MiRNA expression changes in arsenic-induced skin cancer in vitro and in vivo.

Arsenic is a naturally prevalent metalloid. Chronic arsenic ingestion through drinking water causes skin cancer. Arsenic-induced cancer mechanisms are not well defined. Epigenetic changes, including microRNA expression changes, might be playing a role. This dissertation investigates the impact of miRNA expression changes in arsenic-induced skin cancer. MiRNA expression was measure and compared using 3 different techniques, RTq-PCR, hybridization arrays and RNA-sequencing. MiRNAs differential expression in skin lesions was phenotype- and stage-related. Immortalized human keratinocytes (HaCaT) were transformed by chronic low arsenite exposure serving as a model for arsenic-induced skin carcinogenesis. Early changes in miRNAs and target mRNAs contribute to arsenic-induced carcinogenesis. Throughout the time course of arsenic exposure, dysregulation of cells’ growth and cancer-related pathways were identified. Comparisons between the miRNA profiles in lesions and cells predict some miRNAs may serve as biomarkers and/or therapeutic targets for arsenic-induced tumors. This dissertation provides strong evidences of epigenetic changes related to carcinogenesis in arsenic-induced skin cancer

The role of pesticides in non-alcoholic fatty liver disease (NAFLD).

NAFLD, the most common form of liver disease worldwide, is caused by various factors including industrial chemicals and pesticides exposure. Annually, 5.2 billion pounds of pesticides are used worldwide and can contribute to liver disease, but their role is modestly studied. We hypothesize that pesticides contaminating food supply can worsen diet-induced steatosis via xenobiotic receptor activation. Two human and two rodent databases were utilized and 85% of the 330 chemicals identified associated with NAFLD were pesticides. Eight were selected for evaluating hepatic receptor activation in vitro. The majority including DDT activated hPXR/CAR and mPXR. DDT (100 mg/kg) was studied in vivo in a diet-induced obesity (DIO) model. DDT upregulated Cyp2b10 (CAR target) in control diet-fed mice. DDT decreased adiposity, but it did not affect weight gain, food consumption or insulin resistance. In conclusion, DDT improved steatosis, but it did not affect NAFLD, obesity, liver damage or diabetes caused by DIO

Supplementary table 4

Filtered differentially expressed mRNA targets of differentially expressed miRNAs (3 weeks time point

Data from: Differentially expressed mRNA targets of differentially expressed miRNAs predict changes in the TP53 axis and carcinogenesis related pathways in human keratinocytes chronically exposed to arsenic

Background: Arsenic is a widely distributed toxic natural element. Chronic arsenic ingestion causes several cancers, especially skin cancer. Arsenic-induced cancer mechanisms are not well defined, but several studies indicate that mutation is not the driving force and that microRNA expression changes play a role. Chronic low arsenite exposure malignantly transforms immortalized human keratinocytes (HaCaT), serving as a model for arsenic-induced skin carcinogenesis. Hypothesis: Early changes in miRNA expression in HaCaT cells chronically exposed to arsenite will reveal early steps in transformation. Methods: HaCaT cells were maintained with 0/100 nM NaAsO2 for 3 and 7 weeks. Total RNA was purified. miRNA and mRNA expression was assayed using Affymetrix microarrays. Targets of differentially expressed miRNAs were collected from TargetScan 6.2, intersected with differentially expressed mRNAs using Partek Genomic Suite™ software, and mapped to their pathways using MetaCore™ software. MDM2, HMGB1 and TP53 mRNA and protein levels were assayed by western blot. Results: Numerous miRNAs and mRNAs involved in carcinogenesis pathways in other systems were differentially expressed at 3 and 7 weeks. A TP53 regulatory network including MDM2 and HMGB1 was predicted by the miRNA and mRNA networks. Total TP53 and TP53-S15-phosphorylation were induced. However, TP53-K382-hypoacetylation suggested that the induced TP53 is inactive in arsenic exposed cells. Conclusions: Our data provide strong evidence that early changes in miRNAs and target mRNAs may contribute to arsenic-induced carcinogenesis

Supplementary table 2

Filtered differentially expressed 38 predicted targets of differentially expressed miRNA at 3 and 7 weeks

Supplementary table 3

Filtered differentially expressed mRNA targets of differentially expressed miRNAs (3 weeks time point

miRNA expression profiles of premalignant and malignant arsenic-induced skin lesions.

Arsenic, a naturally occurring element, contaminates the drinking water of over 200 million people globally. Chronic arsenic exposure causes multiple cancers including those originating from skin, lung and bladder, and is associated with liver, kidney, and prostate cancers. Skin is the primary target organ for arsenic toxicity; chronic toxicity initially manifests as non-malignant hyperkeratoses (HK) and subsequently advances to malignant lesions, including squamous cell carcinoma (SCC) and basal cell carcinoma (BCC). In this study, we evaluate the miRNA expression profiles of premalignant (3 HK) and malignant (3 BCC and 3 SCC) skin lesions from individuals chronically exposed to high levels of arsenic (59-172 ppb) in their drinking water in West Bengal, India. The lesions were histologically complex requiring histopathologic identification of keratinocytes to be isolated for RNA analyses. Keratinocytes were harvested using Laser Capture Microdissection and miRNA expression profiles were determined using TaqMan® Array Human MiRNA A Card v2.0. Thirty-five miRNAs were differentially expressed among the three lesion types analyzed. Two miRNAs (miR-425-5p and miR-433) were induced in both BCC and SCC relative to HK indicating their association with malignancy. Two other miRNAs (miR-184 and miR-576-3p) were induced in SCC relative to both BCC and HK suggesting selective induction in tumors capable of metastasis. Six miRNAs (miR-29c, miR-381, miR-452, miR-487b, miR-494 and miR-590-5p) were selectively suppressed in BCC relative to both SCC and HK. In conclusion, the differential miRNA expression was both phenotype- and stage-related. These miRNAs are potential biomarkers and may serve as therapy targets for arsenic-induced internal tumors

Supplementary table 1

Differentially expressed mRNAs at both 3 and 7 weeks of exposure to arsenit