The Association Of CALCA Promoter Hypermethylation With The High-risk HPV16 Infection And Its Clinical Outcome

Objective Previous reports showed the promoter hypermethylation of calcitonin-related peptide gene (CALCA) in cervical cancer, and implied that the gene expression maybe downregulated at the transcriptional level and lost at protein level. The persistent infection of human papillomavirus type 16 (HPV16) has been believed to be an important factor in the development of cervical cancer, but its association with CALCA gene expression has not been studied fo far. In this study, we will investigate the dependence of CALCA gene promoter hypermethylation on HPV16-E7 oncoprotein expression by RNAi technic and using the HPV16-positive SiHa cervical carcinoma cells as target cells, and analyze the association of cervical lesion pathogenesis with the alteration of CALCA protein expression using cervical lesion specimens from Uighur women.Methods (1) A recombinant lentiviral siRNA expression vector was constructed using a HPV16-E7 oncogene specific siRNA fragment, and an RNAi cell model stably expressing the HPV16E7-siRNA was established by transfection of 293FT virus-packiging cells with the viral vector followed by infection of HPV16-positive SiHA carcinoma cells with recombinant virus, antibiotica selection and molecular biological characterization.(2) After extraction of genomic DNA from SiHa cells and RNAi cell model, the reversibility of CALCA gene promoter hypermethylation induced by RNAi inhibition of HPV16-E7 oncogene expression was analyzed by PCR amplification, subsequent cloning and sequencing of a CpG-rich target fragment in CALCA gene promoter. The alteration of CALCA gene transcription level was detected by RT-PCR. (3) We collected 104 cases of paraffin-embedded cervical tissue specimens from Uighur women with cervicitis, low grade squamous intraepithelial neoplasia (LSIL), high grade squamous intraepithelial neoplasia (HSIL) and cervical squamous cell carcinoma (CSCC), and detected the protein expression level of the CALCA gene by immunohistochemistry.Results:(1) We successfully established RNAi cell modell stably transcribing the HPV16E7-siRNA fragment, and the inhitory effect on target gene (E7) was also characterized by western blotting and RT-PCR. (2) We found a 365 bp CpG-rich sequence selected in the CALCA promoter region as target fragment containing 19 CpG islands, among them the cytosine of 13 CpG sites was methylated in the genomic DNA of SiHa cells (13/19 CpG sites), whereas all of the methylation was fully reversed in RNAi cell model expressing HPV16E7-siRNA(0/19 CpG sites), and in addition to this, the CALCA gene transcription was upregulated and protein expression remarkably increased after RNAi intervention, provided the evidence that the target fragment methylation was dependent on HPV16-E7 oncoprotein expression, and the methylation had an inhibitory effect on the expression of the gene.(3) The studies on clinical samples showed that CALCA protein expression was altered from normal expression to partial loss or total loss expression with the development of cervical lesion,and the "total loss" rate for cervicitis, LSIL, HSIL and CSCC was 45.5% (15/33),75% (12/16),80.8% (21/26)和82.8%(24/29),respectively, negatively correlated with cervical lesion pathogenesis. Conclusion:The RNAi cell model expressing the HPV16E7 gene-specific siRNA fragment using lentiviral vector was stable, effective and feasible. This study showed that the CALCA gene promoter hypermethylation was directly dependent on the HPV16-E7 oncoprotein expression in cervical carcinoma cells, and consequenntly, the gene hypermethylation was tightly associated with the altered gene expression. The loss of CALCA protein expression may be an important marker of cervical lesion progression in Uighur women, and the epigenetic modifications such as gene promoter hypermethylation may be responsible for the loss expression of the protein.