Nicotine Activates PI3K/AKT Pathway to Induce Cellular Proliferation, Invasion, and Migration in HPV-16 Positive Cervical Cancer SiHa Cells.

This study attempted to investigate the expression and significance of lncRNA HOST2 (human ovarian cancer-specific transcript 2) and microRNA let-7b in human papillomavirus (HPV)-positive cervical cancer (CC) tissues and cell lines. The expression of levels of HOST2 and let-7b were detected by qRT-PCR in HPV-positive CC tissues and cell lines. The HPV-positive CaSki and HeLa cells were divided into the Blank, NC, pcDNA3.0-HOST2, siHOST2, let-7b mimic, and pcDNA3.0-HOST2+let-7b mimic groups. Dual-luciferase reporter gene assay was employed to verify the targeting relationship between HOST2 and let-7b, MTT and flow cytometry to determinate cell proliferation and apoptosis, and wound-healing and transwell assays to evaluate cell migration and invasion capabilities. HOST2 was up-regulated but let-7b was down-regulated in HPV-positive CC tissues and cells. Dual-luciferase reporter gene assay confirmed the targeting relationship between HOST2 and let-7b. Over-expressed HOST2 reduced let-7b expression, promoted proliferation migration and invasion and inhibited the apoptosis of CaSki and HeLa cells; however, silencing HOST2 or overexpressing let-7b enhanced the expression of let-7b, inhibited proliferation migration and invasion, and promoted the apoptosis of CaSki and HeLa cells, and let-7b mimic could reverse the promoting effect of HOST2 on the growth of CC cells. HOST2 was upregulated in HPV-positive CC tissues and cells, which could promote the proliferation, migration and invasion, but inhibit the apoptosis of HPV-positive CC cells via inhibition of let-7b.

BackgroundAlthough evidence has revealed that miR-200a-3p is involved in the malignant progression of various tumors, the regulatory mechanism of miR-200a-3p in the development of cervical cancer (CC) cells with different HPV statuses remains unknown. The present study was to investigate the differential effects of either miR-200a-3p or YAP on tumorous cells’ fate in vitro in HPV-negative and HPV-positive cervical cancer cell models, and to explore if the changes in proliferation, migration, and invasion of the CC cells with different HPV statuses could be attributed to the differential interactions between miR-200a-3p and YAP.MethodsThe colony formation assays, EDU assays and Transwell assays were performed for CC cell proliferation, migration and invasion capacities analysis. The prediction of downstream targets of miR-200a-3p was performed by bioinformatical databases. The dual-luciferase reporter assays were used to validate the binding sites of miR-200a-3p and YAP. The qRT-PCR assays were performed to quantify the mRNA expression of miR-200a-3p and YAP, and the protein levels of YAP were examined by Western blot analysis.ResultsThe results demonstrated that miR-200a-3p overexpression suppressed proliferation, migration, and invasion of the HPV-negative C33A cells but promoted the growth and metastasis of HPV-positive CC cells, while YAP promoted the cell growth and metastasis not only in HPV-negative but also in the HPV-positive CC cells. The suppressive role of miR-200a-3p in C33A cells appeared to be mediated partially by direct interaction with YAP, and YAP might participate in miR-200a-3p-mediated cellular changes in CC cells differing from not only the presence or absence of HPV but even also the subtypes of HPV of CC cells. Meanwhile, we preliminarily revealed that the expression level of miR-200a-3p was significantly decreased in HPV-negative, but not in HPV16-positive cervical neoplasm mucus samples.ConclusionmiR-200a-3p-mediated functional changes of YAP exhibited regulatory effects on cells’ fate differentially in HPV-negative and HPV-positive cervical cancer cells.

Introduction/BackgroundLong non-coding RNAs are emerging to be novel regulators in gene expression. In current study, lncRNAs microarray and lncRNA-mRNA co-expression analysis were performed to explore the alternation and function of...

Cervical cancer is one of the leading causes of death due to cancer in women. Human Papilloma Virus infection is the primary risk factor for developing cervical cancer, however very little is known about HPV negative cervical cancers. Folate fortification of flour for prevention of neural tube defects has been mandatory in North America since 1998; and folate supplementation is recommended during pregnancy. Folate fortification is not mandated in all countries, however, due to concerns about possible adverse effects on cancer incidence or prognosis. There is evidence that folate intake can reduce risk of developing tumors but other contradictory studies indicate that supraphysiologic doses of folate can actually lead to disease progression. Folate receptor alpha is known to be expressed in many cancer cell lines, including HPV positive cervical cancer. There is some evidence that folic acid can promote methylation of the EGFR, leading to a decrease in EGFR expression, and that EGFR might be active in some HPV positive cervical cancers. The purpose of this study is to determine if EGFR plays a role in HPV negative cervical cancer cell growth, and the effects of folic acid on EGFR protein expression and activation. Two HPV negative cervical cancer cell lines were used for this study: C33A and DoTc2‐4510. High doses of folic acid caused a decrease in both C33A and DoTc cell proliferation. Interestingly, qPCR and western blotting analysis indicate that only the DoTc2‐4510 cells expressed EGFR. The DoTc2‐4510 cells were sensitive to the EGFR inhibitor, gefinitib, with an IC50 of 0.01 uM. There was no change in EGFR expression upon folic acid treatment, but interestingly high doses of folic acid did inhibit EGFR activation. Taken together this data indicates that EGFR may be a target for a subset of HPV negative cervical cancers, and that high doses of folic acid may help prevent EGFR positive HPV negative cervical cancer cell growth.

Cervical cancer treatment with a locally insertable controlled release delivery system

A71 Cervical cancer is one of the most prevalent female cancers in world. The treatment for cervical cancer includes chemotherapy. Most potential standard chemotherapeutic agents have side effects. A need-based approach for developing new anti cancer agents from metal compounds with fewer side effects will have enormous implications. Opportunities exist to exploit metal-based drugs in the discovery and development of pharmaceuticals against cancers. Vanadium is a transition metal widely distributed in the environment, which is also a dietary micronutrient. The antidiabetic and anticancer activities of the oxovanadium complexes are recently known. In this study, human cervical cancer cell line SiHa, which is HPV16 - positive has been employed to determine the anticarcinogenic property of two oxovanadium complexes, OVK 49 and OVK 89. Among these OVK 49 proved to be more effective than OVK 89. Both complexes inhibited the growth of SiHa cells in a concentration and time dependent manner. Treatment of cells with oxovanadium complexes caused loss of mitochondrial membrane potential (ΔΨm) and morphological changes characteristic of apoptosis, such as the nuclear condensation. Moreover oxovanadium complex induced apoptosis, which involved release of mitochondrial cytochrome c and activation of caspase 8 and 9. The molecular mechanism behind the apoptotic induction due to these oxovanadium complexes is well established in this study. The E6 oncoprotein of human papillomaviruses has the potential to funtionally antagonize p53. Here it has also been investigated whether this reflects the regulation of p53 expression in HPV positive cervical cells. It has also been found that p21 was efficiently induced and HPV E6, E7 p53 and GADD45 were decreased by OVK 49 and 89 in SiHa cells. These results suggests that inspite of the presence of E6 protein HPV 16 positive cervical cancer cell lines are capable of responding efficiently to DNA damage provoked by metal based drug treatment through a p53 dependent pathway. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A71.

Previous studies confirmed that high-risk human papillomavirus (HR-HPV) infection is a risk factor of cervical cancer, and the infection was associated with significantly reduced miR-34a expression during carcinogenesis. However, the downstream targets of miR-34a and their roles are still not well understood. This study explored the regulative role of miR-34a on E2F3 and survivin expression and the viability and invasion of HPV-positive cervical cancer cells. MiR-34a and survivin expression in 56 cases of HR-HPV-positive patients, 28 cases of HR-HPV-negative patients, and 28 normal cases without HR-HPV infections were measured. Human papillomavirus-18-positive HeLa cervical cancer cells and HPV-16-positive SiHa cells were used to explore the effect of miR-34a on cell viability and invasion. The molecular target of miR-34a was also explored in cervical cancer cells. The results showed that miR-34a overexpression could inhibit HPV-positive cancer cell viability, whereas its downregulation promoted cell viability. E2F3 is a direct target of miR-34a in HPV-positive cervical cancer cells. By targeting E2F3, miR-34a could regulate the expression of survivin. Thus, through regulating E2F3 and survivin, miR-34a could reduce the viability and invasion of HPV-positive cervical cancer cells. This study confirmed a novel miR-34a-E2F3-survivin axis in the tumor suppressor role of miR-34a in cervical cancer.

BackgroundCervical cancer (CC) is one of the most common gynaecological malignancies all around the world. The mechanisms of cervical carcinoma formation remain under close scrutiny. The long non-coding RNAs (lncRNA) and microRNAs (miRNAs) play important roles in controlling gene expression and promoting the development and progression of cervical cancer by acting as competitive endogenous RNA (ceRNA). However, the roles of lncRNA associated with ceRNAs in cervical carcinogenesis remains unknown. In this study, the expression of long non-coding RNA HOTAIR was investigated in HPV16 positive cervical cancer cells, the candidate miRNAs and target genes were identified to clarify putative ceRNAs of HOTAIR/miRNA in cervical cancer cells.MethodsThe proliferation ability of cells was measured by CCK8 and EdU incorporation assays and cell apoptosis was analyzed by flow cytometry. The expression of HOTAIR, miR-214-3p, HPV16 E7 mRNA were detected by qRT-PCR. As for searching for the interaction between miR-214-3p and HOTAIR, the binding sites for miR-214-3p on HOTAIR was predicted by starbase v2.0 database, then dual-luciferase assay was used to verify the binding sites. In addition, Gene Ontology (GO) and protein–protein interaction (PPI) network analysis of target genes of miR-214-3p were performed with bioinformatics analysis. The potential signal pathway regulated by HOTAIR/miR-214-3p was predicted by KEGG enrichment analysis and confirmed by qPCR and WB analysis in cervical cancer cells.ResultsOur results showed that expression of HOTAIR was up-regulated, while that of miR-214-3p was down-regulated in HPV16-positive cervical cancer cells. The expression status of HPV16 E7 played an important role in regulating expression of HOTAIR or miR-214-3p in cervical cancer cells. HOTAIR knockdown could significantly inhibited cell proliferate ability and promote cellular apoptosis, whereas the inhibition of miR-214-3p expression partially reversed such results. Bioinformatics analysis identified 1451 genes as target genes of miR-214-3p. The Gene ontology (GO) and KEGG Pathway enrichment analysis showed that these target genes were mainly related to regulation of cell communication, protein binding, enzyme binding and transferase activity, and Wnt ligand biogenesis. Pathway enrichment analysis results showed that the predicted target genes were significantly enriched in Wnt/β-catenin signaling pathway. Finally, our results confirmed that miR-214-3p could significantly inhibit β-catenin expression in HPV16 positive cancer cells by qPCR and WB analysis.ConclusionHOTAIR could act as a ceRNA through binding to miR-214-3p, promote cell proliferation and inhibit the apoptosis of HPV16 positive cervical cancer. HOTAIR/miR-214-3p/Wnt/β-catenin signal pathway might played important regulated roles in HPV16 positive cervical cancer. Our results provided new insight into defining novel biomarkers for cervical cancer.

Epigenetic modulation is an important mechanism of miRNA dysregulation in cervical cancer. In this study, we firstly studied how this mechanism contributes to miR-375 downregulation in cervical cancer cells. Then, we further studied the association between miR-375 and MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in epithelial mesenchymal transition (EMT) of the cancer cells. HPV-16 positive SiHa and CaSki cells were used as in vitro model. Our data showed that HPV-16 E6 positively modulated DNMT1 expression in both SiHa and CaSki cells. Knockdown of DNMT1 partly restored miR-375 levels in the cells. The following methylation-specific PCR (MSP) assay and qRT-PCR analysis showed that methylation was common in the promoter region of miR-375 in both SiHa and CaSki cells and demethylation partly restored miR-375 levels in the cells. Therefore, we infer that miR-375 is downregulated partly due to promoter hypermethylation mediated by DNMT1 in HPV-16 positive cervical cancer cells. Our bioinformatics analysis showed that MALAT1 has three putative binding sites with miR-375 and the following dual luciferase assay confirmed two of them. QRT-PCR analysis showed that miR-375 overexpression significantly reduced MALAT1 expression, while MALAT1 overexpression reversely suppressed miR-375 levels. Therefore, we infer that there is a reciprocal regulation between miR-375 and MALAT1 in the cells. In SiHa cells, miR-375 overexpression or MALAT1 siRNA partly restored E-cadherin expression, significantly reduced N-cadherin and also reduced invasion capacity of SiHa cells. Therefore, these results suggest that miR-375 and MALAT1 form a functional axis modulating EMT in cervical cancer.

Puerarin is an effective component that is present in high concentrations in the Pueraria lobata plant and is extensively distributed throughout nature. Puerarin possesses a number of pharmacological effects and has strong pharmacological activity with few side effects and extensive clinical applications. The aim of the present study was to explore the effects of Puerarin on the apoptosis of human papillomavirus (HPV)-positive cervical cancer cells and the underlying molecular mechanisms. MTT assay, lactate dehydrogenase activity and Annexin V/fluorescein isothiocyanate/propidium iodide analysis were used to analyze cell growth of HPV-positive HeLa cervical cancer cells treated with Puerarin. Western blotting was performed to measure protein expression in the treated cells. Puerarin significantly reduced cell proliferation and induced apoptosis in HeLa cells. In addition, it was observed that Puerarin significantly enhanced caspase-3/9 activities and significantly increased B-cell lymphoma 2-asscoiate X protein expression in HeLa cells. Puerarin suppressed phosphatidylinositol-3 kinase (PI3K), phosphorylated (p)-protein kinase B (Akt) and p-mammalian target of rapamycin (mTOR) protein expression in HeLa cells. These results indicate that Puerarin induces apoptosis in HPV-positive HeLa cervical cancer cells via inhibiting PI3K/Akt/mTOR signaling.

The effect of andrographolide on Human papillomavirus type 16 (HPV16) positive cervical cancer cells (SiHa)

PurposeCervical cancer is the second most prevalent female malignance, and human papillomavirus (HPV) infection is the main pathogenic factor of cervical cancer. Emerging evidence has revealed that a number of long non-coding RNAs (lncRNAs) play critical roles in the tumorigenesis and progression of cervical cancer. The aim of this study was to further investigate the precise role of lncRNA LINC00511 in HPV-negative and HPV-positive cervical cancer cells and explore the potential regulatory mechanism.MethodsThe expression of LINC00511 in cervical cancer and cell lines was examined by RT-PCR. Fluorescence in situ hybridization analysis (FISH) assay was performed to detect the localization of LINC00511 in cervical cancer cells. Loss-of-function experiments of LINC00511 by siRNA interference were performed to assess its effects on HPV-negative and HPV-positive cervical cancer cells. Dual-luciferase reporter and RNA immunoprecipitation (RIP) assays were used to identify the target of LINC00511. Relative expression of related proteins was detected using Western blot.ResultsHerein, the results showed that LINC00511 was significantly up-regulated in cervical cancer and cell lines and mainly distributed in the cytoplasm of cervical cancer cells. Loss-of-function experiments indicated that silencing of LINC00511 inhibited the proliferation and invasion of both HPV-negative and HPV-positive cervical cancer cells, as well as promoted apoptosis by regulating the Bcl-2/Bax axis and Caspase 3 activation. Bioinformatic analysis, dual-luciferase reporter, and RIP assays showed that LINC00511 was a target of miR-324-5p, while DRAM1 was a direct target of miR-324-5p. The expression of miR-324-5p was down-regulated in cervical cancer, while the expression of DRAM1 was up-regulated. Moreover, the expression of LINC00511 was negatively correlated with miR-324-5p expression in cervical cancer tissues and positively correlated with DRAM1. Further, DRAM1 overexpression promoted both HPV-negative and HPV-positive cervical cancer cell proliferation and invasion, which could be reversed by miR-324-5p mimics or si-LINC00511.ConclusionCollectively, these results suggest that LINC00511 functions as a competing endogenous RNA (ceRNA) to regulate the miR-324-5p/DRAM1 axis, leading to HPV-negative and HPV-positive cervical cancer aggravation.

Cervical cancer is a largely preventable malignancy due to the availability of cytology screening and vaccination against the essential initiation factor of cervical carcinogenesis, human papillomavirus (HPV). However, cervical cancer remains a significant medical burden worldwide, particularly in developing countries where large scale screening or vaccination programs are not financially feasible. Molecular tests such as HPV DNA tests have the potential to improve the speed and sensitivity of cervical cancer screening but suffer from limited specificity. Additional adjunct molecular markers are therefore desirable for enhancing molecular tests. Our previous research has revealed miR-143, a microRNA downregulated in a number of cancers, could be detected in liquid based cytology samples and is significantly reduced in cervical cancer samples and cell lines. Cellular role of miR-143 and mechanism behind its downregulation remain an unknown in cervical carcinogenesis. To explore the cellular roles of miR-143 in cervical cancer, a construct expressing miR-143 was transfected into cervical cancer cell lines HeLa, SiHa and C33A. miR- 143 overexpression was verified by qPCR. The miR-143 overexpressing cell lines were used to conduct a number of cellular function assays. It has been reported that miR-143 is able to suppress cell growth in HPV-positive HeLa. We followed up the findings and revealed miR-143 overexpression in HPV-negative C33A did not suppress cell growth in an MTT cell proliferation assay. ERK5 and KRAS, two targets of miR-143, are downregulated in colon cancer and bladder cancer to suppress cell grwoth. However, mRNA level of ERK5 and KRAS were not altered in all three miR-143 overexpressed cervical cancer cell lines, suggesting that miR-143 may not target ERK5 and KRAS transcriptionally in cervical cancer. Ability of miR-143 in regulating cell differentiation was evaluated by the expression of K10, an early keratinocyte differentiation marker. K10 was upregulated only in miR-143 overexpressed HeLa and SiHa as revealed by qPCR. A parallel increase in hSkn-1a mRNA, a transcription factor of K10, was also observed specifically in the two miR-overexpressed HPV-positive cell lines. miR-143 level is inversely correlated with cytology grading and progression of cervical disease, hinting its role in mediating cell migration and invasion during cancer progression and metastasis. A reduction of cell migration as demonstrated in wound healing assay and in vitro transwell migration assay was observed exclusively in miR-143 overexpressed HeLa and SiHa. miR-143 overexpression in C33A did not introduce any effect in cell migration. A reduction of cell invasion was also observed merely in miR-143 overexpressed HeLa and SiHa as revealed in a transwell invasion assay. Apart from studying the cellular roles of miR-143 in cervical cancer, this study has also explored mechanisms behind miR-143 downregulation in cervical cancer owing to the fact that certain miR-143 mediated cellular functions were observed only in HPV-positive cervical cancer cell lines. We hypothesized that HPV E6 and E7 oncoprotein may downregulate miR-143 in cervical cancer. The hypothesis was supported by our findings where normal cervical epithelial cell line immortalized by E6 and E7 had an undetectable level of endogenous miR-143 level. The same primary cells immortalized by shp16-hTERT expressed residual amounts of miR-143 as revealed by qPCR. Owing to the low miR-143 expression in shp16-hTERTimmortalized normal cervical epithelial cell line, downregulation of miR-143 in cervical cancer cell lines may also be contributed to hTERT overexpression and p16 silencing. Overall, miR-143 plays an important role in suppressing cell proliferation, enhancing keratinocyte differentiation marker expression, reducing migration and invasion in HPV-positive cervical cancer. Downregulation of miR-143 level may be an effect as manifested by E6 and E7 in HPV-positive cervical cancer. Differential cellular effects in miR-143 overexpressed HPV-positive and HPV-negative cervical cancer cell lines suggest that HPV oncoprotein mediates miR-143 cellular functions.

Using conditional cell reprogramming, we generated a stable cell culture of an extremely rare and aggressive neuroendocrine cervical cancer. The cultured cells contained HPV-16, formed colonies in soft agar and rapidly produced tumors in immunodeficient mice. The HPV-16 genome was integrated adjacent to the Myc gene, both of which were amplified 40-fold. Analysis of RNA transcripts detected fusion of the HPV/Myc genes, arising from apparent microhomologous recombination. Spectral karyotyping (SKY) and fluorescent-in-situ hybridization (FISH) demonstrated coordinate localization and translocation of the amplified Myc and HPV genes on chromosomes 8 and 21. Similar to the primary tumor, tumor cell cultures expressed very high levels of the Myc protein and, in contrast to all other HPV-positive cervical cancer cell lines, they harbored a gain-of-function mutation in p53 (R273C). Unexpectedly, viral oncogene knockdown had no effect on the growth of the cells, but it did inhibit the proliferation of a conventional HPV-16 positive cervical cancer cell line. Knockdown of Myc, but not the mutant p53, significantly inhibited tumor cell proliferation. On the basis of these data, we propose that the primary driver of transformation in this aggressive cervical cancer is not HPV oncogene expression but rather the overexpression of Myc.

Objective : The purpose of this study is investigated the differentially expressed genes between normal and cervical cancer cell line. Methods : We used normal human keratinocyte (HaCaT) as a control and HPV-16 positive cervical cancer (SiHa) cell line. Two cell lines were studied differential expressed genes by using GeneFishingTM PCR and analyzed with BLAST search. Results : As compared with normal, cervical cancer cell line was showed 25 up-regulated genes including the S1-2-2, S5-1 and 24 down-regulated genes. Conclusion : GeneFishTM PCR test is very sensitive and effective method for detection of changed gene expression. We could search differentially expressed genes between normal and cervical cancer cell line. In the future, we need to research various genes unction to participate in the process of tumor development and progression.