3.1 Baseline characteristics of patients
The average age of the CA patients and healthy control was 36.0± 8.8 and 31.2± 10.0, respectively. In the CA group, 80.8% of the participants were male, and in the control group, 50% of the participants were male. CA commonly occurs on the prepuce, labium, scrotum, perineum, coronary sulcus and glans of the penis. But in our study, only the foreskin and inner labia as control group epithelial tissue due to the patients’ intention. At baseline, there was no significant difference in age, sex or sampling location between the CA and control groups (all P> 0.05, as shown in Table 1).
3.2 HPV might induce the abnormal proliferation of skin tissue in the genital area
According to normal biopsy procedures, tissue samples were cut, a pathological examination was performed, and then the proliferation marker Ki-67[9] in the tissues was labeled by immunohistochemistry (Fig. 1B). CA is clinically characterized by scattered verrucous projections of the vulva, usually without pigmentation. The surface is usually covered with finger-like projections[10]. The pathological features of CA include hyperkeratosis, parakeratosis, papillated epidermal hyperplasia, hypertrophy of the spinous layer, vacuolar cell presence, keratinaceous granule concentration in the granular layer and the spinous layer, vascular expansion of the superficial dermis and infiltration of lymphocyte-dominant inflammatory cells into the blood vessels[10] (Fig. 1A). The rate of Ki-67-positive staining in the CA group and control group was 8.97±0.74% and 2.72±0.67%, respectively. The difference between the two groups was statistically significant (P<0.05; Fig. 1B).
3.3 Microarray identification of differentially expressed miRNAs in epithelial samples from patients with CA
Cell proliferation is regulated by miRNAs, suggesting that miRNAs may play an important role in the pathogenesis of CA. However, there are few reports on the changes in miRNA expression in CA tissues. In this study, gene chip technology was used to detect miRNA expression in the CA group and control group epithelial tissue. Compared with that in the control group, the expression of 81 miRNAs was changed in the CA group (Fig. 1C). Among the 81 miRNAs, 25 had increased expression, and 56 had decreased expression. According to the information in the microRNA.org database (http://www.microrna.org/), we selected 2 of the 81 miRNAs. The expression of these 2 miRNAs, miR-30a-5p and miR-514a-3p, was reduced in the CA group compared with the control group, respectively (Fig. 1C).
3.4 MiRNA expression by qRT-PCR detection
We further confirmed whether the changes in the expression of the two miRNAs in CA tissue were consistent with those observed in the chip results. qRT‑PCR was used to analyze the expression of miR-30a-5p and miR-514a-3p. The results showed that the expression of miR-30a-5p and miR-514a-3p was reduced in CA epithelial tissue (P<0.05; Fig. 2A). Moreover, the expression of miR-30a-5p and miR-514a-3p was negatively correlated with the expression of Ki-67 (Fig. 2C, D).
3.5 The mRNA expression of target genes and the related proteins LC3 and P62 expression
The target genes of miR-30a-5p and miR-514a-3p are Atg5 and Atg12, Atg3 and Atg12. Therefore, qRT‑PCR was used to analyze the expression of mRNAs; the results showed that the expression of Atg3, Atg5, and Atg12 was increased in the CA epithelial tissue (P<0.05; Fig. 2B). Atg3, Atg5, and Atg12 genes are associated with autophagy, suggesting that the autophagy level of the local skin tissue in CA patients may be changed. Therefore, we used LC3 and P62 to evaluate the changes in autophagy levels in the local skin tissue of patients with CA. The immunohistochemical results showed that the expression of LC3 in the CA group was - (26.92%), + (69.23%) and ++ (3.85%), while that in the control group was - (70%), + (30%) and ++ (0%). The P62 immunohistochemistry showed that the -, +, and ++ of the CA group was 11.54%, 61.54% and 26.92%, respectively, while that of the control group was 40%, 60% and 0%, respectively. The expression of LC3 and P62 was increased in CA epithelial tissue (P<0.05; Fig. 2E, F).