LIV-1 suppression inhibits HeLa cell invasion by targeting ERK1/2-Snail/Slug pathway

It was reported that expression of the estrogen-regulated zinc transporter LIV-1 was particularly high in human cervical cancer cell line HeLa. This result prompted us to study the role that LIV-1 played in human cervical cancer. The results of real-time PCR showed that LIV-1 mRNA was significantly higher in cervical cancer in situ than in normal tissues. RNAi mediated suppression of LIV-1 in HeLa cells significantly inhibited cell proliferation, colony formation, migration, and invasive ability, but had no effect on cell apoptosis. Furthermore, LIV-1 suppression is accompanied by down-regulation of p44/42 MAPK, phospho-p44/42 MAPK, Snail and Slug expression levels. Hence, our data provide the first evidence that LIV-1 mRNA is overexpressed in cervical cancer in situ and is involved in invasion of cervical cancer cells through targeting MAPK-mediated Snail and Slug expression.     

Daidzin inhibits growth and induces apoptosis through the JAK2/STAT3 in human cervical cancer HeLa cells

Daidzin, 4′, 7-dihydroxyisoflavone is an isoflavonic phytoestrogen present in leguminous plants. Traditional Chinese medicine utilizes daidzin to treat various diseases such diarrhea, fever, hepatitis, cardiac problems etc. In current study we examined the anticancer activity of daidzin against human cervical cancer in vitro. HeLa, human cervical cancer cell line was purchased from ATCC and the cells were cultured with DMEM medium. The cytotoxic effect of daidzin against HeLa cell line was analyzed with MTT assay. The IC-50 value was obtained at 20 µM hence the cells were treated with 20 µM of daidzin for further analysis. ROS generation was assessed with DCFH-DA staining and the induction of apoptosis was examined with Rhoadmine-123 staining. Acridine orange and ethidium bromide staining was done to examine the apoptotic and viable cells. Further the matrigel cell adhesion assay was done to analyze the inhibitory property of daidzin against cancer cell adhesion. Apoptotic induction of daidzin was examined by estimating the levels of Caspase 8 & 9 using ELISA technique. Inflammatory and cell proliferation signaling proteins were analyzed with qPCR analysis to confirm the anticancer activity of daidzin against human cervical cancer HeLa cell line. Daidzin significantly generated ROS and altered the mitochondrial membrane permeability in HeLa cell line. The results of AO/EtBr staining prove daidzin induced apoptosis in HeLa cell line and it also inhibited the cell adhesion property of HeLa which is reported in our matrigel cell adhesion assay. It also increased the caspases 8 & 9 which are key regulators of apoptosis. Daidzin significantly decreased the expression of inflammatory gene and cell proliferating signaling molecule. To, conclude our results confirm daidzin effectively decreased inflammation and induced apoptosis in human cervical cancer HeLa cell line.     

Cellular and molecular mechanisms of silibinin induces cell-cycle arrest and apoptosis on HeLa cells

Silibinin, an effective anti-cancer and chemopreventive agent in various epithelial cancer models, has been reported to inhibit cancer cell growth through mitogenic signalling pathways including cervical cancer. However, the underlying mechanisms are still not well elucidated. Here, we assessed the effect of silibinin on human cervical carcinoma cell cycle modulation, apoptosis induction and associated molecular alterations by employing HeLa cell line. Silibinin treatment of HeLa cells resulted in a G2 arrest and induced a decrease in cyclin-dependent kinases involved in both G1 and G2 progression. In addition, silibinin showed a dose-dependent and a time-dependent apoptotic death in HeLa cells in both the mitochondrial pathway and the death receptor-mediated pathway, providing a strong rationale for future studies evaluating preventive and/or intervention strategies for silibinin in cervical cancer pre-clinical models.     

Down-Regulating Overexpressed Human Lon in Cervical Cancer Suppresses Cell Proliferation and Bioenergetics

The human mitochondrial ATP-dependent Lon protease functions in regulating the metabolism and quality control of proteins and mitochondrial DNA (mtDNA). However, the role of Lon in cancer is not well understood. Therefore, this study was undertaken to investigate the importance of Lon in cervical cancer cells from patients and in established cell lines. Microarray analysis from 30 cancer and 10 normal cervical tissues were analyzed by immunohistochemistry for Lon protein levels. The expression of Lon was also examined by immunoblotting 16 fresh cervical cancer tissues and their respective non-tumor cervical tissues. In all cases, Lon expression was significantly elevated in cervical carcinomas as compared to normal tissues. Augmented Lon expression in tissue microarrays did not vary between age, tumor-node-metastasis grades, or lymph node metastasis. Knocking down Lon in HeLa cervical cancer cells by lentivrial transduction resulted in a substantial decrease in both mRNA and protein levels. Such down-regulation of Lon expression significantly blocked HeLa cell proliferation. In addition, knocking down Lon resulted in decreased cellular bioenergetics as determined by measuring aerobic respiration and glycolysis using the Seahorse XF24 extracellular flux analyzer. Together, these data demonstrate that Lon plays a potential role in the oncogenesis of cervical cancer, and may be a useful biomarker and target in the treatment of cervical cancer. Lon; immunohistochemistry; cervical cancer; cell proliferation; cellular bioenergetics.     

稳定干扰核糖体蛋白RPS7基因表达的宫颈癌HeLa细胞株的建立

目的建立稳定抑制RPS7基因表达的宫颈癌HeLa细胞株。方法设计并合成靶向人RPS7基因的shRNA寡核苷酸片段,克隆到逆转录病毒载体pSIREN中,构建重组质粒pSIREN-RPS7-shRNA,转染293T细胞,将包装产生的重组逆转录病毒感染宫颈癌HeLa细胞,经嘌呤霉素筛选获得稳定的细胞克隆,用real-timePCR和Western印迹检测细胞中RPS7mRNA和蛋白表达水平。结果获得了经测序鉴定正确的重组逆转录病毒质粒,逆转录病毒感染HeLa细胞后用嘌呤霉素筛选出的稳定细胞中,RPS7mRNA和蛋白水平均显著低于干扰对照细胞。结论成功构建了靶向人RPS7基因的shRNA逆转录病毒载体,建立了稳定抑制RPS7基因表达的宫颈癌HeLa细胞株．为进一步研究RPS7在宫颈癌中的生物学功能和作用机制提供了可靠的细胞模型。     

Downregulation of long noncoding RNA DGCR5 contributes to the proliferation,migration, and invasion of cervical cancer by activating Wnt signaling pathway

Accumulating research works have reported that long noncoding RNAs (lncRNAs) are involved in various cancers, including cervical cancer. LncRNA DGCR5 has been identified in many cancers. However, the biological role of DGCR5 in cervical cancer remains barely known. We aimed to investigate the biological function of DGCR5 in cervical cancer progression. Here, in our current study, we observed that DGCR5 was downregulated in human cervical cancer cell lines (MS751, SiHa, HeLa, and HT-3) compared with the primary normal cervical squamous cells (NCSC1 and NCSC2). Then, DGCR5 was restrained by transfection with lenti-virus-short hairpin RNA (LV-shRNA) while induced by LV-DGCR5 in HeLa and C33A cells. Silence of DGCR5 obviously induced cervical cancer cell viability and cell proliferation. Reversely, upregulation of DGCR5 inhibited HeLa and C33A cell survival and proliferation. Furthermore, silencing of DGCR5 increased cervical cancer cell colony formation ability and decreased cell apoptosis, whereas its overexpression exhibited an opposite process. Moreover, DGCR5 suppressed migration and invasion capacity of cervical cancer cells. The Wnt signaling is integral in numerous biological processes. Here, we found that Wnt signaling was strongly activated in cervical cancer cells. Downregulation of DGCR5 contributed to cervical cancer progression by activating Wnt signaling. Subsequently, in vivo animal models were used to confirm that DGCR5 suppressed cervical cancer via targeting Wnt signaling. In conclusion, we reported that DGCR5 was involved in cervical cancer progression via modulating the Wnt pathway.     

Plexin-B1 is a target of miR-214 in cervical cancer and promotes the growth and invasion of HeLa cells

Plexin-B1, the receptor for Sema4D, has been reported to trigger multiple and sometimes opposing cellular responses in various types of tumor cells. It has been implicated in the regulation of tumor-cell survival, proliferation, angiogenesis, invasion and metastasis. However, the plexin-B1 gene expression and its regulatory mechanism in cervical cancer remain unclear. The present study shows that plexin-B1 is over-expressed in cervical tumor tissues compared to normal cervical tissues by immunohistochemistry, Western blotting and quantitative RT-PCR. The expression of plexin-B1 is significantly associated with cervical tumor metastasis and invasion according to the analysis of the clinicopathologic data. Plexin-B1 also promotes proliferation, migration and invasion in human cervical cancer HeLa cells. We also found that the plexin-B1 levels are inversely correlated with miR-214 amounts in both cervical cancer tissues and HeLa cells. And miR-214 expression level is also associated with metastasis and invasion of cervical tumor. Furthermore, we demonstrate that plexin-B1 is inhibited by miR-214 through a miR-214 binding site within the 3'UTR of plexin-B1 in HeLa cells. Ectopic expression of miR-214 could inhibit the proliferation capacity, migration and invasion ability of HeLa cells. Our findings suggest that plexin-B1, a target of miR-214, may function as an oncogene in human cervical cancer HeLa cells.     

Adenoviral p53 effects and cell-specific E7 protein-protein interactions of human cervical cancer cells

We investigated the time-course tumor growth suppression effects of recombinant adenovirus expressing p53 on human cervical cancer cells and cell-specific E7 protein-protein interactions in cell lysates using surface plasmon resonance (SPR) biosensor. Six HPV-infected human cervical cancer cell lines (HPV 16-positive cells, CaSki and SiHa cells; HPV 18-positive cells, HeLa and HeLaS3 cells; and HPV negative C33A and HT3 cells) were used. After infection with AdCMVp53, the cell-specific growth inhibition was studied in vitro and in vivo. Also, we produced the recombinant E7 oncoprotein of HPV 16 type and tested chip-based protein-protein interactions with each cell lysate. For each cervical cancer cell, differential cell growth inhibitions were shown via cell count assay and MTT assay. Note that the same trend in suppression levels was shown in CaSki, HeLa and in SiHa, HeLaS3, respectively. In contrast, infection with AdCMVLacZ showed increased cell growth in a manner similar to the negative control group. The levels of p53 protein were notably expressed in CaSki and HeLa more than in SiHa and HeLaS3 for 4 days. In contrast, p53 expression was continually maintained in C33A and HT3 for 6 days. After transfection AdCMVp53 into CaSki- and SiHa-xenografted nude mice, the size of tumor was remarkably decreased in SiHa cells as compared to AdCMVLacZ transfection. The SPR sensor surface was successfully modified with the recombinant E7 oncoprotein and showed cell-specific interactions between E7 and its target proteins from cell lysates. The anti-tumor effects were accomplished via differential role of p53-specific apoptotic cell death, which is dependent upon the cervical cancer cell line. Also, a molecular level understanding of cell-dependent protein interaction effects of recombinant E7 was shown.     

The IFN-γ-IDO1-kynureine pathway-induced autophagy in cervical cancer cell promotes phagocytosis of macrophage

Background: Cervical cancer is a common malignant disease in female patients accompanied by activation of autophagy in tumor cells. However, the exact regulatory factors of autophagy and its effects on the immune response remain unknown.Methods: The induction of autophagy in HeLa and SiHa cells treated with IFN-γ, tryptophan depletion, kynurenine and epacadostat was detected by western blot analysis and by an autophagy detection kit. Following co-culture with pre-treated HeLa and SiHa cells, U937 cells were analyzed by flow cytometry to detect CD80, CD86, CD163 and CD206 expression and the induction of phagocytosis.Results: IFN-γ caused a significant increase in the autophagy levels of HeLa and SiHa cells by promoting indoleamine-2,3-dioxygenase-1 (IDO1) expression. The induction of phagocytosis in HeLa and SiHa cells and the expression levels of CD80 and CD86 in U937 cells were increased significantly following treatment with recombinant human IFN-γ. This effect was associated with the induction of tumor cell autophagy. IFN-γ treatment and IDO1 overexpression promoted tryptophan depletion and kynurenine accumulation in cervical cancer cells. The latter was more potent in inducing autophagy of cervical cancer cells and promoting phagocytosis of macrophages. In vivo, IDO1 overexpression restricted tumor growth in C57 mice and enhanced the induction of phagocytosis in macrophages.Conclusions: IFN-γ promoted induction of autophagy and macrophage phagocytosis in cervical cancer cells possibly via IDO1 expression and kynurenine metabolism.     

Knock-down of glutaminase 2 expression decreases glutathione,NADH, and sensitizes cervical cancer to ionizing radiation

Phosphate-activated mitochondrial glutaminase (GLS2) is suggested to be linked with elevated glutamine metabolism. It plays an important role in catalyzing the hydrolysis of glutamine to glutamate. The present study was to investigate the potent effect of GLS2 on radioresistance of cervical carcinoma. GLS2 was examined in 144 cases of human cervical cancer specimens (58 radioresistant specimens, 86 radiosensitive specimens) and 15 adjacent normal cervical specimens with immunohistochemistry. HeLa cells were treated with a cumulative dose of 50 Gy X-rays, over 6 months, yielding the resistant sub-line HeLaR. The expressions of GLS2 were measured by Western blot. Radioresistance was tested by colony survival assay. Apoptosis was determined by flow cytometry. The levels of glutathione (GSH), reactive oxygen species (ROS), NAD⁺/NADH ratio and NADP⁺/NADPH ratio were detected by quantization assay kit. Xenografts were used to confirm the effect of GLS2 on radioresistance in vivo. The expressions of GLS2 were significantly enhanced in tumor tissues of radioresistant patients compared with that in radiosensitive patients. In vitro, the radioresistant cell line HeLaR exhibited significantly increased GLS2 levels than its parental cell line HeLa. GLS2 silenced radioresistant cell HeLaR shows substantially enhanced radiosensitivity with lower colony survival and higher apoptosis in response to radiation. In vivo, xenografts with GLS2 silenced HeLaR were more sensitive to radiation. At the molecular level, knock-down of GLS2 increased the intracellular ROS levels of HeLaR exposed to irradiation by decreasing the productions of antioxidant GSH, NADH and NADPH. GLS2 may have an important role in radioresistance in cervical cancer patients.     

Ubiquitin B in Cervical Cancer: Critical for the Maintenance of Cancer Stem-Like Cell Characters

Cervical cancer cells exhibit an increased requirement for ubiquitin-dependent protein degradation associated with an elevated metabolic turnover rate. Ubiquitin, which is a small, highly conserved protein expressed in all eukaryotic cells, can be covalently linked to certain target proteins to mark them for degradation by the ubiquitin-proteasome system. Previous studies highlight the essential role of Ubiquitin B (UbB) and UbB-dependent proteasomal protein degradation in histone deacetylase inhibitor (HDACi) -induced tumor selectivity. We hypothesized that UbB plays a critical role in the function of cervical cancer stem cells. We measured endogenous UbB levels in mammospheres in vitro by real-time PCR and Western blotting. The function of UbB in cancer stem-like cells was assessed after knockdown of UbB expression in prolonged Trichostatin A-selected HeLa cells (HeLa/TSA) by measuring in vitro cell proliferation, cell apoptosis, invasion, and chemotherapy resistance as well as by measuring in vivo growth in an orthotopic model of cervical cancer. We also assessed the cancer stem cell frequency, tumorsphere formation, and in vivo growth of human cervical cancer xenografts after UbB silencing. We found that HeLa/TSA were resistant to chemotherapy, highly expressed the UbB gene and the stem cell markers Sox2, Oct4 and Nanog. These cells also displayed induced differentiation abilities, including enhanced migration/invasion/malignancy capabilities in vitro and in vivo. Furthermore, an elevated expression of UbB was shown in the tumor samples of chemotherapy patients. Silencing of UbB inhibited tumorsphere formation, lowered the expression of stem cell markers and decreased cervical xenograft growth. Our results demonstrate that UbB was significantly increased in prolonged Trichostatin A-selected HeLa cells and it played a key role in the maintenance of cervical cancer stem-like cells.     

Overexpression of MALAT1 contributes to cervical cancer progression by acting as a sponge of miR-429

Cervical cancer remains a malignant type of tumor and is the fourth leading cause of cancer-related death among females. MALAT1 has been identified as a tumor oncogene in various cancers. Our present study aimed to explore the biological role of MALAT1 in cervical cancer. We observed that MALAT1 was significantly upregulated in human cervical cancer cell lines compared with the ectocervical epithelial cells. MALAT1 was repressed by transfection with LV-shMALAT1, whereas increased by LV-MALAT1 in HeLa and Caski cells. Silencing of MALAT1 obviously reduced cervical cell viability, induced cell apoptosis, and repressed cell invasion capacity. Conversely, overexpression of MALAT1 exhibited an opposite phenomenon. Furthermore, miR-429 was predicted as a direct target of MALAT1, and it was dramatically decreased in cervical cancer cells. It has been shown that miR-429 plays a crucial role in cervical cancer progression. In our current study, the targeting correlation between MALAT1 and miR-429 was confirmed by luciferase reporter assays and RIP experiments. Finally, in vivo animal models were established, and we indicated that MALAT1 inhibited cervical cancer progression via targeting miR-429. These findings revealed that MALAT1 can sponge miR-429 and regulate cervical cancer pathogenesis in vivo and in vitro. In conclusion, we indicated that the MALAT1/miR-429 axis was involved in cervical cancer development.     

过表达长非编码RNA SLC25A25-AS1可抑制HeLa细胞的增殖、迁移和侵袭

长非编码RNA SLC25A25-AS1在结直肠癌的发展中具有肿瘤抑制作用,然而,其在宫颈癌中作用机制有待深入研究.本文研究了宫颈癌和宫颈上皮内瘤变(cervical intraepithelial neoplasia,CIN)病人血清中SLC25A25-AS1的异常表达,并探讨了SLC25A25-AS1在宫颈癌发展中...     

HeLa细胞中缝隙连接蛋白Cx26/Cx32的表达及其功能调控

目的 在HeLa宫颈癌细胞中研究不同浓度的多西环素对缝隙连接蛋白Cx26/Cx32表达及由其形成的缝隙连接通讯功能的影响.方法 采用Western印迹检测HeLa细胞中Cx26/Cx32的蛋白表达;荧光示踪实验用于检测HeLa细胞中由Cx26/Cx32形成的缝隙连接通讯功能.结果 Western印迹结果显示多西环素在0.01～1 μg/ml的范围内,随着剂量的增加,Cx26/Cx32蛋白表达水平增加;荧光示踪实验结果显示HeLa细胞之间的荧光传递随着多西环素增加也相应增强.结论 采用加入不同浓度多西环素的方法,可制备缝隙连接通讯功能强弱不同的细胞模型.     

Expression and Functional Analysis of Toll-like Receptor 4 in Human Cervical Carcinoma

Toll-like receptors are expressed in human immune cells and many tumors, but the role of toll-like receptor 4 (TLR4) in the development of tumors is controversial. We demonstrated the expression, distribution, and functional activity of TLR4 in tissues of normal cervix, cervical intraepithelial neoplasia (CIN), invasion cervical cancers (ICC), and different human papillomavirus (HPV)-infected cervical cancer cells. The results showed that TLR4 expression was in accordance with the histopathological grade: higher in ICC than in CIN, and low in normal cervical tissues and malignant cervical stroma. Expression was higher in SiHa (HPV16+) than in HeLa (HPV18+) cells, but was not observed in C33A (HPV?) cells. After treatment with its agonist, lipopolysaccharide (LPS), the expression levels of TLR4 was increased and apoptosis resistance was induced in SiHa cells, but not in HeLa or C33A cells. Meanwhile, LPS treatment did not alter the cell cycle distribution in SiHa cells. The mechanism of apoptosis resistance may be related to HPV16 infection and not correlated with the cell cycle distribution. Targeting TLR4 in combination with traditional drug treatment may serve as a novel strategy for more effectively killing cancer cells.     

Induction of apoptosis by casticin in cervical cancer cells: reactive oxygen species-dependent sustained activation of Jun N-terminal kinase

Casticin, a polymethoxyflavone from Fructus viticis used as an anti-inflammatory agent in Chinese traditional medicine, has been reported to have anti-cancer activity. The purpose of this study was to examine the apoptotic activity of casticin on human cervical cancer cells and its molecular mechanism. We revealed a novel mechanism by which casticin-induced apoptosis occurs and showed for the first time that the apoptosis induced by casticin is mediated through generation of reactive oxygen species (ROS) and sustained activation of c-Jun N-terminal kinase (JNK) in HeLa cells. Casticin markedly increased the levels of intracellular ROS and induced the expression of phosphorylated JNK and c-Jun protein. Pre-treatment with N-acetylcysteine and SP600125 effectively attenuated induction of apoptosis by casticin in HeLa cells. Moreover, casticin induced ROS production and apoptotic cell death in other cervical cancer cell lines, such as CasKi and SiHa. Importantly, casticin did not cause generation of ROS or induction of apoptosis in normal human peripheral blood mononuclear cells and embryonic kidney epithelium 293 cells. These results suggest that ROS generation and sustained JNK activation by casticin play a role in casticin-induced apoptosis and raise the possibility that treatment with casticin might be promising as a new therapy against human cervical cancer.