MiRNA-200C expression in Fanconi anemia pathway functionally deficient lung cancers

The Fanconi Anemia (FA) pathway is essential for human cells to maintain genomic integrity following DNA damage. This pathway is involved in repairing damaged DNA through homologous recombination. Cancers with a defective FA pathway are expected to be more sensitive to cross-link based therapy or PARP inhibitors. To evaluate downstream effectors of the FA pathway, we studied the expression of 734 different micro RNAs (miRNA) using NanoString nCounter miRNA array in two FA defective lung cancer cells and matched control cells, along with two lung tumors and matched non-tumor tissue samples that were deficient in the FA pathway. Selected miRNA expression was validated with real-time PCR analysis. Among 734 different miRNAs, a cluster of microRNAs were found to be up-regulated including an important cancer related micro RNA, miR-200C. MiRNA-200C has been reported as a negative regulator of epithelial-mesenchymal transition (EMT) and inhibits cell migration and invasion by promoting the upregulation of E-cadherin through targeting ZEB1 and ZEB2 transcription factors. miRNA-200C was increased in the FA defective lung cancers as compared to controls. AmpliSeq analysis showed significant reduction in ZEB1 and ZEB2 mRNA expression. Our findings indicate the miRNA-200C potentially play a very important role in FA pathway downstream regulation.


Scientific Reports
| (2021) 11:4420 | https://doi.org/10.1038/s41598-021-83884-9 www.nature.com/scientificreports/ cell cycle or following induction of DNA double-strand breaks or interstrand crosslinks [14][15][16] . Any mutation or epigenetic change that disrupts components of the Core Complex also abrogates its E3 ligase function, leading to defective FA pathway. In order to evaluate functionality of the FA pathway, we developed a Triple-Staining Immunofluorescence method (FATSI), which is able to detect FANCD2 foci formation or lack of formation in paraffin embedded tumor tissues 8,13 . In one study, a total of 139 non-small cell lung cancer formalin-fixed paraffin embedded (FFPE) tumor samples were screened; 104 were evaluable for FANCD2 foci status. Eighty-one of the 104 (81/104, 78%) evaluable tumors were found FANCD2 foci positive and 23 (22%) were foci negative 9 . In a separate study, we analyzed total of 643 human solid tumors and one hundred eighty-five of 643 (185/643, 28.7%) screened patients were FANCD2 foci negative 13 , documenting a 19.7% of lung cancers being functionally deficient.
Recent studies showed that MicroRNA (miRNA) expression is associated with DNA repair pathways 24 . MiR-NAs are small, 18 to 25 nucleotide noncoding RNAs, which regulate target genes mRNA levels and direct regulation of protein synthesis of target genes 25 . MiRNAs play important endogenous roles in biological pathways of mammals and multicellular organisms via targeting mRNA [26][27][28][29] . Increasing evidence suggests that microRNAs can function as either oncogenes or tumor suppressor genes 30 . They derive from larger precursors, which form stem-loop structures with the mature miRNA as one arm of the precursor hairpin released by ribonuclease III (RNase III) 31 .
MiRNAs are critical to cancer development due to their regulatory effects on genes involved in apoptosis, cell cycle progression, differentiation, migration, metabolism, epithelial-mesenchymal transition and tumor cell invasion and metastasis [26][27][28][29] . Among these regulations, the most common way is via translational silencing of gene expression via suppression or degradation by binding to target mRNA at the 3′ untranslated region UTR through base pairing 27,32,33 . A single miRNA may target several hundred mRNAs and can function as oncogenes or tumor suppressors 26,[32][33][34] . Many studies have shown that certain miRNAs can serve as potential cancer biomarkers for diagnosis or prognosis 27,[34][35][36] .
We report herein results of our evaluation of regulation and expression of miRNAs in FA pathway deficient cancers and discuss potential implications on their use as predictive or prognostic biomarkers. along with other additional proteins that associate with the FA Core Complex, including FAAP100, FAAP24, FAAP20, and the histone fold dimer proteins MHF1 and MHF2. The FA Core Complex activates FANCD2 and FANCI by mono-ubiquitinating the protein as a response to DNA damage. The activated FANCD2-I heterodimeric proteins are subsequently transported to sub-nuclear foci, thought to be the sites of DNA repair, which also contain FA downstream proteins including the DNA repair proteins FANCD1(BRCA2), D2, I, J, N, O, P, Q, R, S (BRCA1), U, V and W. FFPE tumor tissue was cut at 4 microns, placed on positively charged slides, and stained with hematoxylin-eosin. Additional sections for immunofluorescence staining were processed as described previously 8,9 . Briefly, slides were laced in a 60 °C oven for 1 h, cooled, deparaffinized, and rehydrated through xylenes and graded ethanol solutions to water in standard fashion. The tissue sections were incubated with a primary antibody cocktail of rabbit polyclonal FANC-D2 antibody (Novus Biologicals, Littleton, CO, Catalog #: NB100-182) at a dilution of 1:1000 and a monoclonal anti-Ki67 mouse antibody (Dako, Carpenteria, CA, USA, Catalog #: M7240) at a dilution of 1:150 for 1 h at room temperature. Sections then were co-incubated with a secondary antibody fluorescein isothiocyanate (FITC) conjugated to anti-rabbit immunoglobulin (Ig)G (Novus Biologicals, Littleton, CO, Catalog #: NBP2-30342F) and Alexa Fluor 594 donkey anti-mouse IgG (Invitrogen, Carlsbad, California, Catalog #: A32744 ) at 1:1000 for 1 h at room temperature. All rinses were performed on the auto-stainer with TBS-T. The sections were mounted on glass slides using a DAPI-containing embedding medium (Vysis Dapi 1, Abbott Laboratories, Downers Grove, Ill). The slides were analyzed under a Nikon (Tokyo, Japan) E-400 fluorescence microscope 8 .

FANCD2 defective cells.
The FANCD2 foci defective cells, A549D2D and H1299D2D were created by knocking down FancD2 expression as described previously 9 . Briefly, non-small cell lung cancer cells A549 and H1299 were plated 24 h prior to transduction and when the cells reached 60% confluence, cells were transduced with FANCD2-specific shRNA-lentiviral particles conferring resistance to puromycin. Control shRNA lentiviral particles were also transduced into the cells (Santa Cruz Biotechnology Inc., Santa Cruz, CA, Catalog#: SC-35356-V) according to the manufacturer's protocol. One day after incubation in medium containing polybrene agent, these transduced cells were transferred to a dish that contains normal growth medium. The transduced cells were selected in 4ug/ml puromycin. To create stably transduced cells, 100-200 transduced cells were cultured in a 100 mm dish, and medium was replaced with fresh puromycin-containing medium every 3 days, until resistant colonies were identified. Twenty colonies were picked for each cell line, and then the colonies were expanded. Successful FANCD2 knockdown was confirmed by western blot (Fig. 2). The FA effective cells, A549E and H1299E were A549 and H1299 cells transfected with empty vectors. SV40 transformed empty retroviral vector transduced human FANCD2 defective fibroblasts PD20RV (FancD2 deletion) and their wild-type FANCD2 transduced counterparts PD20D2 (FANCD2 effective) 37 were obtained from the Oregon Health and Science University Fanconi Anemia Cell Repository using as control cells.
Western immunoblot analysis. Western immunoblot analysis was performed as described previously 9 .
Briefly, cells were digested with the cell lysis buffer (Cell Signaling Technology Inc., Danvers, MA, Catalog #: 9803). Protein concentrations were evaluated using the Bradford reagent (Bio-Rad, Hercules, CA, USA). One hundred micrograms of total protein was loaded onto NuPAGE 4-12% Bis-Tris Gel (Invitrogen, Carlsbad, CA, USA). Protein on the gels was electro-transferred onto nitrocellulose membranes and blocked with blocking buffer (5% of non-fat milk, 500 mM of NaCl, 20 mM Tris, and 0.1% Tween 20). The membranes were incubated with primary anti-bodies at 4 °C overnight. After washing with TBS-T (blocking buffer without milk) five times, 10 min each, the membranes were incubated with anti-mouse IgG (Amersham Pharmacia Biotech, Piscataway, NJ, USA, Catalog #: NA931) or anti-rabbit IgG horse radish peroxidase linked to whole secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ, USA, Catalog #: NA934) at room temperature for 1 h. A chemi- Figure 2. Western blot analysis of FANCD2 protein expression. Non-small cell lung cancer cells H1299 and A549 were transduced with FANCD2-specific shRNA-expressing or empty vector and puromycin-resistant lentiviral particles. The transduced cells were selected in 4 ug/ml puromycin to create stably transduced cells with reduced FANCD2 expression, the H1299D2D and A549D2D. H1299E and A549E were transduced with empty lentiviral particles. FANCD2 protein expression was confirmed by western blot analysis. www.nature.com/scientificreports/ luminescent detection system (ECLwestern blotting detection reagents, GE) was used to detect the secondary antibody. Finally, the membranes were exposed to x-ray films. Antibodies used were rabbit polyclonal FANCD2 antibody (NovusBiologicals, Littleton, CO, USA, Catalog #: NB100-182) and anti-Actin monoclonal (Sigma, St. Louis, MI, USA, Catalog #: A2228). NanoString nCounter miRNA expression. nCounter miRNA assays are highly sensitive and based on direct molecular barcoding and digital detection of targeted RNA molecules of interest though target-specific color-coded probe pairs and does not require conversion of RNA to cDNA by reverse transcription via PCR. Probe pairs have a reporter probe carrying signals on 5′ end, and a capture probe with biotin ion at 3′. Hybridization washes away access probes and allows for RNA probe complexed to be aligned and immobilized for data collection of absolute miRNA expression 38  www.nature.com/scientificreports/ to be consistently up-regulated by at least twofold in two of the three cell lines (A549, H1299 and PD20), and a cluster of 3 miRNAs were found to be down regulated by at least twofold in two of the three groups in FANCD2 foci deficient samples as compared to their respective non deficient control samples (Table 1). Among all these miRNAs, miRNA-200C had consistently higher expression (6.9 fold on average) in FANCD2 foci deficient cells compared to matched control cells (Table 1). In addition, miRNA-200C expression was 22.02 folds higher in average in two FANCD2 foci defective lung cancer tissues comparing to expression of this miRNA in matched non-tumor tissues.

Quantitative real time PCR analysis of miRNA-200C expression. To validate the results from
NanoString analysis, quantitative PCR was used with the same cell lines along with 29 tumor and non-tumor pairs (13 FANCD2 foci positive, 16 FANCD2 foci negative). TaqMan miRNA-200C assay was employed along with RNU48 as an internal control. As depicted in Fig. 3  Primers and probes used for the real time PCR analysis were obtained from Applied Biosystems (Foster City, CA). Real-time PCR was carried out in a 96-well plate using a Bio-Rad CFX96 system. Each sample was done in triplicate and each reaction was repeated at least once to ensure reproducibility. Resulting data were quantified and normalized using RNU48 as control.  www.nature.com/scientificreports/ foci negative tumor samples were used with a gene expression panel with specific interest in ZEB1, and ZEB2 expression levels. Both ZEB1 and ZEB2 mRNA expressions were reduced in FANCD2 knockdown lung cancer cells (Fig. 5A). All 10 tumor samples (100%) showed reduced mRNA expression in ZEB1, and 9 out of 10 samples (90%) showed reduction in ZEB2 expression as compared to matched non-tumors (Fig. 5B).

Number of viable cells was reduced in the FANCD2 knockdown lung cancer cells A549D2D and H1299D2D.
To investigate the influence of FANCD2 knockdown in cell proliferation, we investigated viability of the FANCD2 knockdown lung cancer cells (A549D2D and H1299D2D) using MTT assay. The number of viable cells was reduced in the FANCD2 knockdown cells, A549D2D and H1299D2D as comparing to their counterparts the A549E and H1299E respectively (Fig. 6). There were 70% viable cells in the H1299D2D comparing to H1299E, and 88% viable cells in the A549D2D comparing to A549E cell. These results indicate the number of viable cells was reduced in the FANCD2 knockdown lung cancer A549D2D and H1299D2D cells.
Herein we report that miRNA-200C is an important FANCD2 downstream regulator of cell proliferation and cancer cell metastasis. Based on our study, miRNA-200C was upregulated in the FANCD2 foci deficient non-small cell lung cancer cells and tumor tissues. Both, NanoString nCounter data and real time PCR analysis showed overexpression of miRNA-200C in FANCD2 deficient samples compared to their non-deficient counter parts. This is the first study that shows correlation between the Fanconi Anemia pathway and miRNA expression  A recent study showed expression levels of miR-200C was no significant change in human non-small cell lung cancers (NSCLCs) as it is compared to normal adjacent tissues by real-time PCR 50 . In current study, the average of fold change on miRNA-200C in the 16 FANCD2 foci negative non-small cell lung cancer was 2.7 indicating overexpression in the foci negative tumors compared to matched non-tumor lung tissue. However, the averaged fold change on miRNA-200C in 13 FANCVD2 foci positive non-small cell lung cancers was nearly 1 which indicates miRNA-200C expression was no difference between tumor and non-tumor counterpart (Fig. 4A,B). Since the lung cancers reported in the Nourmohammadi et al. 50 were non-FA-associated NSCLC, the miRNA-200C was no significant change. In contrast, the NSCLC samples in the FANCD2 foci negative cohort were FA defective tumors in the current study. In these FANCD2 foci negative tumors, miRNA-200C was upregulated compared to matched non-cancer tissues. Our results suggest that FA deficiency upregulates the miRNA-200C expression in NSCLC.
In A459 cells overexpressing miR-200C resulted in downregulation of EMT through downregulating N-cadherin and upregulating E-cadherin. Furthermore miR-200C has also been shown to significantly reduce cell invasion and migration through inhibition of ZEB2 expression 44 . It has also been reported that downregulation of miR-200C resulted in decreased E-cadherin through upregulation of ZEB1 51 . Reduction in E-cadherin through ZEB1 leads to increase in cell motility; leading to epithelial mesenchymal transition (EMT). In addition, miR-200C overexpression significantly accelerates cell cycle arrest at G0/G1 phase, inhibits cell proliferation, and www.nature.com/scientificreports/ induces apoptosis in A549 cells, possibly by activating the p53/p21 pathway 35 . Thus, the FANCD2 may regulate cell cycle and metastasis indirectly through the regulation of miRNA-200C expression. We have also investigated the viability of the FANCD2 knockdown lung cancer cells (A549D2D and H1299D2D) using MTT assay. We found the number of viable cells was reduced in the FANCD2 knockdown cells A549D2D and H1299D2D as comparing their counterparts the A549E and H1299E respectively (Fig. 6). This indicates FANCD2 knockdown results in a reduction in number of viable cells in lung cancer.
MicroRNAs are found in a variety of body fluids including blood, saliva and urine, where they are quantifiable and extremely stable. For these reasons, miRNAs are excellent candidates as non-invasive biomarkers for early diagnosis of human disease, and for monitoring treatment response in cancer patients 52 . In our current research, the cells (A549 and H1299) and human tissues are from lung cancer patients. The miRNAs reported in this study are related to DNA homologous recombination repair deficiency, thus miRNA-200C and other FA related miRNAs are potential biomarkers of DNA repair deficiency in patients with lung cancer.
We noticed that the miRNA-200C expression was 1.44 folds higher in the A549D2D cell comparing to A549E cell (Table 1). We think this may be associated the incomplete FANCD2 knocking down in the A549 cell. Our knocking down strategy did not result in 100% ablation of FANCD2 expression. Therefore, it is possible that the partial FANCD2 expression was remained in the A549D2D cell. A small amount of the monoubiquitinated FANCD2 (the L-form of the FANCD2) remained in the A549D2D cells (Fig. 2), whereas the monoubiquitinated FANCD2 was absent from the H1299 D2D cell. Therefor we think the insignificant up-regulation of miR-200C in A549D2D cell may be caused by the incomplete knocking down of FANCD2 expression.
In addition to miR200C, other miRNAs (e.g. miRNA-26a, 143, 921, 184, 567, 873, 933, 1282 and 1288) were also found to be up-regulated, and a cluster of miRNAs (e.g. miRNA-20a, 20b, 129-3p and 262) were found to be downregulated in FANCD2 foci defective non-small cell lung cancer cells. It would be very interesting to study the biological function of these miRNAs, in particular the miRNA-20 (a and b), in regulation of cell proliferation in non-small cell lung cancers. In conclusion, the FA pathway regulates downstream genes through microRNAs in lung cancer and that MiRNA-200C appears to play a very important role in this regulation. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.