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@#Introduction: Recent studies have published the roles of exosomal miRNAs in the pathogenesis of various type of malignancies and can be developed as potential biomarkers for diagnostic, prognostic and therapeutic purposes. The aim of this study was to identify the expression level of selected miRNAs (miR-182, miR-301a, and miR-373) in exosomes of the serum and ascitic fluid in patients with non-alcoholic steatohepatitis (NASH)-related liver cirrhosis with or without hepatocellular carcinoma (HCC). Materials and Methods: A literature search was performed to identify potential miRNAs involved in the pathogenesis of HCC. Unpaired serum and ascitic fluid were obtained from 52 patients with NASH related liver cirrhosis (n=26 for each group of with and without HCC). Exosomal miRNA was isolated from all samples. Expression levels of miR-182, miR-301a and miR373 were determined using quantitative real-time PCR. Results: Serum-derived exosomal mir-182, miR-301a and miR-373 were significantly up-regulated with fold change of 1.77, 2.52, and 1.67 (p< 0.05) respectively in NASH-induced liver cirrhosis with HCC as compared to NASH-induced liver cirrhosis without HCC. We identified the expression levels of ascitic fluid-derived exosomal mir-182, miR-301a, and miR-373 were significantly up-regulated with fold change of 1.6, 1.94 and 2.13 respectively in NASH-induced liver cirrhosis with HCC as compared to NASH-induced liver cirrhosis without HCC (p <0.05). There was poor correlation expression of all the selected exosomal miRNA between serum- and ascitic fluid-derived in HCC group. Conclusions: This preliminary data showed significant increase in the expression levels of exosomal miR-182, miR-301a and miR373 in both serum and ascetic fluid suggesting the possible roles of these miRNAs as circulating biomarkers for NASH-induced liver cirrhosis with hepatocellular carcinoma
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Objective To examine the effect of miR-301b in the regulating the differentiation of mesenchymal stem cells into adipocytes.Methods Murine ST2 stromal cells were isolated,cultured and induced with adipogenic agents.The expression of miR-301b was detected by qRT-PCR in adipogenic differentiation group and control group.Stromal ST2 cells were transfected with miR-301b mimics followed by adipogenic treatment.qRT-PCR and Western blotting were conducted to detect the changes of adipocyte specific genes and protein expression levels in the miR-301b mimics transfection group and negative control(NC)transfection group. Results qRT-PCR showed that the expression of miR-301b was decreased after adipogenic treatment in ST2 cells.The relative expression level of miR-301b was less in the adipogenic differentiation group (0.219 ± 0.021) than that of the control group (1.000 ± 0.425, P<0.05). The relative expressions of peroxisome proliferator activated receptor γ(PPARγ),CCAAT/enhancer-binding protein α(C/EBPα)and adipocyte fatty acid binding proteins(aP2)were lower in the miR-301b mimics transfecting group than those in the NC transfecting group(P<0.05).The protein levels of the marker gene aP2 and transcription factors PPARγ and C/EBPα decreased in miR-310b mimics transfecting group compared with those of the NC transfecting group (P<0.05). Conclusion miR-301b can reduce adipocyte differentiation.
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Objective To establish miRNA knockout mouse model using CRISPRICas9 gene targeting technology. Methods According to the gene sequence of miRNA, we designed the primers of the gRNA targeting miR-301a (two targets) and obtained DNA template for in vitro transcription using PCR amplification; then we constructed Cas9 template for in vitro transcription, followed by in vitro transcription for gRNA of Cas9. In vitro transcribed gRNAICas9 mRNA was microinjected into the mouse zygote. T7E1 digestion and gene sequencing were used to detect and characterize the mutation of miRNA. Results PCR amplification, gel electrophoresis and gene sequencing proved that we had obtained the correct DNA template targeting miR-301a for in vitro transcription. By in vitro transcription we obtained gRNAICas9 mRNA, which was successfully microinjected into mouse zygote. The miR-301a mutants were detected by digestion with T7E1, and it was found that 7 of the 8 (87. 5%) neonatal mice were found carrying mutations in miR-301a sites. Gene sequencing results showed that all mice had different levels of nucleotide insertion or deletion mutation, with the maximum number of 31 bases deletion found in No. 4 mouse. Conclusion We have successfully established a mouse model with miR-301a gene knocked out, which lays a solid foundation for related future research.
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Objective To investigate the changes of miR-301a and its host gene expression SKA2 in LNCaP prostate cancer xenografts in the castrated nude mice.Methods LNCaP cells were inoculated subcutaneously in nude mice to establish xenograft models of human prostate cancer.When the tumor volume grew to 200 mm3,the nude mice were randomly divided into the following 4 groups(n =6): 2 groups of nude mice to surgical castration,the other 2 groups of mice as control groups.The growth of those xnografts in nude mice was observed weekly and a growth curve of the xenografts was drawn.A point time during the process of tumor-regressing was selected when a group of castrated nude mice and a group of control mice were killed(the first time).The other 2 groups nude mice were continued to be observed.Another point time in the process of tumor re-growth,the rest castrated nude mice and control mice were killed(the second time).The tumors were weighted and the inhibitory rate was calculated.MiR-301a and SKA2 expression were detected by real-time PCR.Results The growth of the xenografts gradually decreased in LNCaP xenografts in nude mice after castration.After 13 days,the xenografls sizes decreased to(62.5 ±21.5)mm3 and tumor inhibitory rate was 59.8％(t =-3.895,P =0.018)in castration group of nude mice.At the 17th day after castration,tumor volume reached the minimum,and then gradually increased.At the 41st day after castration,tumor volumes in castration group increased to(364.5 ±97.3)mm3 and the tumor inhibitory rate was 62.2％(t =-7.017,P =0.002).MiR-301a and SKA2 in the first time of xenografts from the castrated group were both significantly lower than those of the control group(0.65-fold and 0.50-fold,respectively).However,their expressions in the second time of xenografts from the castrated group increased and were consistent with the control group(P ＞ 0.05).Conclusions Castration could turn prostate cancer xenografts from androgen-dependent into androgen-independent.There could be a close correlation between the characteristic of prostate cancer androgen-dependent and the expressions of miR-301 a and host gene SKA2.
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Background and purpose: The miR-224 in a variety of malignant tumors is overexpression, however, its expression and function in colon cancer are not clear. The aim of this study was to investigate the expression of miR-301 in colon cancer tissues and demonstrate the regulative effects of miR-301 ASO on the proliferation and apoptosis of colon cancer cell in vitro and in vitro. Methods:The expression of miR-301 in 120 colon cancer tissues and their adjacent tissues was detected by real-time quantitative PCR method. After transfection with miR-301ASO, the biological effects of miR-301 in SW620 cells were measured by MTT assay, the colony formation experiment, flow cytometry and the in vivo experiment. Results: The expression level of miR-301 was found to be overexpressed in 63.33% (76/120) of the colon cancer cases (P<0.05). miR-301 expression in SW620 cells (transfection with miR-301 ASO, 0.09±0.01) was significantly less than control group (0.50±0.07, P=0.00). MTT assay results showed that SW620 cells survived rate at 24, 48 and 96 h decreased greatly after transfection with miR-301ASO (P=0.00). Clone formation assay revealed that miR-301 ASO group colony formation rate (5.33%±0.74%) was significantly lower than the control group (33.33%±8.38%, P=0.00). In vivo study further confirmed that miR-301ASO could inhibit the proliferation of SW620 cells (P<0.05), and miR-301ASO group grew substantially slow compared with the negative control group (P=0.00). Flow cytometry indicated that the apoptotic index in miR-301 ASO group (15.68±1.46) was significantly higher than the control group (3.36±0.88, P=0.02). In addition, the Bcl2 mRNA and protein were significantly decreased after reduce the expression of miR-301 (P=0.00, P=0.00). Conclusion:MiR-301 was overexpressed in human colon cancer. Reduce the expression of miR-301 can effectively inhibit the growth of colon cancer cells and promote apoptosis. MiR-301 may become a new target for the regulation of gene expression in colon cancer.