Evaluation of Salivary Carcinogenic microR-21 and miR-125a Expression Associated with Alcohol Consumption and Smoking

Abstract

Objective:

The concept of “lifestyle” encompasses various factors, including nutrition, behavior, stress, physical activity, work habits, smoking, and alcohol consumption. Increasing evidence suggests that environmental and lifestyle factors can influence epigenetic mechanisms, such as DNA methylation, histone acetylation, and microRNA expression. Given that microRNAs (miRNAs) are an emerging focus in cancer research, there is growing interest in understanding how lifestyle choices affect miRNA responses. MiR-21 is well-established as an oncogenic miRNA, while miR-125a is reported as a tumor-suppressive miRNA in different cancers. This study aimed to analyze whether cigarette smoking and alcohol consumption are associated with altered levels of these salivary miRNAs in healthy individuals.

Methods:

Saliva supernatant samples from 50 healthy individuals (10% smokers and 34% alcohol drinkers) were analyzed alongside non-smokers and non-alcohol drinkers using real-time polymerase chain reaction (PCR). The expression levels of miR-21 and miR-125a were compared across samples based on demographic characteristics, social status, and smoking and drinking habits.

Result:

The data showed overexpression of salivary miR-21 in individuals who regularly consumed alcohol and smoked, while miR-125a expression was not significantly affected in either group.

Conclusion:

The differential expression of salivary miR-21 in healthy individuals from a localized population suggests a correlation with common lifestyle risk factors.

Introduction

Lifestyle is the way of living of a society or individual [1]. Epigenetics of an individual could be influenced by different factors such as environmental, genetics, lifestyle, psychological state and many other factors. It has been reported that those factors could impact the epigenetic process, for instance DNA methylation and miRNA expression. Not only could genome function be influenced by epigenetic mechanisms, but further stable propagation of gene activity could be impacted as well [2]. Moreover, P53 gene has been stated to be hypomethylated in blood cells of smoker lung cancer patients [3]. Starkman and colleagues have stated that interested studies in the influence of alcohol on epigenetic expression have been grown [4].

Tobacco and alcohol have been considered to be major risk factors for different types of cancer, especially head and neck cancer, in addition to human papillomavirus (HPV) infection which is considered to be an independent risk factor [5]. On the other hand, alcohol consumption has been suggested to be associated with LINE-1 hypomethylation in gastric cancer patients [6]. Many different human diseases have also been demonstrated to be associated with epigenetic mark alterations [7].

Alcohol can be metabolized by an oxidative pathway that occurs in the liver or non-oxidative pathway that takes place in the other tissues in the body. The oxidative pathway of ethanol metabolism is considered the major pathway in the liver which results in reduced of NAD+ to NADH and produces acetaldehyde, a highly reactive and toxic molecule by cytosolic alcohol dehydrogenase (ADH). Any change in ethanol metabolism could influence the ratio of NADH to NAD+. Hepatic NADH/ NAD+ ratio in the cytoplasm and mitochondria of hepatocytes is signicantly increased in response to the alcohol metabolism with other changes in different molecules in the cell such as upregulation in the ratio of lactate/ pyruvate in the cytoplasm and ratio of γ-hydroxybutyrate/acetoacetate in the mitochondria [8]. Krebs and Veech have stated that significant alteration in different reversible metabolic pathways of the hepatocytes could be revealed by alteration of a redox state [9]. Consequently, availability of oxidizable NADH to the electron transport chain will be severely increased by alcohol oxidation. Cellular reactions are influenced by NAD+. For instance, energy metabolism, histone deacetylation, and cell death could be revealed by enzymes that use NAD+ and NADH. Several studies have reported that the cytosolic free NAD+ to NADH ratio is 700:1, while it has been stated to be 7–8 to 1 in the mitochondria under normal conditions [10, 11]. Several metabolic reactions in the liver such as lipid, carbohydrate, protein, lactate, and uric acid could be changed in response to several hepatic reversible reactions that result from an increase of NADH/NAD+ ratios in both cytosol and mitochondria of the liver cells. As a result, modulation of gene expression could be revealed from any of these metabolic alterations.

MiRNA is a small non-coding RNA molecule containing about 22 nucleotides. miRNA targets mRNA via base-pairing of complementary sequence molecules. As a result, these mRNA molecules are silenced, which could inhibit the expression of the tumour suppressor gene or proto-oncogene [12]. It has been reported that human tumours could be caused by miRNAs [13]. However, other studies have stated that gastric cancer could be correlated with aberrant expression of miRNAs. [14, 15]. Moreover, several studies have reported that miR-21 was upregulated in plasma samples of patients with large B-cell lymphoma, gastric cancer and pancreatic ductal adenocarcinoma [16-18]. In addition, miR-21 has been stated to be upregulated in saliva samples of patients with head and neck cancer and pancreatic cancer [19, 20]. Meanwhile, miR-125a was reported to be up- or down-regulated depending on the type of cancer. For instance, in non-small cell lung cancer, it was down regulated but up regulated in invasive lung cancer cells [21]. Based on a recent study, correlation between deregulation of miRNAs expression and lifestyle manner such as smoking, alcohol consumption, stress, infection and radiation has been concluded [22]. These evidences of association between miRNAs expression and lifestyle factors have resulted in a clear understanding of molecular mechanisms and cancer disease which has helped to create a developed preventative method.

The aim of this study was to investigate the influence of lifestyle and social habits especially alcohol consumption and smoking on the miR-21 and miR-125a expressions, which were diagnosed as significant dysregulated miRNAs in oral cancer patients in our previous study.

Materials and Methods

Subjects

Samples was collected from different ages and genders of 50 healthy individuals who work or study at Griffith University in Gold Cost / Australia. 50 healthy adults also comprised 25 males and 25 females with a mean age of 56.6 ± 8.2 years (range 38-75 years), Consent was obtained from all participants. All participants were recruited from February 1, 2022, to July 30, 2022.

Saliva Collection

A whole saliva sample was collected without stimulation from healthy people as previously described [23, 24]. Prior, participants were prevented from eating, drinking, and using oral hygiene prior to sample collection. To keep samples clear and uncontaminated, participants were asked to wash their mouths before collecting the sample. A round 8 ml of saliva sample was spat into sterile free falcon tube size 50 ml and kept on ice.

Total Salivary RNA extraction

Total RNAs were isolated from 440 µl saliva sample using RNeasy kit (Qiagen, Valencia, CA, USA). The purification of RNAs was 1.5- 2.0 while the quantification was 500-900 ng/µ using NanoDrop spectrophotometer (ND2000; Wilmington, DE, USA).

Reverse transcription

Total RNA (10-50 ng) was reverse transcribed to cDNA via the miRNA reverse transcription kit (Origene, HP). The10-µl RT reaction mixture contained 1 to 2μg of total RNA, 1 μl of poly A tailing buffer , 1 μl of mM ATP, 1 μl of Poly (A) polymerase and was adjusted to 10 µl by nuclease-free water. The polyadenylation reaction continued at 37˚C for two hours. Subsequently, 1μl oligo dT primer was added to the reaction and then the tube was incubated at 70˚C for five minutes and on ice for two minutes. For final cDNA synthesis, 4 μl of 5X MMLV buffer was added, then incubated at 42˚C for 1 hour, 95˚ for 5 minutes and then placed on ice. The reaction mixture was diluted with 200 µl molecular water and stored at -20˚C prior to real-time RT-qPCR assay.

Real-time Quantitative Polymerase Chain Reaction

To validate salivary miR-21 and miR-125a expressions of healthy individuals in different conditions, RT-qPCR was performed. RT-qPCR was performed with SYBR green assay (Bio-RAD, HP 172-5017). RT-qPCR detection for the miRNAs was performed using specific forward primers and a universal reverse primer according to the manufacturer’s protocol (Origene). The primers used in this study are listed in Table 1. PCR amplicons were detected by the level of fluorescence emitted by SYBR green. Each reaction was performed in triplicate and cycling conditions were as follows: 95˚C for 30 seconds then 95˚C for 15 seconds, 55˚C for 10 seconds, and 72˚C for 30 seconds repeated 42 times. At the end of the PCR cycles, melt curve analysis was performed to validate generation of the expected PCR product. The setting of the melting curve was 60.0 to 95.0˚C at increments of 0.5˚C for 0.05 minutes. All Ct values were < 35. A negative template control (NTC) was used as a negative control. The RT-qPCR reactions were carried out in a Quant Studio 6 flex (Applied Biosystems); Δct was calculated and normalized against housekeeping miRNA-16.

Table 1.

Primers and Probes used for miRNA-specific RT-qPCR

Name	Forward primer	Reverse primer
miRNA-21	GCTTATCAGACTGATGTTG	GAACATGTCTGCGTATCTC
miRNA-125a	CCTGAGACCCTTTAACC	GAACATGTCTGCGTATCTC
miRNA-16	AGCAGCACGTAAATATTGG	GAACATGTCTGCGTATCTC

Statistical analyses

Samples were grouped based on demographic characteristics and lifestyle conditions. The expression level of each miR-21 and miR-125a was quantified by its normalized threshold cycle number ΔCt, where ΔCt = [Ct (Target miRNA)]- [Ct (miR-16)], and the relative expression level was calculated as 2^-(ΔCt) which is commonly used in genome-wide profiling studies of miRNAs. Data analyses were performed using Prism software, version 6. The p-value less than 0.05 was considered significant. Data expressed as mean ±SEM.

Results

In total, 50 saliva samples were collected from healthy individuals. Demographic properties were obtained from individuals. The mean age of the individuals who consumed alcohol was 60.5 ± 3.96 years, while the mean age of non-alcohol consuming individuals was 57.3 ± 2.124. The mean age of individuals who smoked was 62.4 ± 4.578 years and 68 ± 2.017 for individuals who were non-smokers. The percentage of males in the smoker group and non-smoker group was (10, 52 %) respectively, with non-significant differences. The alcohol consuming group consisted of 11 males and 6 females and the non-alcohol consuming group consisted of 20 males and 13 females which represented (22, 12, 40, 26 %) respectively.

Salivary miR-21 was selected for analysis of the influence of lifestyle (alcohol and smoking) exposure on the miRNA expression, based on the results from our previous study identifying this salivary miRNA (miR-21) as differentially expressed between OSCC patients and normal head and neck epithelia.

The expression levels of miR-21 significantly differed in saliva samples of healthy people with different habits. Upregulation of miR-21 (p=0.04) was found in alcohol consuming individuals compared with non-alcoholic people (Figure 1). Interestingly, there was statistically significant difference between the two groups in terms of smoker and non-smoker (P <0.05) (Figure 2). In addition, drinking and smoking individuals revealed significant difference in miR-21 expression when compared with non-drinking and smoking group (P=0.009).

Figure 1.

miR-21 and miR125a Expression of Samples According to the Individual’s Drinking Habit, * p<0.05

Figure 2.

miR-21 and miR125a Expression of Samples According to the Individual’s Smoking Habit,*p< 0.05

Unlike miR-21, miR-125a was found to be non-significantly influenced by smoking and alcohol consumption where p>0.05.

Discussion

This study aimed to evaluate the correlation between panel of miRNAs (miR-21 and miR-125a) expression and lifestyle condition particularly alcohol consumption and smoking. The expression of miR-21 and miR-125a, in normal saliva samples was investigated as a first step of the analysis, and then we focused on the correlation between miR-21 expression in various demographic characteristics of the subjects and lifestyle behaviour such as alcohol and smoking. The results have revealed that miR-21 was significantly increased with alcohol consumption and smoking while non-significant alteration was shown in the expression level of miR-125a.

Oral tissue which has direct contact and involved with saliva fluid, can be collected by non-invasive methods. As such saliva samples could be a good source to search for potential biomarkers. Saliva contains different enzymes some of them which degrade RNA, such as ribonucleases, but many types of RNA have been revealed in saliva samples by different studies, the results pointed to the potential presence of some mechanisms that protect salivary RNAs from degradation [25, 26]. Saliva fluid has been described to be directly contact oral tissue and could be easily obtained from individuals by non-invasive method. Several studies have stated that salivary RNAs can be protected from distraction by different processes.

It has been reported that miRNAs have a great influence on the carcinogenesis by targeting the tumor suppressor gene and oncogenic genes [27]. Furthermore, miR-21 has been reported to be upregulated in different types of cancer, for instance pancreas, breast, lung, kidney, bladder and prostate [28]. Inhibition of expressions for several genes could be revealed by miR-21 which is known as oncomiRNA, and associated with the pathogenesis of several types of human cancer [29, 30]. This study has described various factors that influence the epigenetics of humans (Figure 1 and 2). It revealed that expression level of miR-21 was increased and positively correlated with the amount of alcohol consumed and smoking in healthy subjects. An imbalance in the many gene regulation processes could be the result of any kinds of epigenetic factors influencing genes or gene expression networks during life stages. These results concur with previous studies pointing to the influence of alcohol on the different epigenetic factors. For instance, up-regulation of miR-21 expression level was reported in the tissue samples of the smoker and low social status gastric cancer patients [31]. Moreover, hepatic and neuronal tissue were stated to be influenced by the epigenetic of alcohol consumption [32]. It has been stated that methanol could lead to site-selective methylation, acetylation and phosphorylation of histones and DNA hypomethylation and result in reduction of S-adenosyl methionine (SAM) in tissue [32, 33]. Kruman and Fowler have reported that alcohol’s interference with one carbon metabolism (OCM) result in reduced availability of methyl groups and increased aberrant methylation of DNA and gene expression in alcoholic individuals [34].

Another study has also revealed that global DNA hydroxyl methylation in the liver of young mice was reduced by chronic alcohol exposure [35]. Furthermore, epigenetic alteration results from alcohol consumption have led to gastrointestinal- hepatic levels like steatosis, carcinogenesis and endotexemia [36]. Neuro-adaptations like tolerance and dependence induction have been stated to be associated with alcoholic epigenetic expression [37]. Moreover, it has been stated that reduced expression of GAD1,HDAC2,HDAC11 correlated with decreased histone acetylation at GAD1, HDAC2 promoters and enhanced expression of MT1, MT2, EGR1, and associated with upregulation of H3K4me3 at MT2 promoter and a decreased level of H3K27me3 at the MT1 promoter in the cerebral cortex [37]. Other side effects of alcohol have been shown such as an increase in the anxiolytic and motor effects and decrease in alcohol consumption especially in the male progeny of mice [38]. In addition, the same study has found upregulated BDNF expression with DNA hypomethylation in the both genders of mice offspring [38]. Therefore, epigenetic modification could be directly influenced by alcohol or indirect by these epigenetic expressions that result from alcohol consumption.

Based on our results, there are significant differences in miR-21 expression patterns in healthy individuals depending on lifestyle behaviour. This finding can demonstrate better understanding of the role of alcohol consumption and smoking on epigenetic expression to contribute in carcinogenesis. In addition, it suggests examining salivary miR-21 as a potential therapeutic target for diseases and as a new biomarker and stress free sample to evaluate, diagnose and prognosis of human health conditions and diseases.

In conclusion, the relation between imRNAs expression and lifestyle factors such as alcohol consumption and smoking has been investigated by several studies over the past few years. This study has presented the influence of lifestyle on miR-21 expression.

These results revealed that miR-21expression is associated with risk factor exposure. Moreover, high expression of miR-21 is associated with alcohol consumption and smoking in healthy people. Consequently, miR-21 can be considered a significant marker for health indication. Additionally, this study suggests that miR-21 could be used as a significant indicator for disease diagnosis.

Author Contribution Statement

Rushdi Fadhil participated in devising the search strategy, data extraction, quality assessment, and analytical strategy for this study. All authors participated in data interpretation, critically revised the manuscript, and have read and approved the final manuscript.