Global expression profile of telomerase-associated genes in HeLa cells Akhil Varshney a , Suresh K. Ramakrishnan a,b , Amod Sharma a , Baby Santosh a , Jyoti Bala a , Pramod K. Yadava a, ⁎, Rishi Kumar Jaiswal a a Applied Molecular Biology Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi 110067, India b Molecular Genetics Laboratory, Institute of Cytogenetic and Preventive Oncology, Indian Council of Medical Research, Noida, Uttar Pradesh 201301, India abstract article info Article history: Received 10 April 2014 Received in revised form 14 May 2014 Accepted 9 June 2014 Available online 11 June 2014 Keywords: Telomerase Microarray siRNA shRNA Cancer Telomerase is a specialized nucleoprotein enzyme complex that maintains the telomere length. The telomerase reverse transcriptase (TERT) is the catalytically active component of the telomerase complex. In humans, the pro- tein component (hTERT) and RNA component (hTR) are found to differentially express in cancer cells. In contrast to differentiated cells, most of the cancer cells overexpress hTERT, which is needed to maintain the proliferative potential of cells. The overexpression of telomerase is not proportionate to telomere length in cancer cells, sug- gesting that the immortalizing phenotype can be mediated through other factors in addition to telomere length. To investigate the role of hTERT in immortalizing process, loss of gene function studies were carried out. Short interfering RNA (siRNA) and short hairpin RNA (shRNA) against hTERT showed the reduction of hTERT transcript, reduction of telomerase activity and alteration of gene expression in HeLa cells. The molecular basis of prolifer- ative capacity of hTERT was investigated by gene expression microarray. Analysis of microarray data for HeLa cells following siRNA and shRNA mediated knockdown of hTERT showed that 80 genes were upregulated and 73 genes downregulated. Out of these, 37 genes are known to be involved in cancer. Further analyses of previous- ly known genes involved in cancer like KLF4, FGF2, IRF-9 and PLAU by Real Time PCR showed their upregulation. We are documenting for the first time the effect of knocking down hTERT on expression of KLF4 and FGF2. Inter- estingly, it has been earlier reported that KLF4 and FGF2 up-regulate the expression of hTERT in cancer cells. This suggests that hTERT may be subject to its own auto-regulatory effects. © 2014 Elsevier B.V. All rights reserved. 1. Introduction Telomerase is an RNA–protein complex that synthesizes and main- tains telomere length to counter the end replication problem by adding repeated oligomer sequence to the 3′ termini of chromosomal DNA (Greider and Blackburn, 1987). The human telomerase reverse tran- scriptase (hTERT) is the catalytically active component of the telome- rase complex (Nakamura et al., 1997). hTERT catalyzes the telomere elongation that leads to increased genomic stability and enhanced DNA-repair (Sharma et al., 2003). hTERT is found to be overexpressed in stem cells, germ cells and in most of the cancer cells, whereas in normal and differentiated cells there is minimal or undetectable level of hTERT (Poole et al., 2001). Ac- tivated telomerase helps in maintaining telomere length that provides replicative potential to the proliferating cells. As a proof of concept, ex- pression of hTERT in normal cells also expands the life span and number of cell doublings. Therefore, finding out genes associated with inhibition of expression/function of hTERT may provide potential therapeutic tar- get for cancer treatment. Several small-molecule inhibitors (Damm et al., 2001; Hisatake et al., 1999; Naasani et al., 1998, 1999), compounds targeting putative telomeric DNA G-quadruplex structures (Neidle et al., 2000), dominant-negative hTERT genes (Hahn et al., 1999), antisense oligonucleotides targeting hTR or hTERT mRNA (Glukhov et al., 1998), ribozymes targeting hTR (Suresh Kumar et al., 2014; Wan et al., 1998) and hTERT mRNA (Kondo et al., 2001), result in limitation of prolifera- tive potential, genomic instability and induction of apoptosis. The role of telomerase in cellular biology is becoming increasingly complex and includes functions other than telomere maintenance (Choi et al., 2008; Kraemer et al., 2003; Xiao et al., 2005). Telomerase in- fluences normal cellular physiology in cells having long telomeres e.g., mice lacking functional telomerase are unable to maintain proper tissue homeostasis, particularly in tissues of high cell turnover, such as the bone marrow, skin, liver, and gastrointestinal tract (Herrera et al., 1999; Lee et al., 1998; Rudolph et al., 1999). Other studies have demon- strated that ectopic expression of telomerase even in cells that are en- dogenously telomerase positive results in increased resistance to apoptosis (Holt et al., 1999). Non-correlation of telomerase expression status and telomere length in these studies has given enough evidence that telomerase has extra-curricular activities in cellular physiology. Gene 547 (2014) 211–217 Abbreviations: hTR, human telomerase RNA component; hTERT, human telomerase reverse transcriptase; siRNA, short interfering RNA; shRNA, short hairpin RNA; KLF4, Kruppel like factor 4; FGF2, fibroblast growth factor 2. ⁎ Corresponding author. E-mail addresses: pky0200@mail.jnu.ac.in, pkyadava1953@gmail.com (P.K. Yadava). http://dx.doi.org/10.1016/j.gene.2014.06.018 0378-1119/© 2014 Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene