Tailam paramyxovirus C protein inhibits viral replication.

Jeilongviruses are emerging single-stranded negative-sense RNA viruses in the Paramyxoviridae family. Tailam paramyxovirus (TlmPV) is a Jeilongvirus that was identified in 2011. Very little is known about the mechanisms that regulate viral replication in these newly emerging viruses. Among the non-structural viral proteins of TlmPV, the C protein is predicted to be translated from an open reading frame within the phosphoprotein gene through alternative translation initiation. Though the regulatory roles of C proteins in virus replication of other paramyxoviruses have been reported before, the function of the TlmPV C protein and the relevant molecular mechanisms have not been reported. Here, we show that the C protein is expressed in TlmPV-infected cells and negatively modulates viral RNA replication. The TlmPV C protein interacts with the P protein, negatively impacting the interaction between N and P, resulting in inhibition of viral RNA replication. Deletion mutagenesis studies indicate that the 50 amino-terminal amino acid residues of the C protein are dispensable for its inhibition of virus RNA replication and interaction with the P protein.IMPORTANCETailam paramyxovirus (TlmPV) is a newly identified paramyxovirus belonging to the Jeilongvirus genus, of which little is known. In this work, we confirmed the expression of the C protein in TlmPV-infected cells, assessed its function, and defined a potential mechanism of action. This is the first time that the existence of a Jeilongvirus C protein has been confirmed and its role in viral replication has been reported.

The C-Terminal 300 Amino Acid Residues of the G Protein and Putative Open Reading Frame X of the G Gene of Tailam Paramyxovirus (TlmPV) Are Not Required for Replication in Tissue Culture Cells.

Tailam paramyxovirus (TlmPV) was identified in Sikkim Rats in Hong Kong, China in 2011. Its negative sense RNA genome is similar to J paramyxovirus (JPV) and Beilong paramyxovirus (BeiPV), the prototypes of the recently established genus Jeilongvirus. TlmPV genome is predicted to have eight genes in the order 3'-N-P/V/C-M-F-SH-TM-G/X-L-5'. The predicted size of the TlmPV G protein is 1,052 amino acid (aa) residues and much larger than G proteins of typical paramyxoviruses, which are often less than 800 aa. In addition to G open reading frame (ORF) in the G gene, another ORF, termed ORF-X exists in the G gene transcript. Similar ORF-X exists in JPV and BeiPV G gene, but their expression in virus-infected cells has not been confirmed. In this study, we generated infectious TlmPV using a newly developed reverse genetics system. We have found that the G protein of TlmPV is truncated in cultured cells: stop codons emerged in the G open reading frame, resulting in deletions of amino acid residues beyond residue 732. We have obtained infectious TlmPV lacking the C-terminal 307 aa (rTlmPV-G745) and TlmPV lacking the C-terminal 306 aa and the ORF-X (rTlmPV-GΔ746-X). The recombinant TlmPVs lacking the C-terminal 300 aa reach a higher peak viral titer and have improved genome stability in tissue cultured cells. The work indicates that the C-terminal of the G protein of TlmPV and ORF-X are not required for replication in tissue culture cells, and the deletion of the C-terminal confers a growth advantage in tissue culture cells. IMPORTANCE TlmPV is a member of the recently established genus Jeilongvirus. TlmPV encodes a large G protein and its G gene contains ORF-X. In this work, infectious TlmPV was recovered using reverse genetics. Using this system, we have demonstrated that 300 aa of C-terminal of G and the ORF-X are not required for viral replication in tissue culture cells.

Positive Correlative over-Expression between eIF4E and Snail in Nasopharyngeal Carcinoma Promotes its Metastasis and Resistance to Cisplatin.

EIF4E is the rate-limiting factor in the mRNA translation of specific set of oncogenes. Snail is the core transcription factor of epithelial-mesenchymal transition (EMT), a key step of cancer metastasis. The connection between the two oncoproteins has not been well established in the human cancer tissues and in nasopharyngeal carcinoma (NPC). Here we showed that the positive correlative over-expression was seen between eIF4E and Snail in NPC tissues, and the expression was significantly higher in the metastatic NPC than in the un-metastatic NPC. In NPC cells, eIF4E knockdown significantly reduced Snail mRNA and protein levels, increased the mRNA level of E-cad (a direct downstream gene of Snail and a negative EMT marker), attenuated the invasive ability of the cells, and sensitized the cells to cisplatin in invasion. In contrast, enforced the expression of eIF4E significantly increased Snail mRNA and protein levels, and promoted the invasive ability in NPC cells. Under the condition of the high eIF4E expression, Snail knockdown significantly increased E-cad mRNA level and weaken the invasive ability of NPC cells. Finally, eIF4E directly bound Snail mRNA for translation initiation displayed by the RIP assay. Therefore, the results firstly suggested that eIF4E enhanced the Snail expression in both transcription and translation manner in human cancer tissues and targeting the eIF4E/Snail axis might intervene with the EMT and metastasis of NPC. This finding provided a new clue for further understanding the metastatic mechanism of human cancers and for preventing and treating NPC metastasis.

Inhibition of human cervical cancer cell invasion by IL-37 involving runt related transcription factor 2 suppression.

BACKGROUND: IL-37 is a newly anti-inflammatory cytokine whose function is largely unknown in cancer. Our preliminary experiment found IL-37 could inhibit the invasion of human cervical cancer (CC) cells and influence the expression of RUNX family whose function was also unclear in CC. The present study aims to further investigate the effects of IL-37 on cell invasion and runt related transcription factor 2 (RUNX2) expression in CC cell lines. METHODS: Firstly, plasmid overexpressing IL-37 or RUNX2 was transfected into Siha and C33A cells by Hilymax. Then, the effects of IL-37 on the mRNA expression of RUNX1, RUNX2 and RUNX3 gene were detected by quantitative real-time polymerase chain reaction. Protein expression was measured by Western blot and the grayscale scanning analysis. Finally, the effects of IL-37 or RUNX2 on cell invasion were tested by transwell assay. RESULTS: IL-37 inhibited the mRNA expression of RUNX1 and RUNX2, and increased that of RUNX3 in CC cells. Among the three RUNX genes, RUNX2 showed the most significant change in mRNA expression (decreased by78.5% in Siha cells and by 61.5% in C33A cells) and thus was chosen for the following study. Overexpressed IL-37 inhibited cell invasion by 36.23% in Siha cells (P<0.05) and 26.21% in C33A cells (P<0.01). Overexpression of RUNX2 promoted cell invasion. Up-regulation of IL-37 suppressed markedly the mRNA and protein expression of RUNX2. Furthermore, overexpressed RUNX2 partially restored the inhibited cell invasion by IL-37 to 86.62% in Siha cells (P<0.01) and 87.08% in C33A cells (P<0.01). CONCLUSIONS: IL-37 can significantly inhibit the cell invasion of Siha and C33A cells, which involves the suppression of RUNX2.

Y Box-Binding Protein 1 Promotes Epithelial-Mesenchymal Transition, Invasion, and Metastasis of Cervical Cancer via Enhancing the Expressions of Snail.

OBJECTIVE: Y box-binding protein 1 (YB-1) is a potent oncogenic protein. How it regulates Snail in most tumors including cervical cancer is unknown. This article is to study if YB-1 plays a role in cervical cancer via regulating the expression of Snail. METHODS: Immunohistochemical staining of YB-1, Snail, and E-cadherin (E-cad) was performed on tissue specimens including 35 cases of chronic cervicitis (as a control), 35 cases of cervical intraepithelial neoplasm (CIN) I, 35 cases of CIN II/III, 28 cases of unmetastatic cervical squamous cell carcinoma, and 19 cases of metastatic cervical squamous cell carcinoma. RNA interference technique was used to knock down YB-1, E6, and Snail genes. Quantitative polymerase chain reaction, western blot, and transwell experiment were used to detect RNA, protein, and cell invasion of cervical cancer cell lines Hela and C33A, respectively. RESULTS: First, YB-1 knockdown significantly reduced messenger RNA (mRNA) and protein levels of Snail, followed by the increased mRNA and protein levels of E-cad and the decreased invasive ability in both Hela (human papillomavirus [HPV] 18+) and C33A (HPV-) cell lines. Second, YB-1 and Snail protein were correlatively expressed in the group order of metastatic cervical squamous cell carcinoma > unmetastatic cervical squamous cell carcinoma > CINs > cervicitis, with the inverse expression mode of E-cad in the group order, P value less than 0.01, between any 2 groups. Finally, HPV18 E6 knockdown reduced the mRNA and protein levels of YB-1 and Snail in Hela cells. CONCLUSIONS: The results firstly reported that YB-1 whose mRNA expression is regulated by HPV18 E6 promotes epithelial-mesenchymal transition and progression of cervical cancer via enhancing the expressions of Snail, which indicated that YB-1/Snail/epithelial-mesenchymal transition axis could have a potential use in the diagnosis and therapy of cervical cancer metastasis as a cancer marker and molecular target.

HPV18 E7 induces the over-transcription of eIF4E gene in cervical cancer.

OBJECTIVES: Eukaryotic translation initiation factor 4E (eIF4E) is overexpressed in cervical cancer (CC). However, the molecular mechanisms are unclear. This study aimed to investigate the molecular mechanism of eIF4E gene overexpression in CC. MATERIALS AND METHODS: The human papillomavirus (HPV) type 18 E7 and eIF4E mRNAs were measured following knock down or overexpression of E7 gene by RT-PCR and real-time PCR. Cell counting kit-8 assay was used to determine the cell proliferation. Flow cytometry was used to analyze the cell cycle and apoptosis. Transwell system was employed to determine the cell migration. RESULTS: Overexpression of E7 gene increased eIF4E mRNA level by 24.3% (P<0.01) in HPV negative C33A cells. Knock down of E7 decreased markedly eIF4E mRNA by 73% (P<0.01) in HPV18 positive HeLa cells. Under the state of high expression of E7, 1) up-regulation of eIF4E drastically promoted the cell proliferation, cell cycle and cell migration, and inhibited the cell apoptosis. 2) down-regulation of eIF4E significantly inhibited the cell proliferation, cell cycle and the ability of cell migration, and also promoted the apoptosis of cervical cancer cells. CONCLUSION: HPV E7 induced eIF4E gene over transcription which might be a new marker for CC. The finding broadens the understanding of the CC carcinogenesis.

Dehydrocostus Lactone Inhibits Proliferation, Antiapoptosis, and Invasion of Cervical Cancer Cells Through PI3K/Akt Signaling Pathway.

OBJECTIVES: Recent studies found that dehydrocostus lactone (DHC), a traditional Chinese medicine in curing chronic ulcer and inflammation, can inhibit several type of tumor cells. The purpose of this study was to define the role of DHC on cervical cancer cells and to explore its mechanism of action. METHODS: We used DHC alone or in combination with PI3K/Akt-specific inhibitor LY294002 (LY) to treat Hela cells [human papillomavirus (HPV)-18 positive] and C33a cells (HPV negative). The proliferation, apoptosis, and Akt activation were assessed. Cell invasive ability was assayed in transwell chambers. RESULTS: We found that DHC significantly inhibited proliferation, antiapoptosis, and invasion of both cells, and reduced the level of p-Akt phosphorylation in these cells, in a dose- or time-dependent manner. In addition, these inhibitions of DHC were significantly strengthened by LY. CONCLUSIONS: The result suggested that DHC plays a potent role in anticervical cancer in multiple biological aspects through PI3K/Akt signaling pathway, independently of HPV infection. This finding surely adds new knowledge to understand the role of DHC in fighting cancers.

Dehydrocostus lactone suppressed the proliferation, migration, and invasion of colorectal carcinoma through the downregulation of eIF4E expression.

Dehydrocostus lactone (DHC) is the main active ingredient extracted from a traditional Chinese medicine called Radix Aucklandiael. A few studies recently showed that DHC has anticancer potential. However, no reports exist as yet on the effects of DHC on colorectal carcinoma (CRC). This study aimed to determine whether and how DHC functions in CRC cells. After treatment with DHC, both Lovo and SW480 cells were significantly inhibited in their proliferation, cell cycle progression, migration, and invasion abilities in a dose-dependent and/or treatment time-dependent manner. Also, DHC significantly increased the apoptosis rate of SW480 cells, but not Lovo cells. The expression of eukaryotic translation initiation factor 4E (eIF4E), which was originally highly expressed in both cells, was significantly decreased by DHC. The inhibition of proliferation, migration, and invasion was significantly attenuated by the ectopic transfection of eIF4E, and was promoted by the knockdown of eIF4E in Lovo cells. To the best of our knowledge, this is the first time it has been shown that DHC suppressed the proliferation, cell cycle progression, antiapoptosis, and migration and invasion capabilities of CRC cells by the downregulation of eIF4E expression. In terms of the overexpression of eIF4E in many cancers, it was speculated that DHC might also play an anticancerous role by suppressing eIF4E expression. This discovery could lay the foundations for advancing our understanding of the anticancerous mechanism of DHC and developing DHC into a novel and effective natural anticancer therapeutic.

Pokemon enhances proliferation, cell cycle progression and anti-apoptosis activity of colorectal cancer independently of p14ARF-MDM2-p53 pathway.

Pokemon has been showed to directly suppress p14(ARF) expression and also to overexpress in multiple cancers. However, p14(ARF)-MDM2-p53 pathway is usually aberrant in colorectal cancer (CRC). The aim is to confirm whether Pokemon plays a role in CRC and explore whether Pokemon works through p14(ARF)-MDM2-p53 pathway in CRC. Immunohistochemistry for Pokemon, p14(ARF) and Mtp53 protein was applied to 45 colorectal epitheliums (CREs), 42 colorectal adenomas (CRAs) and 66 CRCs. Pokemon was knocked down with RNAi technique in CRC cell line Lovo to detect mRNA expression of p14(ARF) with qRT-PCR, cell proliferation with CCK8 assay, and cell cycle and apoptosis with flowcytometry analysis. The protein expression rates were significantly higher in CRC (75.8%) than in CRE (22.2 %) or CRA (38.1%) for Pokemon and higher in CRC (53.0%) than in CRE (0) or CRA (4.8%) for Mtp53, but not significantly different in CRC (86.4 %) versus CRE (93.3%) or CRA (90.5 %) for p14(ARF). Higher expression rate of Pokemon was associated with lymph node metastasis and higher Duke's stage. After knockdown of Pokemon in Lovo cells, the mRNA level of p14(ARF) was not significantly changed, the cell proliferation ability was decreased by 20.6%, cell cycle was arrested by 55.7% in G0/G1 phase, and apoptosis rate was increased by 19.0%. Pokemon enhanced the oncogenesis of CRC by promoting proliferation, cell cycle progression and anti-apoptosis activity of CRC cells independently of p14(ARF)-MDM2-p53 pathway. This finding provided a novel idea for understanding and further studying the molecular mechanism of Pokemon on carcinogenesis of CRC.

LMP1 stimulates the transcription of eIF4E to promote the proliferation, migration and invasion of human nasopharyngeal carcinoma.

Eukaryotic translation initiation factor 4E (eIF4E) is the rate-limiting translation initiation factor for many oncogenes. Previous studies have shown eIF4E overexpression in nasopharyngeal carcinoma (NPC). We aimed to study whether viral oncogene latent membrane protein 1 (LMP1) stimulates the transcription of eIF4E to promote NPC malignancy. In NPC cell lines (CNE1 and CNE2), ectopic LMP1 significantly increased the mRNA and protein levels of eIF4E and the transcriptional activity of the eIF4E promoter in a LMP1-plasmid-transfected dose-dependent manner. As a backward experiment, knocking down of LMP1 significantly reduced eIF4E mRNA in B95-8 cells. In the high LMP1 expression condition, knocking down of c-Myc significantly reduced eIF4E mRNA in both NPC and B95-8 cells, and knocking down of eIF4E significantly inhibited the tumor proliferation, migration and invasion promoted by LMP1. The results indicated that LMP1 stimulates the transcription of eIF4E via c-Myc to promote NPC. To the best of our knowledge, this is the first evidence that LMP1 stimulates the transcription of eIF4E. This might be an important cause of the overexpression of eIF4E in NPC and be the novel mechanism by which LMP1 initiates cancer. LMP1-stimulated eIF4E initiates the translation of those oncogenes transcriptionally activated by LMP1 to amplify and pass down the carcinogenesis signals launched by LMP1.

HPV E6 induces eIF4E transcription to promote the proliferation and migration of cervical cancer.

Increasing evidence has placed eukaryotic translation initiation factor 4E (eIF4E) at the hub of tumor development and progression. Several studies have reported that eIF4E is over-expressed in cervical cancer; however, the mechanism remains elusive. The results of this study further confirm over-expression of eIF4E in cervical cancer tumors and cell lines, and we have discovered that the transcription of eIF4E is induced by protein E6 of the human papillomavirus (HPV). Moreover, regulation of eIF4E by E6 significantly influences cell proliferation, the cell cycle, migration, and apoptosis. Therefore, eIF4E emerges as a key player in tumor development and progression and a potential target for CC treatment and prevention.

EIF4E over-expresses and enhances cell proliferation and cell cycle progression in nasopharyngeal carcinoma.

Eukaryotic translation initiation factor 4E (eIF4E) is involved in integration and amplification of many carcinogenesis signals in tumors. However, it remains unclear whether eIF4E over-expresses in NPC and whether it is associated with the development of NPC. Here, we analyzed the expression state of eIF4E, c-Myc, and MMP9 in 24 nasopharyngitises and 64 nasopharyngeal carcinomas (NPC) tissues and studied the influences of eIF4E on the proliferation and cell cycle in NPC cell lines. The results indicate that eIF4E might over-express in NPC and the over-expression of eIF4E promotes NPC growth and cell cycle progression through enhancing the translational expression of c-Myc and MMP9. The finding certainly adds new knowledge in the understanding of the carcinogenesis of NPC and provides a potential molecular target for the NPC therapy and prevention.