ObjectiveTo investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor β1(TGF-β1) by targeted regulation of miRNA219-5P (miR219-5P). MethodsThe tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-β1. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours. ResultsAfter TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P<0.05). Intracellular expression of miR219-5P was inhibited by miR219-5P mimics inhibitor, however, the protein expression of Smad4 was significantly increased (P<0.05). Luciferase reporter gene test showed that luciferase activities were significantly decreased in pGL3-WT-Smad4+mimics group, but were significantly increased in pGL3-WT-Smad4+inhibitor group when compared with pGL3-WT-Smad4 transfected group (P<0.05), but no significant difference was found between GL3-MT-Smad4+mimics and pGL3-MT-Smad4+inhibitor groups (P>0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-β1 induced TDFs, however, decreased in TGF-β1 induced TDFs after transfected by miR219-5P inhibitor (P<0.01). ConclusionmiR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-β1 by down-regulating Smad4 expression.
Objective To investigate whether miRNA (miR)-34a mediates oxaliplatin (OXA) resistance of colon cancer cells by inhibiting macroautophagy via the transforming growth factor (TGF)-β/Smad4 pathway. Methods miR-34a expression levels were detected in colon cancer tissues and colon cancer cell lines by quantitative real-time polymerase chain reaction (qRT-PCR). Computational search, functional luciferase assay, and Western blotting method were used to demonstrate the downstream target of miR-34a in colon cancer cells. Cell viability was measured with cell counting kit-8. Apoptosis and macroautophagy of colon cancer cells were analyzed by flow cytometry and transmission electron microscopy, and expressions of Beclin1 and LC3Ⅱ protein were detected by Western blotting method. Results Expression of miR-34a was significantly reduced while expressions of TGF-β and Smad4 mRNA were increased in colon cancer patients treated with OXA-based chemotherapy. OXA treatment also resulted in decreased miR-34a expression levels and increased TGF-β and Smad4 expression levels in both parental cells and the OXA-resistant colon cancer cells. Activation of macroautophagy contributed to OXA resistance in colon cancer cells. Expression levels of Smad4 and miR-34a in colon cancer patients had a significant inverse correlation and overexpressing miR-34a inhibited macroautophagy activation by directly targeting Smad4 through the TGF-β/Smad4 pathway. OXA-induced downregulation of miR-34a and increased drug resistance by activating macroautophagy in colon cancer cells. Conclusion miR-34a mediates OXA resistance of colon cancer by inhibiting autophagy via the TGF-β/Smad4 pathway.
Objective To construct and verify a genetically engineered mouse model which is similar to clinical sporadic colorectal cancer and simultaneously expresses KrasLSL-G12D/- and Smad4loxp/loxp genes. Methods The Krastm4Tyj/J mouse and Smad4tm2.1Cxd/J mouse were transformed into the genetic background, and the genotypes of the offspring mice were identified by the PCR to obtain the mice expressed simultaneously KrasLSL-G12D/- and Smad4loxp/loxp genes. The LentivirusCre-IRES-Luciferase was injected into the submucosa of the model mice and the tumorigenicity was observed under the IVIS system. The tumor tissues of the model mice were sampled and the HE staining was used to verify the tumorigenicity of the model mice. Results The genetically engineered mouse model which could simultaneously express KrasLSL-G12D/- and Smad4loxp/loxp genes was obtained by the breeding and selection. The mouse intestinal epithelial cell carcinogenesis was successfully induced by the viral vector containing Cre recombinase. Conclusion Mouse model expressed simultaneously KrasLSL-G12D/- and Smad4loxp/loxp genes is capable of sporadic tumorigenicity by Cre recombinase and could simulate pathological process of human sporadic colorectal cancer.