| Esophageal cacer is one of the most common malignant tumors in the world, with an estimated 450,000 new cases occurred. And about 50% of the cases occurred in China. Cixian is an high-incidence regions of esophageal cancer in China, with the highest incidence and mortality rate of esophageal cancer in the province of malignant tumors. It is a serious threat to human health. The epidemiological characteristics in Cixian are significant regional differences(500 fold difference in incidence between high-incidence regions and low-incidence regions) and familial aggregation phenomenon. Both environmental and genetic factors may play an important role in the occurrence of esophageal cancer.Micro RNAs(mi RNAs) is an non-coding, single-stranded RNAs of about 23 nucleotides in length and widely exist in human and other eukaryotic organisms. mi RNAs can compose an complex biological control network, with its target genes, upstream transcription factors and various feedback control loop, et al., and participate in the occurrence and development of tumor, as a new target marker for the diagnosis and treatment of tumor.Studies have shown that mi R-196 is associated with the tumorigenesis and prognosis of various tumors, playing a role of oncogene or anti-oncogene. mi R-196 a is overexpression in esophageal cancer, which indicated oncogenic potential of mi R-196 a. rs11614913 C/T SNP, which lies in the sequence of mi R-196a-2, can induce a change from G: C to G:T in the stem region of the pre-mi R-196a-2 precursor. Studies have indicated that this variant might influence the process of pre- mi R-196a-2 to its mature form or change the recognition of target gene, which has tissue specificity. As an endogenous gene, mi R-196 a has its own promoter region. An functional polymorphism(rs35010275), which is present in the promoter region of mi R-196a-2, may affect the expression level of mi R-196a-2. Study has shown that the expression level of mi R-196a-2 in gastric cancer tissues and adjacent normal tissues can be up-regulated by GG genotype.The candidate target genes of mi R-196 a include HOX family, HMGA2, ANXA1 and p27kip1. Several studies have shown that mi R-196 a can inhibits HOXB8 expression in myeloid differentiation of HL 60 cells. mi R-196 a can enhanced the proliferation of gastric cancer cell and cervical cancer cell. In addition, up-regulation of mi R-196 a expression level can induce the down-regulation of ANXA1 m RNA and protein levels, and the inhibition or deletion of ANXA1 expression was associated with the occurrence of esophageal cancer.Thus, we first investigated the association between the two SNPs(rs11614913 and rs35010275) of mi R-196a-2 gene and the risk/prognosis of ESCC in high-incidence regions, using PCR-LDR method. q RT-PCR technology was used to detect the expression of mi R-196 a gene and its target genes HOXB8, HOXC8, p27kip1 and ANXA1 in esophageal cancer tissues, adjacent tissues and normal mucosa tissues. The relationship between mi R-196 a gene and its target gene expression level was analyzed. Then, Eca109 cells or KYSE30 cells were transfected with mi R-196 a mimic, inhibitor or negative control respectively. We analysed the role of mi R-196 a gene in proliferation and invasion of Eca109 cells and KYSE30 cells by MTT proliferation assay, invasion assay and wound healing assay. The First part: Association between mi R-196a-2 gene polymorphism and genetic susceptibility and prognosis to esophageal squamous cell carcinomaObjective: To investigate the relationship between rs11614913 SNP and rs35010275 SNP of mi R-196a-2 gene and ESCC risk with patients in high incidence-region of esophageal cancer, and to find the molecular markers of ESCC of the high risk population.Methods: This study recruited 597 ESCC patients and 597 unrelated healthy control subjects during an endoscopic screening campaign between 2006 and 2012. All the study subjects were ethnically homogeneous of Han descent and permanent residents of Cixian county. The patients had histologically confirmed ESCC. Cancer-free subjects who were confirmed to be without UGIC by endoscopies were selected as the healthy controls. Information about age, sex, smoking habit and family history of UGIC were obtained immediately following blood donation. Matched age- and gender- to the ESCC cases, 597 healthy controls were enrolled in the study. We defined smokers as having formerly been or currently smoking ≥5 cigarettes/day for at least 2 year. The definition of a family history of upper gastrointestinal cancer(UGIC) was individuals with at least one first-degree relative or at least two second-degree relatives with esophageal/cardiac/gastric cancer. Using PCR-LDR method to detecting the genotype of mi R-196a-2 gene rs11614913 C/T SNP and rs35010275 C/G SNP.Results:1 The frequency of a positive family history of UGIC among ESCC(47.6%) patients is 47.6%, which was significantly higher than that in healthy controls(38.4%)(χ2=10.34, P<0.01). This indicated that a family history of UGIC would increase the risk of ESCC(the age- and sex-adjusted OR = 1.46, 95% CI = 1.16-1.84).2 The genotype distribution of mi R-196a-2 rs11614913 polymorphism in the healthy controls did not deviate from the Hardy–Weinberg equilibrium(χ2=0.001, P=0.97). The genotype(CC, CT, TT) distribution frequency of ESCC patients and healthy control were 21.5%, 51.4%, 27.1% and 24.3%, 49.9%, 25.8%. There were no significant associations between rs11614913 polymorphism and ESCC risk(χ2=1.40, P=0.50). Compared in CC genotype, CT/TT genotype can not increase the risk of ESCC, adjusted OR were 1.17(0.88-1.56), 1.20(0.86-1.66)and 1.18(0.90-1.55). Stratified analysis revealed that TT/CT genotype can increase ESCC risk with patients in the groups of age ≤60 years(OR = 1.56; 95% CI, 1.08–2.26) but not in the group of age >60 years(OR = 0.82; 95% CI, 0.55-1.23). Interaction between gene and age with rs11614913 SNP and ESCC risk was found, which indicates that the earlier age of onset, the greater the effect of genetic predisposition.3 The genotype distribution of mi R-196a-2 rs35010275 polymorphism in the healthy controls did not deviate from the Hardy–Weinberg equilibrium(χ2=0.04, P=0.83). The genotype(GG, GC, CC) distribution frequency of healthy control and ESCC patients were 49.1%, 42.2%, 8.7% and 52.9%, 40.9%, 6.2%. There were no significant associations between rs11614913 polymorphism and ESCC risk(p>0.05) in total. Compared in GG genotype, GC, CC, GC/CC genotype can not increase the risk of ESCC, adjusted OR were 0.90(95%CI=0.71~1.14), 0.67(95%CI=0.42~1.05) and 0.86(95%CI=0.68~1.08), respectively. Stratified analysis revealed that compared with GG or GC/GG genotype, CC genotype can decrease ESCC risk with patients in the groups of age ≤60 years, OR = 0.49(95% CI, 0.26-0.92) and 0.51(95% CI, 0.28-0.93).4 There were no significant association between mi R-196a-2 rs11614913 SNP or rs35010275 SNP and prognosis of ESCC.Conclusions:1 rs11614913 C/T SNP of mi R196a2 gene can increase the risk of ESCC in the groups of age ≤60 years.2 rs35010275 G/C SNP of mi R196a2 gene can decrease the risk of ESCC in the groups of age ≤60 years. The Second part: Expression and correlation of mi R-196 a and its target genes in tissues of esophageal cancerObjective: To investigate the expression of mi R-196 a gene and ANXA1 gene, p27kip1, HOXC8, HOXB8 in esophageal cancer tissues, adjacent tissues and normal mucosa tissues. And to analyze the association between mi R-196 a and its candidate target genes, and to explore the possible role of mi R-196 a gene and its target genes in esophageal cancer.Methods: 28 cases of esophageal carcinoma radical operation were collected from the department of thoracic surgery of in fourth hospitals, Hebei Medical University from September 2012 to March 2013. q RT-PCR method was used to detect the expression of mi R-196 a gene and its target genes HOXB8, HOXC8, p27kip1, ANXA1 in esophageal cancer tissues, adjacent tissues and normal mucosa tissues. In addition, the correlation of mi R-196 a and its target gene ANXA1, p27kip1, HOXC8, HOXB8 in expression levels were analyzed.Results:1 Expression of mi R-196 a in the adjacent tissues and cancer tissues were 64.29%(18/28) and 100%(28/28). Difference Expression level of mi R-196 a in normal mucosa tissues, adjacent tissue and cancer tissue was statistically significant(χ2=42.07, P<0.001). The expression level of mi R-196 a in cancer tissues was significantly higher than that in normal mucosa tissues and adjacent tissues.2 The expression levels of target gene ANXA1, p27kip1, HOXB8 in normal mucosa tissues, adjacent tissues and cancer tissues were statistically significant(P values were <0.001). The expression levels of p27kip1, ANXA1 and HOXC8 in the cancer tissues were significantly lower than those in normal mucosa tissues and adjacent tissues. Expression levels of HOXB8 were no differences among normal mucosa tissues, adjacent tissue and cancer tissue(χ2=0.29, P=0.87).3 There were positive correlations of mi R-196 a and target gene ANXA1, p27kip1, HOXC8, HOXB8 in expression level among normal mucosa tissues, normal tissues and cancer tissues, normal tissues and cancer tissues(r=0.80, P<0.001).4 There were negative correlations between mi R-196 a and its target genes ANXA1, p27kip1, HOXB8(P values were <0.001). The expression of ANXA1, p27kip1 and HOXB8 were decreased with the high expression of mi R-196 a..Conclusions:1 mi R-196 a expression level in esophageal cancer tissues was significantly higher than that in adjacent tissues and normal mucosa tissues; The expression of candidate target genes ANXA1, p27kip1 and HOXB8 were significantly lower in esophageal cancer tissues than those in adjacent tissues and normal mucosa tissues; there were no difference of HOXC8 expression in esophageal cancer tissues, adjacent tissues and normal mucosa tissues.2 The expression levels of mi R-196 a were significantly negatively correlated with those of candidate target genes HOXB8, p27kip1, HOXC8, ANXA1, which indicated that mi R-196 a can play a oncogene role through inhibiting the expression of target gene HOXB8, p27kip1 and ANXA1. The Third part: The effect of mi R-196 a on biological behaviors of esophageal cancer cell and the expression of its target genesObjective: To investigate the function and the regulation mechanism of mi R-196 a in esophageal cancer.Methods: Eca109 cells and KYSE30 cells were all treated with mi R-196 a mimic, inhibitor or negative control, respectively. Using q RT-PCR method to detect the expression of mi R-196 a and its target genes ANXA1, p27kip1, HOXC8, HOXB8. To analyse the role of mi R-196 a in esophageal cancer by MTT proliferation assay, transwell assay and wound healing assay.Results:1 After treated with mi R-196 a mimic, inhibitor or negative control in Eca109 cells, the expression of mi R-196 a mimic group were significantly increased than that of negative control group(about 14.45 times. P<0.01). And the expression of mi R-196 a inhibitor group were significantly decreased than that of negative control group(about 0.71 fold, P<0.01). There were no difference in expression of ANXA1, p27kip1, HOXC8 and HOXB8(P<0.05).2 After treated with mi R-196 a mimic, inhibitor or negative control in KYSE30 cells, the expression of mi R-196 a mimic group were significantly increased than that of negative control group(about 55.48 times. P<0.01); And the expression of target gene HOXB8 was significantly decreased than that of negative control group. The expression of mi R-196 a inhibitor group were significantly decreased than that of negative control group(about 0.69 fold, p<0.01); And HOXB8 expression was significantly increased than that of negative control group.3 The results of MMT assay show that, after transfected Eca109 cells 0h, 24 h, 48 h, 72 h and 96 h, cell survival rate of mi R-196 a mimic group were 100%, 103%, 112%, 117% and 111%. There were significantly difference in mi R-196 a mimic group and negative control group(F=5952.77, P<0.001). The cell survival rate of mi R-196 a inhibitor group were 99%, 87%, 91%, 87% and 87%. There were significantly difference in mi R-196 a mimic group and negative control group(F=6622.80, P<0.001). And there were similar results in transfected Eca109 cells and transfected KYSE30 cells.4 The results of healing wound assays showed that transfection of the mi R-196 a mimic significantly enhanced the migration capability of Eca109 cells and KYSE30 cells, while the transfection of mi R-196 a inhibitor significantly inhibited the capability for migration(P<0.05). The results of transwell assays showed that transfection of the mi R-196 a mimic significantly enhanced the invasion capability of Eca109 cells and KYSE30 cells, while the transfection of mi R-196 a inhibitors significantly inhibited the capability of invasion(P<0.05).Conclusions:1 After treated with mi R-196 a mimic in Eca109 cells, the expression of mi R-196 a was up-regulated; After treated with mi R-196 a inhibitor in Eca109 cells, the expression of mi R-196 a were down-regulated; There were no abnormal expression in ANXA1, p27kip1, HOXC8 and HOXB8.2 After treated with mi R-196 a mimic in KYSE30 cells, the expression of mi R-196 a was up-regulated, and its target gene HOXB8 was low expression; After treated with mi R-196 a inhibitor in KYSE30 cells, the expression of mi R-196 a was down-regulated, and HOXB8 was high expression..3 MMT assays show that, after transfected mi R-196 a mimic or inhibitor with Eca109 cells or KYSE30 cells, cell proliferation can be enhanced or inhibited.4 Healing wound assays and transwell assays showed that, after transfected mi R-196 a mimic or inhibitor, the migration and invasion capability of Eca109 cells and KYSE30 cells can be enhanced or inhibited. |
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