Migration Of SiHa Cells Research Articles

AUP1 transcriptionally activated by KDM5B reprograms lipid metabolism to promote the malignant progression of cervical cancer.

Cervical cancer is one of the reproductive malignancies threatening women's lives worldwide. In the present study, it was aimed to explore the role and mechanism of ancient ubiquitous protein 1 (AUP1) in cervical cancer. Through bioinformatics analysis, AUP1 expression in cervical cancer tissues and the correlation between AUP1 and the prognosis of patients were analyzed. AUP1 expression in several cervical cancer cell lines was detected. Following the co‑transfection of short hairpin RNA specific to AUP1 with or without lysine demethylase 5B (KDM5B) overexpression plasmids in SiHa cells, the proliferation and apoptosis of SiHa cells were detected. Additionally, wound healing and Transwell assays were used to detect SiHa cell migration and invasion. Cellular lipid droplets level was detected using the Oil red O staining. Meantime, the levels of triglyceride, cholesterol, oxygen consumption rates and expression of lipid metabolism‑related proteins were detected to assess the lipid metabolism in SiHa cells. Then, the luciferase reporter assay and ChIP assay were used to verify the binding between KDM5B and AUP1. Finally, the effects of AUP1 and KDM5B on the growth and lipid metabolism in SiHa tumor‑bearing mice were measured. AUP1 was significantly upregulated in cervical cancer tissues and cells. AUP1 interference inhibited the malignant biological behaviors and lipid metabolism reprogramming of SiHa cells, which was blocked by KDM5B overexpression. Moreover, KDM5B could transcriptionally activate AUP1 and upregulate AUP1 expression. Furthermore, AUP1 knockdown transcriptionally regulated by KDM5B limited the tumor growth and suppressed the lipid metabolism reprogramming in vivo. Collectively, AUP1 could be transcriptionally activated by KDM5B to reprogram lipid metabolism, thereby promoting the progression of cervical cancer. These findings reveal possible therapeutic strategies in targeting metabolic pathways.

Folate deficiency promotes cervical squamous carcinoma SiHa cells progression by targeting miR-375/FZD4/β-catenin signaling

Epidemiological studies suggest an association between folate deficiency (FD) and cervical squamous cell carcinoma (SCC) progression. However, the underlying mechanism is unclear. Our study showed that FD-driven downregulation of miR-375 promoted proliferation of SCC SiHa cells and progression of xenograft tumors developed from SiHa; however, the exact mechanism of this process remained unclear. The current study aimed to elucidate the underlying mechanisms by which FD promotes the progression of SiHa cells by downregulating miR-375 expression. The results showed that miR-375 acted as a suppressor of SCC and inhibited the proliferation, migration, and invasion of SiHa cells. The FZD4 gene was identified as a target gene of miR-375, which can reverse the anti-onco effect of miR-375 and promote the proliferation and migration of SiHa cells. Furthermore, the regulatory effects of miR-375 and FZD4 on SiHa cells may be achieved by activating the β-catenin signaling pathway. Moreover, FD may regulate the expression of miR-375 by regulating its DNA methylation level in the promoter region. In conclusion, our study reveals that FD regulates the miR-375/FZD4 axis by increasing the methylation of the miR-375 promoter, thereby activating β-catenin signaling to promote SiHa cells progression. This study may provide new insights into the role of folic acid in the prevention and treatment of SCC.

2′,4′-Dihydroxy-6′‑methoxy-3′,5′-dimethylchalcone and its amino acid-conjugated derivatives induce G0/G1 cell cycle arrest and apoptosis via BAX/BCL2 ratio upregulation and in silico insight in SiHa cell lines

We modified the chemical structure of 2′,4′-dihydroxy-6′‑methoxy-3′,5′-dimethylchalcone (DMC, 1), a phytochemical found in the seed of Syzygium nervosum A.Cunn. ex DC., by conjugation with the amino acid L-alanine (compound 3a) or L-valine (compound 3b) to enhance anticancer activity and water solubility. Compounds 3a and 3b had antiproliferative activity in human cervical cancer cell lines (C-33A, SiHa and HeLa), with half-maximal inhibitory concentrations (IC50) of 7.56 ± 0.27 and 8.24 ± 0.14 µM, respectively in SiHa cells; these values were approximately two-fold greater than DMC. We investigated the biological activities of compounds 3a and 3b based on a wound healing assay, a cell cycle assay and messenger RNA (mRNA) expression analysis to determine the possible mechanism of anticancer activity. Compounds 3a and 3b inhibited SiHa cell migration in the wound healing assay. After treatment with compounds 3a and 3b, there was an increase in SiHa cells in the G1 phase, indicative of cell cycle arrest. Moreover, compound 3a showed potential anticancer activity by upregulating TP53 and CDKN1A that resulted in upregulation of BAX and downregulation of CDK2 and BCL2, leading to apoptosis and cell cycle arrest. The BAX/BCL2 expression ratio was increased after treatment with compound 3avia the intrinsic apoptotic pathway. In silico molecular dynamics simulation and binding free energy calculation shed light on how these DMC derivatives interact with the HPV16 E6 protein, a viral oncoprotein associated with cervical cancer. Our findings suggest that compound 3a is a potential candidate for anti-cervical cancer drug development.

Britannin Induces Autophagy-Dependent Apoptosis via the Reactive Oxygen Species/Adenosine 5′-monophosphate-Activated Protein Kinase-unc-51-Like Autophagy Activating Kinase 1 Axis in Cervical Cancer as Detected Using Nanomagnetic Beads

Cervical cancer is a unique and common malignancy that occurs in women. Britannin has been proven to inhibit the progression of human liver and breast cancers. However, its efficacy in cervical cancer is still unclear. Human cervical squamous cell carcinoma SiHa cells were used to analyze the effect of britannin administration on cervical tumors by xenotransplantation of human tumor cells. The Cell Counting Kit-8 was used to determine the half maximal inhibitory concentration (IC50) of britannin on SiHa, and the cell invasion ability was measured using Transwell invasion assay. Cell migration was measured using the scratch-healing assay, while the clonogenic assay was performed to determine cell proliferation. Dichloro-dihydro-fluorescein diacetate was used to label reactive oxygen species (ROS) in cells or tissues. Intracellular autophages were stained with monodansylcadaverine. Western blotting assisted by nanomagnetic beads was performed to study the expression of p62, light chain 3 beta (LC3B), Beclin 1, and autophagy-related protein 5 (ATG5) in cells or tissues, as well as the phosphorylation of AMP-activated protein kinase (AMPK) and unc-51-like autophagy activating kinase 1 (ULK1). Hematoxylin and eosin staining was performed to analyze the pathological changes in the tumor cells. The expression of Ki-67, B-cell lymphoma 2 (Bcl-2), and Bax in the tumor cells was analyzed by immunohistochemistry. The IC50 obtained for britannin against SiHa was 10.01 μM. Britannin inhibited the proliferation, invasion, and migration of SiHa cells, and promoted the generation and autophagy of ROS in tumor cells and tissues. Furthermore, p-AMPK/AMPK and p-ULK1/ULK1, LC3B, Beclin 1, and ATG5 were upregulated, whereas p62 was downregulated in cells and tissues. Tumor development was inhibited; tissue inflammation was reduced; Ki-67 and Bcl-2 expression was downregulated; and Bax expression was upregulated. The expression of cleaved caspase-3, cleaved poly-ADP-ribose polymerases, and voltage-dependent anion-selective channel 1 was upregulated. These effects can be partially reversed by the AMPK inhibitor dorsomorphin dihydrochloride (BML-275). In vivo and in vitro studies showed that britannin upregulated the of ROS content of human cervical squamous cell carcinoma cells, leading to significant autophagy, thereby inhibiting the occurrence and development of cervical squamous cell carcinoma. The mechanism may be related to the ROS/AMPK/ULK1 signaling pathway.

Effects of Forkhead box O1 on lipopolysaccharide-induced mitochondrial dysfunction in human cervical squamous carcinoma SiHa cells.

Persistent infection and chronic inflammation play important roles in the development of cervical squamous cell carcinoma. Forkhead box O1 (FOXO1) is a notable regulator of mitochondrial metabolism, which is involved in the occurrence and development of tumors. The present study explored the effects of FOXO1 in human cervical squamous carcinoma SiHa cells. The expression of FOXO1 was examined using reverse transcription-quantitative PCR, western blotting and immunohistochemical staining. SiHa cell migration and proliferation were detected using Transwell and 3H-TdR assays. Mitochondrial functions were assessed based on reactive oxygen species (ROS) generation and changes in the mitochondrial membrane potential (ΔΨm). The present study revealed that lipopolysaccharide (LPS) stimulation significantly inhibited the expression of FOXO1 in cervical squamous carcinoma SiHa cells; while silencing FOXO1 resulted in the accumulation of mitochondrial ROS, a decrease in the ΔΨm and abnormal morphology of mitochondria. Accordingly, enhancing FOXO1 expression or treatment with metformin, which protects mitochondrial function, reversed LPS-induced mitochondrial dysfunction, cell pyroptosis, migration and proliferation of cervical squamous carcinoma SiHa cells. Overall, the current study indicated that treatment with FOXO1 could potentially be used as therapeutic strategy to prevent LPS-induced cervical squamous cell carcinoma-related dysfunction in a mitochondria-dependent manner.

MiR-613 blocked the progression of cervical cancer by targeting LETM1.

The purpose of this study was to investigate the potential effects of miR-613 on the development of cervical squamous cell cancer (CSCC) and the relevant mechanism. The expression level of miR-613 was detected in CSCC tissues and cells by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Thereafter, online prediction software and Luciferase reporter assays were used to evaluate the possible target of miR-613, and the effects of the miR-613 on SiHa cells were determined in vitro. Then, the changes of protein expression in cells were measured by Western blot. Finally, Cell Counting Kit-8 (CCK-8), wound healing and transwell assays were performed to analyze cell proliferation, invasion, and migration. It was found that compared with that in normal tissues and cells, miR-613 expression was found suppressed in CSCC tissues and cells. Based on bioinformatics and Luciferase results, miR-613 could target and bind to LETM1. Besides, it was found that miR-613 could regulate the expression of LETM1 in SiHa cells, and miR-613 had a valuable suppressive effect on the proliferation, invasion, and migration of SiHa cells through the subsequent experiments. However, when co-transfection of miR-613 mimics and LV-LETM1 into SiHa cells, the anti-cancer effects of miR-613 on SiHa cells vanished. Taken all, this research discovered the function of miR-613 on CSCC by targeting LETM1, revealing that miR-613/LETM1 signal pathway might be a potential therapeutic target for the diagnosis and treatment of CSCC.

The Effects of Hsa-miR-5195-3p on the Proliferation, Migration and Invasion of Human Cervical Cancer SiHa Cells

The Effects of Hsa-miR-5195-3p on the Proliferation, Migration and Invasion of Human Cervical Cancer SiHa Cells

Knockdown of METTL14 inhibits the growth and invasion of cervical cancer

Background: Increasing evidence has revealed that N6-methyladenosine (m6A) modification is implicated in multiple biological functions in mammals. Methyltransferase-like 14 (METTL14), an important component of m6A modification, has been reported to play important roles in the pathogenesis of acute myeloid leukaemia and hepatocellular carcinoma metastasis. However, its role in cervical cancer remains unclear. Methods: Expression of METTL14 was knocked down by shRNA-METTL14 interference in HPV-positive and HPV-negative cervical cancer cell lines SiHa and C33a. CCK8, colony formation, wound-healing, and Transwell assays were performed to evaluate the effects of METTL14 knockdown on the proliferation, migration and invasion abilities of SiHa and C33a cells. Flow cytometry analysis was utilized to detect cell cycle distribution, and the expression of related proteins was examined by western blot analysis. Results: Bioinformatics analysis demonstrated that up-regulation of METTL14 acted as an adverse prognostic factor for overall survival in cervical cancer patients. We demonstrated that down-regulation of METTL14 inhibited the proliferation, migration and invasion abilities of SiHa and C33a cells. Moreover, silencing METTL14 induced cell cycle arrest in cervical cancer cells. METTL14 knockdown suppressed the PI3K/Akt/mTOR signaling pathway by decreasing the phosphorylation of Akt and mTOR, and the expression of downstream apoptosis-related proteins was also impacted. Conclusions: In conclusion, these data suggest an important oncogenic role of METTL14 in the growth and invasion of both HPV-positive and HPV-negative cervical cancer cells.

Expression of BDNF, TrkB, VEGF and CD105 is associated with pelvic lymph node metastasis and prognosis in IB2-stage squamous cell carcinoma.

Brain-derived neurotrophic factor (BDNF), tropomyosin receptor kinase B (TrkB), vascular endothelial growth factor (VEGF) and CD105 are highly expressed in several types of cancer. The present study aimed to determine whether BDNF, TrkB, VEGF and CD105 are associated with the prognosis and metastasis of patients with cervical squamous cell carcinoma (SCC) at the IB2 stage. A total of 79 patients with IB2-stage SCC were enrolled in the present study. The expression levels of BDNF, TrkB, VEGF and CD105 in IB2-stage cervical cancer tissue were detected by immunohistochemistry and their association with clinicopathological indexes or prognostic factors was statistically analyzed. Reverse transcription quantitative PCR was used to detect whether the expression of VEGF was affected in SiHa cells co-cultured with BDNF. In addition, BDNF-induced SiHa cell migration and invasion were examined. BDNF expression in the cervical cancer samples was significantly associated with positive lymphovascular space invasion (P<0.001) and pelvic lymph node metastasis (P<0.05). In addition, microvessel density was verified as an independent prognostic factor for overall survival (P<0.05). In vitro analysis indicated that BDNF significantly induced cellular migration and invasion of SiHa cells in a dose-dependent manner (P<0.001). BDNF induced the expression of VEGF in SiHa cells, which was inhibited by BDNF antibodies or an inhibitor of TrkB receptor (P<0.05). BDNF may be considered a useful indicator of pelvic metastasis, which is involved in the aggressive spread of IB2-stage SCC. BDNF-induced upregulation of VEGF was revealed to act as a pro-angiogenic factor in SCC (Trial registration no. http://apps.who.int/trialsearch/; ChiCTR1800017778).

Curcumin decreases epithelial‑mesenchymal transition by a Pirin‑dependent mechanism in cervical cancer cells.

Curcumin is a natural antioxidant polyphenol, which decreases epithelial‑mesenchymal transition(EMT) and cell migration in cervical cancer cells. However, the mechanism by which such a decrease occurs is unclear. It is well established that cervical cancer can be caused by high‑risk human papillomavirus(HPV), which overexpresses E6andE7oncoproteins. Recent findings have suggested that viral oncoproteins regulate the expression of Pirin, which is an oxidative stress sensor involved in EMT and cell migration. Molecular markers associated with EMT, pirin and HPV were evaluated using reverse transcription‑reverse quantitative PCR and western blotting. In addition, the migratory ability of cells was evaluated using a Transwell assay. In order to evaluate the role of Pirin in curcumin‑mediated inhibition of EMT, SiHa cervical carcinoma cells, which contain two integrated copies of HPV16, were exposed to curcumin. Cell migration, and the expression levels of EMT biomarkers and the pirin protein, which is a product of the PIR gene, were subsequently evaluated. The results demonstrated a significant decrease in EMT following exposure to 20µM curcumin for 72h. This finding was supported by a decrease in the protein expression levels of N‑cadherin, Vimentin and Slug. Furthermore, it was observed that PIR expression and Pirin protein levels were significantly decreased when SiHacells were exposed to curcumin. Subsequently, to analyze the effects of Pirin on EMT, SiHacells were transfected with a small interfering RNA (siRNA) to knockdown PIR. A significant increase in E‑cadherin mRNA expression and a decrease in N‑cadherin protein expression were observed. In addition, a similar decrease was observed when SiHacells were exposed to both PIR siRNA and curcumin. Finally, a significant decrease in SiHacell migration was observed in the presence of 20µM curcumin compared with in the control group. These findings suggested that curcumin may decrease EMT, at least in part by a Pirin‑dependent mechanism. Therefore, Pirin protein may be an important pharmacological target for cervical cancer treatment.

MiR-93-5p promotes cervical cancer progression by targeting THBS2/MMPS signal pathway.

The aim of this study was to investigate the potential effects of microRNA-93-5p (miR-93-5p) on the development of cervical cancer (CC), and to explore the underlying mechanism. The expression level of miR-93-5p in CC tissues and cells was detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Online prediction software and Luciferase reporter assay were used to evaluate the possible target of miR-93-5p. Furthermore, the effects of miR-93-5p on siHa cells were determined by Western blot, Cell Counting Kit-8 (CCK-8), scratch-wound and transwell assays, respectively. In our study, miR-93-5p was found highly expressed in CC tissues and cells. Thrombospondin-2 (THBS2) was predicted and experimentally verified as a direct target of miR-93-5p. Subsequent experiments showed that decreased expression of THBS2 resulting from miR-93-5p up-regulation could significantly promote the proliferation, invasion and migration of siHa cells. At the same time, miR-93-5p remarkably increased the expression of matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9), whereas decreased the expression of extracellular matrix (ECM). Our research discovered the promotion function of miR-93-5p on CC by targeting THBS2/Matrix metalloproteinases (MMPS) signaling pathway. We revealed that miR-93-5p might be a potential therapeutic target for the treatment of CC.

Chloride channel-3 is required for efficient tumour cell migration and invasion in human cervical squamous cell carcinoma

ObjectiveChloride channel-3 (ClC-3) plays significant roles in various physiological and physiopathological activities, including cell migration and invasion ability. The purpose of this study was to evaluate whether ClC-3 influences the migration and invasion of cervical squamous cell carcinoma cells and its possible mechanisms. MethodsParaffin-embedded cervical tissues, including normal cervical tissues, cervical squamous cell carcinoma (SCC) and homologous paracancerous tissues, were collected. The cervical squamous cell carcinoma and matched paracarcinoma fresh tissues specimens were collected from 49 patients with SCC, and the normal cervical tissues were collected from 45 non-cervical squamous cell carcinoma patients. The human cervical squamous carcinoma cell line SiHa was cultured. ClC-3 expression was assessed by real-time RT-PCR, immunohistochemistry and Western blot, and the expression of phospho-PI3K/Akt/mTOR and matrix metalloproteinase-9 (MMP-9) was detected by Western blot. Small interfering RNA (siRNA) technology was used to knockdown ClC-3 expression. SiHa cell migration and invasion ability were measured using Transwell assays with or without Matrigel-coated membranes. ResultsClC-3 mRNA and protein expression in SCC tissues from cervical squamous cell carcinoma patients was significantly upregulated, and no significant difference was noted between the matched paracarcinoma fresh tissue from the same patients and non-cervical cancer patients. SiHa cell migration and invasion and phospho-PI3K/Akt/mTOR and MMP-9 expression were attenuated by knocking down ClC-3 expression using ClC-3 siRNA. ConclusionsClC-3 participates in the processes of SCC cell migration and invasion and regulates MMP-9 expression via the PI3K/Akt/mTOR signaling pathway.

The Roles of HK2 on Tumorigenesis of Cervical Cancer.

Cancer cells undergo metabolic changes that support their malignant growth. These changes are often associated with increased expression of the rate-limiting glycolytic enzyme hexokinase 2. Hexokinase 2 is an enzyme that catalyzes the conversion of glucose to glucose-6-phosphate. In this study, we utilized Gene Expression Profiling Interactive Analysis (GEPIA) database analysis and clinical sample analysis to find that hexokinase 2 was highly expressed in cervical cancer. Furthermore, we found that high hexokinase 2 expression in cervical cancer demonstrated a positive correlation with tumor size (P = .009696), pathological grade (P = .028551), and prognosis (P = .00069) but not with age (P = .956201) or lymph node metastasis (P = .131379). At the cellular level, we knocked down the expression of hexokinase 2 in the human cervical cancer cell line SiHa. The results demonstrated that knockdown of hexokinase 2 inhibited the proliferation and migration of SiHa cells and promoted cell apoptosis. During this process, knockdown of hexokinase 2 inhibited phosphorylation of AKT and mammalian target of rapamycin and promoted p53 expression. At the same time, overexpression of human papillomavirus 18 oncogenes E6 and E7 significantly promoted the expression of hexokinase 2. Most importantly, we discovered a novel upstream regulatory microRNA for hexokinase 2: miR-9-5p. Luciferase reporter assays and Western blot assays demonstrated that hexokinase 2 expression was inhibited by miR-9-5p by directly binding its 3′-untranslated region in SiHa cells. Next, we determined that miR-9-5p could suppress the proliferation and migration of SiHa cells and induce apoptosis. In conclusion, we found that hexokinase 2 serves a carcinogenic role in cervical cancer through the miR-9-5p/hexokinase 2/AKT pathway, which serves as the basis for potential therapeutic targets and prognostic indicators.

The mechanisms involved in miR-9 regulated apoptosis in cervical cancer by targeting FOXO3

As a seriously global health problem, cervical cancer is a great risk to women which threatens their lives. Approximately 30% patients who received definitive treatment may fail to recover from this disease. Accordingly, there is an imperatively need to explore alternative therapeutic approaches for this disease. Several studies have revealed that miR-9 was a critical regulator during cervical cancer growth. Here, we reported that the miR-9 was overexpressed in cervical tumor tissue and exerted a promoting effect on human cervical cancer cell (SiHa) growth. Both in vitro and in vivo experiments confirmed that miR-9 could stimulate the proliferation and migration of SiHa cells. In contrast, inhibition of miR-9 induced apoptosis in SiHa cells. In addition, dual luciferase reporter system assay verified that there was a strong target relationship between miR-9 and FOXO3. Result of western blot assay showed that the inhibition of miR-9 increased the expression of FOXO3. Moreover, miR-9 regulated FOXO3 downstream proteins Bax, Bcl-2 and p-Akt expressions, which suggesting that miR-9 was involved in the SiHa cells apoptosis. In conclusion, our results suggest that the inhibition of miR-9 could induce apoptosis in cervical cancer by targeting FOXO3 and presented a potential molecular target for the treatment of cervical cancer patients.

Effect of Rab11 gene expression on the invasion and migration of cervical cancer cell line SiHa in hypoxia

Objective: To explore the expression of Ras-related protein 11 (Rab11) in hypoxia, the effect of Rab11 on the invasion and migration of cervical cancer cell line SiHa and its possible mechanism. Methods: SiHa cells were divided into 4 groups, the normoxic blank group (normal culture in normoxia), the hypoxic blank group (normal culture in hypoxia), the negative control group [transfection of negative control small interfering RNA (siRNA) in hypoxia], the Rab11-siRNA group (transfection of Rab11 siRNA in hypoxia). Western blot was used to examine the expression of Rab11, integrin α5, integrin β3, phosphorylated focal adhesion kinase (p-FAK), phosphorylated phosphatidylinositol 3 kinase (p-PI3K) protein, together with the expression of Ras correlative C3 creotoxin substrate 1 (Rac1), which was critical in regulating cell invasion. The mRNA expression of Rab11 in the 4 groups was detected by realtime-qPCR. The cell invasion was detected by matrigel assay, while the cell migration was detected by transwell assay. Immunofluorescence was used to identify intracellular location of Rac1 in SiHa cell. Results: (1) The expression of Rab11, intergrin α5, intergrin β3, p-FAK, p-PI3K and Rac1 in the normoxic blank group were 0.56±0.04, 0.33±0.03, 0.32±0.03, 0.36±0.03, 0.35±0.03 and 0.47±0.03, respectively. In the hypoxic blank group, they were 0.73±0.03, 0.74±0.03, 0.61±0.03, 0.62±0.03, 0.60±0.03 and 0.73±0.03, respectively. In the negative control group, their expressions were 0.72±0.03, 0.73±0.03, 0.59±0.03, 0.61±0.03, 0.59±0.03 and 0.72±0.03, respectively. While in the Rab11-siRNA group, they were 0.44±0.03, 0.30±0.03, 0.29±0.03, 0.30±0.03, 0.30±0.03 and 0.34±0.04, respectively. The expressions of Rab11, α5, β3, p-FAK, p-PI3K and Rac1 were significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and were significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (2) The expressions of Rab11-mRNA were 1.000±0.000, 1.454±0.114, 1.442±0.101, 0.570± 0.046 in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group, respectively. It was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (3) By Matrigel, the invasion cell number in the normoxic blank group, the hypoxic blank group,the negative control group and the Rab11-siRNA group were 65±12, 106±16, 104± 17 and 50±11, respectively. The invasion capacity was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (4) By transwell assay, the migration cells in the normoxic blank group, the hypoxic blank group, the negative control group and the Rab11-siRNA group were 127±12, 169±15, 161±13 and 77±13, respectively. The capacity of invasion was significantly higher in the hypoxic blank group than in the normoxic blank group (P<0.05), and was significantly lower in the Rab11-siRNA group than in the hypoxic blank group and the negative control group (P<0.05). (5) The immunofluorescence showed that the red fluorescence intensity around nucleus was significantly increased in the normoxic blank group, the hypoxic blank group and the negative control group than in the Rab11-siRNA group. Conclusions: Hypoxia could promote the invasion and migration of SiHa cells. In hypoxia, the down regulation of Rab11 expression could inhibit the invasion and migration of SiHa cells. This might be due to the decreased expression of the intergrin α5, intergrin β3, p-FAK, p-PI3K and Rac1 protein.

MicroRNA-106b is involved in transforming growth factor β1-induced cell migration by targeting disabled homolog 2 in cervical carcinoma.

BackgroundMicroRNA-106b (miR-106b) was recently identified as an oncogene participating in cancer progression. Transforming growth factor β1(TGF-β1) is an indispensable cytokine regulating the local microenvironment, thereby promoting cervical cancer progression. However, the roles of miR-106b in cervical carcinoma progression and TGF-β1-involvement in the tumorigenesis of cervical cancer remain unknown.MethodsThe expression of miR-106b in human cervical specimens was detected by real-time PCR analysis and in situ hybridization assay. The effect of miR-106b on cell migration was analyzed by scratch and transwell assays. TGF-β1 was used to induce cell migration. The expression of the miR-106b target gene DAB2 in human cervical tissues and cell lines were measured by qRT-PCR, western blot and immunohistochemistry. Dual-luciferase reporter assay was used to identify DAB2 as a miR-106b-directed target gene.ResultsmiR-106b was frequently up-regulated in human cervical carcinoma specimens and cervical cancer cell lines. Over-expression of miR-106b significantly promoted HeLa and SiHa cells migration. Likewise, inhibition of miR-106b decreased HeLa and SiHa cells migration. The multifunctional cytokine TGF-β facilitates metastasis in cervical carcinoma. miR-106b inhibitor treatment decreased the TGF-β1-stimulated migration of HeLa and SiHa cells. DAB2, a predicted target gene of miR-106b, was inhibited by TGF-β1 partly through miR-106b and was involved in TGF-β1-induced cervical cancer cell migration. The expression of DAB2 was low in cervical cancer tissues, and negatively correlated with miR-106b expression. Finally, DAB2 was identified as a miR-106b-directed target gene by dual-luciferase reporter assay.ConclusionOur data suggest that the TGF-β1/miR-106b/DAB2 axis may be involved in the pathogenesis of cervical carcinoma.

Aquaporin 8 involvement in human cervical cancer SiHa migration via the EGFR-Erk1/2 pathway.

Overexpression of aquaporins (AQPs) has been reported in several human cancers. Epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinases 1/2 (Erk1/2) are associated with tumorigenesis and cancer progression and may upregulate AQP expression. In this study, we demonstrated that EGF (epidermal growth factor) induces SiHa cells migration and AQP8 expression. Wound healing results showed that cell migration was increased by 2.79-1.50-fold at 24 h and 48 h after EGF treatment. AQP8 expression was significantly increased (3.33-fold) at 48 h after EGF treatment in SiHa cells. An EGFR kinase inhibitor, PD153035, blocked EGF-induced AQP8 expression and cell migration and AQP8 expression was decreased from 1.59-fold (EGF-treated) to 0.43-fold (PD153035-treated) in SiHa. Furthermore, the MEK (MAPK (mitogen-activated protein kinase)/Erk (extracellular signal regulated kinase)/Erk inhibitor U0126 also inhibited EGF-induced AQP8 expression and cell migration. AQP8 expression was decreased from 1.21-fold (EGF-treated) to 0.43-fold (U0126-treated). Immunofluorescence microscopy further confirmed the results. Collectively, our findings show that EGF induces AQP8 expression and cell migration in human cervical cancer SiHa cells via the EGFR/Erk1/2 signal transduction pathway.

The overexpression of MCPH1 inhibits cell growth through regulating cell cycle-related proteins and activating cytochrome c-caspase 3 signaling in cervical cancer

MCPH1, initially identified as an hTERT repressor, has recently been implicated in mediating DNA damage response and maintaining chromosome integrity. This study is to investigate its potential role in the onset of cervical cancer. In the study, decreased expression of MCPH1 was observed in 19 of 31 cases (61.3%) at mRNA level and 44 of 63 cases (69.8%) at protein level of cervical tumor tissues compared with the paired nontumor tissues. Reduced MCPH1 protein expression was significantly associated with high-tumor grade (1 vs. 3 P = 0.013; 2 vs. 3 P = 0.047). In addition to inhibit SiHa cell migration and invasion, the overexpression of MCPH1 inhibited cervical cancer cells growth through inducing S phase arrest and mitochondrial apoptosis. Further analysis demonstrated cyclinA2/CDK2, CDC25C-cyclinB/CDC2, and p53/p21 pathways were involved in the MCPH1 overexpression-induced S phase arrest. Moreover, the overexpression of MCPH1 activated mitochondrial apoptosis through regulating several apoptosis-related proteins such as p53, Bcl-2, Bax, cytochrome c, caspase-3, and PARP-1. Our findings indicate that downregulated MCPH1 correlates with tumor progression in cervical cancer, and MCPH1 has an important role in regulating cell growth through regulating the cell cycle and apoptosis. Thus, it may be a crucial tumor suppressor gene and a novel candidate therapeutic target for cervical cancer.

Increased Migration and Local Invasion Potential of SiHa Cervical Cancer Cells Expressing Aquaporin 8

Overexpression of several aquaporins (AQPS) has been reported in different types of human cancer but roles in human carcinogenesis have yet to be clearly defined. Here, we up-regulated expression of the AQP8 gene in SiHa human cervical cancer cells with a lentivirus transfection system and investigated its effects as a potential therapeutic target for cervical cancer. Results showed AQP8 overexpression did not affect their substrate adherence and proliferation, but accelerated migration as assessed by transwell migration and wound healing assays. Moreover, AQP8 overexpression significantly enhanced local invasion of SiHa cells in nude mice. These findings altogether indicate that AQP8 overexpression increases migration of SiHa cells and probably participates in the process of tumor local invasion.

Differential effects of miR-34c-3p and miR-34c-5p on SiHa cells proliferation apoptosis, migration and invasion

MicroRNAs (miRNA) regulate expression of several genes associated with human cancer. Here, we analyzed the function of miR-34c, an effector of p53, in cervical carcinoma cells. Expression of either miR-34c-3p or miR-34c-5p mimics caused inhibition of cell proliferation in the HPV-containing SiHa cells but not in other cervical cells irrespective of tumorigenicity and HPV content. These results suggest that SiHa cells may lack of regulatory mechanisms for miR-34c. Monolayer proliferation results showed that miR-34c-3p produced a more pronounced inhibitory effect although both miRNAs caused inhibition of anchorage independent growth at similar extent. However, ectopic expression of pre-miR-34c-3p, but not pre-miR-34c-5p, caused S-phase arrest in SiHa cells triggering a strong dose-dependent apoptosis. A significant inhibition was observed only for miR-34c-3p on SiHa cells migration and invasion, therefore implying alternative regulatory pathways and targets. These results suggest differential tumor suppressor roles for miR-34c-3p and miR-34c-5p and provide new insights in the understanding of miRNA biology.