EGF-mediated reduced miR-92a-1-5p controls HTR-8/SVneo cell invasion through activation of MAPK8 and FAS which in turn increase MMP-2/-9 expression

The members of human miR-17-92 cluster are implicated in several cancers and are known to increase cancer cells invasiveness. The present study reports reduced expression of miR-92a-1-5p in EGF treated HTR-8/SVneo trophoblastic cells by NGS and qRT-PCR. Overexpression of miR-92a-1-5p led to significantly reduced EGF-mediated HTR-8/SVneo cells invasion. MAPK8 and FAS were predicted to be miR-92a-1-5p targets, and confirmed to be reduced by qRT-PCR and Western blotting in trophoblast cells overexpressing miR-92a-1-5p. The binding of miR-92a-1-5p to MAPK8 and FAS 3′-UTR was confirmed by Luciferase reporter assay and Rescue assay. EGF increases MMP-2 & MMP-9 expression and reduces TIMP1 expression in HTR-8/SVneo cells. Inhibition of MAPK8 (by SP600125) reduced EGF-mediated MMP-9/TIMP1 ratio and invasion. Similarly, silencing of FAS by siRNA reduced EGF-mediated MMP-2/TIMP1 ratio and invasion. Treatment of HTR-8/SVneo cells with STAT1/3 inhibitors or siRNAs led to loss of EGF-mediated reduction in miR-92a-1-5p levels. Inserting the predicted binding sites of STAT3 present in promoter region of miR-92a-1-5p upstream of Luciferase promoter reduced its expression in presence of STAT3 expression vector. Thus, EGF leads to reduced miR-92a-1-5p expression which may be regulated by STAT1/STAT3 and controls HTR-8/SVneo cells invasion by targeting MAPK8 and FAS, which in turn increases MMP-2/MMP-9 expression.


Supplementary Materials and Methods miRNA next generation sequencing (NGS)
HTR-8/SVneo cells (0.1 X 10 6 /well) were seeded in 6-well culture plates and were allowed to grow overnight at 37°C in humidified atmosphere of 5% CO2. Next day, the cells were subjected to FBS starvation for 4 h followed by treatment with EGF (10 ng/mL) in DMEM + Ham's F-12 (1:1) medium without FBS for 24 h at 37°C in humidified atmosphere of 5% CO2. After 24 h, the supernatant was discarded and cells were lysed in Tri reagent for RNA isolation. Total RNA was isolated and the samples were outsourced to Next Generation Sequencing Lab, National Institute of Immunology, New Delhi, India. The small RNAs libraries were constructed using Truseq Small RNA sample preparation kit according to the manufacturer's instructions (Illumina, Inc., USA).
Briefly, the total RNA (2 µg) was ligated with 3' adapter using T4 RNA ligase 2 truncated (NEB) and a 5' adapter using T4 RNA ligase 2. The ligation products were reverse transcribed using Superscript II Reverse Transcriptase and amplified with PCR (12 cycle). The PCR product was purified by electrophoresis using 6% Novex TBE PAGE Gel. The purified cDNA library for each sample was analyzed on Agilent 2100 Bioanalyzer using Agilent DNA high sensitivity kit. Deep sequencing of cDNA libraries were performed with the help of Illumina Genome analyzer IIX for 36 cycles.
A total of 2.8 GB of raw sequence data, comprising of untreated control and 24 h EGF treated samples, was imported into the CLC Genomics Workbench 6.5.1. The sequence reads were trimmed for adapter sequences and low quality base. The trimmed raw sequences were subjected to miRNA-sequence analysis, by mapping them to miRBase release 21 accounting for a maximum of two gaps or mismatches in each sequence. Unpaired group comparisons, based on Transcript Per Million (TPM), were chosen as expression values for comparison. Kal's Z test statistical analyses, based on p value < 0.05, fold change ± 1.5 and read count > 10 were used to filter the differentially expressed miRNA.

mRNA NGS
HTR-8/SVneo cells (0.1 X 10 6 /well) were seeded in 6-well culture plates and were allowed to grow overnight at 37°C in humidified atmosphere of 5% CO2. Next day, the cells were subjected to FBS starvation for 4 h followed by treatment with EGF (10 ng/mL) in DMEM + Ham's F-12 (1:1) medium without FBS for 24 h at 37°C in humidified atmosphere of 5% CO2. After 24 h, the supernatant was discarded and cells were lysed in Tri reagent for RNA isolation. Total RNA was isolated and the samples were outsourced to Bionivid Technology Pvt Ltd. (Bangalore, India) for mRNA NGS. The RNA samples were first subjected to quality check (QC) using Bioanalyzer, RNA integrity number (RIN) for all samples was above 8. RNA above RIN value 5.0 is considered suitable for mRNA sequencing experiment. Thereafter, samples were processed for mRNA NGS employing Illumina HiSeq system. The raw data was collected as fastq.gz files. These files were used for data normalization and calculation of p-value to find statistically relevant differentially expressed genes. Normalized data was imported into GSTACK -Genomics cloud software.

Rescue Assay
For the rescue assay, HEK-293T cells (ATCC) were co-transfected with 250 ng each of luciferase reporter plasmid harboring the wild type and the mutated binding sites of MAPK8 & FAS respectively along with 25 nM mimic control/miR-92a-1-5p mimic using Lipofectamine RNAiMAX reagent in OptiMEM. After 48 h of transfection, cells were washed in PBS and lysed in Reporter lysis buffer (Promega), and luciferase activity was measured in a Sirius Tube Luminometer (Berthold Detection Systems, Bad Wildbad, Germany) using the Dual-Luciferase reporter assay kit (Promega) according to the manufacturer's instructions. Firefly luciferase activity was normalized to Renilla luciferase activity, and relative luciferase activity was calculated taking firefly luciferase activity of empty pmirGLO transfected cells as 100 percent.

Fig. S3 -Reduced expression of miR-19a-5p in EGF treated HTR-8/SVneo cells and role of its mimic in invasion.
HTR-8/SVneo cells were treated with EGF (10 ng/mL) for 24 h along with untreated control cells and used to perform qRT-PCR. Panel A shows relative expression of miR-19a-5p in EGF treated as compared to untreated HTR-8/SVneo cells. Values are expressed as mean ± SEM of three independent experiments performed in triplicates. HTR-8/SVneo cells transfected with miR-19a-5p mimic or control mimic were used to perform Matrigel invasion assay in presence or absence of EGF treatment for 24 h. Panel B shows fold change in invasion of EGF treated and untreated miR-19a-5p mimic transfected HTR-8/SVneo cells as compared to EGF untreated control mimic transfected HTR-8/SVneo cells. Values are expressed as mean ± SEM of three independent experiments performed in duplicates. Panel C shows representative photographs of invading cells in various treatment groups on 0.8 µm pore size transwell membranes as observed under microscope after processing for invasion assay. Scale bar represents 5 µm.

Fig S4 -Effect of EGF treatment on HTR-8/SVneo cells invasion in presence/absence of miR-92a-1-5p inhibitor.
HTR-8/SVneo cells transfected with miR-92a-1-5p inhibitor or inhibitor control were used to perform Matrigel invasion assay in presence or absence of EGF treatment for 24 h. Bar graph shows fold change in invasion of the HTR-8/SVneo cells after treatment with EGF in inhibitor control cells and those treated with miR-92a-1-5p inhibitor followed by treatment with and without EGF as compared to cells that were neither treated with EGF nor miR-92a-1-5p inhibitor. Values are expressed as mean ± SEM of three independent experiments performed in duplicates. Representative photographs of invading cells in various treatment groups on 0.8 µm pore size transwell membranes as observed under microscope after processing for invasion assay are appended alongside. Scale bar represents 5 µm.