miR-100 suppresses the proliferation and tumor growth of esophageal squamous cancer cells via targeting CXCR7.

MicroRNAs are highly conserved non-coding RNAs that regulate gene expression at the post-transcriptional level, and play pivotal roles in cancer development and progression. miR-100 has been reported to be significantly downregulated in a variety of cancers, including esophageal cancer. However, the role of miR-100 in human esophageal cancer has not been fully elucidated. We demonstrated that overexpression of miR-100 in esophageal cancer cells markedly inhibited cell proliferation, migration and invasion as well as tumor growth. We subsequently showed that CXCR7 is a direct target gene of miR-100. Our results indicated that miR-100 plays a tumor-suppressor role in esophageal cancer and suggest its potential application for esophageal cancer treatment.

C3-Luc Cells Are an Excellent Model for Evaluation of Cellular Immunity following HPV16L1 Vaccination.

C3 and TC-1 are the two model cell lines most commonly used in studies of vaccines and drugs against human papillomavirus (HPV) infection. Because C3 cells contain both the HPV16 E and L genes, but TC-1 cells contain only the HPV16 E genes, C3 cells are usually used as the model cell line in studies targeting the HPV16 L protein. However, expression of the L1 protein is difficult to detect in C3 cells using common methods. In our study, Short tandem repeat analysis (STR) was used to demonstrate that C3 cells are indeed derived from mice, PCR results show that HPV16 L1, E6 and E7 genes were detected in C3 genomic DNA, and RT-PCR results demonstrated that L1 transcription had occurred in C3 cells. However, the expression of C3 protein was not found in the results of western blot and immunohistochemistry (IHC). Growth and proliferation of C3 were inhibited by mice spleen lymphocytes that had been immunized with a vaccine against HPV16L1. The luciferase gene was integrated into C3 cells, and it was confirmed that addition of the exogenous gene had no effect on C3 cells by comparing cell growth and tumor formation with untransformed cells. Cells stably expressing luciferase (C3-luc) were screened and subcutaneously injected into the mice. Tumors became established and were observed using a Spectrum Pre-clinical in Vivo Imaging System. Tumor size of mice in the different groups at various time points was calculated by counting photons. The sensitivity of the animals to the vaccine was quantified by statistical comparison. Ten or 30 days following injection of the C3-luc cells, tumor size differed significantly between the PBS and vaccine groups, indicating that C3 cells were susceptible to vaccination even after tumors were formed in vivo.

One-prime multi-boost strategy immunization with recombinant DNA, adenovirus, and MVA vector vaccines expressing HPV16 L1 induces potent, sustained, and specific immune response in mice.

Human papillomavirus (HPV) is associated with various human diseases, including cancer, and developing vaccines is a cost-efficient strategy to prevent HPV-related disease. The major capsid protein L1, which an increasing number of studies have confirmed is typically expressed early in infection, is a promising antigen for such a vaccine, although the E6 and E7 proteins have been characterized more extensively. Thus, the L1 gene from HPV16 was inserted into a recombinant vector, AdHu5, and MVA viral vectors, and administered by prime-boost immunization. Virus-like particles were used as control antigens. Our results indicate that prime-boost immunization with heterologous vaccines induced robust and sustained cellular and humoral response specific to HPV16 L1. In particular, sera obtained from mice immunized with DNA + DNA + Ad + MVA had excellent antitumor activity in vivo. However, the data also confirm that virus-like particles can only elicit low levels cellular immunity and not be long-lasting, and are therefore unsuitable for treatment of existing HPV infections.

Prevalence of human papillomavirus in esophageal carcinoma in Tangshan, China.

AIM: To study the prevalence of human papillomavirus (HPV) in esophageal carcinoma in Tangshan, China, a high-incidence area. METHODS: Formalin-fixed, paraffin-embedded tissue specimens from 198 patients who were pathologically diagnosed with esophageal squamous cell carcinoma from 2011 to 2013 were obtained from a pathology department in Tangshan. DNA was extracted from all 198 specimens to detect HPV by polymerase chain reaction (PCR). ß-globin PCR was performed to check the quality of the DNA extraction procedure. PCR was performed to detect a wide range of HPV types, and type-specific PCR was performed to detect HPV types 16 and 18. Negative and positive controls were used for HPV 16 and 18 detection. RESULTS: The DNA extraction method in this study appeared to be more effective than other previously reported methods. After DNA extraction, more than 98% of the tissue specimens had an acceptable result in the DNA qualification test (ß-globin PCR). The overall prevalence of HPV in tumor tissues by GP6+/GP5+ PCR was 79.79%, and the prevalence of HPV types 16 and 18 was 40.40% and 47.47%, respectively. PCR demonstrated the presence of HPV, and direct sequencing confirmed the HPV genotypes. All HPV-positive PCR products were checked by DNA sequence analysis using DNAman and compared with the known HPV sequences listed in the Basic Local Alignment Search Tool database to evaluate the HPV types. This analysis confirmed the presence of HPV types 16 and 18. CONCLUSION: DNA of high-risk HPV types 16 and 18 is present in esophageal tumors, implicating HPV as a possible etiologic factor for esophageal squamous cell carcinoma.

Integrated HPV genomes tend to integrate in gene desert areas in the CaSki, HeLa, and SiHa cervical cancer cell lines.

AIMS: The integration preferences of human papillomavirus (HPV) have been intensively studied and contested over recent years. To disclose the integration preferences of high-risk HPV in cervical cancer, HPV transcriptional sites and features in different cervical cancer cell lines were identified. MAIN METHODS: In this study, three cervical cancer cell lines (CaSki, HeLa, and SiHa) were subjected for HPV genome status determination by amplification of papillomavirus oncogene transcripts (APOT) assay. The numbers of viral copies in human genomes and numbers of viral-human fusion mRNAs in three HPV-integrated cervical cancer cell lines were measured and analysed. KEY FINDINGS: The results revealed that the gene desert region 8q24 of the HPV type 18 integrated HeLa cell line and the 13q21-22 region of the HPV type 16 integrated CaSki and SiHa cell lines were hotspots for HPV integration, and the numbers of viral copies in the human genomes of the three cell lines that we detected were not in accordance with those reported in previous studies. SIGNIFICANCE: Integration of the HPV genome into the host cell chromosome suggests that persistent HPV infection is vital for malignant cell transformation and carcinogenesis. This study provides information to benefit health care professionals seeking more comprehensive and accurate diagnostics for HPV-related disease"? Please check, and amend as necessary.

[Immune response induced by vaccination with pseudotyped rAAV1 expressing HPV16 L1 protein].

To investigate the feasibility of using recombinant adeno-associated virus type 1 vector as prophylactic vaccine against HPV16 infection, rAAV1-mod. HPV16L1, the recombinant AAV1 vector containing codon-modified HPV16 L1 gene, was constructed. C57BL/6 mice were immunized with purified rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based on HPV16 pseudovirus. The result shows that the single dose of rAAV1-mod. HPV16L1 can induce specific neutralizing antibody in serum through both inoculation routes. Compared with intranasal group, intramuscular group can induce higher titer of neutralizing antibody. Eliciting strong and prolonged neutralizing antibody in serum, the rAAV1-mod. HPV16L1 is one of promising HPV16 prophylactic vaccine candidates.

[Construction and immunological evaluation of recombinant adenovirus containing codon-modified HPV 16 L1 gene].

OBJECTIVE: To construct recombinant adenovirus containing codon-modified HPV16L1 gene, and evaluate systemic and mucosal immunological responses induced after immunization with the recombinant virus. METHODS: The recombinant adenovirus rAd-mod.HPV16L1 was constructed by Admax kit. The C57 BL/6 mice were immunized by purified rAd-mod.HPV16L1 through different inoculation routes. The immunological effect was evaluated by testing the specific neutralizing antibodies in sera and vaginal secretions of immunized mice through indirect ELISA and neutralization assay based HPV pseudovirus. RESULTS: The result showed that intramuscular immunization could induce good systemic immunity, but the mucosal immunity was too weak, and immunization via intranasal route could induce satisfactory immunity both in sera and vaginal secretions, while intravaginal immunization failed to induce any specific immunological responses either in sera or vaginal secretions. CONCLUSION: The recombinant adenovirus containing codon- modified HPV16L1 gene was successfully constructed. Immunization through intranasal route could induce satisfactory immunity both in sera and vaginal secretions, while intramuscular immunization could only induce high titer of neutralizing antibodies in sera.

[Immune response induced by recombinant adenovirus combined with recombinant adeno-associated virus type 1 containing HPV16 L1 gene].

OBJECTIVE: To evaluate the immune potency of recombinant adenovirus combined with rAAV1 vector expressing HPV16L1 protein in mice. METHODS: The rAdV and rAAV1 vector containing codon-modified HPV16L1 gene was constructed using Admax and AAVmax packaging system respectively. C57 BL/6 mice were immunized with purified rAdV and rAAV1 vector through intramuscular and intranasal inoculation routes, and the titer of neutralizing antibody was determined by neutralization assay based HPV16 pseudovirus. RESULTS: Intramuscular immunization by rAAV1-mod. HPV16L1 or combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum than that of other groups. The titer of neutralizing antibody of intranasal groups is significantly lower than that of intramuscular group, although the prime-boost strategy using in intranasal group was effective to enhance the specific humoral immunity. CONCLUSION: The rAAV1-mod. HPV16L1 combined with rAd-mod. HPV16L1 can induce higher titer of neutralizing antibody in serum through intramuscular route than that of other groups at the 16th week after the first immunization.