J Biosci (2020)45:104 Ó Indian Academy of Sciences DOI: 10.1007/s12038-020-00075-w (0123456789().,-volV)(0123456789().,-volV) Differential transcriptome analysis in HPV-positive and HPV-negative cervical cancer cells through CRISPR knockout of miR-214 1 1 2 PRAKRITI SEN ,POOJA GANGULY ,KIRTI KKULKARNI , 2 1 ROLI BUDHWAR and NILADRI GANGULY * 1Cancer Biology Lab, School of Biotechnology, KIIT University, Bhubaneswar, India 2Bionivid Technology Pvt. Ltd., Kasturi Nagar, Bengaluru, India *Corresponding author (Email, [email protected]) MS received 14 January 2020; accepted 19 July 2020 In this study we have investigated the effects of a tumour suppressor microRNA, miR-214, on gene expression in HPV-positive (CaSki) and HPV-negative cervical cancer cells (C33A) by RNA sequencing using next generation sequencing. The HPV-positive and HPV-negative cervical cancer cells were either miR-214- knocked-out or miR-214-overexpressed. Gene expression analysis showed that a total of 904 genes were upregulated and 365 genes were downregulated between HPV-positive and HPV-negative cervical cancer cells with a fold change of ±2. Furthermore, 11 differentially expressed and relevant genes (TNFAIP3, RAB25, MET, CYP1B1, NDRG1, CD24, LOXL2, CD44, PMS2, LATS1 and MDM1) which showed a fold change of ±5 were selected to confirm by real-time PCR. This study represents the first report of miR-214 on global gene expression in the context of HPV. Keywords. Cervical cancer; CRISPR; Human papilloma virus; miR-214; RNA transcriptome Abbreviations: 30-UTR, 30-untranslated region; ATCC, American Type Culture Collection; CC, cervical cancer; CIN, cervical intraepithelial neoplasia; CRISPR, clustered regularly interspaced short palindromic repeats; DAVID, the database for annotation, visualization and integrated discovery; DEG, differentially expressed genes; DMEM, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; GO, gene ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; miR, microRNA; NGS, Next Generation sequencing; NIAID, National Institute of Allergy and Infectious Diseases; qRT-PCR, quantitative real-time PCR; RIN, RNA integrity number; RNA-seq, RNA-sequencing; sgRNA, single guide RNA; SRA, sequence read archive. 1. Introduction called Human papillomavirus, or HPV (Sen et al. 2018). Cervical carcinoma development happens from As indicated by World Cancer Report, cervical malig- standard to low-grade squamous intraepithelial lesion nancy or disease of the uterine cervix is the second (LSIL) to high-grade squamous intraepithelial lesion most regular reason for disease and the fourth most (HSIL) to in vitro carcinoma (CIS) and lastly to basic reason for death due to disease in women metastatic disease (Hopman and Ramaekers 2017). worldwide. It has additionally been accounted for that Oncogenic human papillomaviruses (HPV), mostly in the year 2012, an expected 528,000 cases have come HPV types-16, 18, 31, 33, etc., are associated with about among which 266,000 deaths have occurred invasive lesions of cervical cancer. There are over 200 around the world (Bray et al. 2018). It has been seen distinct HPV varieties (Agorastos et al. 2015). Human that the foremost common anorexigenic agent of cer- papillomaviruses (HPV) have their viral genome in the vical malignant growth is by an infection of a virus capsid of the 72-capsomere. The genome is split into Electronic supplementary material: The online version of this article (https://doi.org/10.1007/s12038-020-00075-w) contains supplementary material, which is available to authorized users. http://www.ias.ac.in/jbiosci 104 Page 2 of 25 Prakriti Sen et al. three areas that include the Long Control Region the first report of comparative gene expression on the (LCR) without coding capacity; the Early Protein effect of miR-214 between HPV-negative and HPV- Region (E1–E7) and the Late Protein Region (L1 and positive cervical cancer cells. L2) (Ghittoni et al. 2010) LCR. Early Region and Late Region regulate DNA replication, oncogenesis, cell conversion and viral capsule formation, respectively. 2. Materials and methods The early HPV proteins are used to determine the risk factor engaged in cervical cancer. The HPV found in 2.1 Cell lines and reagents benign tumors are low-risk kinds: HPV-6, 11, 42, 43 and 44; whereas in invasive and malignant tumors the C33A and CaSki cervical cancer-derived cell line was high-risk HPV are found which are HPV-16, 18, 31, 33, obtained from American Type Culture Collection 35, 51, 52 and 58 (Goodman 2015). (ATCC). The C33A cell line was grown as a monolayer MicroRNAs (miRs) are a class of small non-coding and maintained in Dulbecco’s Modified Eagle Medium single-stranded RNAs which are composed of nearly (DMEM) media (GIBCO, Life Technologies) and 19-24 nucleotides. According to different types of CaSki cell line was grown as a monolayer and main- cancers, oncogenic miRs are upregulated and tumor tained in RPMI 1640 media (GIBCO, Life Technolo- suppressor miRs are downregulated (Sochor et al. gies). Along with the media the cells were 2014;Liet al. 2016). There are around 250 micro- supplemented with 1% antibiotics which consists of RNAs which are uncharacteristically expressed in 100 U/ml penicillin and 10 mg/ml streptomycin cervical cancer cells and miR-214 is one of them (GIBCO, Life Technologies), 10% Fetal Bovine Serum (Kawai et al. 2018). This miR-214 in cervical cancer is (FBS) (GIBCO, Life Technologies) and 1% (w/v) reported to be downregulated in all three stages of L-glutamine (GIBCO, Life Technologies) at 37 °Cina cervical intraepithelial neoplasia (CIN) which are stage humidified atmosphere containing 5% CO2. 1, 2 and 3. Cellular activity, such as cell proliferation, apoptosis, cell invasion, metastasis and angiogenesis in cervical carcinoma, is regulated by miR-214 (Penna 2.2 Knockdown of mir-214 using CRISPR/Cas 9 et al. 2015; Tomasetti et al. 2016; Frediani and Fabbri Technique and confirming gene expression by RNA 2016). Recently, profiling of separate miRNAs has extraction followed by quantitative RT-PCR suggested a crosstalk between miR expression and progression of disease in cervical cancer (Berdasco and Using the CRISPR/Cas9 technique mir-214 was Esteller 2017; Abba et al. 2017; Rupaimoole and Slack knocked down in cervical cancer cell lines C33A (C1) 2017). and CaSki (K1). By using CRISPR Design Tool online Transcriptome analysis by using RNA-sequencing (http://crispr.mit.edu/) top and bottom sgRNA oligo (RNA-seq) has emerged as a powerful tool to study inserts for mir-214 were designed (miR-214a forward: gene expression profile (Byron et al. 2016; Uhlen et al. 50-GACTGAGAGCGTTGTCTGTCT-30; miR-214a 2017). The major drawbacks of microarray-based gene reverse: 50-TGCCATCTGTCTGCCTGT-30). The vec- expression analysis such as cross-hybridization tor pSpCas9 (BB)-2A-Puro vector was obtained from between similar sequences can be overcome by tran- Addgene (plasmid ID: 48139) for cloning the sgRNA scriptome analysis (Mao et al. 2017). Also, it offers oligos. First, the top and bottom sgRNAs strands were accurate single nucleotide resolution, which permits the phosphorylated and annealed at 37 °C for 30 min; discrimination between highly related sequences, as 95 °C for 5 min; ramped down to 25 °Cat5°C min-1. well as provide increased sensitivity to detect rare Next, the vector pSpCas9 (BB)-puro-2A, was digested sequences (Jaskowiak et al. 2018). Furthermore, RNA- using the BbsI restriction endonuclease at 37 °C for seq provides a unique advantage in quantitatively 1–16 h. The diluted sgRNAs were then ligated in the analyzing RNA expression levels. Based on the above- vector pSpCas9 (BB)-puro-2A with T4 DNA Ligase at mentioned advantages, NGS technology has replaced 16 °C overnight and then heat-inactivated at 65 °C for microarrays as the tool of choice for RNA analysis 10 minutes. For transformation 5 ll of the above mix (Imam 2016). was added to 50 llofE. coli DH5a chemically com- In this study, we performed a comparative tran- petent cells. A plasmid of positive cloned cells was scriptome analysis between HPV-positive (CaSki) and selected, isolated and purified using the Qiagen Midi- HPV-negative (C33A) cervical cancer cells having Prep Plasmid Isolation Kit. After obtaining purified either overexpression or knockout of miR-214. This is plasmid DNAs they were used for transfection in C33A Differential transcriptome analysis in HPV cervical cancer cells Page 3 of 25 104 (C1) and CaSki (K1) cervical cancer cells. For which cDNAs of C2 and K2 were prepared with the help of cells were seeded overnight in 60 mm cell culture dish SuperScriptTM IV First-Strand Synthesis System Kit at a density of 6 9 105 cells/ml with media consisting (Invitrogen, Thermo Scientific) using gene-specific of only 10% FBS and no antibiotics (100 U/ml peni- primer for mir-214. Gene expression was confirmed cillin and 10 mg/ml streptomycin). A total of 500 ng using PCR as described above. sgRNAs at equimolar ratios was transfected using Lipofectamine 2000. After 48 h, 2 lg/ml mammalian selective antibiotic Puromycin was added to the cells 2.4 Sample preparation followed by Library for screening of positively transfected cells. After 3 Construction and Sequencing days the dose of the antibiotic Puromycin was changed to 1lg/ml and maintained further. For library construction, total RNA was extracted from The gene expression was confirmed by subjecting the samples. After performing quality control (QC), the stable transfected cells C33A knockdown (C3) and passed samples were proceeded with the library con- CaSki knockdown (K3) to total RNA extraction using struction. The extracted RNA with an RNA integrity TRIZOL reagent (Invitrogen). Reverse transcription number (RIN) of 7.0 were used for mRNA purification. was performed using SuperScriptTM IV First-Strand The mRNAs were purified using oligo-dT beads Synthesis System (Invitrogen, Thermo Fisher Scein- (TruSeq RNA Sample Preparation Kit, Illumina) taking tific) as per the manufacturer’s protocol.