Unraveling susceptibility genes: A contemporary overview of autoimmune thyroid diseases.

Autoimmune thyroid diseases (AITDs), including Graves' disease and Hashimoto's thyroiditis, are organ-specific autoimmune disorders characterized by conditions including goiter, autoimmune thyroiditis, hyperthyroidism, and hypothyroidism, which represent the most severe clinical manifestations of AITDs. The prevalence of autoimmune thyroid disorders is on the rise, influenced by increased environmental factors and changes in modern lifestyles. Understanding the pathophysiology of AITDs is crucial for identifying key factors that affect the disease's onset, progression, and recurrence, thereby laying a solid foundation for precise diagnosis and treatment. The development of AITDs involves a complex interplay of environmental influences, immune dysfunctions, and genetic predispositions. Genetic predispositions, in particular, are significant, with numerous genes identified as being linked to AITDs. This article focuses on examining the genes vulnerable to AITDs to deepen our understanding of the relevant genetic contributors, ultimately facilitating the development of effective prevention and treatment methods.

MicroRNA-21: A promising biomarker for the prognosis and diagnosis of non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most common subtype of lung cancer worldwide. The high mortality rate of NSCLC is due to a limited number of diagnosis being made at an early stage of disease. Therefore, the development of a novel biological marker for the diagnosis and prognosis prediction of NSCLC remains urgent. Current literature shows that microRNA-21 (miRNA-21/miR-21), as an oncogenic miRNA, is involved in the growth, metastasis and apoptosis of NSCLC cells through its control of various target molecules and signaling pathways. Notably, a growing body of evidence further shows that miR-21 is closely associated with the prognosis prediction, recurrence and diagnosis of cancer patients, indicating that miR-21 may be a novel promising biomarker for the diagnosis and prognosis prediction of NSCLC. The present review aimed to provide a summary of recent findings on the associated progression toward finding a novel biomarker for NSCLC.

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.

MicroRNA-7: a promising new target in cancer therapy.

The incidence of tumors with life-threatening effects has increased gradually over time; however, the mechanisms involved in tumor development have not been fully elucidated. Recent studies have shown that microRNA-7 (miR-7), which is endogenous non-coding RNA molecules of approximately 23 nucleotides, plays an important role in the occurrence and development of tumors as a key tumor suppressor. Mechanistic evidence showed that miR-7 is closely related to the growth, metastasis, and prognosis of various malignant tumors through regulating different target molecules, which suggest that miR-7 may be a new target for the clinical diagnosis and treatment of various tumors. In this review, we summarize current knowledge of the relationship between miR-7 and tumor development, diagnosis, and treatment.

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

BACKGROUND: Colon 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. METHODS: In 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. RESULTS: We 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. CONCLUSIONS: Therefore, 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.

[Identification of miR-126 knockdown mouse and the change of blood glucose].

OBJECTIVE: To detect the expression of miR-126 in different tissues and organs and the change of peripheral blood glucose in microRNA-126 knock down (miR-126 KD) mouse, and to explore the pathological significance. METHODS: Total RNAs were isolated from twelve kinds of tissues and organs in wild-type mouse (WT) and miR-126 KD mouse respectively. Th en, the expression level of miR-126 was detected by real-time PCR assay. Th e levels of peripheral blood glucose and body weight of miR-126 KD mice were measured. Th e pathologic changes of pancreas and lung tissue were observed by HE staining. RESULTS: Compared with the WT mice, the relative expression of miR-126 in spleen, liver, muscle and lung from the miR-126 KD mice were dramatically decreased respectively (P<0.05). The level of peripheral blood glucose in the miR-126 KD mouse increased significantly at seven week and sixteen week after the birth (P<0.05). HE staining showed that the pathological structure of pancreas and liver were abnormal. The body weight of miR-126 KD mice was increased obviously from thirteen week after birth (P<0.05). CONCLUSION: Peripheral blood glucose levels in the miR-126 KD mouse were dramatically elevated, which might be related to the pathological changes in the structure of pancreas and liver. These results suggest that miR-126 may play an important role in the metabolism of blood glucose and the development of type 2 diabetes mellitus.

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.

[Preparation of polyclonal antibody for heat shock protein 16.3].

OBJECTIVE: To induce the prokaryotic expression of His-heat shock protein 16.3 (Hsp16.3) and prepare its polyclonal antibody. METHODS: DNA primers were designed and synthesized, and Hsp16.3 cDNA was amplified from Mycobacterium tuberculosis H37Rv by PCR. Thereafter, the recombinant vector pET28a-Hsp16.3 was constructed, cloned and induced to express. The His-Hsp16.3 protein was purified using a Ni-NTA column. After identification by SDS-PAGE, the purified protein was used to immunize the New Zealand white rabbits to prepare the polyclonal antibody. The titer and biological activity of the polyclonal antibody were analyzed by ELISA and Western blotting, respectively. RESULTS: The prokaryotic expression vector pET28a-Hsp16.3 was constructed and expressed well in E.coil BL21(DE3). SDS-PAGE showed that the relative molecular mass was about 20 000. The titer of the polyclonal antibody reached 1:800. The purified antibody was proved to have a good specificity. CONCLUSION: The His-Hsp16.3 has been cloned, expressed and purified successfully. Polyclonal antibody of His-Hsp16.3 protein has been obtained as well.

[Over-expressed microRNA-7 inhibits the growth of human lung cancer cells via suppressing CGGBP1 expression].

OBJECTIVE: To investigate the effect of microRNA-7 (miR-7) over-expression on the growth of human lung cancer cells in vivo and in vitro and explore its possible mechanism. METHODS: The eukaryotic expression vector of pcDNA3.1 encoding miR-7 (p-miR-7) was transiently transfected into human lung cancer 95D cells in vitro. The proliferation of cells was detected by MTT assay and colony formation assay. Moreover, the expressions of nuclear antigen Ki-67 and CGG binding protein 1 (CGGBP1) were detected by immunofluorescence assay. Human lung cancer model in nude mice was established. Next, p-miR-7 vector was directly injected into local tumor tissue. Then, tumor size was measured and the survival time of mice was observed. The expression level of miR-7 in the tumor tissue was determined by real-time PCR. Finally, the expressions of Ki-67 and CGGBP1 were detected by immunohistochemistry. RESULTS: Over-expression of miR-7 could significantly inhibit the growth of 95D cells in vitro (P<0.05), accompanied by the remarkably reduced expressions of Ki-67 and CGGBP1 (P<0.05). Moreover, compared with those in control group, the expression level of miR-7 increased significantly in p-miR-7 injected group (P<0.05). Meanwhile, the growth of tumors in injected group was slower than in the control group. Consistently, the survival time of mice was dramatically prolonged in p-miR-7 injected group (P<0.05). The expression levels of Ki-67 and CGGBP1 also remarkably decreased in tumor tissue (P<0.05). CONCLUSION: Over-expression of miR-7 could significantly inhibit the growth of human lung cancer cells in vivo and in vitro, which might be related to the down-regulated expression of tumor growth-associated protein CGGBP1.

Expression of TNF-alpha leader sequence renders MCF-7 tumor cells resistant to the cytotoxicity of soluble TNF-alpha.

Transmembrane TNF-alpha (tmTNF-alpha) contains a leader sequence (LS) that can be phosphorylated and cleaved at its cytoplasmic portion, inducing IL-12 production. We observed that the breast cancer cell line MDA-MB-231 expressing transmembrane TNF-alpha (tmTNF-alpha) at high level was resistant to soluble TNF-alpha (sTNF-alpha)-induced cytotoxicity, accompanied by constitutive NF-kappaB activation. In contrast, MCF-7 cells expressing tmTNF-alpha at very low level were sensitive to sTNF-alpha-induced cell death and had no detectable NF-kappaB activation. Consistently, siRNA-mediated tmTNF-alpha knockdown blocked NF-kappaB activation and rendered MDA-MB-231 sensitive. To test our hypothesis that TNF-LS may play an important role in determining the sensitivity of tumor cells to sTNF-alpha, we stably transfected MCF-7 cells with TNF-LS. We found that transfection of TNF-LS or wild-type TNF-alpha containing LS constitutively activated NF-kappaB and conferred the cytotoxic resistance of MCF-7 cells, while transfection of a mutant tmTNF-alpha lacking the cytoplasmic segment of LS neither activated NF-kappaB nor affected the sensitivity. However, NF-kappaB inhibitor PDTC suppressed NF-kappaB activation and reconstituted sensitivity of TNF-LS/MCF-7 cells. To check whether TNF-LS is required to be cleaved or internalized for NF-kappaB activation to occur, we used signal peptide peptidase inhibitor (Z-LL)(2)-ketone and receptor internalization inhibitor MDC to treat cells. Interestingly, both inhibitors increased TNF-LS expression on the cell surface and enhanced NF-kappaB activation. These results indicate that membrane-anchored TNF-LS contributes to constitutive activation of NF-kappaB and resistance to sTNF-alpha-induced cell death. Therefore, TNF-LS appears to be responsible for tmTNF-alpha-induced resistance in the breast cancer cells.

Transmembrane TNF-alpha mediates "forward" and "reverse" signaling, inducing cell death or survival via the NF-kappaB pathway in Raji Burkitt lymphoma cells.

Interestingly, some lymphoma cells, expressing high levels of transmembrane (tm)TNF-alpha, are resistant to secretory (s)TNF-alpha-induced necrosis but sensitive to tmTNF-alpha-mediated apoptosis. As tmTNF-alpha mediates "forward" as well as "reverse" signaling, we hypothesize that a balanced signaling between forward and reverse directions may play a critical role in determining the fate of cells bearing tmTNF-alpha. Using Raji cells as a model, we first added exogenous tmTNF-alpha on fixed, transfected NIH3T3 cells onto Raji cells to examine tmTNF-alpha forward signaling and its effects, showing that constitutive NF-kappaB activity and cellular inhibitor-of-apoptosis protein 1 transcription were down-regulated, paralleled with Raji cell death. As Raji cells express tmTNF-alpha, an inhibition of their tmTNF-alpha expression by antisense oligonucleotide caused down-regulation of NF-kappaB activity. Conversely, increasing tmTNF-alpha expression by suppressing expression of TNF-alpha-converting enzyme that cleaves tmTNF-alpha led to an enhanced activation of NF-kappaB, indicating that tmTNF-alpha, but not sTNF-alpha, contributes to constitutive NF-kappaB activation. We next transfected Raji cells with a mutant tmTNF-alpha lacking the intracellular domain to competitively suppress reverse signaling via tmTNF-alpha; as expected, constitutive NF-kappaB activity was decreased. In contrast, treating Raji cells with sTNFR2 to stimulate reverse signaling via tmTNF-alpha enhanced NF-kappaB activation. We conclude that tmTNF-alpha, when highly expressed on tumor cells and acting as a receptor, promotes NF-kappaB activation through reverse signaling, which is helpful to maintain tumor cell survival. On the contrary, tmTNF-alpha, when acting as a ligand, inhibits NF-kappaB activity through forward signaling, which is inclined to induce tumor cell death.

Involvement of ATP-sensitive potassium channels in proliferation and differentiation of rat preadipocytes.

This paper was aimed to investigate the effects of ATP-sensitive potassium channels on the proliferation and differentiation of rat preadipocytes. We examined the expression of sulphonylurea receptor 2 (SUR2) mRNA in preadipocytes and adipocytes obtained by inducing for 5 d and the effects of the inhibitor (glibenclamide) and opener (diazoxide) of ATP-sensitive potassium channels on the expression of SUR2 mRNA in preadipocytes by real-time PCR. Preadipocyte proliferation and cell cycle were measured by MTT spectrophotometry and flow cytometer. The content of intracellular lipid was measured by oil red O staining, cell diameter was determined by Image-Pro Plus 5.0 software and the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) mRNA was estimated by RT-PCR. SUR2 mRNA was expressed in both preadipocytes and adipocytes obtained by inducing for 5 d, and the expression in adipocytes was obviously higher than that in preadipocytes. Glibenclamide inhibited the expression of SUR2 mRNA in preadipocyte, promoted preadipocyte proliferation in a dose-dependent manner, increased the cell percentages in G(2)/M + S phase, increased lipid content, augmented adipocyte diameter, and promoted the expression of PPAR-gamma mRNA. But the actions of diazoxide were contrary to those of glibenclamide. These results suggest that ATP-sensitive potassium channels regulate the proliferation and differentiation of preadipocytes, and PPAR-gamma is probably involved in the effect of ATP-sensitive potassium channels.