Effects Of RBMS3 On The Development Of HPV16-related Cervical Squamous Cell Carcinoma

Background The infection of high risk-human papillomaviruses (HR-HPVs) plays a fundamental role in the development of cervical cacinama, and E7 persistent expression, which is one of its early genes, is an key carcinogenic factor of HR-HPVs. E7 protein can lead to abnormal proliferation through different ways. RNA binding motif, single-stranded-interacting protein 3 (RBMS3) is expressed widely in normal tissues, but is down-regulated dramatically in many cancer cell lines and primary tumors. We found that the expression of RBMS3 was up-regulated after silencing E7 expression in HPV16 E7 positive cervical carcinoma cell line by Agilent human gene expression microarray analysis.Objectives The HPV16 E7-positive cervical squamous cell carcinoma tissues, cervical intra-epithelial neoplasia(CIN) tissues, cervical squamous epithelium tissues, cervical carcinoma cell line(cervical carcinoma SiHa cells), and the HPV16 E7-negative cervical squamous cell carcinoma tissues, CIN tissues, cervical squamous epithelium tissues, cervical carcinoma cell line(cervical carcinoma C33-A cells) were used as the objects of study. In present study, the aims are to investigate the expression and role of RBMS3 on the development of HPV16-related cervical squamous cell carcinoma, and provide scientific evidence for further study on the prophylaxis and treatment of HPV16 related cervical carcinoma.Methods1. The expression of RBMS3 in HPV16 E7-positive cervical carcinoma SiHa cells and HPV16 E7-negative cervical carcinoma C33-A cells was tested by Western blottingssay.2. The immunohistochemical(IHC) staining method was used to detected the expression of RBMS3 in HPV16 E7 positive/negative cervical squamous cell carcinoma tissues, CIN tissues, cervical squamous epithelium tissues. Furthermore, the relationship between the expressions of HPV16 E6 and ZNF84 was evaluated.3. The cervical carcinoma C33-A cells were cultured routinely. Then, the total RNA were extracted and quantified. Furthermore, RBMS3 gene coding sequence(CDS) was amplified by reverse transcription-polymerase chain reaction(RT-PCR) technique. Moreover, the eukaryotic expression recombinant plasmid pcDNA3.1-RBMS3 was constructed and identificated, and the recombinant plasmid and pcDNA3.1 plasmid were transfected into HPV16-positive cervical carcinoma SiHa cells via Liposome, respectively. Finally, the effects of stable expression of RBMS3 on the proliferation and apoptosis of cervical carcinoma SiHa cells were observed by growth curve, colony formation experiment, annexin V-FITC/PI combined with flow cytometry.Results1. The results from Western blotting suggested that:â‘  HPV16 E7 was expressed in cervical carcinoma SiHa cells, but not in cervical carcinoma C33-A cells.â‘¡ The expression of RBMS3 in cervical carcinoma C33-A cells was significantly higher than that in SiHa cells (P<0.01).2. The results from IHC indicated that the expression of RBMS3 in HPV16 E7-positive cervical squamous epithelium tissues, CIN tissues and cervical squamous cell carcinoma tissues was significantly lower than that in HPV16 E7-negative corresponding cervical tissues (P<0.01), and the expression of RBMS3 was negatively correlated with HPV16 E7(P<0.01).3.â‘ The results from growth curve and doubling time showed that, compared with SiHa-vect cells, the growth rate of SiHa-RBMS3 cells was significantly slowed(P <0.01), and doubling time was significantly prolonged(P<0.01).â‘¡The results from colony formation experiment displayed that, compared with SiHa-RBMS3, the mean of clone formation were significantly depressed in SiHa-RBMS3 cells(P<0.01), the volume was smaller.â‘¢ The results from annexin V-FITC/PI combined with flow cytometry revealed that, compared with SiHa-vect cells, the mean of apoptotic cells were obviously high in SiHa-RBMS3 cells (P<0.01). The percentages of apoptotic cells in SiHa-RBMS3 cells and SiHa-vect cells were 11.543% and 0.267%, respectively.Conclusions 1. The expression of E7 in HPV16-related cervical carcinoma cell strains and different stages cervical carcinoma tissues are negatively related to RBMS3.2. The stable expression of RBMS3 in HPV16-positive cervical carcinoma SiHa cells may significantly inhibit their proliferation and promote their apoptosis.