Expression And Mechanism Of KCNA1 In Occurrence And Development Of Cervical Cancer

BackgroundFor adult women,due to their specific body structure,gynecological diseases are extremely prone to occur,and cervical cancer is one of the gynecological tumors with a higher incidence.The incidence rate of cervical cancer is about 500,000 cases per year worldwide,and the number of new cases and deaths in China accounts for more than one quarter of the world.At the same time,the age of onset of cervical cancer tends to be younger.The mortality rate of cervical cancer ranks the first among female malignant tumors,therefore making cervical cancer one of the major diseases seriously threatening women's lives.Research on cervical cancer has always attracted much attention.Some studies have shown that the aggressiveness of human cervical cancer is related to many molecular abnormalities,including activation of multiple oncogenes and inactivation of tumor suppressor genes.However,the lack of sufficient genetic and epigenetic data on the pathogenesis and effective targets of cervical cancer hinders the development of new targeted therapy.Previous studies have shown that HPVE6 and E7 fragments inserted into the host gene can induce the abnormal expression of p53 and pRB,which is considered as one of the pathogenesis of cervical cancer.But it can only provide limited information to explain the pathogenesis of tumor through single gene or multi gene.With the development of microarray technology,it is possible to analyze the possible signal pathway and reveal the mechanism of carcinogenesis.In recent years,many studies have found that Wnt/?-Catenin,notch and hedgehog signaling pathways are also closely related to the pathogenesis of hpve6/E7.These signaling pathways may be involved in the occurrence and development of HPV infection related cervical cancer.Disregulation of apoptosis signal is one of the important reasons for the malignant transformation of cells,and the inhibition of endogenous apoptosis pathway mediated by mitochondria is the main way of cell malignant transformation.When the cells receive apoptosis signals,Bax and Bak,the pro-apoptotic factors in the Bcl-2 family undergo oligomerization,transfer to the outer mitochondrial membrane,and act on the voltage-dependent anion channel(VDAC)of the outer mitochondrial membrane,opening the mitochondrial permeability transition pore(mPTP),and releasing cytochrome C activates downstream caspase family proteins,or directly affects the substrate,and cell apoptosis occurs.Mitochondrial potassium conductance affects the opening and closing of mitochondrial permeability transition pore(mPTP).Opening mitochondrial inner membrane potassium channels can help mPTP remain closed,thus maintaining a stable level of mitochondrial membrane potential(??)and can further inhibiting cell death.At present,the relationship between potassium channels and tumor cell proliferation has become a new hotspot in basic research of tumor.Many experiments in cell biology and pharmacology have confirmed that potassium channels play a role in the regulatory mechanism of tumor cell proliferation and survival.In different types of potassium channels,potassium channels(mainly Kirs,Eag),which represent the internal rectifying current,are particularly important for the growth of tumor in vivo.In fact,in addition to maintaining depolarization to relieve the growth inhibition of tumor cells at rest,they also provide favorable environment for tumor cells in the high hypoxia environment caused by accumulation.Potassium channel blockers have been shown to inhibit tumor cell proliferation in vitro and tumor development in vivo.Voltage gated potassium channel subfamily 1(KCNA1/Kv1.1)is an important component of type a potassium channel,which has been found to be a key step in membrane repolarization.Mutations in KCNA1 have been shown to cause type 1 ataxia.In recent years,KCNA1 has also been found to be closely related to the occurrence and development of tumors.However,there is no study on the function of KCNA1 in cervical cancer.The purpose of this study is to explore the role of KCNA1 in cervical cancer and its molecular mechanism.It includes:Part ? Study on the Expression of KCNA1 in Cervical CancerPurpose:1.To study the expression of KCNA1 in cervical cancer tissues and adjacent normal tissues.2 To study the relationship between the expression of KCNA1 and the stage in cervical cancer patients.3.To establish a stable HeLa cell line of KCNA1 knockdown and overexpression,and to study the effect of KCNA1 on the tumor size of nude mice in vitro.Methods:1.The mRNA expression of KCNA1 was analyzed by real-time fluorescencequantitative PCR in cervical cancer and adjacent tissues.2 The protein levels of KCNA1 in cervical cancer cell lines and normal cervical epithelial cell line was analyzed by Western blot,and the cell line suitable for the next experiment was selected.3.Stable HeLa cell lines with knockdown and overexpression of KCNA1 were established by lentivirus infection.Real time fluorescent quantitative PCR and Western blot were used to verify whether the cell lines were constructed successfully.4.The tumorigenesis of nude mice was observed twice a week by subcutaneous injection of cervical cancer cells suspension(KCNA1 knockdown,overexpression and control HeLa cells).To analyze the effect of KCNA1 knockdown and overexpression of cervical cancer cells on the tumor size of nude mice.Results:1.The mRNA expression of KCNA1 was significantly up-regulated in cervical cancer tissues compared with the adjacent tissues.The results of real-time fluorescent quantitative PCR showed that the level of KCNA1 mRNA in 17 cases of cervical cancer were more than 2 times higher than that in the normal control group.2 The statistical results showed that the expression of KCNA1 increased with the stage in cervical cancer patients,indicating that the expression of KCNA1 was significantly related to the poor prognosis of patients.3.The results of Western blot showed that the expression of KCNA1 in cervical cancer cell lines were significantly higher than that in human normal cervical cell lines,and the expression of KCNA1 was the highest in HeLa cell lines.4.The results of real-time fluorescent quantitative PCR and Western blotting showed that the HeLa cell lines with low and over expression of KCNA1 were successfully constructed.5.In nude mice,HeLa cells with KCNA1 knockdown produced smaller tumors while HeLa cells with overexpression of KCNA1 formed larger tumors.Conclusion:1.The results showed that KCNA1 was highly expressed in cervical cancer tissues and cell lines.Therefore,KCNA1 can be used as a new potential diagnostic marker for cervical cancer.2.The results of this study showed that the overexpression of KCNA1 was significantly related to the poor prognosis of the patients,and the tumorigenesis experiment in nude mice also confirmed our results.Therefore,the detection of KCNA1 expression in cervical cancer tissue is expected to be used to predict the prognosis of patients.Part ? Study on the Effect of KCNA1 on the Function of Cervical Cancer cellPurposeThe aim of this study was to explore the effect of KCNA1 on the growth,proliferation,migration and invasion of cervical cancer cells.Methods:1.we examined cell number of the previous established stable KCNA1 knockdown and overexpression HeLa cells2.we examined proliferation of the previous established stable KCNA1 knockdown and overexpression HeLa cells using CCK-8 assay.3.we examined migration of the previous established stable KCNA1 knockdown and overexpression HeLa cells using cell migration assay.4.we examined invasion of the previous established stable KCNA1 knockdown and overexpression HeLa cells using Transwell assay.Results:1.The results of cell count showed that KCNA1 knockdown inhibited the growth of HeLa cells,while KCNA1 overexpression promoted the growth of HeLa cells.2.The results of CCK-8 cell proliferation experiment show that KCNA1 knockdown inhibited HeLa cell proliferation,while KCNA1 overexpression promoted HeLa cell proliferation.3.Cell migration assay showed that KCNA1 knockdown could inhibit HeLa cell migration,while KCNA1 overexpression promoted HeLa cell migration.4.Transwell experiment showed that KCNA1 knockdown decreased HeLa cell invasion,while KCNA1 overexpression increased HeLa cell invasion.Conclusion:1.KCNA1 knockdown can significantly inhibit cervical cancer cell growth and proliferation,Migration and invasion.2.This part of the study found new possibilities for the role of KCNA1 in tumors.Therefore,KCNA1 can be a potential therapeutic target for clinical treatment of cervical cancer.Part ? Study on the Regulatory Mechanism of KCNA1 in Cervical CancerPurpose1.To study whether the expression of KCNA1 could promote the occurrence and development of tumor by activating the signaling pathways of Hg,Wnt and notch.2.To study whether the expression of KCNA1 could induce abnormal apoptosis of tumor cells and promote the occurrence and development of tumors through affectting the function of mitochondria.Methods:1.Protein levels of Hedgehog(Hhg),Wnt and Notch were detected by Western blot in KCNA1 knockdown and overexpression HeLa cells.2.The mitochondrial capacity of KCNA1 knockdown and overexpression HeLa cells was examined by immunostaining with MitoTracker Red CMXRos.we examined the expression of mitochondrial marker(MT)in the three groups using the mitochondria monoclonal antibody.3.Using real-time fluorescent quantitative PCR and Western blotting,we analyzed the expression of mitochondrial markers in KCNA1 knockdown and overexpression HeLa cells.Results:1.Western blotting results showed that knockdown of KCNA1 decreased the protein levels of Hhg and Wntl,and overexpression of KCNA1 increased the protein levels of Wntl and Notch2.The results of mitotracker red cmxros immunostaining showed that the knockdown of KCNA1 decreased the positive staining of mitochondria,and the over expression of KCNA1 showed the opposite effect.3.The results of real-time fluorescent quantitative PCR and Western blotting showed that the mRNA and protein expression levels of mitochondrial markers were up-regulated in HeLa cells overexpressing KCNA1,but down regulated in HeLa cells knockdown by KCNA1.Conclusion:1.Our results showed that knockdown of KCNA1 significantly reduced the protein levels of Hhg and Wntl,while over expression of KCNA1 increased the protein levels of Wntl and notch.KCNA1 may affect the function of cervical cancer cells by regulating the signaling pathways of Hhg,Wnt and notch.However,in these three signaling pathways,KCNA1 may play an important role in regulating Wntl level.2.We speculate that KCNA1 may regulate the proliferation,migration and invasion of cervical cancer cells by regulating the intracellular potassium concentration to change the mitochondrial capacity and affect the apoptosis of cervical cancer cells.