Identification and characterization of dysregulated P-element induced wimpy testis-interacting RNAs in head and neck squamous cell carcinoma

Abstract

It is clear that alcohol consumption is a major risk factor in the pathogenesis of head and neck squamous cell carcinoma (HNSCC); however, the molecular mechanism underlying the pathogenesis of alcohol-associated HNSCC remains poorly understood. The aim of the present study was to identify and characterize P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) and PIWI proteins dysregulated in alcohol-associated HNSCC to elucidate their function in the development of this cancer. Using next generation RNA-sequencing (RNA-seq) data obtained from 40 HNSCC patients, the piRNA and PIWI protein expression of HNSCC samples was compared between alcohol drinkers and non-drinkers. A separate piRNA expression RNA-seq analysis of 18 non-smoker HNSCC patients was also conducted. To verify piRNA expression, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed on the most differentially expressed alcohol-associated piRNAs in ethanol and acetaldehyde-treated normal oral keratinocytes. The correlation between piRNA expression and patient survival was analyzed using Kaplan-Meier estimators and multivariate Cox proportional hazard models. A comparison between alcohol drinking and non-drinking HNSCC patients demonstrated that a panel of 3,223 piRNA transcripts were consistently detected and differentially expressed. RNA-seq analysis and in vitro RT-qPCR verification revealed that 4 of these piRNAs, piR-35373, piR-266308, piR-58510 and piR-38034, were significantly dysregulated between drinking and non-drinking cohorts. Of these four piRNAs, low expression of piR-58510 and piR-35373 significantly correlated with improved patient survival. Furthermore, human PIWI-like protein 4 was consistently upregulated in ethanol and acetaldehyde-treated normal oral keratinocytes. These results demonstrate that alcohol consumption may cause dysregulation of piRNA expression in HNSCC and in vitro verifications identified 4 piRNAs that may be involved in the pathogenesis of alcohol-associated HNSCC.

Keywords: alcohol; epigenetics; head and neck squamous cell carcinoma; piwi-interacting RNA.

Figure 1.

Analysis of RNA-sequencing data of HNSCC samples. (A) Heatmap representing the piRNA FPKMs of the entire patient cohort (n=40). Patients were divided into drinking and non-drinking cohorts and further subdivided by tobacco usage. Only piRNAs that were expressed in >20 patients are shown. (B) Kaplan Meier curves indicate that low expression of piR-58510 and piR-35373 is associated with longer survival times. Log rank tests were performed to determine the P-values shown. (C) Cox proportional hazard ratios further demonstrate the correlation between piRNA-58510 and piR-35373 expression and overall prognosis. The hazard ratios presented for both univariate and multivariate tests are based on the low expression of each piRNA. Vertical lines represent censored patients. HNSCC, head and neck squamous cell carcinoma; PIWI, P-element-induced wimpy testis; piRNA, PIWI-interacting RNA; FPKM, Fragments per Kilobase of transcript per Million mapped reads; HR, hazard ratio; CI, confidence interval.

Figure 2.

Long term ethanol exposure and acetaldehyde treatment leads to dysregulation of identified piRNAs in vitro. Quantitative reverse-transcription polymerase chain reaction demonstrated that demonstrate that (A) ethanol and (B) acetaldehyde treatment of normal OKF4 and OKF6 oral epithelial cell cultures increases piRNA 35373, −266308, −58510 and −38034 in both cell lines. Data are presented as the mean ± standard deviation. PIWI, P-element-induced wimpy testis; piRNA, PIWI-interacting RNA. *P<0.05 vs. control.

Figure 3.

Long term ethanol exposure and acetaldehyde treatment leads to dysregulation of PIWIL4 in vitro. Quantitative reverse transcription-polymerase chain reaction demonstrated that ethanol and acetaldehyde treatment of normal OKF4 and OKF6 oral epithelial cell cultures increases the expression of PIWIL4 in both cell lines. Error bars represent standard deviation. Data are presented as the mean ± standard deviation. *P<0.05 vs. control. PIWI, P-element-induced wimpy testis; PIWIL4, PIWI-like protein 4.