, we conclude that HSP60 and HSP70 could have key roles in the etiopathogenesis of the OLP and CLP lesions. The overexpression of both HSP60 and HSP70 in the erosive OLP group compared to the non-erosive OLP group emphasized the possible roles of HSPs in the pathogenesis and premalignant changes of OLP lesions. Copyright:

Introduction

Lichen Planus (LP) is a relatively common chronic inflammatory disease that affects the skin and the mucosal surfaces, or both. LP as the T-cell-mediated disease with unclear etiology and the pathogenesis can present varied clinical appearances depending on its time of evolution, localization, and severity.[1234] The exact prevalence of LP is unknown, however, its estimated prevalence is between 0.22 and 5% worldwide.[5] It affects mostly 30–60-year-old adults, and is more common among women.[13] LP has two major clinical forms including cutaneous LP (CLP) and oral LP (OLP). CLP has several subtypes including popular (non-erosive), hypertrophic, vesiculobullous, actinic, annular, atrophic, linear, follicular, LP pigmentosus, and LP pigmentosus-inversus. Non-erosive CLP is the most common form of CLP and presents as small pruritic violaceous papules.[36] OLP (atrophic-erosive) is considered as a potentially precancerous lesion.[67] Several subtypes of OLP have been described including reticular, erosive, atrophic, papular, plaque-like, and bullous.[58] The development of squamous cell carcinoma is the most important complication of OLP.[9]

Heat shock proteins (HSPs) also termed as 'stress proteins' and 'molecular chaperons' are proteins mostly known for the maintenance of cellular homeostasis, have been implicated as the antigenic stimulus in autoimmune diseases.[21011] HSPs are expressed by most living cells and their synthesis that induce by a variety of naturally occurring stresses and abnormal conditions in order to enable the cellular survival.[212] HSP70 is expressed constitutively in most human cells and involves in antigen presentation, cell recognition, and secretion of pro-inflammatory factors like cytokine. In addition, HSP70 has a protective role as an anti-pro inflammatory agent.[13] HSP60 transport proteins cross the membrane by folding them. Both HSP60 and HSP70 release the cytokine from lymphocyte and participate in the pathogenesis of autoimmune diseases and chorionic inflammation.[14] It appears that OLP exogenous agents cause HSP overexpression following the stressed oral keratinocytes. This provides a T-lymphocyte reaction that results in tissue damage and lesions.[15] OLP can transform to malignancy that develops from lichenoid dysplasia to oral carcinoma.[1617] So, the assessment of LP molecular pathogenesis can increase our awareness of the role of inflammatory factors in promoting malignant changes in OLP. Although prior studies have shown that HSPs act as critical molecules in the immune and inflammatory responses of the skin,[218] the role of HSPs in the pathogenesis of only limited skin diseases has been studied so far. A few investigations have been performed about HSPs expression and their probable role in LP. They provide both supporting and opposing evidences for the participation of HSPs in the LP.[2111415] However, most of them support the hypothesis of HSPs play roles in the pathogenesis of LP. In the current study, we aimed to evaluate the expression levels of two genes, HSP60 and HSP70 in oral and cutaneous forms of LP comparing normal biopsies, and separately in erosive and non-erosive subtypes of OLP, and inquire their probable roles in the etiopathogenesis and behavior of the disease.

Materials and Methods

Study participants

In this study, we collected paraffin-embedded blocks from 112 samples of LP lesions containing 56 OLP samples (28 samples of non-erosive subtype and 28 samples of erosive subtype) and 56 CLP samples stored in the pathology archives of the faculty of dentistry, the Mashhad University of Medical Sciences (MUMS) and Imam Reza Hospital, MUMS, Mashhad, Iran. The oral biopsies were taken from the OLP diagnosed patients who were referred to the Oral and Maxillofacial Department, faculty of dentistry, MUMS, Iran, during June 2014 to September 2019. Furthermore, the cutaneous biopsies were taken from the LP-diagnosed patients referred to the Dermatology ward of the Imam Reza Hospital, MUMS, Iran, from the year 2016 to 2019. Erosive lichen planus (ELP) is a variant of lichen planus which involves chronic and painful ulceration of the skin and mucosal surfaces. Cutaneous lichen planus (CLP) most commonly involves the flexor surfaces of the extremities and presents as small, itchy, violaceous papules in middle-aged adults. The cutaneous type of LP is classified to classic, hypertrophic, atrophic, bullous, linear, annular, eruptive, pigmentosus, ulcerative, actinic, and lichen planoplaris.[3192021]

The inclusion criteria considered were patients with CLP or OLP without any other systemic diseases and autoimmune diseases, any previous history about smoking and alcohol using, no tissue necrosis in related lams, and samples with adequate quality. The samples with inadequate quality and quantity, samples with inexplicable PCR results for analysis, and samples with another microscopic diagnosis except OLP and CLP were excluded. Fifty-six samples from normal oral and normal cutaneous samples were obtained from the participants who were referred to our clinics for cosmetic surgery and the normal mucosa obtained from traumatic fibroma without epithelial hyperplasia and inflammatory infiltration. To prevent any possible fault, the samples were examined by two pathologists for the confirmation of the diagnosis. This study was approved by the Ethical Committee of Mashhad University of Medical Science, Mashhad, Iran.

RNA extraction, cDNA synthesis and qRT-PCR

First, five to six pieces of each tissue paraffin block was cut at 5 μm thicknesses, then de-paraffinized by 800 μL xylene, and placed in the incubator for 5 min. After adding 400 μL of 96% ethanol, the microtubes were centrifuged and the supernatant was discarded. The total RNA was extracted from tissues using the High Pure RNA Paraffin Kit (Roche Company, Germany) following the manufacture's protocol. The quality of RNA extracted was evaluated by %1.5 agarose gel electrophoresis and its quantity was determined by NanoDrop (Thermo 2000, USA) in absorbance at 260 nm in relation to absorbance at 280 nm. In the next step, a defined amount of RNA (200 ng) was applied to synthesis cDNA by Revert Aid First Strand cDNA Synthesis Kit (Thermos Scientific, Germany) according to the manufacturer's instruction: the reaction was performed on total 20 μL volume containing 5X reaction buffer 4 μL, Ribolock Rnase inhibitor 1 μL, dNTP mix 10 mMol 2 μL and reverse transcriptase 1 μL by ABI thermocycler (One Step, USA).

The level of HSP60 and HSP70 gene expression was quantified by real-time PCR system (ABI, USA). A total 2 μL of cDNA was mixture to 10 μL SYBER Green master mix (Thermos Scientific, Germany) and added to 0.7 pM of each primer and 7 μL DEPC water. The cycling parameters were related to the respected processes; initial holding (94°C for 10), denaturation (40 cycles at 94°C for 30 s), annealing (62°C for 45 s), extension (72°C for 30 s), and final extension (72°C for 30 s).

The △△CT method[36] was used for relative expression of mRNAs gene expression and also for RT-PCR analysis by considering HSP60 and HSP70 as target genes and the housekeeping β-Actin gene was used to normalize for the amount and quality of the RNAs [Figures 1 and 2]. The sequences of primer for the target genes are listed in Table 1.

Figure 1

Amplification plot for HSP60 and HSP70 expression. Amplification plot for HSP60 and HSP70 expression. Each curve in this diagram describes the gene expression in one of the patients. The gene expression curves on the left of the graph show the increase in gene expression and the curve on the right shows the decrease in gene expression.

Figure 2

Melt curve for HSP60 and HSP70 and β-actin expression. Melt curve for HSP60 and HSP70 and β-actin expression. Each curve shows the expression of a gene in the tissues of patients.

Table 1

Primer sequences of genes HSP 60, HSP 70 and β-actin

Gene	Forward primer	Reverse primer
HSP 60	5’- GAGTAGAGGCGGAGGGAG -3’	5’- AGTGAGATGAGGAGCCAGTA-3’
HSP 70	5’- CAGATGAGGCCGTGGCTTAT-3’	5’- GGGAGTCACATCCAACAGCAA-3’
β-actin	5’- AGCGGGAAATCGTGCGTG -3’	5’- GGGTACATGGTGGTGCCG-3’

Statistical analyses

The continuous variables including age and gene expressions of HSP60 and HSP70 were expressed as means ± standard deviation and categorical variables were presented as numbers (percentage). The gene expressions between the dummy variables were analyzed using the Mann–Whitney test and between categorical variables were compared by the ANOVA analysis of variance test. The comparisons between groups according to categorical variables were done using a two-sided Chi-square test. The statistical analysis was performed using the software SPSS, ver. 18 (IBM SPSS Statistics for Windows). A P value less than 0.05 was considered as the level of statistical significance.

Results

This study was performed on 112 samples of the lesions diagnosed with LP containing 56 samples of CLP and 56 samples of OLP (28 non-erosive and 28 erosive lesions). The patients enrolled were 56 males (40.3%) and 83 females (59.7%). The average age of the study population was 42.49 ± 13.56. The locations of the oral lesions were in the oral mucosa (41.1%), gum (32.1%), tongue (17.9%), lips (5.4%), and mouth floor (3.6%) respectively. Age and gender differences related to the studied groups are presented in Table 2. There were not any significant differences between the studied groups related to age and gender (P > 0.05).

Table 2

Demographic characteristics of participants related to different studied groups

Variable	Studied groups			P
	CLP	OLP	Normal
Age, years (Mean±SD)	39.96±14.31	46.42±11.89	43.51±11.87	*0.37
Gender
Male	21 (37.5%)	24 (42.9%)	29 (51.7%)	**0.13
Female	35 (62.5%)	32 (57.1%)	27 (48.21%)

SD: Standard deviation, P value calculated by *ANOVA Test of variance and by** chis-square test respectively

HSP-60 expression in CLP and OLP lesions

Figure 3 shows that the overexpression of HSP60 was prominent for the CLP samples compared to others (P = 0.001). The expression of HSP60 among different studied samples is shown in Table 3. This expression shows that there was a significant difference between HSP60 expression in the non-erosive and erosive OLP samples (P = 0.001). Although, the expression of HSP60 was higher in females compared to the male participants, there was no significant difference between the HSP60 expression in the male and female participants (P > 0.05, Table 4). Moreover, there was not any significant difference of the HSP60 expression among different locations of OLP lesion (P > 0.05, Table 4).

Figure 3

Expression of HSP 60 in studied groups

Table 3

Distribution of expression of HSP 60 and HSP 70 in studied groups

Variable	Variable	Mean±SD	P
HSP60	CLP	36.78±5.31	*0.01
	OLP	28.90±2.43
	Normal	0.99±0.38
HSP70	CLP	87.81±11.43	*0.001
	OLP	71.07±5.24
	Normal	1±0.75
OLP	non-erosive	22.39±3.48	**0.001
HSP60	Erosive	35.41±2.96
OLP	non-erosive	61.50±8.77	**0.001
HSP70	Erosive	80.64±5.31

SD: Standard deviation, P value calculated by *ANOVA Test of variance and by **Mann-Whitney Test respectively

Table 4

Expression of HSP 60 and HSP 70 in studied groups related to gender and different oral locations

Variable			Mean±SD	P
HSP60	CLP	Female	37.09±6.60
		Male	36.25±9.06	*0.94
	OLP	Female	29.68±3.75
		Male	27.85±2.74	*0.71
	Normal	Female	1.17±0.42	*0.49
		Male	0.83±0.24
HSP70	CLP	Female	85.97±13.84	*0.80
		Male	92.09±20.90
	OLP	Female	70.14±7.34	*0.84
		Male	72.31±7.48
	Normal	Female	0.84±0.16	*0.24
		Male	1.14±0.20
HSP60	Location	Oral mucosa	29.09±3.87	**0.94
		Gums	29.52±4.24
		Tongue	27.61±4.02
		Lips	22.88±11.23
		Mouth flour	36.56±3.42
HSP70	Location	Oral mucosa	72.43±8.81
		Gums	71.51±9	**0.84
		Tongue	72.62±9.42
		Lips	74.78±35.42z
		Mouth flour	38.21±3.98

SD: Standard deviation, P value calculated by *Mann-Whitney Test of variance and **by ANOVA Test respectively

HSP70 expression in CLP and OLP lesions

The expression of HSP70 was noticeable for CLP samples compared to CLP and normal samples (P = 0.001, Figure 4). Table 3 shows the expression of HSP70 among CLP, OLP, and normal studied samples. This expression shows that there was a significant difference between HSP60 and HSP70 expression in the non-erosive and erosive OLP samples. Also, the expression of HSP60 and HSP70 shows that there was a significant difference between CLP and OLP compared to the normal participants samples (P = 0.001). The expression of HSP60 and HSP70 genes was high in both groups of CLP and OLP patients, but the amount was not significantly different between the two groups.The female patients had a higher HSP70 expression than the male patients but this difference was not meaningful (P > 0.05, Table 4). Moreover, respected to different locations of the OLP lesions, mouth flour had the expression of HSP70 than the other locations. However, this difference of HSP70 expression was not significant among the different locations of OLP samples (P > 0.05, Table 4).

Figure 4

Expression of HSP70 in three studied groups. Numbers in the vertical axis represent the relative ratio of HSP70 gene expression

Discussion

Even though the lesions of LP have prominent clinical and histopathological structures but its etiology and pathogenesis is still not clear.[13] Along with the role of HSPs into inducing innate and adaptive immunity,[222324] their roles in the pathogenesis of some autoimmune diseases were reported.[25] Particularly, two studied subtypes of HSPs including HSP60 and HSP70 are highly capable for T-cell activation.[26]

The relationship between the expression of HSPs and LP could be maintained by the ability of the HSPs to mediate unknown antigen presentation process and interaction between dendritic and T-cells by over-expression of the T cells CD40+. Previously, it was assisted that HSP70 chaperon caused the activation of both T-helper cells CD4+ through triggering the NF-κB-dependent activation of CD4+ CD45RA memory T-cells[2728] Moreover, it was reported that HSP mediates the apoptosis induction, so its role in the pathogenesis of OLP was supported. Also, overregulation and down-regulation of HSPs were observed to be associated with the different disease phenotypes.[10] Studies about the pathological features of LP could be helpful to make better therapeutic strategies. In the present investigation, for the first, we measured the expression of the HSPs genes including HSP60 and HSP70 in CLP and OLP participants comparing with healthy participants.

Previously, different studies have investigated the altered levels of HSPs in LP. The results of the current study are consistent with the last researches on the regulation of HSPs in LP which found high levels of studied HSPs and implied a probable role for HSPs in its pathogenesis.[9293031] The last study on 45 OLP patients and 35 healthy individuals reported that the serum HSP70 values in the OLP cases were significantly higher than the normal participants, which is similar to our findings.[29] In another study by Tyagi et al., 30 OLP cases, grouped as non-erosive, atrophic, and acanthotic based on the epithelial thickness were studied for the expression of HSP70 using the immune-histochemical (IHC) technique in paraffin-embedded tissues.[13] They reported that the expression of HSP70 in all the epithelial layers with significantly higher count of stained cells in the atrophic and acanthotic groups compared to the normal group.[13] This point might be implied the possible correlation between the histological nature/state of epithelium and aggravation in the disease process. They concluded that HSP70 could be recognized as a tool in the modification of the antigenic profiles within keratinocytes in OLP. However, there are studies which found that there was down-regulation or non-significant difference related to the expressions of HSPs molecules,[18] in the oral mucosal[32] and CLP lesions.[1031] Indeed, based on the immune-histochemical analysis, the pro-inflammatory role of HSP70 in LP is apparent. This might be probable that due to the stress-induced gene dysfunction, HSP70 of oral keratinocytes may act as an autoantigen before the other HSPs and mediate the “outbreak” of inflammation as part of cellular immune response in the early phase of LP pathogenesis. Fortunately, in present study, the detection the expression of HSPs in normal biopsies comparing to OLP and CLP lesions were performed. In this study, the expression of HSP60 and HSP70 genes was significantly high in both groups of CLP and OLP patients. This increase in expression may be due to the common inflammatory mechanisms in these two diseases.[3334] So, the exact over-expression pattern of HSP60 and HSP70 was detected, compared to their normal even down-regulated expression in the normal biopsies in inflammatory phase.

Therefore, the overexpression of both HSP60 and HSP70 genes in CLP and OLP compared to the normal biopsies, might be hypothesized their possible roles in the pathogenesis of this disease. Moreover, we found a higher expression of HSP60 and HSP70 in the erosive subtypes of OLP compared to non-erosive OLP subtypes. As mentioned before, cancers arise mostly from erosive, atrophic and plaque lesions of OLP. Therefore, significantly higher rates of over-expressions of both HSP60 and HSP70 in erosive-atrophic subtypes compared to non-erosive cases of OLP could be implies a probable role of these genes in premalignant and possibly malignant changes of erosive OLP. These finding shows them to be probably useful for differentiation and detection of those lesions. For as much as oral cavity is open to traumas, oral mucosa is easily ulcered and as HSP60 and HSP70 were found to be significantly higher in erosive subtype, there propounds a possibility of microbial HSPs participating in the pathogenesis of LP. Although, the immunohistochemical detection or quantitative evaluations of HSPs in every case of CLP or OLP don't recommend due to upregulations of HSPs didn't recognize as a diagnostic tool. But, detection of roles of HSP60 and HSP70 into pathogenesis of LP lesions could be the basis for using it in therapeutics purposes.[30]

Therefore, the increased expression of both HSP60 and HSP70 in CLP and OLP lesions separately and compared to the normal biopsies, as shown for the first time in our study, possibly represent a “fingerprint” of a generalized immune response which was occurred in immune-mediated diseases, like the other immune mediated disease, recurrent aphthous ulceration or Behcet disease.[35] The significant overexpression of both HSP60 and HSP70 in erosive form of OLP comparing to non-erosive form indicates a role in the systemic regulation of immune response, actually in self-limitation, and “chronicity” of OLP. Besides, it might be hypothesized the probable roles of studied HSPs into the expansion of tissue inflammation and etio-pathogenesis of OLP.

Conclusion

The current study, for the first was carried with an aim of assessing the overall altered qualitative expression of both HSP60 and HSP70 genes in cutaneous and OLP comparing to normal healthy biopsies by real-time RT-PCR. A comparison between expression of HSP60 and HSP70 into different sampled biopsies revealed that there are their over expressions in LP lesions compare to the healthy biopsies. Therefore, key roles of HSP60 and HSP70 in etio-pathogenesis of OLP and CLP lesions are established. Moreover, overexpression of both HSP60 and HSP70 in erosive subtypes of OLP compare to non-erosive subtypes of OLP could be highlighted the possible roles of HSPs into pathogenesis of LP lesions from premalignant to cancerous variations of lesions. Further molecular investigations could be helpful to make better therapeutic strategies for this disease.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.