Screening And Preliminary Validation Of MiRNAs With The Regulation Of HTERT In Colorectal Cancer

Screening of miRNAs regulating telomerase reverse transcriptase(hTERT) in colorectal cancer(colorectal cancer, CRC) is the third most common cancer in the world, and with an increasing incidence rate. Most cases are diagnosed in an advanced stage. Even the use of multimodal treatment combining surgery, chemotherapy, radiotherapy and target therapy for CRC is applied, the overall survival is not satisfactory. The abnormal activation and function of relevant factors including telomerase is a condition for genesis and proliferation of CRC. Further research into these relevant factors is important for us to understand the mechanisms of CRC, the prognostic factors in CRC, and the choice of treatment in CRC.Human telomerase reverse transcriptase(hTERT) is the catalytic ratelimiting subunit of the enzyme telomerase, it consists an RNA component that serves as a template for the telomere repeat. Previous studies have showed that hTERT is involved in the genesis and metastasis of CRC, and it is closely related with the lymph node metastases, TNM stage and survival in CRC. The transcriptional regulatory mechanism of the hTERT is not clear, so the understanding of the mechanism in CRC and the search of the inhibitor ofhTERT have great significance.Micro RNA(miRNA) is a small non-coding RNA molecule(containing about 18-24 nucleotides) encoded by eukaryotic nuclear DNA. Mi RNAs function via base-pairing with complementary sequences within mRNA molecules, and these mRNAs are silenced by less efficient translation of the mRNA or cleavage of the mRNA strand into two pieces. It plays an important role in cell proliferation, cell differentiation, apoptosis, gene regulation and oncogenesis. Up to now 3 miRNAs(mi R-138, mi R-1266, mi R-1207-5p) have been identified which are involved in the regulation of hTERT on the posttranscription level in gastric cancer and thyroid adenocarcinoma, while no such findings have been reported. Because the same group of target genes of the same miRNA may be regulated differently in different tumors, while the same miRNA may regulate hundreds target genes, and the same target gene may be regulated by hundreds miRNAs, it is suggesting that there are other miRNAs involved in hTERT transcriptional regulation. This paper is divided into four parts:(1) Quantitative real-time polymerase chain reaction(q RT-PCR) analysis was performed to detect the hTERT-regulating mRNA in CRC tissue, analysis of its association with the clinicopathological features of patients with CRC.(2) Bioinformatics technique was used to predict the possible miRNAs which might take a role in regulating hTERT.(3) q RT-PCR analysis was performed to detect the probable hTERT-regulating miRNA in CRC tissue, analysis of its association with the clinicopathological features of patients with CRC.(4)Western-blot was performed to detect the hTERT protein, and analysis of the correlation between the hTERT-regulating miRNAs and the hTERT protein. Then the probable miRNA targeting hTERT will be verified in the next step.PART 1 ANALYSIS OF hTERT MIRNA EXPRESSION IN CRC AND ITS CLINICAL SIGNIFICANCEPurpose: To analyze the correlation between hTERT mRNA expression level and clincopathological parameters in CRC, and its significance in the prognosis of CRC patients.Methods: q RT-PCR was performed to detect hTERT mRNA expression level in 84 CRC cases, and the expression level was analyzed in combination with clincopathological parameters and a follow-up.Results: 1. The expression level of hTERT mRNA was down-regulated in 10 cases(RQ<1), while it was up-regulated in 64 cases(RQ>1), the upregulation of hTERT mRNA was 76.19%.2. The rate of high expression of hTERT mRNA was significantly higher in CRC patients with lymph node metastases compared with CRC patients without lymph node metastases(92% vs 52%, P=0.011). The rate of high expression of hTERT mRNA was significantly higher in stage III/IV patients compared with stageⅠ/Ⅱ patients(92.3% vs 50%, P=0.006). While the high expression level of hTERT mRNA was not associated with the patients’ age, the tumor size, the differentiation grade, the tumor site, the infiltration depth, and distant metastases(P>0.05).3. Overall survival was significantly different between patients with the expression of hTERT mRNA and patients without the expression of hTERT mRNA(P=0.014).Conclusion: The expression of hTERT mRNA was significantly higher in CRC tissue compared with normal tissue, and was correlated with the patients’ TNM stage, lymph node metastases and overall survival. It suggested that hTERT plays an important part in the genesis and development of CRC.PART 2 SCREENING OF hTERT-REGULATING MIRNAPurpose: High-throughput data of miRNAs were downloaded and bioinformatics technique was used to predict the possible miRNAs which might take a role in regulating hTERT.Methods: Expression data of miRNAs(GSE10259, GSE33127, GSE35602, GSE38389, GSE39845, GSE18392, GSE35982) and the corresponding clinical data for CRC patients were downloaded from the Gene Expression Omnibus(GEO) database. An extensive text-mining search and mi RWalk were used to predict hTERT-regulating miRNAs.Results: A total of 8 hTERT-regulating miRNAs were down-regulated in CRC samples, including mi R-124, mi R-138, mi R-150, mi R-378, mi R-133 a, mi R133 b, mi R-29 c, mi R-442 a.Conclusion: With the combination of text-mining search and mi RWalk, the possible hTERT-regulating miRNAs were defined.PART 3 THE EXPRESSION OF HTERT-REGULATING MIRNAS IN CRC SAMPLES AND ITS CLINICAL SIGNIFICANCEPurpose: To investigate the expression of hTERT-regulating miRNAs in CRC samples and its corresponding normal tissue, and analyze its clinical significance.Methods: q RT-PCR analysis was performed to validate the expression of the 8 miRNAs in 84 CRC samples, and the data were analyzed in combination with clinical parameters and follow-up.Results: 1. There was no significant difference in miRNA-29c-3p expression between CRC samples and its corresponding normal tissue(P=0.260), while the expression of mi R-133a-3p, mi R-133 b, mi R-422 a, mi R-150-5p, mi R-378a-3p, mi R-124-3p, mi R-138-5p were significantly lower in CRC samples compared with their corresponding normal tissue(P<0.05).2. There was no significant correlation between the expression of mi R-133a-3p, mi R-133 b, mi R-150-5p, mi R-124-3p and the patients’ age, gender, tumor site, differentiation grade, infiltration depth, lymph node metastases, distant metastases and TNM stage(P>0.05).3. The rate of low expression of mi R-138-5p was significantly higher in CRC patients with distant metastases compared with patients without distant metastases(88.89% vs 40.91%, P<0.000). The re was no significant correlation between the expression of mi R-138-5p and the patients’ age, gender, tumor site, differentiation grade, infiltration depth, lymph node metastases and TNM stage(P>0.05).4. The rate of low expression of mi R-378a-3p was significantly higher in CRC patients with distant metastases compared with patients without distant metastases(77.78% vs 42.42%, P=0.008). The rate of low expression of mi R-378a-3p was significantly higher in colon cancer patients compared with rectal cancer patients(66.67% vs 37.5%, P=0.008). There was no significantcorrelation between the expression of mi R-138a-3p and the patients’ age, gender, tumor site, differentiation grade, infiltration depth, lymph node metastases and TNM stage(P>0.05).5. The rate of low expression of mi R-422 a was significantly higher in CRC patients with lymph node metastases compared with patients without lymph node metastases(59.26% vs 33.33%, P=0.023). There was no significant correlation between the expression of mi R-422 a and the patients’ age, gender, tumor site, differentiation grade, infiltration depth, lymph node metastases and TNM stage(P>0.05).Conclusion: A total of 7 miRNAs(mi R-124-3p, mi R-133a-3p, mi R-133 b, mi R138-5p, mi R-150-5p, mi R-378-3p, mi R-422a) were down-regulated in CRC samples. While mi R-378a-3p, mi R-138-5p, mi R-422 a were correlated with distant metastases or lymph node metastases in CRC patients, and mi R-422 a was correlated with the overall survival in CRC patients.PART 4 CORRELATION STUDY BETWEEN MIRNAS AND HTERT PROTEINPurpose: To investigate the relationship between the low expression of miRNAs above and hTERT protein, for the further functional research of micro RNAs possibly targeted regulation of hTERT.Method: Detection of hTERT protein expression in 62 cases of CRC through techniques of Western blotting, correlation studies between the 7 lowexpression of miRNAs and hTERT protein.Result:(1)Kaplan-Meier survival curves demonstrated that there is no significance between high-expression group vs. low-expression group of hTERT, mi R-422 a, mi R-378a-3p, mi R-150-5p, mi R-138-5p, mi R-133 b, mi R-133a-3p, mi R-124-3p in 62 patients with CRC( P=0. 272, P= 0. 062, P=0.130, P=0.707, P=0.174, P=0.845, P=0.575, P=0.127, respectivelly). But the survival curve also show that the two curves is obviously separated between high group and the low expression of mi R-422 a, mi R-150-5p, mi R- 124-3p, mi R-138-5p.(2) The expression of mi R-124-3p, mi R-150-5p was significantly associated with the other five micro RNAs, while no significance associated with hTERT. The expression of mi R-133a-3p was negative significantly associated with hTERT, but associated with the other five micro RNAs. The expression of mi R-138-5p is no significantly associated with mi R-133a-3p, while associated with the other five micro RNAs and negative significantly associated with hTERT. The expression of mi R-133 b, mi R-378a-3p, mi R-422 a is associated with the other six micro RNAs, especially, while negative significantly associated with hTERT.Conclution: Mi R-422 a, mi R-133 b, mi R-378a-3p and mi R-138-5p may coregulated the protein expression of hTERT. For the four micro RNAs, especially mi R-422 a, further functional study associated with hTERT is necessary.