| Background Nasopharyngeal carcinoma is a nasopharyngeal epi-thelial-orginated tumor,which is epidemic in several provinces of Southern China.At present,radiotherapy is a primary treatment for NPC,chemotherapy is a significantly auxiliary treatment means.In spite of continous improvement of radiotherapy and chemotherapy technology,the curative effect of NPC is still unsatisfactory.One of the most important reasons is generating of chemoresistance in NPC. Therefore,exploring an gene,which is closely related with NPC occurrence,development and chemosensitivity,by means of detecting and targeted modulating this effective gene,which would contribute to dignosing NPC, increasing chemosensitivity,improving curative effect,enhancing survival rate,and there is potential value of clinical application.Erythropoietin-producing hepatocellular receptor A2(EphA2)was overexpressed in many malignant tumors,and expression level of EphA2was closely related with T classification,clinical stage,metastasis and prognosis,which participated in regulating growth,proliferation, migration, invasion and metastasis process of tumor cells.At present,most of studies about EphA2were focused on its effection on invasion and metastasis of many malignant tumors at home and abroad. However,there are rare reports about EphA2modulating chemosensitivity.Paclitaxel is a frequently-used chemotherapitics,and targeted modulating chemosensi-tivity of paclitaxel would contribute to enhancing curative effect to carcinoma.This study will be divided into the following three stages to explore the roles of EphA2in processes of growth,proliferation,invasion in NPC,and further study the roles of EphA2on modulating NPC chemosensitivity to paclitaxel and its molecular mechanism.Charpter1Clinical significance of EphA2expression in Nasopharyngeal carcinomaPurpose To evaluate the expression of EphA2in tissue specimens and cell lines of NPC,and further correlate EphA2expression with clinicopathological parameters in NPC.Method EphA2protein expression in47NPC fresh tissue specimens and21nasopharyngeal non-carcinoma tissue specimens and4NPC cell lines were detected by Western Blot,correlation between the protein expression and clinicopathological parameters was analyzed statistically.Result Expression level of EphA2protein was more upregulated in NPC tissues than nasopharynx non-carcinoma tissues and EphA2protein overexpressed in NPC cell lines,and the expression of EphA2protein was closely correlated with T classification,clinical stage and neck nodal metastasis of NPC,there is statistical significance in diffierence between groups(all P<0.05).Conclusion Overexpression of EphA2in NPC tissues and cell lines was closely correlated with T classification,clinical stage and neck nodal metastasis, promping significant effection of EphA2in tumorigensis and development process of NPC.Charpter2Knockdown and upregulation of EphA2had influences in cell proliferation, cycle and invasion in nasopharyngeal carcinoma cell linesPurpose To investigate whether downregulating and upregulating of EphA2affect cell proliferation,apoptosis,cell cycle and invasivity in NPC cell lines.Methods Steadily EphA2silencing and upregulating cell lines was established by transfecting NPC5-8F cell lines with lentivirus induced EphA2shRNA and EphA2cDNA,screening with puromycin.Western blot was performed to verify the silencing and upregulating result of EphA2. CCK-8assays,flow cytometry,transwell invasion assays were employed to detect growth inhibition rate,apoptosis rate and cell cycle,and invasivity respectively.Results1.EphA2was successfully knockdowned or upregulated in NPC5-8F cell lines by lentivirus transfecting technology,and steadily EphA2silencing and overexpressing cell lines were established.2. Multiplication capacity were markedly decreased in EphA2silencing5-8F cell lines,compared to5-8F cell lines, there was statistical difference in growth inhibition rate from the time of48h(P<0.05);EphA2upregulating increased multiplication capacity of5-8F cell in vitro,and the difference of growth inhibition rate between EphA2upregulating group and5-8F group was significant(P<0.05).3.G1/G2stage cell proportion(silencing group:74.38±3.71VS control group:53.82±2.19)was increased,and S stage cell proportion (silencing group:17.89±2.21VS control group:30.41±1.55) and G2/M stage cell proportion (silencing group:7.72±2.24VS control group:15.80±3.72) were decreased in EphA2silencing cell lines,respectively (P>0.05). G1/G2stage cell proportion(upregulating group:47.39±3.75VS control group:53.89±2.22) of EphA2upregulating group was decreased (P<0.05),although S stage cell proportion (upregulating group:38.80±7.11VS control group:38.63±4.15) had no change (P>0.05) and G2/M stage cell proportion (upregulating group:14.15■3.00VS control group:7.81±2.36) was incresed (P<0.05).Compared to normal NPC cell lines,invasivity was significantly decreased in EphA2silencing cell lines (EphA2silencting group:59±4VS control group:103±12)(P<0.05).Besides,apoptosis rate had no difference compared EphA2silencing group cell lines with control group cell lines(P>0.05),but EphA2upregulating decreased cell apoptosis rate compared with control group (EphA2upregulating group:4.94±1.45VS control group:8.99±0.84)(P<0.05)Conclusion These results suggest that EphA2modulated cell cycle development,affected cell proliferation ability and cell invasivity in vitro. In brief,EphA2may play a critical role in aggressive transformation and progression in NPC.Charpter3Modulation of EphA2on paclitaxel chemosensitivity and its molecular mechanism in nasopharyngeal carcinoma cell linesPurpose:This study aimed to investigate function of EphA2on paclitaxel chemosensitivity and its molecular mechanism in NPC cell lines.Methods:5-8F cell lines were transiently transfected with EphA2cDNA and EphA2vector plasmid, respectively; Western Blot were employed to estimate EphA2expression after72hours.5-8FEphA2-RNAi+,5-8FEphA2cDNA+,5.8FEphA2vector and5-8F cell lines were treated with different concentration of paclitaxel, respestively; CCK-8assays were applied to measure OD value, then survival rate and IC50of paclitaxel were caculated in each cell lines after48hours.5-8F EphA2cDNA+cell lines,5-8FEphA2vector cell lines and5-8F cell were treated with IC30of paclitaxel; flow cytometry were adopted to evaluate cell apoptosis rate and cell cycle after48hours, respectively.5-8F EphA2cDNA+cell lines,58FEphA2vector cell lines and5-8F cell lines were treated with paclitaxel in IC30concentration; Western Blot were utilized to measure expression of EphA2, cell cycle regulatory proteins (CDK2, Cyclin E, p21, p27, Rb and p-Rb) and PI3-K/Akt signal pathway proteins (Akt, p-Akt, GSK-3β, p-GSK-3(3) after48hours, respectively.5-8F EphA2cDNA+cell lines were treated with LY294002(a PI3-K/Akt pathway inhibitor) in concentration of20nM, lOnM and OnM, and then treated with different concentration of paclitaxel; CCK-8assays were employed to evaluate OD value, then cell survival rate were caculated and survival curve were depicted.5-8F EphA2cDNA+cell lines which treated with LY294002were treated with IC30of paclitaxel subsequently; western blot were adopted to estimate expression of EphA2, PI3-K/Akt signal pathway proteins and cell cycle modulation proteins, respectively, and flow cytometry were applied to estimate changes in cell cycle and apoptosis rate, respectively.Results:1. EphA2expression were successfully upregulated in5-8F cell lines which were transiently transfected with EphA2cDNA and vec-tor plasmid, and EphA2overexpressed5-8F cell lines were established.2. CCK-8assays showed that, IC50of paclitaxel in5-8FEphA2RNAi+cell lines and normal5-8F cell lines were1.6nM and5.6nM, which indi-cated that paclitaxel chemosensitivity was significantly increased in5-8F EphA2RNAi+cell lines by silencing EphA2expression compared to5-8F cell lines (P<0.5). IC50of paclitaxel in5-8FEphA2cDNA+cell lines,5-8FEphA2vector cell lines and5-8F cell lines were5nM,0.7nM and0.6nM, which demonstrated that paclitaxel chemosensitivity was significantly decreased by upregulating EphA2expression compared to5-8F cell lines (P<0.5).3. Flow cytometry revealed that apoptosis rate (%) in5-8F EphA2cDNA+cell lines,5-8FEphA2vector cell lines and5-8F cell lines after treated with IC30of paclitaxel were9.84±2.08,8.66±1.59,7.74±1.34, respect-tively,which had no significant difference (P>0.5). G0/G1stage cell pro-portion (%) in5-8FEphA2cDNA+cell lines was significant lower than that in5-8FEphA2vector cell lines and5-8F cell lines (45.25±3.89VS65.85±2.28,64.52±3.31, P<0.5). S stage cell proportion in5-8FEphA2cDNA+cell lines was significant higher than that in5-8FEphA2vector cell lines and5-8F cell lines (31.56±1.59VS25.76±1.89,24.55±3.64, P<0.5). G2/M stage cell proportion in5-8FEphA2cDNA+cell iines was significant higher than that in5-8FEphA2vector cell lines and normal58F cell lines (23.10±4.55VS8.39±0.81,10.94±3.27, P<0.5). Different stage cell proportion in5-8F EphA2vector cell lines had no significant difference compared to that in5-8F cell lines (P>0.5).4. Western Blot manifested that p-Akt and p-GSK-3β expression was upregulated in EphA2overexpressed cell lines after treated with paclitaxel, but Akt and GSK-3β expression had no change. Meanwhile, expression of cell cycle progresssion retardant proteins p21and p27were inhibited,;and inhibitor of cell cycle progression Rb was actived and p-Rb were increased,promoting cell cycle progression; however, expression of cell cycle launching proteins CDK2and Cyclin E had no significant changes.5. Western Blot testified that expression of p-Akt was downregula-ted with Akt phosphorylation inhibited by PI3-K/Akt inhibitor LY294002, but Akt expression had no change. Meanwhile, expression of cell cycle progression retardant proteins p21and p27was increased, and expression of Rb was inactived and p-Rb were downergulated, however, expression of cell cycle launching proteins CDK2and Cyclin E had no significant changes.5-8F EphA2cDNA+cell lines treated with IC30paclitaxel were subsequently treated with PI3-K/Akt inhibitor LY294002in concentration of20nM,10nM and OnM. Flow cytometry revealed that cell apoptosis rate (%) in5-8F EphA2cDNA+cell lines treated with LY294002in20nM and10nM were9.55±0.84and9.92±2.13, which were lower than that in5-8F EphA2cDNA+cell Hnes treated with LY294002in OnM (11.67±2.30), but there was no significant difference (P>0.5). G0/G1stage cell proportion (%) in5-8F EphA2cDNA+cell lines treated with LY294002in20nM and lOnM were65.69±2.78and58.61±2.13, which were significantly increased compared to that in5-8F EphA2cDNA+cell lines treated with LY294002in OnM (46.62±1.77, P<0.5). S stage cell proportion in5-8F EphA2cDNA+cell lines treated with LY294002in20nM and10nM were significantly lower than that in5-8F EphA2cDNA+cell lines treated with LY294002in OnM (24.37±3.70,27.08±2.19VS32.14±1.98, P<0.5). G2/M stage cell proportion in5-8F EphA2cDNA+cell lines treated with LY294002in20nM and lOnM were significantly lower than that in5-8F EPhA2cdna+cell lines treated with LY294002in OnM (9.94±3.97,14.31±1.61VS21.24±1.47, P<0.5). All these results indica-ted that PI3-K/Akt inhibitor LY294002increased paclitaxel chemo-sensitivity in5-8FEphA2cDNA+cell lines.Conclusion:EphA2affected cell cycle regulatory proteins expres-sion through PI3-K/Akt induced signal pathway, and subsequently modulated paclitaxel sensitivity in NPC cell lines.EphA2may be a hopeful molecular target modulating paclitaxel chemosensitivity to NPC,which has important significance to improve curative effect of the NPC.
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