Data on the function of CDH17 in pancreatic cancer growth

Data presented in this article are related to the research article entitled “Disruption of oncogenic liver-intestine cadherin (CDH17) drives apoptotic pancreatic cancer death”. To investigate the influence of CDH17 on human pancreatic cancer (PC), we performed gain and loss of CDH17 function with siRNA and recombinant plasmid to evaluate its impact on PC cell proliferation, colony formation, and migration. The data can be valuable for researchers interested in the study of oncogenic activity related to the CDH17 gene in PC growth and motility.


Data
The shared data are a summary of the impact of CDH17 in PC through in various assays. The mRNA expression of various cadherin family members, including CDH17, in PC and compared to healthy pancreatic tissue was taken from the online database GEPIA (Fig. 1). Using the results from GEPIA, the CDH17 gene was selected for further investigation. siRNA-mediated CDH17 knockdown was performed in mouse Panc02-H7 cells to determine the lasted efficacy of the siRNA treatment over the course of 10 days (Fig. 2). CDH17 knockdown and overexpression was then performed in human PC cell line Panc-1 and mRNA expression of CDH17 measured for treatment performance (Fig. 3). Gain and loss of function was used to investigate the impact of CDH17 on human PC cell proliferation (Fig. 4), cell colony formation (Fig. 5), and cell migration (Fig. 6) using the Panc-1 cell line. The mouse Panc02-H7 cell line underwent CDH17 CRISPR-mediated knockout for seeding into WT C57BL/6 mice. After the orthotopic tumors formed, cancerous tissue was extracted for IHC of cadherin family members (Fig. 7).

Experimental design, materials, and methods
Below is a brief description of the experimental methods used to acquire data in this paper. For a more detailed and thorough report, please refer to the related research article [1].

Cell culture
The mouse panc02-H7 cell line was a gift from the MD Anderson Cancer Center. The human cell line Panc-1 was purchased from the American Type Culture Collection (ATCC). The panc02-H7 cell line was maintained in Dulbecco's Modified Eagle Medium (DMEM; Cellgro) containing 100 U/mL penicillin, 100 mg/mL streptomycin, 2 mmol/L L-glutamine, 10 mmol/L HEPES, and 10% fetal bovine serum (FBS) at 37 C in a 5% CO 2 humidified incubator. Panc-1 human cell line was cultured in DMEM containing 10% FBS, 2.5% equine serum, 100 U/mL penicillin, and 100 mg/mL streptomycin at 37 C in a 5% CO 2 humidified incubator.

CDH17 siRNA transfection
Mouse and human cell lines were grown to 50% confluence, then underwent siRNA transfection with RNAi-MAX Lipofectamine reagent (Invitrogen). Eight hours post-transfection medium was replaced with fresh complete medium. The designed mouse or human CDH17-siRNAs, and their negative control siRNAs, were purchased from IDT. Value of the data The data presented here was acquired to assess any influence of CDH17 on human PC. Therefore, the data might contain valuable information on the clinical significance of CDH17 in human cancer. Beneficiaries of these data include those searching for new therapeutic targets for PC or those seeking greater understanding through mechanistic studies. These data are the first to report the effect of CDH17 on PC. Further, the methods used may provide further insight for the exploration of other therapeutic targets for cancer.

CDH17 recombinant plasmid transfection
Human PC cells were grown to 70% confluence, and then received 2.5 mg human CDH17 recombinant plasmid (RC211298, OriGene) or negative control plasmid (PS100001, Origene) transfection with Lipofectamine 3000 reagent (Thermo Fisher Scientific). Eight hours post-transfection medium was replaced with fresh medium. Transfection was validated through qPCR using the VP1.5 and XL39 primers provided by OriGene.

CRISPR mediated CDH17 knockout
LentiCas9-EGFP plasmids were transfected into Panc02-H7 cells for establishment of Cas9expressing cell clones. The crRNAs and tracrRNAs used were designed online (http://crispr.mit.edu) and synthesized through IDT. crRNAs and tracrRNAs suspended in nuclease-free water were heated to 95 C and then cooled down to room temperature in order to form guide RNA duplex (gRNA).  The lasted efficacy of siRNA-mediated CDH17 knockdown. As described in the materials and methods, Panc02-H7 cells were grown to 50% confluence in 6-well plate, then transfected with 5 pmol of siRNAs by using RNAi-MAX Lipofectamine reagent. The cells were harvested over the indicated times post-transfection to extract total RNA. qPCR detected CDH17 suppression even ten days after siRNA transfection. n ¼ 3, error bars represent mean ± SD. The formed gRNA, 5 0 -GATGATCCGGCTACTCCCAATGG-3 0 , was transfected into Cas9-Panc02-H7 stable cells using the RNAi-MAX Lipofectamine (Invitrogen). Single cell clones were generated with limited dilutions. The genomic DNAs were extracted from each clones with SV genomic DNA kit (Promega). PCR was used to amplify CDH17 DNA fragments with specified primers. These DNA fragments were digested using an endonuclease for evaluation of cutting efficiency mediated by Cas9 and the gRNA. Sanger sequencing was performed to identify the base-depleted site in the targeted DNA fragment. qPCR was used to measure CDH17 mRNA expression in each CDH17 knockout clone.

Proliferation assay
Human PC cells were seeded into 96-well plates at a density of 2 Â 10 3 /well. 24 and 48 hours later, cell proliferation was measured with the Proliferation Assay Kit (Promega) according to the manufacturer's instructions.

Colony formation assay
Human PC cells were seeded into 6-well plates at a density of 200 cells/well. 10 days later, cells were rinsed and then stained with 0.05% crystal violet for colony counting. Negative control for E-Cadherin and N-Cadherin by staining tumor section with isotype IgG as well as a positive control for CDH16 by staining kidney section with high expression of CDH16 was shown in lower panel.