Differential mRNA Expression of IRAK1, TRAF6, KAL1 and GAN in Response of HPV16 Infection
TLR9 and TLR4 recognize HPV16 particles as their ligands and then activate and translocate NFkB via MyD88-dependent pathway. After a short time (10min to 30min) of HPV16 VLP infection, human adult keratinocytes cells (HaCaTs) internalize these VLPs and undergo morphological changes such as formation of filopodia.
IRAK1 (Interleukin-1 receptor-associated kinase 1) and TRAF6 (TNF receptor associated factor 6) are two important proteins which participate in MyD88-dependent pathway of TLR signaling.
KAL1 gene encodes protein Anosmin-1 that can increase cancer cell mobility, bind to fibroblast growth factor receptor 1 (FGFR1) and enhance the activity of this signaling pathway. GAN gene encodes a protein named Gigaxonin which functions in neurofilament architecture and may also play a crucial role in the crosstalk between the intermediate filaments and the membrane network.
The object of this study is to identify differential mRNA expression of IRAK1, TRAF6, KAL1 and GAN. Data of this project indicated that after infection of HPV16 PsVs for 30 minutes and 4 hours, IRAK1 and TRAF6 did not show changes in their mRNA levels whereas the mRNA of KAL1 increased for about 50% and GAN mRNA decreased by 20%. These findings may indicate that cells need longer time to induce TLR activity and that HPV16 infection may introduce changes of cell mobility and migration in a short time period.
Materials and Methods
Cell culture and HPV16 PsVs infection:
HaCaT cells were plated in a 6-well plate. After 24 hours of 37℃ incubation. Cells were then treated with serum free medium for 1 hour on ice. Then HPV16 PsVs were added to cells and bound in serum free medium for another hour on ice. Unbounded PsVs were removed by washing 2 times with appropriate medium. Cultures were then incubated at 37℃ for 30min and 4hr, respectively.
RNAs were extracted with RNeasy kit following the manufacturer’s instruction.
Primers design and RT-PCR:
RT-PCR primers were designed to locate in different exons to exclude amplification from potential DNA in RNA samples. ActinβmRNA level was also tested as an quantitative control. Annealing temperatures and cycle numbers of amplification used in RT-PCR were adjusted based on pre-experiments.
Data analysis and bar graph generating:
More than 3 times of experiments were done and gel pictures were scanned. Software ImageJ was used to count the pixel numbers in gel pictures of 3 experiments. Data collected were normalized to pixel numbers of ActB mRNA band.
Using the RNA templates extracted from HaCaT cells infected by HPV16 PsVs for 30min and 4hr, from the RT-PCR result, no significant change on mRNA level of neither IRAK1 nor TRAF6 was observed. Similar assay was used to test KAL1 and GAN mRNA expression level.
The mRNA levels of IRAK1 and TRAF6 did not respond to HPV16 VLP infection in 4 hours. Activation of TLR signaling MyD88-dependent pathway may occur at later time point.
Interestingly, after both 30min and 4hr infection, KAL1 mRNA expression increased. For the 4hr sample, an increase of about 50% was observed. At the same time, GAN gene showed a decrease by 20% on its mRNA expression level. This indicates that virus infection may change cell mobility via FGFR1 signaling and cytoskeleton reorganization.
Data of this project showed that mRNA levels of IRAK1 did not respond to HPV16 PsV infection after 4 hours. This may indicate that at the very early stage of virus infection and internalization, TLR signaling keeps the normal endogenous level as cell’s natural defense towards pathogens. Longer time period may be needed for the signaling pathway to respond and the following expression of virus encoded proteins may lay more regulatory functions on the players in this pathway.
On the other hand, it is quite possible that IRAK1 and TRAF6 show increasing protein expression or their activity can be increased without overexpression of mRNA. Western Blot can be done to test the protein level of them and further experiments can be processed to test the kinase activity of IRAK1.
Interestingly, findings in this project demonstrated that KAL1 had an increase on its mRNA level as well as GAN mRNA had been down regulated. Current researches of these two genes focused on their relationships with the corresponding diseases; studies upon their correlation with virus infection, immune response and other cellular biological functions are still lacking. Changes of the mRNA level of these two genes, which were traditionally considered functioning in embryonic neuronal development, suggest that they may also participate in cell signaling and cancer development. Based on the previously published researches, KAL1 may protect cancel cells from apoptosis and increase cancel cell mobility, it is reasonable to presume that this gene has similar performance in response to virus infection. Moreover, KAL1 expression and anosmin-1 secretion were regulated by TGF-β. Combinding with results of this project, it is possible that virus infection can be responded to through FGFR1 pathway and TGF-β signaling as well as other cell rapid response.
Also, it is known that GAN encodes a Gigaxonin protein which is a member of the cytoskeletal BTB/kelch repeat family. Gigaxonin functions in neurofilament architecture and may play a crucial role in the cross-talk between the intermediate filaments and the membrane network. Results of this project that GAN mRNA decreased by 20% in response to HPV16 infection may then indicate that GAN is used in cell response to HPV infection to function and modulate cell mobility, migration, and even reorganization of cytoskeleton.
Figure 1-Toll-like Receptor signaling pathway. Both TRIF-dependent and MyD88-dependent pathways are shown.
Human papillomavirus type 16 (HPV16) is one of the major disease inducing papillomavirus and also the cause of 99.7% of the cases of cervical cancer. Previous studies have indicated that HPV infection can induce cell morphological changes as well as the activation of Toll-like receptor (TLR) signaling via MyD88-dependent pathway. This project tested mRNA expression levels of 4 genes, IRAK1, TRAF6, TAL1 and GAN, which participate importantly in either TLR signaling and the communication between cell and its external matrix. After 4 hours of infection, human adult keratinocytes cells (HaCaT cells) did not show expression difference of IRAK1 and TRAF6 on mRNA level, whereas KAL1 mRNA increased about 50% and GAN mRNA decreased for 20%.
I thank Dr. Berish Rubin for guidance in the development of this project and giving me an opportunity to accomplish this project; thank Dr. Patricio Meneses for generously providing the HaCaT cell line, HPV16 PsV, guidance and encouragements; thank Bo Liu and Xie Xie for their time, assistance, and patience; and thank all my classmates who I spent a very happy time together with.