Impact of microRNA-375 and its target gene SMAD-7 polymorphism on susceptibility of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) has a high morbidity and mortality. Many studies reported that mir-375 is frequently down-regulated in many cancers including esophageal cancer, hepatocellular carcinoma, breast cancer and leukemias. AIM: Our aim was to study the expression of microRNA-375 and its target gene SMAD-7 polymorphisms (rs4939827) in CRC patients in comparison to control subjects and to correlate these results with clinical data of patients to elucidate their role in pathogenesis and early diagnosis of CRC. MATERIAL AND METHODS: The present study was conducted on 122 subjects divided into 86 patients with CRC and 36 age- and sex-matched controls. The followings were done to all subjects: full history taking, full clinical examination, complete blood picture, serum (ALT, AST), serum albumin, CEA, TLC, PLT, and creatinine. Gene expression of miRNA-375 from serum was done by real-time PCR. Gene polymorphism SNPs of SMAD7 (rs4939827) was also done in DNA extracted from blood by real-time PCR. RESULTS: As regards the polymorphism of SMAD7, we found that CC (wild) genotype has high percentage in controls compared to CRC cases (36.1% vs 15.1%). Meanwhile, the mutant and heterozygotes genotypes showed high percentage among cases compared to controls (33.7%, and 51.2% respectively) vs (22.2%, and 41.7% respectively) with no significant statistical analysis. There was a statistically significant high T-allelic frequency among cases and C-allelic frequency among controls. There was a statistically significant association between fold change in micro RNA (-375) and the susceptibility to CRC as there is down-regulation of the microRNA-375 in CRC group with fold change in 0.42±0.27. CONCLUSION: Micro RNA-375 and rs4939827 SNP in SMAD7 could be considered as potential markers for detecting and early diagnosing CRC patients.

In vivo study on the effect of adenovirus mediating Smad 7 gene expression regulated by radiation via Egr-1 promoter in C57BL mice implanted with Lewis lung cancer / 中华放射肿瘤学杂志

Objective Objective To study the effect of adenovirus mediating Smad 7 gene regulated by radiation via Egr-1 on the primary tumor and lung metastasis in C57BL mice implanted with Lewis lung cancer.Methods The radio-inducible elements from the Egr-1 gene promoter were inserted upstream to a cDNA encoding Smad7 and integrated into a replication-defective adenovirus to generate recombinant adenovirus(AD.Egr-Smad 7).270 mice implanted with Lewis lung cancer in the hind legs were used and the experiment was started when the transplanted tumor diameter reached 0.8 to 1.0cm.Then three investigations were undertaken,each demanding 90 mice implanted with Lewis lung cancer respectively.To each group,90 mice models were randomized into 3 groups: the normal control group;the NS control group;and the implanted AD.Egr-Smad 7 group.Every 6 mice in each group were irradiated by different single dose to study the following: 1.The maximal and minimal diameters of the tumor were recorded to observe the tumor growth tendency,the tumor growth delay and the mice survival time,2.The incidence of lung metastasis two weeks after the radiation was recorded.3.The incidence of lung metastasis when the tumor volume was four times as large as that at the beginning of radiation was recorded.Results The adenovirus mediating Smad 7 gene expression regulated by irradiation via Egr-1 in C57BL mice implanted with Lewis lung cancer was able to inhibit the progression of the primary tumor and prolong the survival of the mice significantly as compared with the control group(P0.05).Conclusions The gene expression of AD.Egr-Smad 7 regulated by radiation is not risky in promoting the local progression and distant metastasis of Lewis lung cancer in mice.On the other hand,the gene expression of AD.Egr-Smad 7 regulated by radiation could inhibit the progression of the primary tumor and prolong the survival time of the mice significantly.It is safe,to some extent,of using AD.Egr-Smad 7 to block the signal transduction of TGF-?locally as a novel strategy for gene therapy aiming at the prevention of radiation-induced lung

In vitro study of Smad 7 gene therapy for preventing radiation-induced pulmonary fibrosis / 中华放射肿瘤学杂志

Objective To study whether the expression Smad 7 protein by the recombinant adenovirus with Egr-1 promoter and Smad 7 cDNA in fibroblast cell can block the signal transduction pathway of transforming growth factor-beta1 (TGF-?1) under irradiation thereby inhibiting collagen synthesis in vitro. Methods The location of endogenous Smad 7 and exogenous Smad 7 protein in recombinant adenovirus infected fibroblast cells(3T6) were determined by immunocytochemical method. The infected 3T6 cells were irradiated and then cultured with TGF-?1 4 hours after irradiation. The activity of preventing radiation-induced fibrosis by expression Smad 7 protein was evaluated by the amount of collagen synthesis and proliferation of 3T6 cells. The amount of collagen synthesis was shown by the coruscant per minute (cmp) through the 3?H-Proline incorporation technique. Results The endogenous Smad 7 and exogenous Smad 7 protein both were located in the cytoplasm. When cultured with TGF-?1 4 hours after irradiation, the amount of collagen synthesis in the 3T6 cells infected with the recombinant adenovirus was significantly less than that in the cells without infecting adenovirus after irradiation(P=0.001), But, there was no difference in the proliferation of 3T6 cells between those with and without adenovirus infection (P= 0.312 ). Conclusions The Egr-1 promoter in the recombinant adenovirus can regulate the expression of downstream Smad 7 cDNA in 3T6 cells. The expression Smad 7 protein could block the TGF-?1 signal transduction pathway thereby inhibiting the collagen synthesis. The mechanism of inhibiting the collagen synthesis may be accomplished at the transcription level.

In vivo study on adenovirus mediating Smad 7 gene expression regulated by radiation via Egr-1 promoter / 中华放射肿瘤学杂志

Objective To investigate the time-effect relationship and dose-effect relationship of the expression of adenovirus mediating Smad 7 gene regulated by irradiation via Egr-1 promoter in the lung of C57BL mice. Methods Recombinant adenovirus (AD.Egr-Smad 7) was made up through replication-defective adenovirus enclosed radio-inducible elements from the Egr-1 gene promoter and cDNA encoding Smad 7. Mice infected by intratracheal instillation with AD.Egr-Smad 7 were irradiated to their whole lungs after 24 hours. 324 mice were randomly divided into 6 groups, and the lungs were harvested at different time points following irradiation with single dose of 8?Gy to observe the time-effect relationship of Smad7 gene expression with different time intervals. 108 mice randomly divided into 3 groups were irradiated respectively by different single irradiation dose and the lungs were harvested 5 hours after radiation to evaluated the dose-effect relationship of Smad 7 gene expression with different radiation doses. The expression of exogenous Smad 7 was detected by Western blot analysis. Results The expression of adenovirus mediating Smad 7 gene regulated by Egr-1 promoter could be induced by radiation markedly as compared with the control groups(P