GAPDH expression as a measurement of transfection efficiency for p^16INK4a gene silencing (siRNA) in senescent human diploid fibroblasts

Suzana Makpol^1*, Azalina Zainuddin¹, Kien Hui Chua²

¹Department of Biochemistry Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
²Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.

DOI: 10.4236/ajmb.2012.24041                

Abstract

Human diploid fibroblasts (HDFs) undergo a limited number of cell divisions in culture. After certain population doublings, they reach a state of irreversible growth arrest known as replicative senescence. Senescent HDFs showed several molecular and cytological changes such as large flat morphology, expression of senescence-associated β-galactosidase (SA β-gal) activity and altered gene expression. Small interfering RNA (siRNA) has been demonstrated to be a potential research tool to analyse gene function and pathway. Expression of an appropriate housekeeping or reference gene can be used as a measurement of transfection efficiency in siRNA. Therefore this study was designed to determine the suitability of GAPDH expression as a measurement of transfection efficiency for p^16INK4a gene silencing in HDFs aging model. GAPDH knockdown with an appropriate transfection reagent was measured by quantitative real time RT-PCR while cellular senescence was characterized based on morphological changes, expression of SA β-gal and p16^INK4a expression levels. Our findings showed that GAPDH knockdown represents silencing efficiency and down regulation of p16^INK4a in senescent transfected HDFs caused morphological alterations which results in the formation of spindle shaped fibroblasts. This study demonstrated the suitability of GAPDH expression as a measurement of transfection efficiency for p^16INK4a gene silencing in HDFs aging model.

Keywords

GAPDH; Transfection Efficiency; p^16INK4a siRNA; HDF Aging model

1. INTRODUCTION

Cellular senescence is recognized as a general response to a variety of oncogenic and genotoxic stresses. It was originally observed in cultures of primary Human Diploid Fibroblasts (HDFs) as they reached the end of their proliferative life span [1]. Normal HDFs enter the senescence state after about 55 - 60 population doublings [2]. Senescent cells have been shown to accumulate with age in human tissues and thus have been proposed to contribute to organismal aging [3]. Senescent cells are resistant to mitogen-induced proliferation, express senescence-associated β-galactosidase (SA β-gal) activity and have an enlarged and flattened morphology [4-7]. Consequently, senescent cells exhibit a gradual loss of replicative potential that results in reduced cell harvest and saturation densities [8].

A distinctive feature of senescent HDFs is that they express elevated levels of p16^INK4a and p21^CIP1 CyclinDependent Kinase (CDK) inhibitors [9-13].

Different roles of p21 and p16 in cellular senescence of human diploid fibroblasts have been reported. Up‐ regulation of p16 might be essential for maintaining cell cycle arrest in cellular senescence, whereas p21 might be responsible for the inactivation of both cyclin E and cyclin D1-associated kinase activity at the early stage of replicative senescence. A series of cyclin-dependent kinases (CDKs) such as CDK2, CDK4 and CDK6 has been re- ported to play a critical role in the phosphorylation of pRb [14,15] which results in pRb loses its ability to bind to E2F/DP transcription factor complexes. As a consequence, cells commit to S-phase of the cell cycle. However, in senescent cells the activity of CDKs is inhibited by CDK inhibitors such as p^21Cip1 and p16^INK4a [10,11,16]. Induction of p16^INK4a and p^21Cip1 prevents phosphorylation of pRb and results in a stable G1 arrest [17]. Thus, CDK inhibitors have the capacity to arrest cells in the G1- phase of the cell cycle and its probable physiological role is in the implementation of irreversible growth arrest termed cellular senescence [18]. In addition, CDK inhibitors specifically p16^INK4a can induce some features of cell senescence [19-23]. Therefore, CDK inhibitor p16 was suggested as an important player in aging and agerelated diseases. Previous study showed that p16 expression was very low or absent in young organisms but was increased with advancing age [24].

Over expression of any of the CDK inhibitors will induce G1 cell cycle arrest. The INK4 (inhibitors of CDK4) family of CDK inhibitors including p15, p16, p18 and p19, bind to and inhibit CDK4 and CDK6. In contrast, the Cip/Kip (CDK2-interacting protein) family of CDK inhibitors (p21, p27 and p57) exhibits a broad specificity for CDKs [25].

CDK inhibitor p16 has emerged as an important player in aging and age-related diseases. p16^INK4a can induce some features of cell senescence [19-23]. p16 expression was very low or absent in young organisms but was increased with advancing age [24].

In recent years, advances in the development of technologies based on short RNAs that silence gene expression and knockdown the production of proteins have been enormous. The initial report on the development of a potent and sequence specific gene silencing by injecting double-stranded RNA (dsRNA) into Caenorhabditis elegans in 1998 sparked the phenomenon known as interference RNA (RNAi) [26,27]. Application of RNAi was restricted to basic research on the mechanisms of gene expression regulation in nematode and flies, until it was found that gene expression in mammalian cells was silenced by transfecting the cells with “short” RNA inhibitors also known as siRNA [28]. Therefore, inhibition of gene expression using the RNAi has become the method of choice for studying gene function in mammalian cells. However, successful knockdown of the target gene requires efficient delivery of short or small interfering RNAs [29].

The efficiency of siRNA delivery can vary between cell types with different delivery methods. Therefore, identifying a suitable housekeeping gene as a positive control for optimizing gene silencing is important to facilitate successful delivery of siRNA without affecting cell viability.

Housekeeping genes or reference genes are essential endogenous regulatory genes that are involved in various processes in the cell such as metabolism, cell structure, gene transcription and homeostasis and are therefore constitutively expressed [30].

The RNA encoding for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) is universally expressed. GAPDH catalyzes the oxidative phosphorylation of glyceraldehyde 3-phosphate to 1,3-bisphosphoglycerate during glycolysis as well as the reverse reaction in tissues involved in gluconeogenesis. GAPDH has also been implicated in other ubiquitous processes such as DNA replication and repair as well as in apoptosis [31]. In this study, we determined the transfection efficiency for p^16INK4a gene silencing by using GAPDH siRNA as a positive control. The silencing effect of p16^INK4a gene in senescent HDFs was also observed.

2. MATERIALS AND METHODS

2.1. Cell Culture and Induction of Cellular Senescence

This research has been approved by the Ethics Committee of Universiti Kebangsaan Malaysia (Approval Project Code: FF-328-2009). Primary HDFs were derived from the circumcision foreskins of 9 to 12 year-old boys. Written informed consents were obtained from parents of all subjects. The samples were aseptically collected and washed several times with 75% alcohol and phosphate buffered saline (PBS) containing 1% antibiotic-antimycotic solution (PAA, Austria). After removing the epidermis, the pure dermis was cut into small pieces and transferred into a falcon tube containing 0.03% collagenase type I solution (Worthington Biochemical Corporation, USA). Pure dermis was digested in the incubator shaker at 37˚C for 6 - 12 h. Then, cells were rinsed with PBS before being cultured in Dulbecco Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS) (PAA, Austria) and 1% antibiotic-antimycotic solution at 37˚C in 5% CO₂ humidified incubator. After 5 - 6 days, the cultured HDFs were harvested by trypsinization and culture-expand into new T25 culture flasks (Nunc, Denmark) with expansion degree of 1:4. When the subcultures reached 80% - 90% confluence, serial passaging was done by trypsinization and the number of population doublings (PDs) was monitored until HDFs reached senescence. For the subsequent experiments, cells were used at passage 4 (young cells, population doubling; PD < 12), passage 20 (pre-senescent cells, 30 < PD < 40), or passage 30 (senescent cells, PD > 55).

The number of population doubling of the cells was determined by cell count using trypan blue exclusion dye by using the following formula:

Example:

Initial cell seeding—N₀: 100, 000.

Total cell count when culture confluent—N₁: 800,000.

Therefore 2ⁿ = 800,000/100,000 = 8.

n log2= log8.

The number of cell doubling in culture; n = 3.

Figure 1 shows the arrangement of cells treatment for this study.

Conflicts of Interest

The authors declare no conflicts of interest.

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