MiR-210Inhibits Proliferation Of Esophageal Carcinoma Eca109Cells And Correlated Mechanisms

Esophageal carcinoma is one of the most common malignant diseases inChina, with high incidence and high mortality characteristics.90%of esopha-geal tumors are esophageal squamous cell carcinoma (ESCC) in in Asia-pacific region. Currently radiotherapy is the mainstay in the treatment ofesophageal cancer, but the local failure has remained a major concern, withpersistent or recurrent disease being reported in around60-80%of patients,and an overall5-year survival rate of10%.Intratumoral hypoxia is a hallmark of most solid tumors and results fromincreased oxygen consumption and/or insufficient blood supply. Evidenceobtained from radiochemical and radiobiological studies has revealed theseproblems to be caused, at least in part, by a tumor-specific microenvironment,hypoxia. Many of the hypoxia-induced cellular responses are mediatedthrough the hypoxia-inducible factors (HIFs), which regulate genes involvedin angiogenesis, survival, cell metabolism, invasion and other functions.Recently, a number of miRNAs induced during hypoxia have been identified.One of these miRNAs, miR-210, is strongly induced by HIF-lα and haspleiotropic effects. Several recent studies also demonstrated that miR-210inhibited tumor cell proliferation by inducing G0/G1and/or G2/M phase cellcycle arrest. In contrast to other solid tumors, miR-210is frequently under-expressed in ovarian cancers, which potentially leads to increased expressionof E2F transcription factor3(E2F3) which participates in the regulation of thecell cycle. Likewise, miR-210is down-regulated in esophageal squamous cellcarcinoma. MiR-210inhibits cancer cell proliferation and induces G0/G1phase cell cycle arrest by derepressing fibroblast growth factor receptor-like1(FGFRL1) that in turn accelerates cell cycle progression. However, onecannot generally state that miR-210induction in hypoxia negatively regulates cell cycle progression and proliferation. In hepatic cancer cells, miR-210activates the myc pathway, via downregulation of the c-Myc antagonist MNT,and loss of MYC abolished miR-210-mediated override of hypoxia-inducedcell cycle arrest. Therefore, the net impact of miR-210on cell cycle regulationseems to be context dependent.In previous studies, the miR-210up/down-regulated expression isreported in other malignancy tissues, such as cancers of the head and neck,pancreas, breast, and ovarian. Recently, studies on the role of circulating miR-210in cancer and its potential utility as prognostic markers have emerged.Lately, the correlation of circulating miR-210levels with breast cancermortality is a striking one. Eun-Jung Jung and colleagues reported plasmamiR-210levels correlate with sensitivity to trastuzumab, tumor presence, andlymph node metastases in breast cancer patients. Another two studies showeda statistically significant four-fold increase of circulating miR-210in expres-sion in pancreatic and clear cell renal cancer patients compared with normalcontrols. Although the results of these three studies were obtained from smallcohorts, the presence of elevated miR-210in the plasma of cancer patientssuggests that this may be an important feature of these diseases, which mayfurther advance our understanding of the underlying pathogenesis of cancers.In present study, we investigated the functional role of miR-210in thegrowth of carcinomas and the mechanism by which it acts. Next, we analyzedthe serum miR-210expression level in ESCC patients for the first time.Furthermore, our work also revealed the potential role of serum miR-210levelas response markers during radiotherapy for ESCC patients.Part1The expression of miR-210in hypoxic esophageal carcinomaEca109cells and its effects on biological behiviorObjective: To detect the expression of miR-210in hypoxic esophagealcarcinoma Eca109cells and explore its effects on proliferation, cell cycle andapoptosis.Methods: After being treated with hypoxia, miR-210at different hypoxicculture phases were detected by RT-PCR. Furthermore, and the proliferation, cycle distribution and apoptosis of Eca109cells were detected with CCK-8,EdU and flow cytometry after transfection.Results: Under hypoxia, the level of miR-210increased obviously.Transfection of miR-210significantly decreased the proliferation of cancercells in CCK-8test and reduced the uptake of EdU, respectively. Transfectionof miR-210resulted in a significant increase in the proportion of cells in G2/Mphase. No significant apoptotic changes were found.Conclusion: miR-210was inducted by hypoxia in ESCC cells; miR-210may inhibit proliferation of mainly by inducing cell cycle arrest in G2/Mphase.Part2miR-210targeted the expression ofPlk1geneObjective: To explore the candidate target of miR-210that reugulatesG2/M arrst.Methods: The candidate target of miR-210that reugulate G2/M arrst waspredicted by bioinformation.The dual luciferase vector which contains3’-UTRof the target was constructed, and was regared as the binding sites withmiR-210. The expression of the target gene in Eca109cells was investigatedafter transfection by Western blot.Results: Plk1was predicted as the specific target gene of miR-34a bybioinformation method.3’-UTR of Plk1which was contained in the dualluciferase recombinant vector was successfully constructed and vertified byenzyme digestion and gene sequence methods. The results of luciferaseWhenmiR-210oligos were transfected into293T/17cells with the reporter constructpmiR-RB-Report_Plk1, luciferase activity was repressed more than50%compared with transfection of scramble oligos. The level of Plk1proteinexpression in transfected cells with miR-210oligos was significantly decree-ased compared with that of cells with stable integration of the scramblesequence after48hours.Conclusion:3’-UTR of Plk1was the binding sites with miR-210andmiR-210inhabited its protein expression, which suggested Plk1was the targetgenes ofmiR-210. Part3The effect of radiotherapy on plasma miR-210and miR-21inesophageal cancer patientsObjective: To analyze the miR-210and miR-21expression level inesophageal cancer patients and reveale the potential role of their level asresponse markers during radiotherapy.Methods: Plasma miR-210and miR-21expression level of ESCCpatients during radiotherapy and healthy controls were measured by usingreal-time RT-PCR.Results: A totle of55plasma samples from22ESCC patients before＆after radiotherapy and15healthy controls were measured. Plasmaconcentration of miR-210and miR-21in ESCC patients were significantlyhigher than that in healthy controls (P=0.0024;P=0.0004). No significantassociation was found between the levels of the two miRNAs and sex, age,tumor location and differentiation. Furthermore, a significant elevation in theserum miR-210and miR-21levels were observed in the postradiotherapysamples versus the preradiotherapy samples (P=0.0025;P=0.0316).Conclusion: The results suggested that a statistically significant increaseof plasma miR-210miR-21expression in esophageal cancer patientscompared with normal controls.Their potential utility as prognostic andresponse markers during radiotherapy markers had emerged.