Overexpression of miR-221 inhibits proliferation and promotes apoptosis of human astrocytoma cells

microRNAs (miRNAs) play tumor-promoting roles in a variety of tumors. This study investigated the expression of miRNA-211 (miR-221) in human astrocytoma, and its effect on proliferation and apoptosis of human astrocytoma cells in vitro. miR-221 expression was detected in 10 astrocytoma tissues and 4 adjacent tissues by real-time quantitative PCR (qRT-PCR). miR-221 expression in situ was significantly higher in astrocytoma tissues than in adjacent tissues (P<0.05). To determine whether the upregulation of miR-221 could be associated with tumor development or progression, a synthetic miR-221 mimic was transiently transfected into U251 astrocytoma cells in vitro. qRT-PCR confirmed that the mimic significantly increased the expression of miR-221 in these cells. An MTT colorimetric assay indicated that proliferation was significantly higher in U251 cells transfected with miR-221 mimic than in scramble-transfected control cells (P<0.05). Further analysis of miR-221 transfected cells by flow cytometry revealed an altered cell cycle progression, with more cells in S and G1 phase, as well as an inhibition of apoptosis (P<0.05). These findings indicate that the upregulation of miR-221 in astrocytoma tissues may be associated with development or progression of these tumors. Thus, miR-221 should be explored as a potential molecular marker for the diagnosis and treatment of astrocytoma.     

MiR-29b-3p affects growth and biological functions of human extravillous trophoblast cells by regulating bradykinin B2 receptor

IntroductionThis study investigated miR-29b-3p’s effects and mechanisms in preeclampsia development.Material and methodsIn this study, we analysed the pathology and expression of miR-29b-3p and B2R mRNA from normal and preeclampsia placenta tissues using hematoxylin and eosin staining and RT-qPCR assay. For cell experiments, we used transwell assay CCK-8, flow cytometry and wound healing assay to determine the effects and correlation of miR-29b-3p and B2R in HTR-8/SVneo cell proliferation, apoptosis, cell cycle, cell invasion and migration in a preeclampsia cell model. Moreover, the mechanisms were determined using Western blot or immunofluorescence in different groups.ResultsClinical analysis revealed that miR-29b-3p gene expression dramatically increased with increasing degree of preeclampsia (p < 0.001 or p < 0.05, respectively). The HTR-8/SVneo cell biological activities of the model group were significantly depressed (p < 0.001). However, with miR-29b-3p inhibitor or B2R transfection, the HTR-8/SVneo cell biological activities significantly recovered (p < 0.001). Western blot assay showed that B2R, VEGF-A, CCND-1, MMP-2 and MMP-9 levels were suppressed in the model group, compared with those in the NC groups (p < 0.001, respectively). With miR-29b-3p inhibitor or B2R transfection, the protein expression levels of B2R, VEGF-A, CCND-1, MMP-2 and MMP-9 dramatically increased (p < 0.001, respectively).ConclusionsThe down-regulation of miR-29b-3p could improve HTR-8/SVneo cell biological activities in a preeclampsia cell model by targeting B2R.     

MicroRNA 144 inhibits cell migration and invasion and regulates inflammatory cytokine secretion through targeting toll like receptor 2 in non-small cell lung cancer

IntroductionMicroRNAs (miRNAs) are endogenous small noncoding RNA molecules involved in modulation of cancer progression. Here, we investigated the possible role of miR-144 in non-small cell lung cancer (NSCLC) development.Material and methodsThe expression of miR-144 and TLR2 in NSCLC tissue and cell lines was determined by quantitative real-time PCR (qPCR). The TargetScan database was used to predict potential target genes of miR-144. Luciferase assay was used to verify the interaction between TLR2 and miR-144. TLR2 protein expression was measured by western blot. The secretion of interleukin (IL)-1β, IL-6 and IL-8 in A549 cells was detected by an ELISA kit. Cell migration and invasion were evaluated by wound healing assay and transwell assay, respectively.ResultsOur results showed that miR-144 was downregulated in NSCLC tissue and cell lines when compared with the normal tissues and cell line (p < 0.05). The protein level of TLR2 in NSCLC tissue and cell lines was significantly higher than that in normal lung tissues. Dual luciferase reporter gene assay showed that miR-144 could bind to the 3ʹUTR of TLR2 specifically. Up-regulation of miR-144 significantly decreased the expression of TLR2. Up-regulation of miR-144 or down-regulation of TLR2 could decrease cell migration, invasion and secretion of IL-1β, IL-6 and IL-8 in A549 cells. Moreover, overexpression of TLR2 rescued the inhibitory effects of miR-144 on migration, invasion and inflammatory factor secretion of A549 cells.ConclusionsmiR-144 could inhibit the migration, invasion and secretion of IL-1β, IL-6 and IL-8 through downregulation of TLR2 expression in A549 cells.     

MicroRNA-200a inhibits epithelial-mesenchymal transition in human hepatocellular carcinoma cell line

Objective: Our study investigated the role of microRNA (miR)-200a and its molecular targets in hepatocellular carcinoma (HCC) cells. Methods: An inhibitor of miR-200a was transiently transfected into the hepatocellular carcinoma cell line, MHCC-97L. The effect of this transfection on mRNA levels of epithelial-mesenchymal transition (EMT)-related genes was measured by fluorescence-based quantitative real-time polymerase chain reaction (qRT-PCR). Further, protein levels of EMT-related genes, cell proliferation and apoptosis-related markers were assessed by Western blot analysis in these transfected cells. MTT and wound-healing assay were used to evaluate the proliferation and migration of MHCC-97L cells in presence and in absence of miR-200a inhibitor. Results: Compared with miR-NC control group, qRT-PCR results in anti-miR-200a group revealed a significant reduction in the mRNA levels of E-cadherin, with a concomitant increasing in vimentin mRNA level (all P < 0.05). Western blot results showed higher E-cadherin and Caspase-3 protein expressions in anti-miR-200a group compared to miR-NC group (P < 0.05). In addition, vimentin and Ki-67 protein expression was found sharply decreased in anti-miR-200a group compared to miR-NC group (P < 0.05). Consistent with this, wound-healing and MTT assay showed that migration and proliferation capacity of MHCC-97L cells in anti-miR-200a group is significantly increased compared with miR-NC group (both P < 0.05). Conclusion: Our study reveals an important role of miR-200a in inhibiting EMT, proliferation and migration in HCC cells, suggesting the possibility of miR-200a-based therapeutics in HCC.     

Expression of miR-32 in human non-small cell lung cancer and its correlation with tumor progression and patient survival

Introduction: miR-32 has recently been found to be implicated in many critical processes in various types of human cancer. However, its clinical significance in human non-small cell lung cancer (NSCLC) has not yet been elucidated. In the present study, we investigated the expression of miR-32 in NSCLC and analyzed its association with clinical features and prognosis of NSCLC patients. Methods: Quantitative real-time PCR (qRT-PCR) was used to measure expression level of miR-32 in lung cancer cell lines, normal bronchial epithelial cells, 90 pairs of tumor samples and adjacent non-tumor tissues. To determine its prognostic value, overall survival was evaluated using the Kaplan-Meier method. Univariate and multivariate analysis were performed using the Cox proportional hazard analysis. Results: The expression of miR-32 was significantly decreased in lung cancer cell lines and NSCLC tissues compared with normal bronchial epithelial cells and adjacent non-tumor tissues (P < 0.05). This reduction of miR-32 was associated with tumor stage and lymph node metastasis (P < 0.05). Moreover, Kaplan-Meier analysis demonstrated that patients with low miR-32 expression had shorter overall survival time than those with high miR-32 expression (P < 0.05). Univariate analysis revealed statistically significant correlations between overall survival and miR-32 level, tumor stage and lymph node metastasis (P < 0.05). Furthermore, miR-32 levels, tumor stage and lymph node metastasis were independently associated with overall survival (P < 0.05). Conclusions: Our results provided the first evidence that down-regulation of miR-32 was correlated with NSCLC progression, and miR-32 might be a potential molecular biomarker for predicting the prognosis of patients.     

miR-126 regulated breast cancer cell invasion by targeting ADAM9

Accumulating evidence has shown that microRNAs (miRNAs) deregulation is commonly observed in human malignancies and crucial to cancer metastasis. Herein, we demonstrated that miR-126 play a suppressor role in human breast cancer cells invasion through the direct repression of a disintegrin and metalloprotease 9 (ADAM9). MiR-126 expression was investigated in forty cases of breast cancer specimens by real-time PCR. Transwell assay was conducted to explore the effects of miR-126 on the invasion of human breast cancer cell lines. The impact of miR-126 overexpression on putative target ADAM9 was subsequently confirmed by Western blot analysis. Our results indicated that miR-126 expression was frequently down-regulated in breast cancer specimens compared with adjacent normal tissues (P<0.05). Overexpression of miR-126 significantly reduced (P<0.05) the protein levels of ADAM9, further suppressed (P<0.05) breast cancer cell invasion in vitro. Meanwhile, knockdown of ADAM9 by small interfering RNA (siRNA) also inhibited (P<0.05) breast cancer cell invasion. Thus, our study revealed that miR-126 may act as a tumor suppressor via inhibition of cell invasion by downregulating ADAM9 in breast cancer development.     

Effects of miR-489 targeting on SOX4 gene on proliferation and apoptosis of human hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma is one of the most common malignant tumors found all over the globe. Despite advances in surgery and chemotherapy, the five-year survival rate of patients with hepatocellular carcinoma is still low. It is known that the proliferation of hepatocellular carcinoma cells is closely related to the occurrence, development and prognosis of hepatocellular carcinoma. The present work investigates the expression of microRNA-489 (miR-489) in human hepatocellular carcinoma cells and its effect on the biological behavior of human hepatocellular carcinoma cells.MethodsThe expression of miR-489 by fluorescence quantitative PCR detection in 30 patients with hepatoblastoma of liver cancer tissues and adjacent tissues was studied. Also, the determination of hepatoblastoma in four cell lines with different metastatic potential (HR8348, HCT116, HT29 and HEPG2) and the expression of miR-489 during miR-489 simulation process was studied. MTT assay, flow cytometry and Western blot analysis were performed to know the cell proliferation to detect the changes in cell cycle, apoptosis of cells, and SOX4 gene expression respectively.ResultsRT-PCR results showed that the cells compared with pre-cancerous tissue, the expression level of miR-489 in hepatocellular carcinoma tissues than in adjacent tissue significantly decreased (P<0.05), and with liver cancer cell metastasis increased (P<0.05); analogue transfection constructed miR-489 overexpressing HEPG2 cell line by microRNA. MTT results showed that miR-489 can inhibit the proliferation of HEPG2 cells, the differences were statistically significant (P<0.05); flow cytometry results showed that miR-489 mimics was transfected into HEPG2 cells at 48 hours had no significant effect on cell cycle distribution (P > 0.05); but miR-489 expression could induce apoptosis, compared with the control group, the apoptosis of miR-489 mimics was significantly increased and the difference was statistically significant (P < 0.05).ConclusionIn conclusion, miR-489 can significantly inhibit the occurrence and development of hepatocellular carcinoma cells. The mechanism may be down regulated by the expression of SOX4 and inhibit cell proliferation. Further this study showed that the tumor cells SOX4 gene as a regulatory factor target the genes of miR-489 in hepatocellular carcinoma.     

Adsorption of miR-218 by lncRNA HOTAIR regulates PDE7A and affects glioma cell proliferation,invasion, and apoptosis

Objective: To evaluate the role of targeted adsorption of miR-218 by long-chain non-coding RNAHOTAIR to regulate PDE7A on glioma cell proliferation, invasion, and apoptosis. Methods: The expressions of lncRNA HOTAIR, miR-218, and PDE7A in glioma tissues and normal parcancer tissues, NHA and glioma cell lines were determined, and correlations among the three genes were analyzed. The subcellular localization of lncRNA HOTAIR was determined by fluorescent in situ hybridization. Dual-luciferase reporter assay was used to validate the targeted relationship between lncRNA HOTAIR/miR-218/PDE7A. Glioma cells were grouped to receive intervention of lncRNA HOTAIR or miR-218. MTT, transwell, and flow cytometry were performed to determine the proliferation, invasion, and apoptosis of cells. Results: Compared with the normal tissues and cells, the expression of lncRNA HOTAIR was increased while miR-218 was suppressed in glioma tissues samples and cells (all P<0.05). Inhibition of lncRNA HOTAIR expression, was able to induce apoptosis and suppress the proliferation and invasion of cells (all P<0.05). LncRNA HOTAIR is mainly localized in the cytoplasm, and is able to adsorb miR-218 as ceRNA. The effect of knockdown of HOTAIR on glioma cells could be partially rescued by miR-218 inhibitor. The expression of PDE7A was enhanced in glioma tissues and cells compared to normal tissues and cells (all P<0.05), which positively correlated with the expression of HOTAIR (r=0.546, P<0.05) and negatively correlated with the expression of miR-218 (r=0.363, P<0.05). The targeted relationship between miR-218 and PDE7A was validated: Overexpression of miR-218 was able to suppress the proliferation and invasion of glioma cells and restrain apoptosis compared to the miR-NC group (all P<0.05). The effect of miR-218 on glioma cells could be partially rescued by PDE7A. Conclusion: lncRNA HOTAIR can adsorb miR-218 to regulate expression of PDE7A and promote the malignant biologic behavior of glioma cells.     

Aberrant SATB1 expression is associated with Epstein-Barr virus infection,metastasis and survival in human nasopharyngeal cells and endemic nasopharyngeal carcinoma

Special AT-rich sequence-binding protein 1 (SATB1) has been identified as a key factor in the progression of some cancers, functioning as a global genome organizer and chromatin regulator. We examined the levels of SATB1 mRNA expression in NPC cell lines 5-8F (high metastasis) and 6-10B (low metastasis) and immortalized human nasopharyngeal epithelial cells NP69-SV40T by quantitative real-time PCR. We also examined the protein expression levels of SATB1 in 72 cases of nasopharyngeal carcinoma (NPC) tissues and 30 cases of normal nasopharyngeal (NNP) tissues by immunohistochemistry, and then assessed the correlations between SATB1 expression and clinicopathological factors. The expression level of SATB1 mRNA in 5-8F was much higher than those in 6-10B and NP69-SV40T (P < 0.05). The expression level of SATB1 mRNA in 6-10B was higher than in NP69-SV40T, but the difference was not statistically significant (P > 0.05). The positive expression rates of SATB1 protein in NPC (38/72, 52.8%) were significantly higher than in NNP (4/30, 13.3%) (P < 0.05). SATB1 protein levels in NPC were not associated with gender, age, and T stage (P > 0.05), but positively correlated with the titers of EBVCA-IgA, metastasis (N and M stage), recurrence, and survival (P < 0.05). Multivariate analysis showed that the overexpression of SATB1 protein is an independent prognostic factor for NPC. The expression levels of SATB1 were obviously upregulated in primary NPC tissues and human NPC cell lines. Therefore, SATB1 may be a valuable predictor in assessing the metastasis, recurrence, and prognosis of NPC.     

Knock-down of ABCE1 gene induces G1/S arrest in human oral cancer cells

Purpose: This study aims to explore the clinical characteristics of ATP binding cassette E1 (ABCE1) in oral squamous cell carcinomas (OSCC) and its roles in the proliferation, invasiveness, migration and apoptosis of the human oral squamous cell carcinoma cells CAL-27. Methods: The expression of ABCE1 and its target protein-RNase L, were first studied in tumor tissues of OSCC and adjacent non-tumor tissues. Moreover, CAL-27cells were transfected by ABCE1-specific shRNA, then MTT assay, the transwell and scratch assay were used to study cell proliferation and migration activity; the apoptosis rate and cell cycle distribution were tested by flow cytometry. Western blot and RT-PCR assay were adopted to measure their silencing efficacy. Results: ABCE1 expression is low in the adjacent non-tumor tissues while the expression is high in the oral cancer; the expression is reversely proportional to the differentiation degrees. The expression of RNaseL was in contrary to ABCE1. After transfected with ABCE1-siRNA, the proliferation, invasiveness and migration capabilities of cells decreased significantly whilst the apoptosis rate enhanced greatly (P < 0.01). Meanwhile, the expression of ABCE1 in CAL-27 cells was blocked (P < 0.01) while the expression of RNase L increased significantly (P < 0.01). Conclusion: ABCE1 is closely connected with the pathogenesis and development of oral cancer, which acts through the cellular pathways of 2-5A/RNase L.     

Down-regulation of microRNA-124 is correlated with tumor metastasis and poor prognosis in patients with lung cancer

Introduction: MicroRNA-124 (miR-124) has been proven dysregulated in several human malignancies and correlated with tumor progression. However, its expression and clinical significance in non-small cell lung cancer (NSCLC) is still unclear. Thus, the aim of this study was to investigate the clinical significance of miR-124 expression in NSCLC. Methods: Expression levels of miR-124 in 92 pairs of NSCLC and adjacent non-tumor tissues were detected by quantitative real-time PCR (qRT-PCR). In order to determine its prognostic value, overall survival (OS) and disease-free survival (DFS) were evaluated using the Kaplan-Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. Results: miR-124 expression level was significantly lower in NSCLC tissues compared with adjacent non-tumor tissues (P < 0.05). The 5-year OS of low miR-124 expression group was significantly shorter than that of high miR-124 expression group (P < 0.05). Moreover, the 5-year DFS of low miR-124 expression group was also significantly shorter than that of high miR-124 expression group (P < 0.05). In a multivariate Cox model, we found that miR-124 expression was an independent prognostic factor for both 5-year OS and 5-year DFS in NSCLC (P < 0.05). Conclusions: Our results offer the convincing evidence that miR-124 may play key roles in the progression of lung cancer and that the down-regulated expression of miR-124 may be independently associated with shorter OS and DFS of patients, suggesting that miR-124 might be a potential marker for further risk stratification in the treatment of lung cancer.     

miR-144 regulates transforming growth factor-β1 iduced hepatic stellate cell activation in human fibrotic liver

Objectives: Activation of hepatic stellate cells (HSCs) into collagen producing myofibroblasts is critical for pathogenesis of liver fibrosis. Transforming growth factor-β1 (TGF-β1) is one of the main profibrogenic mediators for HSC transdifferentiation. Recent studies have shown effect of microRNAs (miRNAs) on regulating TGF-β1-induced HSC activation during liver fibrosis. Here, we aimed to explore the roles of miR-144 and miR-200c in human liver fibrosis. Methods: Expression of TGF-β1 was detected in 42 fibrotic and 18 normal human liver tissues by quantitative real time polymerase chain reaction (qRT-PCR) and immunohistochemistry, and its correlation with α-smooth muscle actin (α-SMA) was calculated. miR-144 and miR-200c expression level in fibrotic liver tissues were also detected by qRT-PCR. The correlation of TGF-β1 expression with miR-200c and miR-144 in the fibrotic liver was analyzed. Results: The results showed that TGF-β1 expression was much higher in fibrotic liver than that in normal liver tissues (P<0.05). TGF-β1 protein high expressing liver fibrosis showed α-SMA positive cells in the liver parenchyma indicating activated HSCs. Expression of TGF-β1 in fibrotic liver was significantly correlated with α-SMA expression (R=0.633, P<0.001). Furthermore, miR-144 was less expressed in liver fibrosis (P<0.05) and was significantly correlated with expression of TGF-β1 in fibrotic liver tissues (R=-0.442, P<0.01). However, miR-200c did not show significant difference between normal and fibrotic liver (P=0.48) and correlation with TGF-β1 expression (R=0.106, P=0.51). Conclusion: All the results indicate that miR-144 can be a novel regulator of TGF-β1-induced HSC activation during liver fibrosis.     

JMJD5 is a potential oncogene for colon carcinogenesis

Objective: To observe the effects of Jumonji C domain-containing (JMJD) 5 depletion on colon cancer (CC). Methods: A short-hairpin RNA targeting JMJD5 was transfected into a lentivirus to make Lv-shJMJD5 for infection into the Caco-2 human cell. Besides, a negative control shRNA was constructed. The mRNA and protein levels of JMJD5 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting, respectively. Cell proliferation, migration, and invasion were assessed by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), soft agar colony assay and transwell assay, respectively. In addition, immunohistochemical (IHC) staining was performed to investigate the expression of JMJD5 in adjacent normal tissues and tumor tissues from patients with CC. Results: Compared with control group, mRNA and protein levels of JMJD5 was significantly reduced after infection with Lv-shJMJD5 (P<0.05), and Caco-2 cell proliferation, migration, and invasion were all obviously inhibited (P<0.05). The results of IHC showed that JMJD5 was significantly up-regulated compared with normal tissues (P<0.01). Additionally, follow-up data demonstrated that the survival rate of patients with high expression of JMJD5 was obviously lower than that with low expression (P<0.01). Conclusions: JMJD5 depletion could significantly inhibit human CC cell proliferation, migration, and invasion, implying that JMJD5 might be a potential oncogene.     

Depleting ABCE1 expression induces apoptosis and inhibits the ability of proliferation and migration of human esophageal carcinoma cells

Objective: This study aims to explore the clinical characteristics of ABCE1 in esophageal cancers and its roles in the proliferation, invasiveness, migration and apoptosis of the esophageal carcinoma Eca109 cell line. Methods: The expression of ABCE1 and its target protein-RNase L, were first studied in tumor tissues of esophageal carcinoma and adjacent non-tumor tissues. The siRNA green fluorescent protein (GFP) expression vector of ABCE1 was prepared and transfected into the esophageal carcinoma Eca109 cells, then the fluorescence microscope was used to study the transfection efficiency. The MTT assay, cell invasion, the transwell and scratch assay were used to study cell proliferation and migration activity; the apoptosis rate was tested by flow cytometry. Western blot and RT-PCR assay were adopted to measure their silencing efficacy. Results: ABCE1 expression is low in the adjacent non-tumor tissues while the expression is high in the esophageal carcinoma; the expression is reversely proportional to the differentiation degrees. The expression of RNase L was in contrary to ABCE1. After transfected with ABCE1-siRNA, the proliferation, invasiveness and migration capabilities of cells decreased significantly whilst the apoptosis rate enhanced greatly (P<0.01). Meanwhile, the expression of ABCE1 in Eca109 cells was blocked (P<0.01) while the expression of RNase L increased significantly (P<0.01). Conclusion: ABCE1 is closely connected with the pathogenesis and development of esophageal carcinoma, which act through the cellular pathways of 2-5A/RNase L.     

Long non-coding RNA TP73-AS1 predicts poor prognosis and regulates cell proliferation and migration in cervical cancer

IntroductionCervical cancer is one of the most common malignant tumors in women, which seriously affects women’s health, especially in developing countries. This study aims to investigate novel molecular markers for poor prognosis of cervical cancer to achieve correct guidance of clinical treatment, accurate assessment of prognosis, and provide a new basis for the choice of reasonable treatment options for cervical cancer patients.Material and methodsQRT-PCR was employed to investigate the expression of lncRNA TP73-AS1 in cervical cancer tissues and cell lines. COX multivariate analysis showed the relationship between TP73-AS1 expression and clinicopathological features of patients with cervical cancer. Colony formation and MTT assay detected the effect of TP73-AS1 on proliferation of cervical cancer cells. The effect of TP73-AS1 on migration and invasion of cervical cancer cells was determined by the wound-healing assay and transwell assay. Western blot was performed to assess the expression of EMT markers.ResultsThis study showed that lncRNA TP73-AS1 was up-regulated in cervical cancer tissues and cell lines (p < 0.001), and high expression of TP73-AS1 could be considered as an independent prognostic factor (p < 0.05). Moreover, lncRNA TP73-AS1 promotes cervical cancer cell migration and invasion, and knockdown of TP73-AS1 inhibits the growth of cervical cancer cells (p < 0.001).ConclusionsOur results indicated that lncRNA TP73-AS1 was up-regulated in cervical cancer tissues and cell lines, predicting poor prognosis of cervical cancer and regulating cell proliferation and migration.