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Spermatogonial stem cells （SSCs） can produce numerous male gametes after transplantation into recipient testes, presenting a valuable approach for gene therapy and continuous production of gene-modified animals. However, successful genetic manipulation of SSCs has been limited, partially due to complexity and low efficiency of currently available genetic editing techniques. Here, we show that efficient genetic modifications can be introduced into SSCs using the CRISPR-Cas9 system. We used the CRISPR-Cas9 system to mutate an EGFP transgene or the endogenous Crygc gene in SCCs. The mutated SSCs underwent spermatogenesis after transplantation into the seminiferous tubules of infertile mouse testes. Round spermatids were generated and, after injection into mature oocytes, supported the production of heterozygous offspring displaying the corresponding mutant phenotypes. Furthermore, a disease-causing mutation in Crygc （Crygc-/-） that pre-existed in SSCs could be readily repaired by CRISPR-Cas9-induced nonhomologous end joining （NHEJ） or homology-directed repair （HDR）, resulting in SSC lines carrying the corrected gene with no evidence of off-target modifications as shown by whole-genome sequencing. Fertilization using round spermatids generated from these lines gave rise to offspring with the corrected phenotype at an efficiency of 100%. Our results demonstrate efficient gene editing in mouse SSCs by the CRISPR-Cas9 system, and provide the proof of principle of curing a genetic disease via gene correction in SSCs.

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin β1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin β1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3'-UTRs of integrin β1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin β1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin β1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.

Magnolol is a traditional Chinese medicine from the root and bark of Magnolia officinalis . It has long been used to treat anxiety, cough, headache and allergies, as well as a variety of inflammations. Lung inflammation is a key event in the pathogenesis of asthma and chronic obstructive pulmonary disease. The present study sought to examine the effects of magnolol on tumor necrosis factor (TNF)-α-induced upregulation of intercellular adhesion molecule-1 (ICAM-1), activation of the nuclear factor (NF)-κB and mitogen-activated protein kinase (MAPK) signaling pathway in cultured human pulmonary epithelial cells, and adhesion of human macrophage-like U937 cells to A549 cells. A549 cells were incubated with magnolol at 25 and 50 μmol/l. Then, 20 ng/ml TNF-α was used to activate the cells. Magnolol inhibited the growth of human pulmonary epithelial A549 cells in a dose- and time-dependent manner. Magnolol suppressed the adhesion of U937 cells to TNF-α-induced A549 cells. In cultured human pulmonary epithelial A549 cells, magnolol decreased TNF-α-induced upregulation of ICAM-1. Magnolol repressed TNF-α-induced activation of NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways in A549 cells by inhibiting phosphorylation of NF-κB, p38, extracellular signal-regulated kinase (ERK) 1/2, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). These findings support the hypothesis that magnolol inhibits the inflammatory process in lung epithelial A549 cells by suppressing the ICAM-1 and NF-κB and MAPK signaling pathways. Taken together, these results indicate that magnolol offers significant potential as a therapeutic treatment for inflammatory diseases of the lungs including asthma, sepsis, and chronic obstructive pulmonary disease.

C-kit positive (c-kit + ) cells are usual tissue-specific stem cells. However, in postnatal testis, undifferentiated spermatogonial stem cells (SSCs) are c-kit negative (c-kit − ) and activation of c-kit represents the start of SSC differentiation, leaving an intriguing question whether other c-kit + cells exist and participate in the postnatal development of testis. To this end, a feasible system for testicular reconstitution, in which a specific type of cells can be manipulated, is needed. Here, we first establish de novo morphogenesis of testis by subcutaneous injection of testicular cells from neonatal testes into the backs of nude mice. We observe testicular tissue formation and spermatogenesis from all injected sites. Importantly, functional spermatids can be isolated from these testicular tissues. Using this system, we systemically analyze the roles of c-kit + cells in testicular reconstitution and identify a small population of cells (c-kit + :CD140a + :F4/80 + ), which express typical markers of macrophages, are critical for de novo morphogenesis of testis. Interestingly, we demonstrate that these cells are gradually replaced by peripheral blood cells of recipient mice during the morphogenesis of testis. Thus, we develop a system, which may mimic the complete developmental process of postnatal testis, for investigating the testicular development and spermatogenesis.

Triple-negative breast cancer (TNBC) exhibits poor prognosis, with high metastasis and low survival. Long non-coding RNAs (lncRNAs) play critical roles in tumor progression. Here, we identified lncRNA MIR100HG as a pro-oncogene for TNBC progression. Knockdown of MIR100HG decreased cell proliferation and induced cell arrest in the G1 phase, whereas overexpression of MIR100HG significantly increased cell proliferation. Furthermore, MIR100HG regulated the p27 gene to control the cell cycle, and subsequently impacted the progression of TNBC. In analyzing its underlying mechanism, bioinformatics prediction and experimental data demonstrated that MIR100HG participated in the formation of RNA–DNA triplex structures. MIR100HG in The Cancer Genome Atlas (TCGA) and breast cancer cell lines showed higher expression in TNBC than in other tumor types with poor prognosis. In conclusion, our data indicated a novel working pattern of lncRNA in TNBC progression, which may be a potential therapeutic target in such cancers.

Aberrant alternative splicing has been highlighted as a potential hallmark of cancer. Here, we identify TDP43 (TAR DNA-binding protein 43) as an important splicing regulator responsible for the unique splicing profile in triple-negative breast cancer (TNBC). Clinical data demonstrate that TDP43 is highly expressed in TNBC with poor prognosis. Knockdown of TDP43 inhibits tumor progression, including proliferation and metastasis, and overexpression of TDP43 promotes proliferation and malignancy of mammary epithelial cells. Deep sequencing analysis and functional experiments indicate that TDP43 alters most splicing events with splicing factor SRSF3 (serine/arginine-rich splicing factor 3), in the regulation of TNBC progression. The TDP43/SRSF3 complex controls specific splicing events, including downstream genes PAR3 and NUMB. The effect of reduced metastasis and proliferation upon the knockdown of TDP43 or SRSF3 is mediated by the splicing regulation of PAR3 and NUMB exon 12, respectively. The TDP43/SRSF3 complex and downstream PAR3 isoform are potential therapeutic targets for TNBC.

IL-2 is critical to the activation, growth, and survival of T cells and NK cells, and maintains the delicate balance between auto-immunity and anti-neoplasm surveillance. High IL-2 doses have clear antitumor capabilities, but also have severe side effects that limit its clinical use. Side effects include the vascular leak syndrome (VLS), which results in lung edema and liver damage. Therefore, a new version of IL-2 that does not induce organ toxicity would improve IL-2-based immunotherapy. We conducted a systematic screening by changing one amino acid at a time at the interaction area of IL-2 with its receptor IL-2R to select one particular mutant IL-2, FSD13, in which the proline at position 65 was substituted by lysine (P65L). FSD13 had a greater ability than wild-type IL-2 in stimulating CD4 + T, CD8 + T, and NK cell proliferation, enhancing the expression of CD69, CD183, CD44, and CD54 in these cells, and triggering cancer cell apoptosis. FSD13 had three-time lower than wild-type IL-2 in inducing CD4 + T to Tregs. Compared with wild-type IL-2, FSD13 greatly limited the growth, invasion into adjacent tissues, and metastasis of melanoma metastatic into the lung. In contrast to wild-type IL-2, high dose of FSD3 did not alter structures and induce any pathogenic changes in the liver and lung. Thus, we generated a novel the IL-2 mutant, FSD13, by targeting a different area than previously reported. FSD13 surpasses the wild-type IL-2’s ability in stimulating the antitumor immune cell functions, but exerts much less systemic toxicity.

Our pilot study using miRNA arrays found that miRNA-29c (miR-29c) is differentially expressed in the paired low-metastatic lung cancer cell line 95C compared to the high-metastatic lung cancer cell line 95D. Bioinformatics analysis shows that integrin [beta]1 and matrix metalloproteinase 2 (MMP2) could be important target genes of miR-29c. Therefore, we hypothesized that miR-29c suppresses lung cancer cell adhesion to extracellular matrix (ECM) and metastasis by targeting integrin [beta]1 and MMP2. The gain-of-function studies that raised miR-29c expression in 95D cells by using its mimics showed reductions in cell proliferation, adhesion to ECM, invasion and migration. In contrasts, loss-of-function studies that reduced miR-29c by using its inhibitor in 95C cells promoted proliferation, adhesion to ECM, invasion and migration. Furthermore, the dual-luciferase reporter assay demonstrated that miR-29c inhibited the expression of the luciferase gene containing the 3'-UTRs of integrin [beta]1 and MMP2 mRNA. Western blotting indicated that miR-29c downregulated the expression of integrin [beta]1 and MMP2 at the protein level. Gelatin zymography analysis further confirmed that miR-29c decreased MMP2 enzyme activity. Nude mice with xenograft models of lung cancer cells confirmed that miR-29c inhibited lung cancer metastasis in vivo, including bone and liver metastasis. Taken together, our results demonstrate that miR-29c serves as a tumor metastasis suppressor, which suppresses lung cancer cell adhesion to ECM and metastasis by directly inhibiting integrin [beta]1 and MMP2 expression and by further reducing MMP2 enzyme activity. The results show that miR-29c may be a novel therapeutic candidate target to slow lung cancer metastasis.

Magnolol is a traditional Chinese medicine from the root and bark of Magnolia officinalis. It has long been used to treat anxiety, cough, headache and allergies, as well as a variety of inflammations. Lung inflammation is a key event in the pathogenesis of asthma and chronic obstructive pulmonary disease. The present study sought to examine the effects of magnolol on tumor necrosis factor (TNF)-[alpha]-induced upregulation of intercellular adhesion molecule-1 (ICAM-1), activation of the nuclear factor (NF)-[kappa]B and mitogen-activated protein kinase (MAPK) signaling pathway in cultured human pulmonary epithelial cells, and adhesion of human macrophage-like U937 cells to A549 cells. A549 cells were incubated with magnolol at 25 and 50 [mu]mol/l. Then, 20 ng/ml TNF-[alpha] was used to activate the cells. Magnolol inhibited the growth of human pulmonary epithelial A549 cells in a dose- and time-dependent manner. Magnolol suppressed the adhesion of U937 cells to TNF-[alpha]-induced A549 cells. In cultured human pulmonary epithelial A549 cells, magnolol decreased TNF-[alpha]-induced upregulation of ICAM-1. Magnolol repressed TNF-[alpha]-induced activation of NF-[kappa]B and mitogen-activated protein kinase (MAPK) signaling pathways in A549 cells by inhibiting phosphorylation of NF-[kappa]B, p38, extracellular signal-regulated kinase (ERK) 1/2, and stress-activated protein kinase (SAPK)/c-Jun N-terminal kinase (JNK). These findings support the hypothesis that magnolol inhibits the inflammatory process in lung epithelial A549 cells by suppressing the ICAM-1 and NF-[kappa]B and MAPK signaling pathways. Taken together, these results indicate that magnolol offers significant potential as a therapeutic treatment for inflammatory diseases of the lungs including asthma, sepsis, and chronic obstructive pulmonary disease.[PUBLICATION ABSTRACT]