The Role Of FHL2 And Its Possible Mechanism In The Progression Of Cervical Cancer

BackgroundCervical cancer is the most common gynaecological malignancy.Worldwide,cervical cancer is both the fourth-most common cause of cancer and the fourth-most common cause of death from cancer in women.About 70%of cervical cancers occur in developing countries.In low-income countries,it is one of the most common causes of cancer death.Worldwide,the incidences of cervical cancer among different countries are significantly different.According to a survey by the International Agency of Research on Cancer(IARC),in 2012,about 85%of the cervical cancer occurred in less well-developed regions,accounting for 12%of female cancer.However,in developed countries,the incidence of cervical cancer accounted for only 3%of female cancer.In China,because of the large population and the unbalanced development of economics among different areas,the incidence of cervical cancer in different areas also show significant differences.For the western or remote mountainous areas,because of the lack of the economic,medical health facilities,and the medical staff,it is difficult to widely employ the cervical cancer screening strategy that is suitable for developed countries or areas.The most significant cause of cervical cancer is persistent human papillomavirus infection.HPV is detected in 99%of cervical tumors,particularly the oncogenic subtypes such as HPV16 and 18.In some developed countries,nationwide cytological screening has dramatically reduced death rates from cervical cancer.However,according to the recommendation of the World Health Organization(WHO),only when the cervical cancer screening coverage surpasses 80%could the aim of reducing the incidence and mortality of cervical cancer be achieved.It is more important to increase the coverage of cervical cancer screening than to increase screening frequency.In the developing world,however,absence of organized screening programs correlates with high cervical cancer death rates.A comprehensive approach that includes early diagnosis of CC can reduce the high mortality rate of this disease globally(Cervical Cancer:World Health Organization,2017).Thus the identification of novel prognostic and predictive biomarkers is an important factor in the management of patients during therapy and in the early diagnosis of cervical cancer.The Four-and-a-half LIM(FHL)-only protein subfamily belongs to the LIM-only protein family.The proteins within the group might originated by gene duplicate from a common ancestor and are sharing a high degree of homology all over their amino acid sequence.The name LIM was derived from the first letter of the transcription factors LIN-11,ISL-1 and MEC-3,from which the domain were originally characterized.LIM domain proteins are important mediators of protein-protein interactions,and thus,they are thought to act as docking sites for multi-protein complex assembly based on their highly conserved cysteine-rich zinc finger-like interaction motifs.FHL2 is one of the FHL protein families and one of the most studied ones so far.The multiplicity of molecular pathways affected by FHL2 suggests an important role in several physiological and pathological events.The function of FHL2 in cancer is particularly intriguing,since it may act as an oncoprotein or as a tumour suppressor in a tissue-dependent fashion.Recently,numerous studies have shown that FHL2 might act as a tumour suppressor or an oncoprotein in a cell-type dependent manner in human cancers.FHL2 was first elucidated in 2003 as a molecular culprit critical for the pathogenesis of breast cancer by Yan et al.FHL2 is highly expressed in gastrointestinal cancers such as colon cancer in which FHL2 is the cell cycle and growth modulator of cancer cells.FHL2 is always downregulated in the clinical samples of hepatocellular carcinoma,suggesting a tumor-suppressive property of FHL2.FHL2 is crucial to cancer cell invasion,migration and adhesion to extracellular matrix.However,the role of FHL2 in CC has not been elucidated until now.In this study,we aimed to investigate the prognostic significance of FHL2 expression in cervical cancer and its effects on cell proliferation,apoptosis and tumorigenicity via in vivo and in vitro studies.Furthermore,we preliminarily explored the role of FHL2 and the possible molecular signalling pathway underlying its involvement in cervical cancer development.According to the experiments,this study includes the following three parts:Part 1 Expression of FHL2 in cervical cancer tissues and its effect on the prognosis of patients with cervical cancerObjective1.To detect the expression of FHL2 in cervical cancer tissues and adjacent tissues,and to analyze the correlation between the expression level of FHL2 in cervical cancer tissues and the clinicopathological parameters of cervical cancer patients.2.To explore the correlation between the expression level of FHL2 in cervical cancer tissues and the prognosis of patients with cervical cancer.MethodsThe cervical cancer tissues and corresponding adjacent tissues of 52 patients with cervical cancer who underwent surgical treatment from January 2011 to December 2012 were collected from the Department of Obstetrics and Gynecology,Qilu Hospital of Shandong University.The clinical and pathological data of the above patients were collected and passed the postoperative patient.The follow-up was performed to collect statistical data on patient survival within 5 years after surgery for subsequent analysis.Immunohistochemistry was used to detect the expression of FHL2 in cervical cancer tissues and adjacent tissues,and the results were statistically analyzed after then.The correlation was analyzed between the expression level of FHL2 in cervical cancer tissues and clinicopathological parameters such as the age,histological type,degree of differentiation,tumor size,and degree of interstitial infiltration,lymph node metastasis,lymphatic infiltration,and FIGO stage in cervical cancer patients.The Kaplan-Meier survival curve was used to analyze the correlation between FHL2 expression and cervical survival time in patients with cervical cancer,and to judge the effect of FHL2 expression on the prognosis of cervical cancer patients.Results1.Immunohistochemical staining(200x)showed that FHL2 was mainly expressed in the nucleus of tumor tissues.2.Quantitative real-time PCR results showed that the expression level of FHL2 mRNA in cervical cancer tissues was significantly higher than that in adjacent tissues.3.Western blotting results showed that the expression level of FHL2 in cervical cancer tissues was significantly higher than that in adjacent tissues.4.Analysis of clinical basic data showed that different degrees of differentiation,depth of interstitial infiltration,lymph node metastasis,lymphatic vessel infiltration and the expression of FHL2 in cervical cancer cells in different FIGO stages were statistically different(P<0.05),and there was no significant difference in FHL2 expression levels among patients of different ages,histological types,and tumor size.5.The Kaplan-Meier survival curve showed that the overall survival time of the FHL2 high expression group was shorter.Conclusions1.The expression of FHL2 in cervical cancer tissues is significantly higher than that in adjacent tissues.21.The expression of FHL2 in cervical cancer is related to the degree of tumor differentiation,depth of interstitial infiltration,lymph node metastasis,lymphatic vessel infiltration and FIGO staging,and may be involved in the development of cervical cancer as an oncogene.3.Patients with high expression of FHL2 have poor prognosis.FHL2 may be used as an indicator of prognosis.Part 2 Effect of FHL2 on apoptosis and proliferation of cervical cancer cells and the research of its mechanismObjective1.To detect the effect of down-regulation of FHL2 expression on the biological behavior of cervical cancer cells2.To detect the effect of up-regulation of FHL2 on the biological behavior of cervical cancer cells3.To explore the possible mechanism of FHL2 affecting the biological behavior of cervical cancer cellsMethodsFour groups of human cervical cancer cell lines(HeLa,CaSki,SiHa and C33a)were selected for culture.The levels of FHL2 mRNA in four groups of tumor cells were detected by quantitative real-time PCR,and FHL2 in four groups of tumor cells were detected by Western blotting.The expression level of the protein was analyzed and the results of the two methods were analyzed to determine the cervical cancer cell line for subsequent studies.Three groups of FHL2 siRNA were selected and transfected into selected cervical cancer cells.The interference efficiency of three groups of FHL2 siRNA was detected by Western blotting.The best interference group of FHL2 siRNA was used for subsequent study.Then,flow cytometry assay,Western blotting and CCK-8 were used to detect the apoptosis and proliferation of cervical cancer cells in different groups when FHL2 expression was down-regulated and up-regulated.Finally,Western blotting was used to detect the expression levels of p-AKT and p-Mtor in AKT/mTOR signaling pathways in cervical cancer cells transfected with FHL2 siRNA,and to explore the possible mechanism of FHL2 affecting the biological behavior of cervical cancer cells.Results1.Quantitative real-time PCR showed that the levels of FHL2 mRNA in HeLa group and CaSki group were significantly higher than those in SiHa group and C33a group in four groups of cervical cancer cell lines.Western blotting results showed that HeLa group and CaSki group in four groups of cervical cancer cell lines The FHL2 protein level was significantly higher than the SiHa group and the C33a group.Therefore,the HeLa cell line and the CaSki cell line were selected for subsequent studies.2 Quantitative real-time PCR showed that the three groups of transfected siRNAs had significant interference effects compared with the negative control group,among which siRNA955 had the highest interference efficiency in HeLa cell line and CaSki cell line(FHL2 had the lowest protein expression level),and siRNA955 The level of FHL2 was significantly reduced in the transfected cell lines,so siRNA 955 was selected for subsequent studies to inhibit the effects of FHL2.3.Flow cytometry assay showed that the apoptosis level of HeLa cells and CaSki cells transfected with siRNA955 was significantly higher than that of the control group.Western blotting showed that siRNA955 transfected HeLa cells and CaSki cells were compared with the control group.There were significant differences in apoptosis-related proteins,in which Bax protein(promoting apoptosis)was significantly increased,and Bcl2 protein(inhibiting apoptosis)was significantly reduced.4.CCK8 assay showed that the proliferation of cervical cancer cells was significantly inhibited in HeLa cells and CaSki cells transfected with siRNA955 compared with the control group.5.Western blotting showed that FLAG-NC(negative control)and FLAG-FHL2 transfected HeLa cells and CaSki cells showed significantly increased FHL2 expression in FLAG-FHL2-transfected cells compared with the control group,demonstrating that FLAG-FHL2 can induce Overexpression of cervical cancer cell FHL2.CCK8 assay showed that the proliferation of cervical cancer cells was significantly promoted in FLAG-FHL2-transfected HeLa cells and CaSki cells compared with the control group.6.Western blotting showed that the expression of p-AKT and p-Mtor was significantly decreased in HeLa cells and CaSki cells transfected with siRNA955 compared with the control group.Conclusions1.The expression of FHL2 is up-regulated in cervical cancer cell lines and can be inhibited by siRNA.2.Down-regulation of FHL2 can induce apoptosis and inhibit proliferation of cervical cancer cells,while up-regulation of FHL2 can promote the proliferation of cervical cancer cells,suggesting that FHL2 may act as an oncogene.3.The possible signaling pathway for FHL2 to play a role in the regulation of apoptosis and proliferation of cervical cancer cells is AKT/mTORPart 3 Effect of down-regulation of FHL2 expression on tumorigenesis in nude miceObjective1.The cervical cancer cell line transfected with FHL2 shRNA was cultured,and a nude mouse model of xenograft was constructed to observe the effect of FHL2 inhibition on the tumor formation of nude mice.2.To detect the expression level of FHL2 in xenograft tumor tissues。MethodsThe FHL2 shRNA was used to transfect Hela cells,and the expression of FHL2 was observed by fluorescence microscopy and Western blotting.The transfection effect was judged accordingly.The transfected Hela cells were then inoculated subcutaneously into the right axilla of 6-week-old female athymic nude mice for a specified number of days,and the growth trend of the tumor was observed,and the difference between the two groups was compared.Western blotting was used to detect the expression of FHL2 protein in FHL2 shRNA-transfected xenograft tumor tissues compared with the control group.Results1.Fluorescence microscopy and Western blotting showed that FHL2 protein expression was significantly decreased in FHL2 shRNA transfected Hela cells,and the transfection effect was good.2.After the FHL2 shRNA-transfected HeLa cells were inoculated into nude mice,the growth of cervical tumors was inhibited compared with the control group,and the volume and quality of the tumor tissues were significantly reduced compared with the control group.3.Western blotting showed that the expression level of FHL2 protein in Xenograft tumor tissues transfected with FHL2 shRNA was significantly lower than that of the control group.Conclusions1.The expression of FHL2 in HeLa cells can be inhibited by shRNA.2.In the nude mouse model,down-regulation of FHL2 can inhibit the growth of cervical tumors,suggesting that FHL2 may become a target for clinical treatment of cervical cancer combined with the first two conclusions.