| Background:Cervical cancer is a complex process involving multiple factors,and its pathogenesis needs further clarification.At present,HPVE6 and E7 carcinogens are thought to inhibit p53-related and Rb-related signaling pathways,resulting in uncontrolled cell cycle,aneuploidy formation,avoiding apoptosis of aneuploidy cells so as to continue to proliferate and leading to cervical cancer.Mad2 and BubR1 are two key spindle checkpoint proteins and the last line of defense against aneuploidy and chromosome instability.Studies have shown that Mad2 and BubR1 are closely related to aneuploidy and human tumorigenesis,and affect the sensitivity of cancer cells to anti-microtubule drugs.Studies have confirmed that Mad2 is the downstream molecular of p53,and p53 affects chromosomal instability by inhibiting the expression of Mad2.P63 gene is one of the members of p53 family.Some studies have shown that?Np63,a subtype of p63,may antagonize the anti-cancer effect of p53 by competitive binding to p53 DNA target region,thereby participating in the occurrence and development of cervical cancer and playing the role of carcinogenic genes.Therefore,the study of the interaction between ?Np63 and Mad2 and BubR1,and their role in the pathogenesis of cervical cancer has aroused our interest.Aim:the purpose of this study was to clarify the relationship between ?Np63 and Mad2,BubR1 and the effects of this relationship on the malignant potential of cervical cancer cells and the sensitivity of cervical cancer cells to antimicrotubule drugs.Methods:Immunohistochemistry was used to detect the expression of Mad2,?Np63,and BubR1 in cervical lesion tissues of different grades.The results of immunohistochemistry were divided into high expression(++and+++)and low expression(-and+)according to the semi-quantitative score of Immunohistochemistry.The recombinant plasmids pEGFP-Mad2 and pEGFPBubR1 overexpressing Mad2 and BubR1 were constructed.PEGFP-Mad2 and pEGFP-BubR1 were transfected into SiHa cells alone or co-transfected(the cotransfection group was named pEGFP-Mad2-BubR1).The siRNA sequence of?Np63(siRNA-?Np63)was synthesized and transfected into SiHa cells.Real time PCR and western blot were used to detect the expression of ?Np63,Mad2 and BubR1 in SiHa cells.CCK-8 was used to detect cell proliferation.Transwell was used to detect cell migration and invasion.Fourteen hours after nocodazole treatment to activate spindle detection points,PI staining flow cytometry was used to detect cell cycle.Twenty four hours after nocodazole treatment,Annexin V-FITC/7-AAD double staining flow cytometry was used to detect apoptosis.Results:The expression of ?Np63 protein in HSIL and cervical squamous cell carcinoma is higher than that in chronic cervicitis.The expression of Mad2 protein gradually decreased in chronic cervicitis,HSIL and cervical squamous cell carcinoma.However,there was no difference in the expression of BubR1 in the above tissues.The expression of ?NP63 and Mad2 was negatively correlated.Overexpression of Mad2 and BubR1 can slow down the proliferation of SiHa cells,enhance the G2/M arrest induced by nocodazole activation,and enhance the sensitivity of SiHa cells to anti-microtubule drug nocodazole.The above effects of Mad2 and BubR1 were synergistic.Mad2 can significantly inhibit the migration and invasion of SiHa.The effect of BubR1 on cell migration and invasion and its synergistic effect with Mad2 are still unclear.Knockdown of ?Np63 can up-regulate the expression of Mad2 without affecting the expression of BubR1.Interference with the expression of ?Np63 can inhibit the capabilities of proliferation,migration and invasion of SiHa cells,enhance the G2/M arrest induced by the activation of nocodazole,and enhance the sensitivity to the anti-microtubule drug nocodazole.It was found that the effects of ?Np63 knockdown on the function of SiHa cells were highly consistent with that of overexpression of Mad2.Conclusion:In cervical squamous cell carcinoma and HSIL,the expression of ?Np63 was high,the expression of Mad2 was low,and the expression of BubR1 was not different significantly;?Np63 was negatively correlated with Mad2,but not with BubR1;Overexpression of Mad2 and BubR1 could reduce the malignant potential of SiHa cells and the resistance to microtubule drugs,and there was synergistic effect.Knockdown of ?Np63 could up-regulate the expression of Mad2,without affecting the expression of BubR1,and affect the function of SiHa cells which was highly consistent with overexpression of Mad2.Therefore,we suggest that ?Np63 may increase the malignant potential of cervical squamous cancer cells by inhibiting the expression of Mad2,which may be a new molecular mechanism for the occurrence and development of cervical cancer.?Np63 may enhance the resistance of cervical squamous cancer cells to microtubule drugs by inhibiting the expression of Mad2,and may be used as a candidate target for the study of cancer chemotherapeutic resistance. |
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