| Gastric cancer is one of the most common malignant tumors in the world. The incidence of gastric cancer in malignant tumors is the fourth place and the second leading cause of cancer deaths. 40% of total global gastric cancer-related death occurred in China. The prognosis of gastric cancer patients is very poor. Standard clinical management includes complete surgery and chemotherapy, but the five-year survival rate of gastric cancer patients with successful surgery and chemotherapy is only 35-40%. This may be attributed to the fact that although gastric cancer is a heterogeneous disease, patients are generally grouped together and treated similarly. Compared to the traditional therapy such as radiotherapy, chemotherapy and surgery, targeted tumor therapy brings cancer treatment in a new stage. The development of new and effective tumor biomarker has become one of the focuses in the field of targeted tumor therapy. Micro RNAs(mi RNAs) are noncoding, single-stranded RNAs of 21 to 25 nucleotides in length that posttranscriptionally regulate gene expression by combination of the mi RNA seed sequence and its complementary sequence within the 3’ untranslated regions(UTRs) of its target m RNAs. And it is estimated that mi RNAs can regulated the expression of approximately 30% of messenger RNA(m RNA) in humans. Many reports shown mi RNAs play critical roles in initiation and progression of malignancies. Thus, mi RNAs most likely become biomarkers of targeted tumor therapy.The micro RNA(mi R)-200 family comprises five members(mi R-200 a,-200 b,-200 c,-141 and-429) that are clustered and expressed as two separate polycistronic pri-mi RNA transcripts, mi R-200b-200a-429 and mi R-200c-141, located on human chromosomes 1(1p36.33) and 12(12p13.31), respectively. mi R-200 family were reported to be involved in several malignancies. They play critical roles in initiation and progression of tumors. However, studies have shown that their functions are tissue-specific. Few studies to date have addressed the dysregulation and function of mi R-200 family members in gastric cancer progression. The expression, functions and the underlying mechanism of action still remains elusive.We detected the status of mi R-200 family of gastric cancer specimens and the expression of mi R-200 c and mi R-141 of human gastric carcinoma cell lines MKN-28, SGC-7901, BGC-823, HGC-27 and the gastric epithelial cell line GES-1 by quantitative q RT-PCR. Then we analyzed the relationship between mi R-200 c or mi R-141 and clinicopathological features, DFS, and gastric cancer biomarkers(CA50, CEA, CA199 and CA724). SGC-7901 cells were treated with mi R-200c/141 mimics, inhibitors or scramble control respectively. The bio-function of mi R-200c/141 in gastric cancer was also assayed by CCK-8 proliferation assay, wound healing assay and invasion assay. The predicted target genes zinc finger E-box-binding homeobox1/2(ZEB1/2) were determined by Luciferase report. We detected the effects of mi R-200 c and mi R-141 on ZEB1/2 and E-cadherin in SGC-7901 by q RT-PCR and Western-blot. We detected the expression of ZEB1/ZEB2 in gastric cancer specimens and analyzed the relationship between the expression level of mi R-200 c or mi R-141 and ZEB1/2. The status of TGF-β and methylation of mi R-200c/141 Cp G island of gastric cancer specimens were evaluated by q RT-PCR and MSP method. We detected the effects of TGF-β and decitabine on the expression of mi R-200 c and mi R-141 in SGC-7901 by q RT-PCR. The main research contents and results were shown as follows: Part ⅠThe expression of mi R-200 family and its relationship withclinicopathological features and DFS in gastric cancerObjective: To investigate the relationship between mi R-200 family expression and the clinical parameter and DFS of gastric cancer through detecting the expression of mi R-200 family in gastric cancer specimens and gastric cancer cell lines.Methods: q RT-PCR was adopted to detect the expression of mi R-200 family at the transcription level in gastric cancer specimens and gastric cancer cell lines. Retrospectively analysis was used to analyze the relationship between mi R-200 c or mi R-141 expression and clinicopathological features and DFS. clinicopathological features including age, gender, pathological differentiation(well-moderate differentiated adenocarcinoma and poorly differentiated adenocarcinoma), TNM stage, tumor invasion depth(T), Tumor embolus, degree of lymph node invasion(N) and distant metastasis(M) age of the patients. In addition, the relationship between mi R-200 c or mi R-141 and gastric cancer biomarkers(CA50, CEA, CA199 and CA724) were analyzed.Results:1 The expression of mi RNA-200 family in gastric cancer specimens.In 64 pairs of gastric cancer tissue and their matched adjacent tissue, the downregulated rate of mi R-200 a, mi R-200 b, mi R-200 c, mi R-141 and mi R-429 was 53.1%(34/64), 48.4%(31/64), 78.1%(50/64), 76.2%(48/63, 1 one sample data was lost) and 50%(32/64), respectively. mi R-200 c and mi R-141 were dramatically decreased in gastric cancer(P<0.001, P<0.001) when compared to the adjacent tissue, and no significant difference was found in mi R-200 a, mi R-200 b and mi R-429(P=0.258, P=0.501, P=0.870). Interestingly, there was a positive correlation between the expression of mi R-200 c and mi R-141(r=0.840, P<0.001).2 The expression of mi R-200 c and mi R-141 in gastric cancer cell lines.Compared with normal cell line GES-1, the expression of mi R-200 c in gastric cancer cell lines MKN-28, SGC-7901, BGC-823 and HGC-27 were significantly downregulated(P=0.003, P=0.003, P=0.002, P=0.008). Similarly, the expression of mi R141 was reduced in gastric cancer cell lines, too(P=0.001, P=0.002, P=0.001, P<0.001).3 The ralationship between mi R-200c/141 expression and clinicopathological features of gastric cancer.In the 63 patients with gastric cancer, mi R-200 c expression was associated with TNM stage, T, M, and tumor embolus(P=0.021, P=0.008, P=0.001, P=0.050) but was not associated with gender, age, pathological differentiation, or N(P=0.062, P=0.906, P=0.218, P=0.139). Mi R-141 expression was associated with T(P=0.004) but was not associated with gender, age, pathological differentiation, TNM stage, N, M, or tumor embolus(P=0.937, P=0.444, P=0.373, P=0.160, P=0.172, P=0.057, P=0.630). To analyze the relationship between mi R-200 c expression with TNM stage, T, M, and tumor embolus, a multiple linear regression analysis was conducted using these clinicopathological features. Mi R-200 c expression was inversely correlated with TNM stage, T and venous invasion(Beta=-0.424,-0.219,-0.453, t=-2.687,-1.398,-3.003, P=0.004, 0.009, <0.001). Mi R-141 was negatively associated with CA724(r=-0.258, P=0.047).4 The relationship between mi R-200c/141 expression and DFS of gastric cancer.The patients with low mi R-200 c expression had shorter median months of DFS than patients with high mi R-200 c expression(13.5 : 22.0, P=0.002). The patients with low mi R-141 expression had shorter mean months of DFS than patients with high mi R-141 expression(13.5 : 21.3, P=0.012).Conclusions:1 mi R-200 c and mi R-141 were dramatically decreased in gastric cancer when compared to the adjacent tissue. Similarly, the expression of mi R-200 c and mi R141 was reduced in gastric cancer cell lines. These data suggest they may play a protective role in gastric cancer progression.2 On clinical samples, the expression of mi R-200 c and mi R-141 was inversely correlated with tumor invasion depth(T), tumor embolus. These data suggest they may play a protective role in invasion and metastasis of gastric cancer.3 mi R-200 c and mi R-141 may therefore be a valuable factor of prognosis.Part ⅡThe effect of mi R-200 c and mi R-141 on biological behaviors ofgastric cancer cell and the regulation mechanismObjective: To investigate the function and the regulation mechanism of mi R-200 c and mi R-141 in gastric cancer.Methods: SGC-7901 cells were treated with mi R-200c/141 mimics, inhibitors or scramble control, respectively. The bio-function of mi R-200c/141 in gastric cancer was also assayed by CCK-8 proliferation assay, wound healing assay and invasion assay. The candidate targets of mi R-200c/141 were predicted by bioinformation and their functions in gastric cancer cells. The predicted target genes were determined by Luciferase report. We detected the effects of mi R-200 c and mi R-141 on ZEB1/2 and E-cadherin in SGC-7901 by q RT-PCR and Western-blot. We detected the expression of predicted target genes of mi R-200c/141 in gastric cancer specimens and analyzed the relationship between the expression level of mi R-200 c or mi R-141 and predicted target genes.Results:1 The effects of mi R-200 c and mi R-141 on proliferation of gastric cancer cell SGC-7901.The results of CCK-8 test show that, after transfected 48 h, there was no significant difference between control group and All Stars Negative Control si RNA group, mi R-200 c mimic group, mi R-200 c Inhibitor group, mi R-141 mimic group, mi R-141 Inhibitor group, mi R-200c/141 mimic group or mi R-200c/141 Inhibitor group(P=0.859, 0.057, 0.103, 0.100, 0.094, 0.072, 0.141). mi R-200 c and mi R-141 do not regulate the proliferation in gastric cancer cells.2 The effects of mi R-200 c and mi R-141 on migration and invasion of gastric cancer cell SGC-7901.The results of healing wound showed that cotransfection of the mi R-200c/141 mimics significantly inhibited the migration capability of SGC-7901 cells(P < 0.001), while the co-transfection of mi R-200c/141 inhibitors significantly enhanced the capability for migration(P<0.001). Individual overexpression or inhibition of mi R-200 c or mi R-141 had negligible effects on migration.The results of transwell showed that cotransfection of the mi R-200c/141 mimics significantly inhibited the invasion capability of SGC-7901 cells(P<0.001), while the co-transfection of mi R-200c/141 inhibitors significantly enhanced the capability of invasion(P<0.001). Individual overexpression or inhibition of mi R-200 c or mi R-141 had negligible effects on invasion. mi R-200c/141 significantly inhibited the migration and invasion capability of SGC-7901 cells.3 mi R-200c/141 directly targets ZEB1 and ZEB2 in SGC-7901.Compared to negative control, single transfections with mi R-200 c or mi R-141 mimics/inhibitors showed few effects on luciferase activity; however, co-transfection of mi R-200c/141 mimics exhibited a striking reduction of luciferase activity.4 The effects of mi R-200 c and mi R-141 on the expression of ZEB1/2 and E-cadherin in gastric cancer cell SGC-7901.m RNA and protein levels of ZEB1/2 in gastric cancer cells treated with mi R-200/141 mimics were reduced and the level of E-cadherin was increased. Consistently, mi R-200c/141 inhibitors increased the expression of ZEB1/2 and reduced the expression of E-cadherin in gastric cancer cells.5 The expression of ZEB1/2 in gastric cancer tissues and the relationship between mi R-200c/141 and ZEB1/2.In 64 gastric cancer tissues, both ZEB1 and ZEB2 were upregulated in 68.75%(44/64) of the tumor samples. Expression of ZEB1 and ZEB2 in gastric cancer tissue was also significantly higher compared to that in para-carcinoma tissue(P=0.001, P=0.004). mi R-200 c expression was negatively related to the expression of ZEB1 and ZEB2 in gastric cancer specimens, while mi R-141 expression was negatively related to ZEB2.Conclusions:1 mi R-200c/141 inhibited the migration and invasion capability of SGC-7901 cells.2 mi R-200c/141 inhibited the migration and invasion capability of SGC-7901 cells through induced upregultion of E-cadherin by inhibiting the expression of ZEB1 and ZEB2.3 mi R-200c/141 played anti-metastatic function in gastric cancer patients may probably through targeting ZEB1 and/or ZEB2. Part Ⅲ Increased TGF-β and mi R-200c/141 methylation inhibit theexpression of mi R-200c/141 in gastric cancerObjective: To detect the levels of TGF-β and mi R-200c/141 methylationin gastric cancer and investigate the relationship with mi R-200c/141 expression.Methods: The status of TGF-β and mehtylation of mi R-200c/141 Cp Gisland of gastric cancer specimens were evaluated by q RT-PCR and MSPmethod. We analyzed the relationship between the expression level ofmi R-200 c or mi R-141 and the status of TGF-β or mehtylation ofmi R-200c/141 Cp G island. We investigated the effects of TGF-β anddecitabine on the expression of mi R-200 c and mi R-141 in SGC-7901 byq RT-PCR.Results:1 The expression of TGF-β in gastric cancer tissues and the relationshipbetween mi R-200c/141 and TGF-β.In 63 gastric cancer tissues, TGF-β was upregulated in 66.67%(42/63) ofthe tumor samples. Expression of TGF-β in gastric cancer tissue was alsosignificantly higher compared to that in para-carcinoma tissue(P=0.005).TGF-β expression was negatively related to the expression of mi R-200 c andmi R-141 in gastric cancer specimens(r=-0.364,-0.526, P=0.003, <0.001).2 The status of mi R-200c/141 Cp G island methylation in gastric cancertissues and the relationship with mi R-200c/141.In 63 gastric cancer tissues, mi R-200c/141 Cp G island methylation wasupregulated in 84.13%(53/63) of the tumor samples. The status ofmi R-200c/141 Cp G island methylation in gastric cancer tissue was alsosignificantly higher compared to that in para-carcinoma tissue(P<0.001).mi R-200c/141 Cp G island methylation was negatively related to theexpression of mi R-200 c and mi R-141 in gastric cancer specimens(r=-0.303,-0.522, P=0.016, <0.001).3 The effects of TGF-β and decitabine on the expression of mi R-200 c andmi R-141 in SGC-7901TGF-β treatment decreased mi R-200c/141 expression(P=0.006, P=0.005); however, decitabine increased mi R-200c/141 expression(P=0.013, P=0.027) and alleviated the suppression of mi R-200/141 induced by TGF-β(P=0.005, P=0.007).Conclusions:1 TGF-β expression, as well as mi R-200c/141 Cp G island methylation, was upregulated in most of the tumor samples.2 TGF-β treatment decreased mi R-200c/141 expression.3 The upregulation of mi R-200c/141 Cp G methylation inhibits mi R-200c/141 expression in gastric cancer.4 Decitabine increased mi R-200c/141 expression and alleviated the suppression of mi R-200/141 induced by TGF-β. |
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