| Cervical cancer is one of the most common gynecological malignancies,ranking fourth in the incidence of female malignancies worldwide and second in the mortality rate of female malignancies in developing countries.In recent years,A growing body of research shows that cervical cancer occurs at a younger age,and the number of patients under 25 years old suffering from cervical cancer is gradually increasing.Cervical cancer has become one of the important risk factors threatening the life quality and safety of women.The occurrence and development of cervical cancer is a long-term process,and early detection and treatment are the keys to reduce mortality.There are many causes of cervical cancer,of which persistent human papillomavirus(HPV)infection is the main cause of cervical cancer.HPV can be detected in 99.7%of cervical tumors,especially two high-risk virus subtypes,HPV16 and HPV18.Continuous infection of high-risk human papillomavirus(HR-HPV)will lead to normal cell metaplasia,viral gene integration with human cell genome,and high expression of oncogenes HPV E6 and E7,which integrate into host chromosome to generate E6 and E7 proteins and eventually lead to canceration.Studies have shown that E7 protein can combine with PRB to achieve carcinogenic effects,while E6 can bind with P53 protein,which makes the cell regulation of PRB lost,resulting in a long cell survival time,and some cells will be transformed into malignant cells in this process.The expression of E6 and E7 proteins is the key to carcinogenesis after HPV infection,however,the specific carcinogenic mechanism is still unclear,which has become a research hotspot in recent years.Currently,epigenetic abnormalities are considered to be important carcinogenic mechanisms.Epigenetic regulation of DNA and protein modification is increasingly well known,but RNA modification is still uncharted territory.N6-methyladenine(m6A)is the most common and abundant post-transcriptional RNA modification on eukaryotic mRNA.The presence of m6A on transcripts affects different basic cellular functions such as pre-mRNA splicing,nuclear trafficking,stability,translation and microRNA biogenesis,implying that m6A is associated with many human diseases.The methylation level of m6A in tumors often depends on the level of methyltransferase complex,demethylase and reading protein.Therefore,studying the expression level of m6A related protease gene in tumors is helpful for us to understand the occurrence and development of tumors and to establish a new method for early diagnosis and prognostic analysis of tumors.In recent years,the research on m6A demethylase fat obesity-related protein(FTO)has been increasing gradually,and FTO is closely related to the occurrence and development of lung cancer,breast cancer,endometrial cancer and other malignant tumors.However,there are few studies on FTO in cervical cancer tissues,and its various regulatory mechanisms are still unclear.It is common for tumor cells to use the energy produced by glycolysis to provide their own energy supply.As the first key enzyme in the glycolysis pathway,hexokinase(HK)is also the rate-limiting enzyme for glycolysis in tumor tissues.Tumors have a characteristic glucose metabolism,and these enzymes that metabolize glucose act like other regulators of biological behavior in tumors.Hexokinase 2(HK2)plays dual roles in tumor cells:one is to induce glycolysis,and the level of cellular glycolysis is positively correlated with the expression and activity HK2;the other is binding with volt-dependent anion channel(VDAC)in the outer membrane of mitochondria to inhibit apoptosis.Studies have confirmed that HK2 expression is increased in breast cancer,head and neck cancer,pancreatic cancer,lung cancer,colorectal cancer,liver cancer and cervical cancer.Application of HK2 inhibitors such as 3-bromopyruvate(3-BP)can inhibit the proliferation of various tumor cells,such as human glioma cells,breast cancer cells,multiple myeloma cells and liver cancer cells.HK2 is essential for the proliferation,migration and invasion of cancer cells.But,the specific role and mechanism of HK2 in cervical cancer cells remain unclear.Studies have shown that the methylation level of m6A is correlated with the expression level of HK2.M6A methyltransferase complex can promote the expression of HK2 and activate glycolysis,thus it can promote the proliferation of tumor cells and reduce the apoptosis of tumor cells.The stability of HK2 expression depends on the regulation of m6A reading protein.However,the relationship between m6A demethylase and HK2 remains unclear.Studies have shown that PI3K/AKT/mTOR signaling pathway is correlated with the expression levels of both HK2 and FTO.AKT/mTOR signaling pathway can promote the increase of intracellular HK2 expression and promote the binding of HK2 to mitochondria to generate efficient energy and promote cell growth.In endometrial cancer,estrogen induces FTO nuclear aggregation and FTO overexpression through PI3K/AKT/mTOR signaling pathway,thus enhancing the proliferation and invasion of endometrial cancer cells.Will HK2 and FTO be differentially expressed in cervical cancer cells?Is the abnormal expression related to HPV E6/E7 protein?Is there a regulatory relationship between the three?The purpose of this study was to analyze the expression of HK2 and m6A demethylation enzyme FTO in cervical cancer through in vivo and in vitro experiments,to analyze their interaction and relationship with HPV16 E6/E7 protein,and to explore in detail their effects on cervical tumor tumorigenicity and cancer cell growth,invasion,migration and apoptosis.To explore the possible molecular signaling pathway of HPV16 E6/E7-FTO-HK2 in the development of cervical cancer.According to the experimental purpose and design,this subject can be divided into three aspects:Part Ⅰ:The expression level of HK2 in cervical cancer tissues and its effect on the function of cervical cancer cellsPurpose:1.To detect the differential expression of HK2 in cervical and paracancer tissues and its effect on the prognosis of patients with cervical cancer.2.To observe the effect of different expression of HK2 on the function of cervical cancer cells.3.To discuss the possible molecular mechanism of HK2 affecting the function of cervical cancer cells.Materials and MethodsCervical cancer tissues and corresponding para-cancerous tissues of 36 patients with cervical cancer were collected.Patients who received surgical treatment in the Department of Gynecology of Shandong University Qilu Hospital from January 2015 to June 2017 were selected,and their clinicopathological data were collected at the same time.Firstly,immunohistochemistry was used to detect the expression of HK2 in cervical cancer tissues and adjacent tissues,and to further analyze the relationship between the expression level of HK2 in cervical cancer tissues and the age,tumor diameter,lymph node metastasis and pathological grade of cervical cancer patients.The expression levels of HK2 mRNA and protein in SiHa cervical cancer cells were detected by real-time quantitative PCR and Western blotting assay.The growth,invasion and apoptosis of cervical cancer cells down-regulated by HK2 siRNA were detected by CCK8,Transwell,Wound healing,Western blotting and flow cytometry,respectively.Finally,Western blotting experiments were conducted to detect the expression levels of key proteins of AKT/mTOR signaling pathway,Bax,Bcl-2 and caspase3 in cervical cancer cells down-regulated by HK2.Results1.Immunohistochemical results suggested that HK2 mainly existed in the cytoplasm of cervical cancer.HK2 is highly expressed in cervical cancer tissues and low expressed in normal cervical epithelium,which is closely related to tumor diameter and pathological grade.The results indicate that the level of HK2 expression is related to tumor growth and prognosis.2.SiHa cell lines were transfected with siHK2 to construct HK2 knockout cervical cancer cell lines.The results suggested that down-regulation of HK2 gene could reduce the proliferation and migration ability of SiHa cells,up-regulate the expressions of pro-apoptotic proteins Bax and caspase3,and down-regulate the expression of anti-apoptotic protein Bcl-2.The results indicated that down-regulation of HK2 gene could lead to increased apoptosis of cervical cancer cells.3.Western blot results indicated that down-regulation of HK2 gene could significantly reduce the expression of p-AKT and p-mTOR,while significantly increase the expression of P53.Conclusions1.HK2 is located in the cytoplasm of tumor tissues and is highly expressed in cervical cancer tissues.2.HK2 knockdown can inhibit the growth of cervical cancer cells and induce the apoptosis of cervical cancer cells.3.HK2 regulates the apoptosis and proliferation of cervical cancer cells through AKT/mTOR signaling pathway.Part Ⅱ:HPV16 E6/E7 regulates the expression of HK2 by acting on m6A demethylase FTOPurpose:1.To detect the effect of HPV16 E6/E7 on HK2 gene expression in cervical cancer cells.2.After enhancing the expression of HPV16 E6/E7 in cervical cancer cell lines,the changes of m6A methylation level and the expression of m6A related enzymes were detected.3.To detect FTO expression in cervical cancer tissues.4.To detect the effect of FTO on HK2 gene expression in cervical cancer cells.5.To detect the expression relationship among HPV16 E6/E7,FTO and HK2 in cervical cancer cells.6.To analyze the underlying mechanism of FTO regulation of HK2 gene.7.To analyze the potential mechanism of HPV16 E6/E7 regulation of FTO.Materials and MethodsFirstly,two groups of cervical cancer cell lines,C33A and SiHa cell lines,were cultured.After enhancing the expression of HPV16 E6/E7,qPCR and Western blotting were used to detect the expression level of HK2 and m6A,and the expression levels of m6A demethylation enzyme FTO and ALKBH5 were further detected.Twenty-two cervical cancer tissues and their adjacent tissues were collected from cervical cancer patients admitted from June 2019 to June 2021.The expression of FTO in the selected 22 cervical cancer tissues and their adjacent tissues was detected by Western blotting experiment.After overexpression or knockdown of FTO in two cervical cancer cell lines C33 A and SiHa,qPCR and Western blotting were used to detect the mRNA and protein expression of HK2,and to further understand the expression relationship between FTO and HK2.The effects among HPV16 E6/E7,FTO and HK2 in two cervical cancer cell lines were detected by qPCR and Western blotting assays:experimental grouping:1)HPV16 E6/E7 overexpression,2)FTO overexpression,3)HPV16 E6/E7 and FTO co-overexpression,and the cellular RNA and protein were collected to detect the changes of FTO and HK2 gene and protein expression in each group of cells.The cell nucleus and cytoplasm of cervical cancer cells were isolated.After the overexpression of FTO in cervical cancer cells,the expression of HK2 in the cytoplasm of the two groups of cervical cancer cells was detected by qPCR.After E6/E7 expression was enhanced,GSK3β expression was detected by qPCR and Western blotting.After transfection with GSK3β,total proteins were collected for IP determination,and then Western blotting method was used to detect the expression of FTO protein in the two groups of cervical cancer cells overexpressing GSK3β.Results1.The results of qPCR showed that the expression of HPV16 E6/E7 in cervical cancer cell lines C33A and SiHa in the two groups was increased,compared with the control group,HK2 mRNA expression was significantly increased.Western blotting results showed that the expression of HK2 protein was also significantly increased compared with the control group.ELISA and DOTBLOT results showed that overexpression of HPV16 E6/E7 could increase m6A expression level.2.Both qPCR and Western blotting results showed that the expression levels of FTO and ALKBH5 mRNA and protein decreased significantly after HPV16 E6/E7 expression was enhanced in cervical cancer cells..3.Western blotting results showed that in cervical cancer tissues,FTO was highly expressed in paracancer tissues and low expressed in cervical cancer tissues.4.qPCR and Western blotting results showed that with the increase of FTO expression,the expression of HK2 was significantly decreased,while with the decrease of FTO expression,the expression of HK2 was significantly increased.Further qPCR results showed that after the increase of FTO expression,the expression of HK2 in the cytoplasm decreased.5.In the C33A and SiHa groups of cervical cancer cell lines,qPCR and Western blotting showed consistent results,overexpression of HPV16 E6/E7 decreased the mRNA and protein expression of FTO,and increased the mRNA and protein expression of HK2;overexpression of FTO could increase the mRNA and protein expression of FTO,and decrease the mRNA and protein expression of HK2;if HPV16 E6/E7 and FTO were co-overexpressed,the mRNA and protein expression of FTO and HK2 were close to those of the control group.6.qPCR and Western blotting results were consistent in C33A and SiHa cervical cancer cell lines,and the results showed that HPV16 E6/E7 could enhance the expression of GSK3β.The results of ubiquitination assay and Western blotting suggested that enhanced GSK3βexpression could increase FTO ubiquitination and significantly reduce the expression level of FTO.Conclusions1.In cervical cancer cells,HPV16 E6/E7 overexpression could promote the expression of HK2 and m6A.2.In cervical cancer cells,HPV16 E6/E7 overexpression could decrease the expression of FTO and ALKBH5.3.FTO was lower expressed in cervical cancer tissues.4.In cervical cancer cells,HPV16 E6/E7 overexpression promoted HK2 expression,while m6A demethylase FTO inhibited HK2 expression.5.FTO overexpression down-regulated the expression of HK2 in the cytoplasm,indicating that FTO has a regulatory effect on HK2 expression.6.HPV16 E6/E7 enhanced the expression of GSK3β in cervical cancer cells,accelerated the degradation of FTO protein,thus,the expression level of FTO protein in cells is reduced,so it has a regulatory effect on the expression of FTO.Part III:FTO affects the function of cervical cancer cells and tumorigenesis in nude mice by regulating HK2 expressionPurpose1.To confirm the effect of the differential expression of FTO and HK2 on the function of cervical cancer cells.2.To explore the effect of FTO overexpression on tumorigenesis in nude mice.Materials and MethodsThe cell function experiments were divided into 4 groups,1)control group,2)HK2 overexpression group,3)FTO overexpression group,4)HK2+FTO co-overexpression group.CCK-8,clonogenesis,Transwell and flow cytometry were used to detect the effects of HK2 and FTO on the function of cervical cancer cells,including proliferation,migration and apoptosis.A stable cervical cancer cell line overexpressing FTO was constructed and subcutaneously implanted in nude mice.After 4-6 weeks,the tumors of nude mice were collected,and the expression levels of FTO and HK2 proteins were detected by immunohistochemistry and Western blotting.Results1.Compared with the control group,the CCK8 results showed that the OD value of the group with increased HK2 expression increased,and the OD value of the group with increased FTO expression decreased,while the OD value of the group with increased HK2+FTO expression was similar to the control group.The results showed that the number of cloned cells increased with the increase of HK2 expression and decreased with the increase of FTO expression,while the number of tumor cells cloned with the increase of HK2+FTO expression was basically the same as that in the control group.The Transwell results showed that the number of tumor cells migrated increased after HK2 expression was positive,the number of tumor cells migrated decreased after FTO expression was increased,and the number of tumor cells migrated after HK2+FTO expression was increased was basically the same as the control group.Flow cytometry data indicated that the apoptosis rate of tumor cells in the increased HK2 expression group was decreased,while that in the increased FTO expression group was increased.The apoptosis rate of tumor cells was basically the same as that in the control group after the increased HK2+FTO expression..2.A stable cervical cancer cell line overexpressing FTO was constructed and subcutaneously implanted in nude mice.After 4-6 weeks,the tumors of nude mice were collected,the results of immunohistochemistry and Western blotting showed that the expression of FTO protein was significantly increased in cervical cancer tumor tissues,while the expression of HK2 protein was significantly decreased.Conclusions1.Overexpression of FTO inhibited the proliferation and metastasis of cervical cancer cells,induced apoptosis,and the effects of increased HK2 expression on the functions of cervical cancer cells such as proliferation,metastasis and apoptosis were reversed.2.In nude mouse tumor experiments,FTO overexpression can inhibit the growth of cervical tumors and inhibit the expression of HK2 protein.Combined with the previous research conclusions,it is suggested that FTO and HK2 may be the targets for clinical treatment of cervical cancer. |
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