| Objective To investigate the effects of hypoxia on cisplatin sensitivity in EC9706 cells and to investigate the mechanism of serum containing Qige Powder(SQ)for increasing cisplatin sensitivity based on miR-21 in EC9706 cells in hypoxia.Methods1 The EC9706 cells viability were evaluated by MTT assays after cells were treated with cisplatin at lower concentration(0.35?g·m L-1),middle concentration(0.70?g·m L-1),or higher concentration(1.18?g·m L-1)under normoxia or hypoxia respectively.2 The EC9706 cell cycle distribution was measured using PI staining by flow cytometry(FCM)after cells were treated with cisplatin at different concentrations under normoxia or hypoxia respectively.3 The EC9706 cell apoptosis were detected using Annexin V/PI assays by FCM after cells were treated with cisplatin at different concentrations under normoxia or hypoxia respectively.4 The EC9706 cells viability was evaluated by MTT assays after cells were treated with serum containing Qige Powder(SQ),cisplatin alone,or serum containing Qige Powder combined with cisplatin(SQP)at different concentrations under hypoxia respectively;5 The EC9706 cell cycle distribution and cell apoptosis were measured using PI staining or Annexin V/PI assays by FCM after cells were treated with SQ(v/v,8%),cisplatin alone(0.5?g·m L-1,LP or 1.0?g·m L-1,HP),or SQ combined with cisplatin(0.5?g·m L-1,LSQP or 1.0?g·m L-1,HSQP)(under hypoxia respectively;6 The expression of miR-21,programmed cell death protein 4(PDCD4)m RNA and phosphatase and tensin homologdeleted on chromosome ten(PTEN)m RNA were detected using quantitative reverse transcription-polymerase chain reaction(q RT-PCR)after EC9706 cells were treated with SQ,LP,HP,LSQP or HSQP under hypoxia respectively;7 The expression of PDCD4 and PTEN were detected using Western blot after EC9706 cells were treated with SQ,LP,HP,LSQP or HSQP under hypoxia respectively.Results1 The results from MTT assays showed that the absorbance value(A)was decreased with the rise of cisplatin treated for 48 h under normoxia or hypoxia respectively.All absorbance value in hypoxia were higher than that of in normoxia under the same cisplatin concentrations(P <0.05).The IC50 value for EC9706 cells under normoxia or hypoxia treated with cisplatin for 48 h were 1.18 ± 0.05?M or 2.68 ± 0.06?M respectively(P <0.05).2 The DNA distribution in S phase treated with cisplatin was significantly higher followed by a significant decrease in G1 phase than the control group under normoxia(P<0.05),furthermore,a more significant higher of DNA distribution in S phase and a more significant decrease of DNA distribution in G1 phase were detected treated by cisplatin at low or middle concentration compared with the control in normoxia respectively(P<0.05).Under hypoxia,the DNA distribution in S phase treated with cisplatin at different concentrations was significantly higher that of in normoxia respectively(P<0.05).3 The EC9706 cell apoptosis was significantly increased treated with cisplatin at different concentrations than the control in hypoxia or the control in normoxia respectively in a dose-dependent manner(P<0.05).Furthermore,the apoptosis was significantly decreased treated with cisplatin in hypoxia than that of in normoxia under the same concentrations(P < 0.05).4 Under hypoxia,the absorbance value(A)of LSQP or HSQP was significantly decreased compared with that of SQ or cisplatin alone in EC9706 respectively.The coefficient of drug in interaction(CDI)of LSQP or HSQP was 0.90 or 0.94 respectively.5 Under hypoxia,the EC9706 DNA distribution in S phase was significantly increased treated by LP,HP,SQ,LSQP or HSQP compared with the control respectively(P <0.05).6 Under hypoxia,compared with the control,the EC9706 apoptosis was significantly increased in the other groups(P<0.05).Compared with LP and HP,the apoptosis was significantly increased treated by LSQP or HSQP respectively(P<0.05),and HSQP group compared with LSQP group,the apoptosis was significantly increased(P<0.05).7 Under hypoxia,compared with the control,the expression of miR-21 was significantly decreased in HP,LSQP and HSQP groups respectively(P<0.05).The expression of miR-21 in HSQP group was significantly decreased than that of in LSQP group(P<0.05).8 Under hypoxia,compared with the control,the expression of PDCD4 m RNA was significantly decreased in HP group or SQ group respectively(P<0.05).Compared with the cisplatin alone group,the expression of PDCD4 m RNA or PTEN m RNA was significantly increased in HSQP group or LSQP group respectively(P<0.05).The expression of PDCD4 m RNA or PTEN m RNA in HSQP group was significantly increased than that of in LSQP group(P<0.05).9 Under hypoxia,compared with the control,the expression of PTEN was significantly increased in HSQP group or LSQP group respectively(P<0.05),and the expression of PDCD4 or PTEN in HSQP group or LSQP group was significantly increased than that of in HP group or LP group respectively(P<0.05).Compared with the control,the expression of PDCD4 was significantly increased in the other groups(P<0.05).The expression of PDCD4 was significantly increased than that of both in cisplatin alone group and LSQP group respectively(P<0.05).Conclusion1 The higher EC9706 cells viability treated with cisplatin in hypoxia than that of in normoxia was related to the cycle significant arrest in S phase and apoptosis treated with cisplatin in hypoxia than that of in normoxia.2 Under hypoxia,SQ has synergistic effect with cisplatin on inhibition effect for EC9706 viability.3 Under hypoxia,SQ has synergistic effect with cisplatin on apoptosis-induced effect in EC9706.4 Under hypoxia,the synergistic effect mechanism of SQ and cisplatin to inhibit EC9706 viability may be related to increase the expression of PDCD4 and PTEN by inhibiting miRNA-21 expression,and then induce the EC9706 apoptosis. |
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