Study On The Mechanism Of HIF-1α Regulation Of Hippo Pathway Target Gene YAP/TAZ In Malignant Development Of Cervical Cancer

Objective: To investigate the expression and clinicopathological significance of hypoxia inducer factor(HIF-1α),YAP and TAZ proteins in cervical intraepithelial neoplasia(CIN)and cervical squamous cell carcinoma(CSCC)its correlation with clinical malignant phenotype and prognosis.To understand the correlation between the expression of HIF-1α in cervical cancer cells and transcription coactivator YAP/TAZ,and to clarify the main mechanism of HIF-1α regulating transcription coactivator YAP/TAZ expression and affecting the invasion and metastasis of cervical cancer.Methods:(1)The expression levels of HIF-1α,YAP and TAZ in cervical squamous cell carcinoma(CSCC),cervical intraepithelial neoplasia(CIN)tissues and healthy groups were detected and their clinicopathological significance were determined.(2)the expression of HIF-1α m RNA and protein levels in Si Ha,C33 a cells and H8 cells of cervical cancer were detected by real-time fluorescence quantitative PCR and westernblot.(3)The construction of HIF-1αrecombinant lentivirus was constructed and transfected to up-regulate HIF-1α in C33 a cells and transfected sh-HIF-1α to sliencing HIF-1α in Si Ha cell respectively,Then HIF-1α examined by Western blot and RT-PCR.The expression of HIF-1α in cervical cancer Si Ha and C33 a cells was detected,and the effects of HIF-1α expression on growth curve,migration,invasion and proliferation of tumor cells were detected.(4)To detect the effect of HIF-1α expression on YAP and TAZ expression in cervical cancer cells Si Ha and C33 a by lentivirus transfection technique.(5)Construction of xenograft tumor model in nude mice: Based on previous studies at tissue level and in vitro cell level,in vivo experiments were conducted to further verify the effects of altered HIF-1α expression on tumor formation,proliferation rate,tumor volume and tumor weight in nude mice,so as to verify the effect of altered HIF-1α expression on tumor development.Results:(1)The positive expression rates of HIF-1α,YAP and TAZ in normal cervical tissues were significantly lower than in CIN and CSCC tissues.The positive expression rates of HIF-1α,YAP and TAZ in normal cervical tissues were 20%,23.3% and 26.7%,respectively.The positive expression rates of CIN were 56.9%,58.6% and 62.1%,respectively.In CSCC,the positive expression rates of HIF,YAP,and TAZ were 65.0%,72.5%,and 67.5%respectively,with statistically significant differences(P<0.05).The positive expression rates of HIF-1α in the low-differentiation group and the lymph node metastasis group were higher than those in the high-differentiation group and the non-metastasis group although the difference were not statistically significant(P>0.05).YAP is associated with the differentiation of CSCC.The positive expression rates of YAP protein in high differentiated and poorly differentiated CSCC were 57.9% and 85.7% respectively,with statistically significant differences(P<0.05).TAZ is correlated with node metastasis of CSCC.The positive expression rates of TAZ protein were 86.7% and 56.0% respectively in the group with and without lymph node metastasis,with statistically significant differences(P< 0.05).HIF-1α was positively correlated with YAP expression in CSCC(r=0.487,P<0.05).(2)The expression of HIF-1α in cervical normal cells H8 and cervical cancer cell lines Si Ha and C33 a was detected by real-time quantitative PCR and Western blot.The expression of HIF-1α was high in cervical cancer Si Ha cells and low in cervical cancer C33 a cells.(3)After HIF-1α expression was upregulated,both m RNA and protein levels were up-regulated,and tumor cell proliferation,invasion and migration were promoted(P < 0.001).After HIF-1α expression was down-regulated,both m RNA and protein levels were down-regulated,and tumor cell proliferation,invasion and migration were inhibited(P<0.001).(4)Upregulation of HIF-1α promoted the expression of YAP and TAZ in C33 a cells(P < 0.001).After HIF-1α expression was down-regulated,the expression of YAP and TAZ in Si Ha cells was decreased(P<0.001).(5)The knockdown and overexpression of HIF-1α Si Ha and C33 a cells were injected into the left axillary subcutaneously of nude mice respectively.Tumor formation was observed 1 week later,and the nude mice were sacrificed 4 weeks later to measure the volume of transplanted tumor.The results showed: The volume of subcutaneous grafted tumor in nude mice with interference of HIF-1α was smaller than that in the control group,and the difference was statistically significant(P < 0.05),indicating that interference of HIF-1α inhibited the growth of the tumor in nude mice.After overexpression of HIF-1α,the volume of subcutaneous grafted tumor in nude mice was larger than that in control group,and the difference was statistically significant(P<0.05).Conclusion:(1)HIF-1 α,YAP and TAZ are highly expressed in CSCC tissues,and their increased expression may be involved in the occurrence and development of CSCC,and has important significance for clinical prognosis.(2)HIF-1 α can up-regulate the expression of YAP/TAZ protein,a transcription coactivator of Hippo pathway,in cervical cancer cells.The expression of HIF-1α is positively correlated with the expression of transcription coactivator YAP/TAZ.(3)The expression of HIF-1α in cervical cancer cells affects the expression of YAP and TAZ and the proliferation,invasion and metastasis of cervical cancer cells,which provides new ideas for the treatment of anti-tumor drugs and metastasis of cervical cancer.