| Objective: Ovarian cancer has the third highest incidence of ovarian cancer,the highest mortality of gynecological malignancies.ovarian cancer surgery combined with cisplatin and paclitaxel chemotherapy is the standard treatment for ovarian cancer.But the recurrence rate as high as 70%-80%,five-year survival The rate as low as 15%-38.7%,which is highly invasive and resistant to chemotherapy.Looking for the specific oncogene of ovarian cancer,in-depth study of multidrug resistance mechanisms of ovarian cancer,targeting the implementation of precision treatment is to improve the survival rate of the key.Our previous study found that mi R-1307 can regulate DAPK3(death-associated protein kinase-3)mediated resistance in ovarian cancer by Micro RNA microarray and q PCR.DAPK3(death-associated protein kinase 3)a member of the DAPK family,because of its highly but also involved in a variety of pathophysiological processes,such as inhibition Tumor growth,regulation of apoptosis,autophagy,membrane foaming stress fiber formation,has received widespread attention.The specific mechanism of DAPK3 is complex and changeable,and has received wide attention.This study will DAPK3 gene on the occurrence,development and resistance mechanism of ovarian cancer.Micro RNAs(mi RNAs)are a class of highly conserved 20-25 bp single-stranded non-coding RNAs that affect the life cycle of cells mainly through post-transcriptional modifications,such as mi RNAs Expression and the prognosis of malignant tumors are closely related.Our previous study predicted by software that mi R-650 target gene may be DAPK3.mi R-650 is expressed only in humans,located on chromosome 22,has been studied in chronic leukemias,colon and gastric cancers,and has not been studied in ovarian cancer.We study the interaction between mi R-650 and DAPK3 and their functions in ovarian cancer,and provide new ideas for the early diagnosis of ovarian cancer and targeted precise treatment.Methods:Immunohistochemistry and immunofluorescence detection of DAPK3 in 20 cases of benign serous ovarian tumors,20 cases of borderline tumors,30 cases of ovarian chemoresistance,28 cases of chemotherapy-sensitive tissue expression,analysis of DAPK3 expression and ovarian Relationship between chemoresistance in serous carcinoma and its clinicopathological characteristics in patients with ovarian cancer.Western blotting and q RT-PCR methods were used to detect the expression of DAPK3 in 18 cases of ovarian benign serous tumors,11 cases of borderline tumors,20 cases of ovarian chemoresistance and 20 cases of chemosensitive tissues.Kaplan-Meier analysis of DAPK3 protein expression in patients with The relationship between DAPK3 expression in ovarian epithelial cells Hosepic,ovarian cancer-resistant paclitaxel cells SKOV3-TR30 and its parental cells SKOV3,ovarian cancer cisplatin cells COC1 / ddp and its parental cells COC1 were detected by Western bolting and q RT-expression.Using Targetscan(Targetscan7.1(http://www.targetscan.org),mi RNABSE and other commonly used bioinformatics software to predict mi R-650 may bind to 3,UTR of Dapk3.To construct the pmir-DAPK3 and pmir-DAPK3-mut vector,mi R-650 overexpression vector and empty vector were constructed.The recombinant vector was identified by PCR and gene sequencing to prove that the recombinant vector Build successfully.The recombinant plasmids were co-transfected into 293 T cells with mi R-650 mimics or Negative Control(NC)respectively using transfection reagent.The dual luciferase reporter assay was used to detect mi R-650 on the PMIR-DAPK3 reporter gene binding inhibition.The expression of mi R-650 was detected by q RT-PCR in 18 normal ovarian tissues,11 ovarian border tumors and 40 ovarian serous epithelial tissues(chemo-sensitivity and chemo-resistance in each of 20 cases).Kaplan-Meier analysis of mi R-650 Relationship between expression and patient prognosis.The m RNA expression of DAPK3 was detected by q RT-PCR at 48 hours after ovarian cancer cell line SKOV3-TR30 was transfected with mi R-650mimic(mimic NC).DAPK3 protein expression was detected by Western bolting assay and Annexin V-FITC apoptosis assay reagent Cells were stained and apoptosis was detected by flow cytometry.PI cell cycle kit test detects cell cycle changes.Result:Immunohistochemistry showed that DAPK3 was highly expressed in ovarian serous carcinoma tissues(75.8%).The expression of DAPK3 in the cytoplasm wasfound by immunofluorescence.The results of histochemical analysis showed that DAPK3 was significantly increased in ovarian serous chemoresistant tissues(p =0.007).The relationship between DAPK3 expression and the clinicopathological features of ovarian cancer patients was analyzed.The results showed that DAPK3 expression and stage(P = 0.018),lymph node metastasis P = 0.022),which was significantly correlated with tumor differentiation(P = 0.002),but not with age(P>0.05).The results of q RT-PCR showed that DAPK3 was highly expressed in ovarian cancer tissues,which was significantly higher than that in normal ovarian tissues(P =0.0003),significantly higher than that in borderline ovarian tumors(P≤0.0001).The expression of m DAPK3 in chemoresistant tissues was significantly higher than Chemotherapy-sensitive tissue.q RT-PCR showed that m DAPK3 expression in ovarian cancer cells was significantly higher than that in ovarian epithelial Hosepic cells(P≤0.01),m DAPK3 expression in ovarian cancer-resistant paclitaxel-resistant cell line SKOV3-TR30 was significantly higher than that in parental cell SKOV3(P≤0.01)The expression of m DAPK3 in COC1 / DDP-resistant cisplatin cell line was not significantly different from that in parental COC1 cells(P = 0.103).The expression of DAPK3 protein in ovarian epithelial serous carcinoma tissues was significantly higher than that in ovarian epithelial serous carcinoma tissues(P = 0.001)by Western bolting assay;-TR30)was significantly higher than ovarian cancer chemosensitive tissues and cells(Figures 4,5).Western bloting was used to analysis of40 cases of ovarian serous carcinoma tissue DAPK3 expression and prognosis,Kaplan-Meier analysis by Log-Rank test found that overexpression of DAPK3 ovarian serous carcinoma patients overall survival rate was lower than the low expression(P <0.05).The results of luciferase reporter assay showed that the relative fluorescence of luciferase reporter gene was significantly lower in co-transfected mi R-650 mimics +wild-type DAPK3 plasmid compared with wild-type DAPK3 plasmid and empty vector(11.4001 and 9.8707,P = 0.018).However,luciferase reporter assay and luciferase activity of firefly luciferase reporter gene were detected by luciferase reporter gene between cotransfected mi R-650 NC+ DAPK3 mut and pmir-mi R-650 mimic and pmir-DAPK3-mut group The difference was not significant.The expression of mi R-650 in normal ovarian tissue was higher than that in ovarian serous carcinoma(p≤0.0001)by q RT-PCR.The expression of mi R-650 in ovarian tissue was significantly higher than that in chemotherapy-resistant tissue(P = 0.002).q RT-PCR results showed that mi R-650 was higher in ovarian epithelial Hosepic cells than in various ovarian cancer cells(P <0.01).The expression of mi R-650 in ovarian cancer cell line SKOV3 was significantly higher than that in SKOV3-TR30 ovarian cancer cells(P≤0.01).The difference of mi R-650 expression in ovarian cancer cell COC1 / ovarian cancer cisplatin cell line COC1 / DDP Not significant(P> 0.05).Kaplan-Meier analysis showed that the overall survival rate of mi R-650 group was higher than that of low expression group(P <0.01)by Log-Rank test.The expression of m DAPK3 by q RT-PCR was not significantly different at 48 hours after mi R-650 mimic was transfected into SKOV3-TR30 cells(P> 0.05).The DAPK3 protein expression was significantly decreased by western bolting(P <0.01).Flow cytometry showed that the apoptosis of SKOV3-TR30 transfected with mi R-650 mimic significantly increased compared with that of NC group(P <0.05).Overexpression of mi R-650 in SKOV3-TR30 cells enhanced the apoptosis of ovarian cancer Dead effect.The results of cell cycle analysis showed that the proportion of G1phase(P = 0.0138),the proportion of S phase(P = 0.0057),G2(P = 0.0044)was decreased in mi R-650 mimic transfected cells and G2 phase.DAPK3 in ovarian cancer was significantly higher than ovarian borderline tumors,ovarian benign epithelial serous tumors,DAPK3 high expression of poor prognosis.The expression of DAPK3 in ovarian cancer cell lines was significantly higher than that of ovarian cancer epithelial cells;the expression of DAPK3 and paclitaxel-resistant cells in ovarian cancer cells was significantly higher than that in parental cells,but not in cisplatin-resistant cell lines and their parental cells Not significant,DAPK3 high expression and paclitaxel resistance.mi R-650 and DAPK3,UTR region directly.Conclusion:The expression level of mi R-650 in ovarian cancer cells and tissues was lower than that in chemotherapy-resistant tissues;the expression level of mi R-650 in paclitaxel-resistant cell lines was lower than that of parental cells;The parent cell expression difference was not significant.The negative correlation between the expression of mi R650 and the malignant degree of ovarian cancer may be related to the resistance of paclitaxel to chemotherapy.Mi R-650 negatively regulates DAPK3 expression at the post-transcriptional level.Overexpression of mi R-650 in paclitaxel-resistant cells can induce apoptosis of ovarian cancer cells and arrest the G2 phase of ovarian cancer cells.The lack of expression of mi R-650,resulting in overexpression of DAPK3 may be the molecular mechanism of paclitaxel resistance in ovarian cancer.Overexpression of mi R-650 may be a target of reversal of paclitaxel resistance in ovarian cancer. |
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