Overexpression of LINC00037 represses cervical cancer progression by activating mTOR signaling pathway

Long non-coding RNAs have been reported to play crucial roles in tumorigenesis including cervical cancer. LINC00037 has been identified as a significant regulator in several cancers. Our study was aimed to investigate the function of LINC00037 in cervical cancer progression. LINC00037 was significantly downregulated in human cervical cancer cells (HeLa, HCC94, HT-3, Caski, and SiHa cells) compared with the ectocervical epithelial cells (End1/E6E7 cells). Overexpression of LINC00037 was able to inhibit cervical cancer cell proliferation, induce cell apoptosis, and restrain the cell migration/invasion capacity. Reversely, knockdown of LINC00037 exhibited an opposite process in vitro. mTOR has been recognized as an atypical serine/threonine kinase that is involved in regulating significant cellular functions. In our present study, we observed that the mTOR signaling pathway was strongly activated in human cervical cancer cells. Meanwhile, upregulation of LINC00037 contributed to the inactivation of mTOR signaling whereas downregulation of LINC00037 activated the pathway. Subsequently, in vivo animal models using SiHa cells were established and we proved that LINC00037 repressed cervical cancer progression via targeting the mTOR signaling pathway. All these findings implied that LINC00037 could regulate cervical cancer pathogenesis via mTOR signaling. In conclusion, a novel role of LINC00037 was manifested in cervical cancer progression.     

过表达miR-455抑制宫颈癌细胞SiHa的生长

研究表明,microRNA(miRNA)可作为癌基因或抑癌基因发挥功能、调控细胞增殖和凋亡等生物学行为,与肿瘤的发生发展密切相关. 在本研究中,我们检测了miR- 455在宫颈癌组织中的表达变化及其对宫颈癌SiHa细胞生物学功能的影响. Real- time PCR实验结果显示,miR-455在宫颈癌组织样本中较正常宫颈组织表达明显降低. 瞬时转染miR-455 mimics使其在SiHa细胞中过表达. CCK-8及流式细胞术分析显示, 过表达miR-455明显抑制细胞增殖,促进细胞凋亡,导致细胞G1/S期阻滞. Real- time PCR分析显示,PI3KR1,BCL2L2 mRNA明显降低.上述研究结果表明, miR-455可显著降低SiHa细胞存活能力,是一个潜在的抑癌基因.     

miR-503 expression is downregulated in cervical cancer and suppresses tumor growth by targeting AKT2

Previous studies have reported that microRNAs function as key regulators in tumor development and progression. This study aims to investigate the functional effects of miR-503 expression in cervical cancer (CC) progression. We detected the expression of miR-503 in CC tissues and cell lines using quantitative real-time polymerase chain reaction. Synthesized miR-503 mimics or inhibitors were used to upregulate or downregulate the expression of miR-503 in HeLa or SiHa cells. Cell Counting Kit-8 and colony formation assay were used to detect the ability of cell proliferation. Furthermore, luciferase assay and Western blot were applied to confirm the target of miR-503 in CC cells. Here, we demonstrated that miR-503 expression was significantly downregulated in CC tissues, compared with adjacent normal tissues. miR-503 expression was significantly associated with tumor size and International Federation of Gynecology and Obstetrics stage. Furthermore, increasing miR-503 expression in CC cells dramatically inhibited cell proliferation, colony formation ability of CC. However, reducing miR-503 had reverse effects on these malignant behaviors. Moreover, we demonstrated that miR-503 inhibited cell proliferation by targeting AKT2 3′-untranslated region and affected its expression. Overexpression of AKT2 rescued the effects induced by miR-503 on cell proliferation. Therefore, our results indicated that miR-503 may serve as a tumor suppressor in CC and provide a potential value for CC treatment.     

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.     

SPAG5 upregulation predicts poor prognosis in cervical cancer patients and alters sensitivity to taxol treatment via the mTOR signaling pathway

Previously, we found that sperm-associated antigen 5 (SPAG5) was upregulated in pelvic lymph node metastasis–positive cervical cancer. The aim of this study is to examine the role of SPAG5 in the proliferation and tumorigenicity of cervical cancer and its clinical significance in tumor progression. In our study, SPAG5 expression in cervical cancer patients was detected using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry; cervical cancer cell function with downregulated SPAG5 in vitro was explored using tetrazolium assay, flow cytometry, and colony formation and Transwell assays. SPAG5 was upregulated in tumor tissue compared with paired adjacent noncancerous tissues; SPAG5 upregulation in tumor tissues indicated poor disease-free survival, which was also an independent prognostic indicator for cervical cancer patients. In vitro study demonstrated that SPAG5 downregulation inhibited cell proliferation and growth significantly by G2/M arrest and induction of apoptosis, and hindered cell migration and invasion. Under SPAG5 downregulation, the sensitivity of cervical cancer cells differed according to taxol dose, which correlated with mammalian target of rapamycin (mTOR) signaling pathway activity. In general, SPAG5 upregulation relates to poor prognosis in cervical cancer patients, and SPAG5 is a regulator of mTOR activity during taxol treatment in cervical cancer.     

LncRNA CRNDE promotes cell proliferation,migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis

Ovarian cancer seriously threatens the health of women. LncRNA CRNDE is known to be upregulated in ovarian cancer. However, the mechanism by which CRNDE regulates the progress of ovarian cancer is largely unknown. MTT assay was applied to measure the cell viability. Colony formation assay was used to measure the cell proliferation. Cell migration was tested by wound healing, and Transwell assay was performed to detect cell invasion. In addition, the expression of miR-423-5p, CRNDE and FSCN1 were detected by RT-qPCR and western blotting, respectively. Meanwhile, dual-luciferase reporter assay and RIP assay were performed to explore the correlation between miR-423-5p and CRNDE (or FSCN1). CRNDE and FSCN1 were upregulated in ovarian cancer cells (SKOV3, CAOV-3, IGROV1, A2780 and C13K), while miR-423-5p was downregulated. Moreover, silencing of FSCN1/CRNDE significantly decreased proliferation, migration and invasion of ovarian cancer cells (SKOV3 and CI3K) via suppressing MMP-2 and MMP-9. In addition, CRNDE could sponge miR-423-5p, and FSCN1 was confirmed to be the direct target of miR-423-5p. Furthermore, CRNDE knockdown-induced inhibition of FSCN1 was notably reversed by miR-423-5p downregulation. Knockdown of CRNDE inhibited cell proliferation, migration and invasion of ovarian cancer via miR-423-5p/FSCN1 axis. Thus, CRNDE may serve a new target for ovarian cancer.

     

Role and mechanism of miR-4778-3p and its targets NR2C2 and Med19 in cervical cancer radioresistance

The aim of this study was to investigate the effect of miR-4778-3p on the radiosensitivity of cervical cancer cells and to elucidate the underlying mechanism. Tissue samples were collected from eight patients with cervical cancer prior to chemoradiotherapy. MicroRNA chip analyses, RT-PCR, gene transfection, CCK8, wound healing and Transwell assays, colony-forming assay, western blot, and the Dual-Luciferase Reporter Assay System were used to evaluate the role of miR-4778-3p in cervical cancer radiosensitivity and its relationships with target molecules NR2C2 and Med19. Thirty-two differentially expressed miRNA molecules (fold-change?>?2; p?<?0.05) associated with cervical cancer radioresistance were identified. The expression of miR-4778-3p was significantly lower in recurrent or metastatic patients than in control subjects. In vitro studies using radioresistant HeLa and SiHa cervical cancer cell lines showed that miR-4778-3p upregulation significantly inhibited cell proliferation, invasiveness, and migration after irradiation. There was also a significant increase in apoptosis and a significant decrease in the proportion of cells at the G2/M phase. Further, miR-4778-3p upregulation led to increased expression of apoptosis-related molecules, such as Bax, Caspase-3, Caspase-8, and Caspase-9. Reporter gene assays showed that miR-4778-3p bound specifically to NR2C2 and Med19 and negatively regulated their expression. Thus, miR-4778-3p reduces the vitality, proliferation, and migration of radioresistant cervical cancer cells and may regulate the radiosensitivity of cervical cancer by targeting and regulating NR2C2 and Med19 expression.     

Retracted: miR-145 modulates epithelial-mesenchymal transition and invasion by targeting ZEB2 in non–small cell lung cancer cell lines

Lung cancer is the leading cause of cancer-related deaths worldwide. Epithelial-mesenchymal transition (EMT) is a major event that drives cancer progression. Here we aim to investigate the role of microRNA, miR-145, in regulating EMT of the highly invasive non–small cell lung cancer (NSCLC). Quantitative real-time polymerase chain reaction analysis indicated that miR-145 was downregulated in cancer tissue compared with that in adjacent normal tissue. NSCLC cell lines, namely H1299, PC7, and SPCA-1, also demonstrated miR-145 downregulation, which is correlated well with their invasive ability, assessed by the Matrigel invasion assay. miR-145 overexpression resulted in downregulation of N-cadherin, and downregulation of vimentin and E-cadherin, suggesting a decreased EMT activity. TargetScan analysis predicted that a binding site exists between miR-145 and an oncogene, ZEB2, which was verified using the dual-luciferase assay. Alteration of miR-145 expression also induced inverse effects on ZEB2 expression, and a negative correlation exists between ZEB2 and miR-145 in human tissues. ZEB2 and miR-145 also exerted antagonizing effects on the invasion of NSCLC cells. Therefore, miR-145 is an important molecule in NSCLC that regulates cancer EMT through targeting ZEB2.     

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.     

MicroRNA-140 inhibit prostate cancer cell invasion and migration by targeting YES proto-oncogene 1

Prostate cancer (PCa), which is an aggressive malignancy of the male genitourinary system. In the present study, the effects of microRNA-140 (miR-140) on PCa were determined. We transfected miR-140 mimics or negative control into PCa cells, and we used MTT, wound healing, and Transwell assays for determining the capacities of miR-140 in cell proliferation, migration, and invasion, respectively. We also confirmed the relationship between miR-140 and YES proto-oncogene 1 (YES1) using Luciferase reporter assay. The results showed that miR-140 was downregulated in PCa cells and tissues, and overexpression of miR-140 could significantly suppress their capacities of proliferation, migration, and invasion. Moreover, YES1 was shown to be a direct target of miR-140. Moreover, miR-140 expression is negatively correlated with YES1 levels. miR-140 exhibits significant tumor-suppressive effects in PCa by inhibiting YES1. The study indicated that miR-140 and YES1 could be the potential targets for PCa therapy.     

microRNA-143 is associated with the survival of ALDH1+CD133+ osteosarcoma cells and the chemoresistance of osteosarcoma

This study investigated the role of miR-143 in the chemoresistance of osteosarcoma tumor cells and the associated mechanisms. Real-time PCR was used to measure miR-143 levels. Western blot was used to detect protein expression. Cell proliferation was analyzed by MTT assay and Matrigel colony formation assay. Forced miR-143 expression was established by adenoviral vector infection. Cell death was detected by Hoechst33342 staining. Loss of miR-143 expression was observed in osteosarcomas, which correlated with shorter survival of patients with osteosarcomas underlying chemotherapy. In chemoresistant SAOS-2 and U2OS osteosarcomas cells, miR-143 levels were significantly downregulated and accompanied by increases in ATG2B, Bcl-2, and/or LC3-II protein levels, high rate of ALDH1⁺CD133⁺ cells, and an increase in Matrigel colony formation ability. H₂O₂ upregulated p53 and miR-143, but downregulated ATG2B, Bcl-2, and LC3-I expression in U2OS cells (wild-type p53) but not in SAOS-2 (p53-null) cells. Forced miR-143 expression significantly reversed chemoresistance as well as downregulation of ATG2B, LC3-I, and Bcl-2 expression in SAOS-2- and U2OS-resistant cells. Forced miR-143 expression significantly inhibited tumor growth in xenograft SAOS-2-Dox and U2OS-Dox animal models. Loss of miR-143 expression is associated with poor prognosis of patients with osteosarcoma underlying chemotherapy. The chemoresistance of osteosarcoma tumor cells to doxorubicin is associated with the downregulation of miR-143 expression, activation of ALDH1⁺CD133⁺ cells, activation of autophagy, and inhibition of cell death. miR-143 may play a crucial role in the chemoresistance of osterosarcoma tumors.     

miR-140-3p impedes the proliferation of human cervical cancer cells by targeting RRM2 to induce cell-cycle arrest and early apoptosis

Cervical cancer is a critically malignant tumor with the second mortality of females worldwide. MicroRNAs (miRNAs) are short but regulatory non-coding RNAs playing a pivotal role in many biological processes including tumorigenesis. However, the exact role of miR-140-3p in cervical cancer remains to be elucidated. Here we identified that miR-140-3p was significantly reduced in cervical cancer tissues by comprehensive analysis of TCGA data, hinting that higher expression level of miR-140-3p predicted a good clinical prognosis. Quantitative real-time PCR (RT-qPCR) assay was performed to confirm the negative correlation between miR-140-3p expression level and human cervical cancer tissues as well as various cervical cancer cell lines. To clarify the certain role of miR-140-3p, forced expression by microRNA mimics was applied in Caski and C33A cells, showing that miR-140-3p overexpression significantly impeded the proliferation of cervical cancer cells by cell count kit (CCK-8) assay. Western blot analysis of cell cycle-related proteins Cyclin A, Cyclin B1 and Cyclin D1 have further confirmed the cell cycle arrest was induced by the ectopic expression of miR-140-3p. Annexin-V based FACS analysis also found the simultaneous appearance of early apoptotic cell population in miR-140-3p overexpression cells. The protein level of BCL-2 was attenuated in accompany with elevated Bax and Cleaved caspase-3 protein, indicating miR-140-3p overexpression induced early apoptosis. Mechanistically, we demonstrated that miR-140-3p could target the 3′UTR of RRM2 which has been proved to be highly involved in the onset of cancer. Furthermore, upregulation of miR-140-3p and RRM2 failed to inhibit the proliferation of human cervical cancer cells, revealing that RRM2 served as the target downstream gene of miR-140-3p abolishing its ability as a tumor suppressor. Overall, we figured out the new role of miR-140-3p in cervical cancer and concluded that miR-140-3p was a candidate of cancer control in preclinical.     

microRNA-448 inhibits the progression of non-small–cell lung cancer through regulating IRS2

Recently, microRNA-448 (miR-448) has been reported to be a tumor-associated miRNA in many human cancers. In this study, we investigated the function of miR-448 in non-small–cell lung cancer (NSCLC) progression and confirmed the relationship between miR-448 and insulin receptor substrates 2 (IRS2). First, downregulation of miR-448 and upregulation of IRS2 were detected in NSCLC using the quantitative real-time polymerase chain reaction (qRT-PCR) assay. Furthermore, the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay showed that miR-448 inhibited cell viability in NSCLC. Transwell and Western blot assays indicated that the upregulation of miR-448 inhibited cell metastasis and epithelial-to-mesenchymal transition (EMT) in NSCLC. And it was found that overexpression of miR-448 reduced the adhesion of A549 cells to HUVEC cells using the adhesion assay. Furthermore, the dual luciferase assay indicated that miR-448 directly targeted IRS2 in NSCLC. In addition, it was found that IRS2 silencing had an inhibitory effect on the progression of NSCLC, and the upregulation of IRS2 partially impaired the inhibitory effect of miR-448 in NSCLC. Briefly, overexpression of miR-448 inhibited cell proliferation, metastasis, and EMT by suppressing IRS2 expression in NSCLC.     

microRNA-802 inhibits cell proliferation and induces apoptosis in human cervical cancer by targeting serine/arginine-rich splicing factor 9

microRNAs (miRNAs) play crucial roles in cancer development and progression by targeting mRNAs for degradation and/or translational repression. microRNA-802 (miR-802) has been reported as a tumor suppressor and its deregulation is observed in various human cancers. However, the prognostic value of miR-802 and its underlying mechanisms involved in human cervical cancer are poorly investigated. The purposes of this study were to explore the role of miR-802 in cervical cancer and to clarify the regulation of serine/arginine-rich splicing factor 9 (SRSF9) by miR-802. Here, we found that miR-802 was downregulated in both cervical cancer tissues and cell lines. Transfection of a miR-802 mimic into cervical cancer cells inhibited their proliferation and colony formation, and promoted cell cycle arrest at the G0/G1 phase and cell apoptosis. In addition, we found that miR-802 could directly target the 3′-untranslated region of SRSF9 and suppress SRSF9 expression. Rescue experiments revealed that overexpression of SRSF9 partially reversed the inhibition effect of miR-802 in cervical cancer cells. Overall, these findings demonstrate that miR-802 functions as a tumor suppressor in cervical cancer by targeting SRSF9, suggesting that miR-802 might serve as a potential therapeutic target in cervical cancer.