Regulatory light chains modulate in vitro actin motility driven by skeletal heavy meromyosin

Phosphorylation and Ca²⁺-Mg²⁺ exchange on the regulatory light chains (RLCs) of skeletal myosin modulate muscle contraction. However, the relation between the mechanisms for the effects of phosphorylation and metal ion exchange are not clear. We propose that modulation of skeletal muscle contraction by phosphorylation of the myosin regulatory light chains (RLCs) is mediated by altered electrostatic interactions between myosin heads/necks and the negatively charged thick filament backbone. Our study, using the in vitro motility assay, showed actin motility on hydrophilic negatively charged surfaces only over the HMM with phosphorylated RLCs both in the presence and absence of Ca²⁺. In contrast, good actin motility was observed on silanized surfaces (low charge density), independent of RLC phosphorylation status but with markedly lower velocity in the presence of Ca²⁺. The data suggest that Ca²⁺-binding to, and phosphorylation of, the RLCs affect the actomyosin interaction by independent molecular mechanisms. The phosphorylation effects depend on hydrophobicity and charge density of the underlying surface. Such findings might be exploited for control of actomyosin based transportation of cargoes in lab-on-a chip applications, e.g. local and temporary stopping of actin sliding on hydrophilic areas along a nanosized track.     

lncRNA NEAT1 facilitates melanoma cell proliferation,migration, and invasion via regulating miR-495-3p and E2F3

Melanoma contributes a lot to skin cancer-related deaths. lncRNAs are implicated in various diseases, including melanoma. lncRNA NEAT1 is frequently dysregulated and can play important roles in multiple cancers. Nevertheless, little has been studied about the function of NEAT1 in melanoma progression. In our present research, we displayed NEAT1 was overexpressed in melanoma cells. A series of functional assays showed that overexpression of NEAT1 promoted the proliferation, migration, and invasion of melanoma cells. By contrast, NEAT1 knockdown obviously restrained melanoma cell progression. Mechanistically, it was revealed that NEAT1 could directly bind with miR-495-3p, which led to a negative effect on miR-495-3p levels. In addition, miR-495-3p was significantly decreased in melanoma cells. Furthermore, E2F3 was postulated as the target of miR-495-3p and overexpression of this miR could suppress the levels of E2F3. Meanwhile, it was exhibited that melanoma cell proliferation, migration, and invasion induced by E2F3 silence was abrogated by miR-495-3p. Moreover, an in vivo xenograft nude mice model was established using A375 cells and it was indicated that NEAT1 promoted melanoma progression in vivo via regulating the miR-495-3p/E2F3 axis. In conclusion, we suggest that NEAT1 exerts an oncogenic effect on melanoma development via inhibition of miR-495-3p and induction of E2F3. NEAT1 might serve as a crucial prognostic biomarker of melanoma.     

LncRNA CASC11 promoted gastric cancer cell proliferation,migration and invasion in vitro by regulating cell cycle pathway

In this study, we aimed to investigate the effects of lncRNA CASC11 on gastric cancer (GC) cell progression through regulating miR-340-5p and cell cycle pathway. Expressions of lncRNA CASC11 in gastric cancer tissues and cell lines were determined by qRT-PCR. Differentially expressed lncRNAs, mRNAs and miRNAs were screened through microarray analysis. The relationship among CASC11, CDK1 and miR-340-5p was predicted by TargetScan and validated through dual luciferase reporter assay. Western blot assay examined the protein level of CDK1 and several cell cycle regulatory proteins. GO functional analysis and KEGG pathway analysis were used to predict the association between functions and related pathways. Cell proliferation was determined by CCK-8 assays. Cell apoptosis and cell cycle were detected by flow cytometry assay. CASC11 was highly expressed in GC tissues and cell lines. Knockdown of CASC11 inhibited GC cell proliferation, promoted cell apoptosis and blocked cell cycle. KEGG further indicated an enriched cell cycle pathway involving CDK1. QRT-PCR showed that miR-340-5p was down-regulated in GC cells tissues, while CDK1 was up-regulated. Furthermore, CASC11 acted as a sponge of miR-340-5p which directly targeted CDK1. Meanwhile, miR-340-5p overexpression promoted GC cell apoptosis and induced cell cycle arrest, while CDK1 overexpression inhibited cell apoptosis and accelerated cell cycle. Our study revealed the mechanism of CASC11/miR-340-5p/CDK1 network in GC cell line, and suggested that CASC11 was a novel facilitator that exerted a biological effect by activating the cell cycle signaling pathway. This finding provides a potential therapeutic target for GC.     

Protein tyrosine phosphatase receptor-type O (PTPRO) is co-regulated by E2F1 and miR-17-92

PTPRO is often silenced by DNA hypermethylation in primary human tumors and cancer cell lines and functions as a tumor suppressor. Here we show that PTPRO is a target of E2F1. In addition, the microRNA cluster miR-17-92, another target of E2F1, participates in PTPRO regulation. PTPRO mRNA was up-regulated during S phase in synchronized HeLa cells and in vitro PTPRO promoter activity is high in early S phase while the PTPRO 3'UTR reporter activity is low in late S phase. This study provides evidence that the PTPRO gene is co-regulated by both E2F1 and miR-17-92.     

NEAT1 promotes colon cancer progression through sponging miR-495-3p and activating CDK6 in vitro and in vivo

Recently, increasing evidence has indicated lncRNAs are powerful regulators in the progression of multiple tumors. Dysregulation of lncRNA NEAT1 has been recognized in many cancer types. Meanwhile, the studies on NEAT1 function have suggested that NEAT1 can serve as a crucial oncogene. Nevertheless, the investigation of NEAT1 in colon cancer is still few. In our study, the function of NEAT1 was studied in colon cancer. As we observed, NEAT1 level was obviously elevated in colon cancer cells. Then, HCT-116 and SW620 cells were stably infected with shRNA-NEAT1 for 48 hr. As exhibited, silence of NEAT1 could greatly repress colon cancer cell progression. Apoptosis of colon cancer cells was triggered and the cell cycle progression was remarkably inhibited by downregulation of NEAT1. Interestingly, as exhibited, miR-495-3p was obviously decreased in colon cancer cells and it significantly suppressed colon cancer progression. Subsequently, miR-495-3p was predicted as a target of NEAT1. CDK6 was speculated as the target of miR-495-3p and miR-495-3p modulated its expression negatively. Finally, it was indicated that NEAT1 promoted colon cancer development through modulating miR-495-3p and CDK6 in vivo. Taken these together, we reported that NEAT1 could sponge miR-495-3p to contribute to colon cancer progression through activating CDK6.     

miR-129-5p inhibits proliferation,migration, and invasion in rectal adenocarcinoma cells through targeting E2F7

microRNAs (miRNAs), a kind of small noncoding RNAs, are considered able to regulate expression of genes and mediate RNA silencing. miR-129-5p was shown to be a cancer-related miRNA. However, the influence of miR-129-5p in rectal adenocarcinoma (READ) development remains to be determined. Based on the TCGA data, downregulation of miR-129-5p in READ samples was observed. Manual restoration of the miR-129-5p in SW1463 and SW480 cell lines significantly inhibited invasion, migration, and proliferation of READ cell lines, while the apoptosis ability was enhanced. Meanwhile, we found E2F7 acted as a potential target of miR-129-5p and was upregulated in READ samples. E2F7 upregulation reversed the repression of miR-129-5p on READ development. Finally, in vivo experiments showed that inhibition of tumor growth in nude mice was achieved through upregulating miR-129-5p. Overall, our findings suggest increasing of miR-129-5p leads to the suppression of READ progression through regulating the expression of E2F7, which may provide novel insights into the treatment of READ.     

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.     

MiR-142-3p functions as a tumor suppressor by targeting RAC1/PAK1 pathway in breast cancer

MicroRNA-142-3p (miR-142-3p) was previously investigated in various cancers, whereas, it's role in breast cancer (BC) remains far from understood. In this study, we found that miR-142-3p was markedly decreased both in cell lines and BC tumor tissues. Elevated miR-142-3p expression suppressed growth and metastasis of BC cell lines via gain-of-function assay in vitro and in vivo. Mechanistically, miR-142-3p could regulate the ras-related C3 botulinum toxin substrate 1 (RAC1) expression in protein level, which simultaneously suppressed the epithelial-to-mesenchymal transition related protein levels and the activity of PAK1 phosphorylation, respectively. In addition, rescue experiments revealed RAC1 overexpression could reverse tumor-suppressive role of miR-142-3p. Our results showed miR-142-3p could function as a tumor suppressor via targeting RAC1/PAK1 pathway in BC, suggesting a potent therapeutic target for BC treatment.     

LBX2-AS1 promotes ovarian cancer progression by facilitating E2F2 gene expression via miR-455-5p and miR-491-5p sponging

LBX2-AS1 is a long non-coding RNA that facilitates the development of gastrointestinal cancers and lung cancer, but its participation in ovarian cancer development remained uninvestigated. Clinical data retrieved from TCGA ovarian cancer database and the clinography of 60 ovarian cancer patients who received anti-cancer treatment in our facility were analysed. The overall cell growth, colony formation, migration, invasion, apoptosis and tumour formation on nude mice of ovarian cancer cells were evaluated before and after lentiviral-based LBX2-AS1 knockdown. ENCORI platform was used to explore LBX2-AS1-interacting microRNAs and target genes of the candidate microRNAs. Luciferase reporter gene assay and RNA pulldown assay were used to verify the putative miRNA-RNA interactions. Ovarian cancer tissue specimens showed significant higher LBX2-AS1 expression levels that non-cancerous counterparts. High expression level of LBX2-AS1 was significantly associated with reduced overall survival of patients. LBX2-AS1 knockdown significantly down-regulated the cell growth, colony formation, migration, invasion and tumour formation capacity of ovarian cancer cells and increased their apoptosis in vitro. LBX2-AS1 interacts with and thus inhibits the function of miR-455-5p and miR-491-5p, both of which restrained the expression of E2F2 gene in ovarian cancer cells via mRNA targeting. Transfection of miRNA inhibitors of these two miRNAs or forced expression of E2F2 counteracted the effect of LBX2-AS1 knockdown on ovarian cancer cells. LBX2-AS1 was a novel cancer-promoting lncRNA in ovarian cancer. This lncRNA increased the cell growth, survival, migration, invasion and tumour formation of ovarian cancer cells by inhibiting miR-455-5p and miR-491-5p, thus liberating the expression of E2F2 cancer-promoting gene.     

lncRNA CEBPA-AS1靶向miR-455-3p调控胃癌细胞生物学行为的分子机制研究

摘要 目的：探讨lncRNA CEBPA-AS1对胃癌细胞生物学行为的影响及其可能作用机制。方法：qRT-PCR法检测胃癌组织、癌旁组织与正常人胃上皮GES1细胞和人胃癌SNU-1、AGS、HS-746T细胞系中lncRNA CEBPA-AS1、miR-455-3p的表达量。si-NC、si-lncRNA CEBPA-AS1、miR-NC、miR-455-3p mimics、si-lncRNA CEBPA-AS1与anti-miR-NC、si-lncRNA CEBPA-AS1与anti-miR-455-3p分别转染至SNU-1细胞（分别命名为si-NC组、si-lncRNA CEBPA-AS1组、miR-NC组、miR-455-3p组、si-lncRNA CEBPA-AS1+anti-miR-NC组和si-lncRNA CEBPA-AS1+anti-miR-455-3p组）后,MTT实验与平板克隆形成实验分别检测细胞增殖及克隆形成能力,Transwell小室实验检测细胞迁移及侵袭能力,双荧光素酶报告基因实验与qRT-PCR实验验证lncRNA CEBPA-AS1与miR-455-3p的靶向调控关系,Western blot法检测MMP2、MMP9蛋白表达情况。结果：与癌旁组织比较,胃癌组织中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,差异均有统计学意义（均P＜0.05）。与GES1细胞比较,SNU-1、AGS、HS-746T细胞中lncRNA CEBPA-AS1的表达量显著升高,miR-455-3p的表达量显著降低,其中SNU-1细胞的lncRNA CEBPA-AS1表达量最高,差异均有统计学意义（均P＜0.05）。与si-NC组比较,si-lncRNA CEBPA-AS1组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。与miR-NC组比较,miR-455-3p组细胞活力降低,细胞克隆形成数、迁移及侵袭细胞数减少,MMP2、MMP9蛋白表达水平降低,差异均有统计学意义（P＜0.05）。lncRNA CEBPA-AS1可靶向结合miR-455-3p,并可负调控miR-455-3p的表达。与si-lncRNA CEBPA-AS1+anti-miR-NC组比较,si-lncRNA CEBPA-AS1+anti-miR-455-3p组细胞活力升高,细胞克隆形成数、迁移及侵袭细胞数增多,MMP2、MMP9蛋白表达水平升高,差异均有统计学意义（P＜0.05）。结论：干扰lncRNA CEBPA-AS1表达可通过靶向调控miR-455-3p而抑制胃癌细胞增殖、克隆形成、迁移及侵袭。     

Long noncoding RNA FLVCR1-AS1 aggravates biological behaviors of glioma cells via targeting miR-4731-5p/E2F2 axis

Long noncoding RNAs (lncRNAs) display essential roles in cancer progression. FLVCR1-AS1 is a rarely investigated lncRNAs involved in various human cancers, such as hepatocellular carcinoma and lung cancer. However, its function in glioma has not been clarified. In our study, we found that FLVCR1-AS1 was highly expressed in glioma tissues and cell lines. And upregulation of FLVCR1-AS1 predicted poor prognosis in patients with glioma. Moreover, FLVCR1-AS1 knockdown inhibited proliferation, migration and invasion of glioma cells. Through bioinformatics analysis, we identified that FLVCR1-AS1 was a sponge for miR-4731-5p to upregulate E2F2 expression. Moreover, rescue assays indicated that FLVCR1-AS1 modulated E2F2 expression to participate in glioma progression. Altogether, our research demonstrates that the FLVCR1-AS1/miR-4731-5p/E2F2 axis is a novel signaling in glioma and may be a potential target for tumor therapy.     

Long noncoding RNA lncBRM promotes proliferation and invasion of colorectal cancer by sponging miR-204-3p and upregulating TPT1

Long noncoding RNAs (lncRNAs) are characterized as a type of noncoding RNAs over 200 nucleotides with little or none protein-coding potential. In the past years, lncRNAs have been proved to participant in many physiological and pathological processes. However, the role of lncRNAs in colorectal cancer (CRC) still needs more attentions. In our study, we found that lncBRM was highly expressed in CRC samples and the expression level of lncBRM was correlated with metastasis and advanced stage in CRC patients. And also, we showed that high expression of lncBRM predicted poor prognosis. Furthermore, we found that knockdown of lncBRM impaired the proliferation, migration and invasion of CRC cells while overexpressing of lncBRM promotes the proliferation, migration and invasion of CRC cells. Mechanically, we found that lncBRM served as a sponge of miR-204-3p that targeted TPT1. Highly expressed TPT1 can promote the proliferation, migration and invasion of CRC cells. In conclusion, we found that lncBRM was highly expressed in CRC and sponged miR-204-3p to modulate the expression of TPT1.     

Retracted: LncRNA MT1JP functions as a tumor suppressor via regulating miR-214-3p expression in bladder cancer

Growing evidence suggested that the long noncoding RNAs (lncRNAs) regulate several pathophysiological processes in tumorigenesis and may be new biomarkers for tumor therapy. In this study, we studied the expression and role of lncRNA MT1JP in the development of bladder cancer. We demonstrated that the expression of MT1JP was downregulated in bladder tumor samples and cell lines. Ectopic expression of MT1JP suppressed cell proliferation, cycle, and invasion in bladder cancer. In addition, our result suggested that miR-214-3p overexpression decreased the luciferase activity of wild-type MT1JP but not mutated-type MT1JP and elevated expression of MT1JP decreased miR-214-3p expression in the bladder cancer cell. Furthermore, we indicated that the expression of miR-214-3p was upregulated in bladder tumor samples and cell lines. Ectopic expression of miR-214-3p promoted cell proliferation, cycle, and invasion in bladder cancer. MT1JP suppressed cell proliferation, cycle, and invasion via negative modulation of miR-214-3p in bladder cancer. These data suggested that lncRNA MT1JP acts a tumor suppressor gene in bladder cancer progression, considering MT1JP as a new therapeutic target in bladder cancer.