Mengmeng Guo

Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells

BackgroundColon carcinoma is one of the commonly tumors that threaten human beings as its highly morbidity and mortality. Recent evidences suggested that microRNA-21 (miR-21) played an important role in the development of colon carcinoma and might be a potential biological marker for the diagnosis and prognosis of colon carcinoma. However, the potential effect of miR-21 based therapeutic studies in colon carcinoma remains to be fully elucidated.MethodsIn present study, we constructed an eukaryotic expression vector encoding antisense oligonucleotides against miR-21 (termed as p-miR-21-ASO) and the expression of miRNA-21 in human colon cancer was detected by Real-time PCR. To assess its possible effect on the proliferation and migration capacity of human colon carcinoma cells in vitro, CCK-8 assay, colony formation assay and cell invasion, as well as migration assay, were performed respectively. Moreover, PTEN, one of target molecules of miRNA-21, was analyzed by Western blot and Fluorescence activated cell sorter assay. Finally, the transduction of AKT and ERK pathways in human colon carcinoma cells was determined by Western blot.ResultsWe found that transiently transfection of p-miR-21-ASO could efficiently decrease the relative expression of miR-21 in human colon carcinoma HCT116 cells, accompanied by impaired proliferation and clone formation. Furthermore, we found that down-regulation of miR-21 also could significantly abrogate the invasion and migration capacity in vitro, as well as the expression of vascular endothelial growth factor which is critical for the metastatic capacity of colon carcinoma cells. Mechanistic evidence showed that down-regulation of miR-21 increased the expression of its target molecule PTEN in HCT116 cells. Finally, we revealed that the expression level of both phosphor-ERK1/2 and phosphor-AKT also were altered.ConclusionsTherefore, our data suggested miR-21 ASO against miR-21 might be a useful strategy to alter the expression of miR-21 in colon carcinoma cells, which was helpful for the development of miR-21-based therapeutic strategies against clinical colon carcinoma.

MiR-21 controls in situ expansion of CCR6⁺ regulatory T cells through PTEN/AKT pathway in breast cancer.

Our recent evidence showed that prior expansion of CCR6+ Foxp3+ regulatory T cells (Tregs) was important for their dominant enrichment in tumor tissue, which was closely related to poor prognosis of breast cancer patients. However, the underlying regulation mechanism of expansion of CCR6+ Tregs in situ remains largely unknown. In this study, we reported that miR‐21 was highly expressed in CCR6+ Tregs in tumor tissues from a murine breast cancer model. And silencing of miR‐21 could significantly reduce the proliferation of CCR6+ Tregs in vitro. Adoptive cell‐transfer assay further showed that silencing of miR‐21 could alter the enrichment of CCR6+ Tregs in the tumor mass and endow effectively antitumor effect of CD8+ T cells using a murine breast cancer model. Mechanistic evidence showed that silencing of miR‐21 enhanced the expression of its target phosphatase and tensin homolog deleted on chromosome ten (PTEN) and subsequently altered the activation of Akt pathway, which was ultimately responsible for reduced proliferation activity of CCR6+ Tregs. Finally, we further revealed that miR‐21 was also highly expressed on CCR6+ Tregs in clinical breast cancer patients. Therefore, miR‐21 can act as a fine tuner in the regulation of PTEN/Akt pathway transduction in the expansion of CCR6+ Tregs in tumor sites and provided a novel insight into the development of therapeutic strategies for promoting T‐cell immunity by regulating distinct subset of Tregs through targeting specific miRNAs.

MicroRNA-7 Deficiency Ameliorates the Pathologies of Acute Lung Injury through Elevating KLF4

Recent evidence showed that microRNA-7 (miR-7) played an important role in the pathologies of lung-related diseases. However, the potential role of miR-7 in acute lung injury (ALI) still remains poorly understood. Here, we assessed the effect of miR-7 deficiency on the pathology of ALI. We, first, found that the expression of miR-7 was upregulated in lung tissue in murine LPS-induced ALI model. Notably, we generated miR-7 knock down mice by using miRNA-Sponge technique and found that miR-7 deficiency could ameliorate the pathologies of lung as evidenced by accelerated body weight recovery, reduced level of bronchoalveolar lavage (BAL) proinflammatory cytokines and decreased number of BAL cells in ALI mice. Moreover, the proportion and number of various immune cells in BAL, including innate immune cell F4/80+ macrophages, γδT cells, NK1.1+ T cells, and CD11c+DCs, as well as adaptive immune cell CD4+ T cells and CD8+ T cells, also significantly changed, respectively. Mechanistic evidence showed that KLF4, a target molecule of miR-7, was upregulated in lung tissues in ALI model, accompanied by altered transduction of NF-κB, AKT, and ERK pathway. These data provided a previously unknown role of miR-7 in pathology of ALI, which could ultimately aid the understanding of development of ALI and the development of new therapeutic strategies against clinical inflammatory lung diseases.

Targeted Expression of miR-7 Operated by TTF-1 Promoter Inhibited the Growth of Human Lung Cancer through the NDUFA4 Pathway

Targeted expression of gene technique is an important therapeutic strategy for lung cancer. MicroRNA-7 has been well documented as a promising tumor suppressor but never been test in specific gene-promoter-targeted expression in cancer gene therapy. Here, we first evaluated the efficacy of miR-7 expression operated by the promoter of TTF-1, a lineage-specific oncogene in lung cancer, in vitro using an eukaryotic vector of TTF-1-promoter-operated expression of miR-7 (termed as p-T-miR-7). Interestingly, using a nude mice model, the growth and metastasis of human lung cancer cells in vivo were significantly reduced in remote hypodermic injection of the p-T-miR-7 group, accompanied by increased expression of miR-7 and reduced transduction of the Akt and Erk pathway in situ. Mechanism aspect, global gene expression analysis showed that downregulation of NDUFA4, a novel target of miR-7, contributed to the effects of miR-7 expression operated by TTF-1 promoter on the growth and metastasis of human lung cancer cells, as well as altered transduction of the Akt and Erk pathway. Finally, there was no significant difference in weight or histopathology of other organs. These data provided a basis for development of novel modality of miRNA-based targeted expression therapy against clinical lung cancer.

The Role of MicroRNAs in Aβ Deposition and Tau Phosphorylation in Alzheimer's Disease.

Alzheimer’s disease (AD), with main clinical features of progressive impairment in cognitive and behavioral functions, is the most common degenerative disease of the central nervous system. Recent evidence showed that microRNAs (miRNAs) played important roles in the pathological progression of AD. In this article, we reviewed the promising role of miRNAs in both Aβ deposition and Tau phosphorylation, two key pathological characters in the pathological progression of AD, which might be helpful for the understanding of pathogenesis and the development of new strategies of clinical diagnosis and treatment of AD.

MicroRNAs expression profile in CCR6(+) regulatory T cells.

Backgroud. CCR6+ CD4+ regulatory T cells (CCR6+ Tregs), a distinct Tregs subset, played an important role in various immune diseases. Recent evidence showed that microRNAs (miRNAs) are vital regulators in the function of immune cells. However, the potential role of miRNAs in the function of CCR6+ Tregs remains largely unknown. In this study, we detected the expression profile of miRNAs in CCR6+ Tregs. Materials and Methods. The expression profile of miRNAs as well as genes in CCR6+ Tregs or CCR6- Tregs from Balb/c mice were detected by microarray. The signaling pathways were analyzed using the Keggs pathway library. Results. We found that there were 58 miRNAs significantly upregulated and 62 downregulated up to 2 fold in CCR6+ Tregs compared with CCR6- Tregs. Moreover, 1,391 genes were observed with 3 fold change and 20 signaling pathways were enriched using the Keggs pathway library. Conclusion. The present data showed CCR6+ Tregs expressed specific miRNAs pattern, which provides insight into the role of miRNAs in the biological function of distinct Tregs subsets.

Promoter-operating targeted expression of gene therapy in cancer: current stage and prospect

The technique of targeted expression of interesting genes, including distinct delivery systems and specific gene promoter-operating expression, is an important strategy for gene therapy against cancers. Up to now, extensive literature documented the efficacy of distinct delivery systems, such as the liposome system, nano-particle system, polyetherimide (PEI) system, and so on, in cancer gene therapy. However, a related document on the potential value of using a specific gene promoter, such as a tumor suppressor, in cancer gene therapy was still scary. The main obstacle might be that the selection of an ideal gene promoter to operate interesting gene expression in cancer gene therapy is still not fully understood. Therefore, many efforts need to be done in order to make it a real power tool for the human clinical treatment of cancer patients. The purpose of this review is to clarify the current state and some problematics in development of promoter-operating targeted expression of interesting genes and highlight its potential in cancer gene therapy.

Antisense Oligonucleotides against miR-21 Inhibit the Growth and Metastasis of Colorectal Carcinoma via the DUSP8 Pathway

Accumulating research has documented that microRNA-21 (miR-21) plays an important role in the development of human colorectal carcinoma (CRC). Our recent work also showed that antisense oligonucleotides (ASOs) against miR-21 can impair the growth of CRC cells in vitro. However, the potential role of miR-21 in gene therapy against CRC remains to be fully elucidated. Here, we further observed the effect of ASOs against miR-21 on the growth and metastasis of CRC in vivo using a xenograft model of human CRC. We found that ASOs could effectively inhibit the growth and metastasis of CRC in vivo, accompanied by downregulated expression of miR-21 and reduced transduction of the AKT and ERK pathway. Mechanically, global gene expression analysis showed that the expression of DUSP8, a novel target of miR-21, was upregulated in tumor mass. Furthermore, overexpression of DUSP8 could remarkably suppress the proliferation and migration of CRC cells in vitro. Finally, downregulation of DUSP8 could abrogate the effects of ASOs against miR-21 on the proliferation and migration of CRC cells, as well as altered transduction of the AKT and ERK signaling pathway. Together, these data suggest that ASOs against miRNAs are an attractive and potential therapeutic for the treatment of human CRC and warrant further development.

MicroRNA-126 Deficiency Affects the Development of Thymus CD4 + Single-Positive Cells through Elevating IRS-1

Background: MicroRNA-126 (miR-126), a distinct miRNA family member, has been reported to be involved in the development and function of some types of immune cells. However, the potential role of miR-126 in the development of CD4+ T cells remains to be elucidated. Objectives: To investigate the potential role of miR-126 in the development of CD4+ T cells in the thymus and explore its significance. Methods: The relative expression level of miR-126 in thymus CD4+ single-positive (SP) cells was detected by Real-Time PCR assay. The possible change in thymus tissue was assessed by histopathology. The total cell number of thymocytes and the expression of activation-associated molecules including CD62L, CD69, and CD44, as well as proliferation-associated nuclear antigen Ki-67, in CD4+ SP cells were assessed by flow cytometric analysis. The expression of IRS-1 and related signaling pathways including Akt and Erk were determined by flow cytometric analysis. Results: Compared with that in wild-type (WT) mice, the total cell number of thymocytes in miR-126 knockdown (KD) mice increased significantly. Moreover, the proportion and absolute cell number of thymic CD4+ SP cells decreased significantly in miR-126 KD mice. Further analysis showed that the frequencies of activation-associated molecules including CD62L, CD69, and CD44, as well as proliferation-associated nuclear antigen Ki-67 in CD4+ SP cells also changed significantly, respectively. Mechanism aspect, the expression level of IRS-1, a putative target of miR-126, increased significantly in CD4+ SP cells in miR-126 KD mice. Moreover, the expression levels of the signaling molecules phosphorylated (p)-Akt and p-Erk also changed significantly. Conclusions: Our work is the first to reveal a previously unknown role of miR-126 in the development of CD4+ SP cells in the thymus, which might ultimately benefit studies on development of thymocytes.

MicroRNA-126 deficiency enhanced the activation and function of CD4+T cells by elevating IRS-1 pathway

Recent evidence has shown that microRNA‐126 (miR‐126) has been involved in the development and function of immune cells, which contributed to the pathogenesis of related clinical diseases. However, the potential role of miR‐126 in the development and function of CD4+ T cells remains largely unknown. Here we first found that the activation and proliferation, as well as the expression of interferon (IFN)‐γ, of CD4+T cells from miR‐126 knock‐down (KD) mice using the miRNA‐sponge technique were enhanced significantly in vitro, compared with those in CD4+ T cells from wild‐type (WT) mice. To monitor further the possible effect of miR‐126 deficiency on the function of CD4+ T cells in vivo, we used dextran sulphate sodium (DSS)‐induced murine model of acute autoimmune colitis and found that miR‐126 deficiency could elevate the pathology of colitis. Importantly, the proportion of CD4+ T cells in splenocytes increased significantly in miR‐126KD mice. Moreover, the expression levels of CD69 and CD44 on CD4+ T cells increased significantly and the expression level of CD62L decreased significantly. Of note, adoptive cell transfer assay showed that the pathology of colitis was more serious in carboxyfluorescein succinimidyl ester (CFSE)‐labelled miR‐126KD CD4+ T cell‐transferred group, compared with that in the CFSE‐labelled WT CD4+ T cells transferred group. Consistently, the expression levels of CD69 and CD44 on CFSE+ cells increased significantly. Furthermore, both the proliferation and IFN‐γ secretion of CFSE+ cells also increased significantly in the CFSE‐labelled miR‐126KD CD4+ T cell‐transferred group. Mechanistic evidence showed that the expression of insulin receptor substrate 1 (IRS‐1), as a functional target of miR‐126, was elevated in CD4+ T cells from miR‐126KD mice, accompanied by altered transduction of the extracellular regulated kinase, protein B (AKT) and nuclear factor kappa B (NF‐κB) pathway. Our data revealed a novel role in which miR‐126 was an intrinsic regulator in the function of CD4+ T cells, which provided preliminary basis for exploring further the role of miR‐126 in the development, function of CD4+ T cells and related clinical diseases.