| Esophageal Cancer is one of the most common malignancies in the world. Due to poor understanding of the m olecular mechanisms underlying the m alignancy of esophageal, the prognosis of ESCC is very poor though rapid progresshas been m ade in surgery and chemotherapy. The metabolic network of tumor cells is different from normal cells. Therefore, it is very promissing to identify therapeutic targets from the aspect of metabolism. The m evalonate pathway(MVA) is a com plex biochemical pathway and is requiredfor produci ng several fundam ental end-products includingcholesterol, isoprenoidsandisopentenyladenine. These end-products play very important roles in the composition and physiology function of the cells. However, the functions of MVA pathay in the tumorigensis of remain unknown.In this study, we cloned the 3-Hydroxy- 3-methylglutaryl coenzyme A reductase(HMGCR), and stud ied the f unction as well a s the m olecular mechanisms in the tumorigenesis of ESCC. W e collected clinical samples of ESCC, cultured the ESCC cell lines a nd examined the prote in level and m RNA level of HMGCR in ESCC tissues and cell lines using W estern blot and Realtime PCR. In the c ellular level, we establishesd the stable c ell lines overexpressing HMGCR by stable transfection with the help of lipofectam in, down-regulated the expression of HMGCR in ESCC cells using RNA interference(RNAi). We inve stigated the effects of HMGCR on the growth, migration and colony for mation of ESCC cells using MTT assay, Boyde n chamber assay and soft agar assay, and e xplored the the ef fect of HMGCR on the activation of ERK-Ras signaling using GST-pull down assay and Western blot. At the end of the s tudy, we modulated the expre ssion of c-Myc in the ESCC cell lines nd examined the effects of c-Myc on the protein and m RNA levels of HMGCR.Our study showed that the m RNA and protein levels of HMGCR were up-regulated in ESCC tissues compared with the normal esophageal tissues. Also, the protein level of HMGCR was higher in ESCC cell lines compared with the immortalized esophageal epithelial cells. Forced expression of HMGCR in Eca109 cells(ESCC cell lin e) and SHEE cells(imm ortalized esophageal epithelial cell line) promoted the growth, migration and colony formation on soft agar. Wth respect to the molecular mechanism, we found that HMG CR promoted the activation of Ras and up-regulated the phosphorylation level of ERK. Moreover, in ESCC cells, we have found that c-Myc positively regulated the expression of HMGCR both at the m RNA level and the protein level.Taken together, our study de monstrated the important functions of HMGCR in the tumorigenesis of ESCC. At the sa me time, our study i mplied the possible therapeutic effects of Statin, the inhibitor of HMGCR, in the treatment of ESCC. Our study can be divided into the following three parts.PartⅠThe expression of HMGCR was up-regulatedin ESCC tissues and cell linesObjective: To investigate the expression pattern of HMGCR in ESCC tissues and cell lines.Methods: Real-time PCR analysis was perf ormed to study the m RNA level of HMGCR in 40 ESCC tissues and paired norm al tissue; Western blot analys is was performed to study the protein level of HMGCR in 6 randomly chosen ESCC tissues and paired norm al tissue; the protein level of HMGCR in ESCC cell lines and esophageal normal epithelial cells was examined using Western blot analysis.Results: Real-time PCR analys is showed tha t the m RNA level of HMGCR was elevated in ESCC tissues com pared with the paired normal tissues; Western blot analysis showed that the protein level of HMGCR was elevated in six random ly chosen ESCC tissues compared with the m atched normal tissues, the stron ger expression of HMGCR was observed in ESCC cell lines com pared with the esophageal normal epithelial cells by the Western blot analysis.Conclusion: The strong expression of HMGCR was observed in both ESCC tissues and cell lin es. Compared with the matched normal tissues and norm al cells, the expression of HMGCR was up-regulated in ESCC tissues and cell lines. The seobservations suggested that HMGCR might play an important role in the progression of ESCC.PartⅡThe functions of HMGCR in the growth, migration and colony formation of ESCC cellsObjective:To examine the effects of HMGCR on the aggressive behaviors(including growth, migration and colony for mation) of ESCC cells and investigate the roles of HMGCR in ESCC progressionMethods:The expression vector of HM GCR(myc-HMGCR, with myc tag) was transfected into the ESCC cells Eca109 a nd esophageal normal epithelial cells SHEE with the help of Lipofectamine. The role of CIZ1 in the growth of ESCC cells Eca109 and esophageal normal epithelial cells SH EE was analyzed using MT T assay. The function of HMGCR on the colony for mation of ESCC cells was analyzed using soft agar assay. The effect of CIZ1 on the migration of ESCC cells Eca109 and esophageal normal epithelial cells SHEE was analyzed using Boyden chamber assay. At the same time, the lentivirus was prepared to knock down the expression of HM GCR in ESCC cells. The effect of knocking down the expression of HMGCR on the colony formation of ESCC cells was analyzed by so ft agar assay. The effect of knocking down the expression of HMGCR on the m igration of ESCC cells was analyzed by Boyden chamber assay.Results:By the stab le transfection using L ipofectamin, the stab le cell lines was established which were forced to expr ess the myc-tagged HMGCR in Eca109 and SHEE cells. The MTT assay showed that forced expression of HMGCR promoted the growth of Eca109 and SHEE cells. The m igration assay using the Boyden cham ber showed that forced expression of HMGC R promoted the migration of Eca109 and SHEE cells. The colon y formation assay sho wed that u p-reguation of HMGCR enhanced the anchorage-indep endent growth of Eca109 and SHEE cells. Additionallly, we constructed the lenti-vi rus-based RNAi vectors and successfully knocked down the expression of HMGCR in ESCC cells. The Boyden chamber assayshowed that knocking down the expression of HMGCR inhibited the m igration of ESCC cells. The colony for mation assay showed that knocking down the expression of HMGCR in ESCC cells attenuated the anchorage-independent growth of ESCC cells.Conclusions: HMGCR prom oted the growth, m igration and colony form ation of ESCC cells, while knocking down the expression of HMGCR inhibited the anchorage independent growth and m igration of ESCC cells. HMGCR plays im portant roles in the progression of ESCC.PartⅢ HMGCR activated Ras-ERK signaling in ESCC cellsObjective:To investigate the mechanism through which HMGCR regulated the aggressive behaviors(including growth, migration and colony for mation) of ESCC cellsMethods:The effect of HMGCR on the activation of Ras and the phosphorylation of ERK was analyzed by GST-pull down assay. Th e effect of c-Myc1 on the expression of HMGCR was analyzed by Western blot analysis and Real-time PCR.Results : The GST-pull down assay showed that over-expression of HMGCR promoted the activation of Ras and th e phosphorylation of ERK, whi le knocking down the expression of HMGCR down-regul ated the phosphorylation of ERK. The Real-time PCR analysis and W estern blot analysis showed that over-expression of c-Myc up-regulated the m RNA level and protein level of HMGCR, while knockdown the expression of c-M yc down-regulated the m RNA level and protein level of HMGCR.Conclusions:HMGCR activated the Ras-ERK signaling in ESCC cells and c-Myc positively regulated the expression of HMGCR in ESCC cells. |
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