| Objective:Cervical cancer(CC)is a common gynecological tumor,which seriously threatens the life and health of women.According to the Cancer Epidemic Statistics published by the International Agency for Research on Cancer in 2018,there are approximately 528,000 new cases and 266,000 deaths of cervical cancer worldwide each year.Eighty-five percent of cervical cancers occur in developing countries with relatively poor health resources.China’s annual cervical cancer cases account for more than 28%,so the government attaches great importance to the prevention of cervical cancer and has carried out free cervical cancer screening program,but it has not been fully popularized,and the number of people receiving screening is very limited.In addition,the early diagnosis and treatment of patients and the weak awareness of primary and secondary prevention directly lead to many cervical cancer patients are already in the late stage.At present,surgery,radiotherapy and chemotherapy are the most commonly used treatment methods for cervical cancer.Although most of the early and middle cervical cancer patients have good effects,but for advanced cervical cancer patients,even if radiotherapy or chemotherapy,the treatment results are unsatisfactory and the five-year survival rate is low.In recent years,molecular targeted therapy has become one of the new directions of tumor therapy.Therefore,it is very important to carry out research on new treatment methods of cervical cancer,find new therapeutic targets and prognostic biomarkers,and explore measures to improve the prognosis of patients with advanced cervical cancer,so as to improve the survival rate of patients with advanced cervical cancer.Heart and neural Derivatives RNA2(HAND2)is a key regulator of sympathetic nervous system development.As the antisense RNA of HAND2-HAND2-AS1 can regulate the development of the central nervous system.Recent studies have found that HAND2-AS1 is low-expressed in a variety of malignant tumor tissues,plays the role of tumor suppressor genes,significantly down-regulation can induce the occurrence of various tumors,and through the hypothesis of competitive endogenous RNA(ce RNA),regulate Lnc RNA-miRNA-m RNA networks,and interfere with the expression of target genes and the conduction of signaling pathways.According to the literature review,there is few researchs report on the role of HAND2-AS1 in cervical cancer.Therefore,this study intends to preliminarily explore the occurrence and development of cervical cancer through HAND2-AS1 regulating the downstream miRNA and m RNA and the possible molecular mechanism.1.By detecting the expression of HAND2-AS1 in cervical squamous cell carcinoma and paracancerous tissues,the relationship between the expression level of HAND2-AS1 and the clinicopathological features and survival prognosis of cervical cancer was analyzed.2.To detect the role of HAND2-AS1 in the proliferation,apoptosis,invasion and metastasis of cervical cancer.3.By analyzing and verifying the downstream target genes of HAND2-AS1,the interaction and regulatory relationships among HAND2-AS1,miR-21-5p and TIMP3 were discussed,and the molecular mechanism and possible signaling pathways of the HAND2-AS1 / miR-21-5p /TIMP3 signaling axis inhibiting the proliferation,invasion,migration and apoptosis of cervical cancer cells were clarified.Materials and Methods:1.The expression level and difference of HAND2-AS1 in cervical squamous cell carcinoma and paracancerous tissues were detected by qRT-PCR and RNA in situ hybridization,and the relationship between HAND2-AS1 expression and clinicopathological characteristics and survival prognosis of cervical cancer patients was analyzed.At the same time,the expression and differences of HAND2-AS1 in human cervical cancer cell lines Siha,Caski,He La,C33 A and normal cervical epithelial cells H8 were detected.2.The overexpressed plasmid HAND2-AS1 and the silencing plasmid sh-HAND2-AS1 / sh-HAND2-AS2 / sh-HAND2-AS3 were constructed and transfected into Si Ha,Ca Ski and C33 A cells of cervical cancer.qRT-PCR was used to detect the expression of HAND2-AS1 in Si Ha,Ca Ski and C33 A cells after transfection.The proliferation,apoptosis,invasion and migration of Si Ha,Ca Ski and C33 A cells in cervical cancer after overexpression or downregulation of HAND2-AS1 were detected by CCK8 experiment,flow cell experiment,scratch test and Transwell assay.3.The downstream miRNA and target gene of HAND2-AS1 were predicted by bioinformatics,and the target miRNA(miR-21-5p)and target gene TIMP3 were obtained.Expression levels and differences of miR-21-5p and TIMP3 in cervical squamous cell carcinoma tissues and adjacent tissues were detected by qRT-PCR.A miR-21-5p overexpressed plasmid mimic and silencing plasmid miR-21-5p inhibitor were constructed.The silencing plasmid sh-TIMP3 was constructed.The dual luciferase reporter assay and RNA Binding Protein Immunoprecipitation(RIP)assay were used to verify the target-setting relationship between Lnc RNA HAND2-AS1 /miR-21-5p and miR-21-5p /TIMP3.CCK8 experiment,flow cell experiment,scratch test and Transwell experiment further verified whether Lnc RNA HAND2-AS1 regulated TIMP3 expression through competitive binding of miR-21-5p,thus affecting the proliferation,invasion,migration and apoptosis of cervical cancer.4.qRT-PCR and Western blot were used to detect the expression of Src,e NOS and EMT related markers E-cadherin,N-cadherin and MMP-9 in the VEGF signaling pathway after the overexpression of HAND2-AS1.5.The Si Ha and Ca Ski cells overexpressing HAND2-AS1 and the control cells(empty plasmid pc DNA)were subcutaneously injected into nude mice to establish the model of cervical cancer metastasis in nude mice.To observe the metastatic tumor of two groups of nude mice.The expression levels of EMT-related markers(E-cadherin,N-cadherin,MMP-9)and key factors in VEGF signaling pathways(TIMP3,VEGFA)were detected by qRT-PCR and Western blot in the two groups of metastatic tumors.Results:1.Analysis of GEO data set GSE63678 showed that the expression level of HAND2-AS1 in cervical cancer tissues was lower than that in normal cervical tissues(P =0.002).TCGA database also showed that compared with normal cervical tissues,the expression level of HAND2-AS1 in cervical cancer tissues was lower(P< 0.05).qRT-PCR was used to detect the expression level of HAND2-AS1 in 58 cases of cervical cancer and paracancer tissues.The results showed that compared with paracancer tissues,the expression level of HAND2-AS1 in cervical cancer tissues was significantly decreased(P < 0.05).In addition,the expression of HAND2-AS1 in Si Ha,Ca Ski,Hela and C33 A cervical cancer cell lines was significantly lower than normal human cervical epithelial cell lines H8(P < 0.05).RISH test showed that the staining intensity of HAND2-AS1 in cervical cancer tissues was significantly lower than that in paracancerous tissues.The expression level of HAND2-AS1 was correlated with lymph node metastasis(P =0.018)and tissue stratification(P < 0.001),but not with age(P =0.377),tumor volume(P=0.521)or FIGO stage(P =0.142).Kaplan-meier survival curve showed that the overall survival of patients with low expression of HAND2-AS1 was shorter(P=0.04).2.qRT-PCR confirmed the successful construction of Si Ha,Ca Ski and C33 A cells with overexpression and down-regulation of HAND2-AS1 and their respective control cells,which could be used for subsequent experiments.The results of CCK-8 showed that,compared with the control group,the proliferation ability of cells in the HAND2-AS1 group was significantly reduced(P< 0.05),while that in the sh-HAND2-AS1 group was significantly increased(P <0.05).Flow cytometry results showed that the number of apoptosis in the HAND2-AS1 group was significantly higher than that in the control group(pc DNA)(P < 0.01).The number of apoptosis in sh-HAND2-AS1 group was significantly lower than that in the control group(sh-NC)(P < 0.01).The results of scratch test and Transwell assay confirmed that the cell migration and invasion ability of the HAND2-AS1 group was significantly lower than that of the control group(P <0.01).The cell migration and invasion ability of sh-HAND2-AS1 group was significantly higher than that of the control group(P < 0.01).3.The miRNA downstream of HAND2-AS1 was predicted by bioinformatics analysis,and only one target miRNA(miR-21-5p)was finally obtained.The expression levels of miR-21-5p in 58 cervical cancer tissues and adjacent tissues were detected by qRT-PCR.The results showed that the expression levels of miR-21-5p in cervical cancer tissues were significantly increased(P < 0.01)and negatively correlated with the expression levels of HAND2-AS1(r=-0.643).P <0.01).Analysis on the Biological Prediction site revealed that there was a specific binding region between the HAND2-AS1 gene and the sequence of miR-21-5p.Dual luciferase reporter gene experimental results show that miR-21-5p can significantly inhibit HAND2-AS1-WT luciferase activity changes and activity(p < 0.01),while the HAND2-AS1-MUT luciferase activity and expression of no statistical significance(p > 0.05),and thus prove HAND2-AS1 and miR-21-5p binding sites,the combination of both,and target relationship between HAND2-AS1 can be directly adsorption miR-21-5p.RIP experimental results showed that,compared with Ig G group and input group,anti-Ago2 antibodies could precipitate both HAND2-AS1 and miR-21-5p at the same time,indicating that both HAND2-AS1 and miR-21-5p could bind to Ago2,so as to further verify the targeting relationship between HAND2-AS1 and miR-21-5p.In further experiments,qRT-PCR results showed that:Compared with the control group(pc DNA+ mimic-NC),the expression level of miR-21-5p in cervical cancer cells was decreased by upregulation of HAND2-AS1(HAND2-AS1 +mimic-NC)(P < 0.05),and the expression level of miR-21-5p was increased by upregulation of miR-21-5p(pc DNA+miR-21-5p mimic)(P < 0.05).After simultaneously upregulating HAND2-AS1 and miR-21-5p(HAND2-AS1+miR-21-5p mimic),the increased expression level of miR-21-5p could be rescued(P < 0.05).The results of scratch test and Transwell test confirmed that compared with the control group,the migration and invasion ability of cells in miR-21-5p mimic group was significantly enhanced(P < 0.05).After upregulation of HAND2-AS1,the enhanced migration and invasion ability of cells in miR-21-5p mimic group was restored(P < 0.05).CCK-8 results showed that the proliferation ability of cells in miR-21-5p mimic group was significantly enhanced(P < 0.05).After the up-regulation of HAND2-AS1,the proliferation ability of cells in miR-21-5p mimic group was restored by the up-regulation of HAND2-AS1(P <0.05).Flow cytometry results showed that the number of apoptosis in miR-21-5p mimic group was significantly lower than that in control group(P < 0.05).After the overexpression of HAND2-AS1,the number of cell apoptosis was significantly increased(P < 0.05).The above experiments indicated that HAND2-AS1 could negatively regulate miR-21-5p and affect the proliferation,migration,invasion and apoptosis of cervical cancer cells.4.Bioinformatics prediction was conducted on the downstream target genes of miR-21-5p,and TIMP3 was finally selected as the target gene.The expression level of TIMP3 in 58 cervical cancer tissues and adjacent tissues was detected by qRT-PCR,and the expression level of TIMP3 in cervical cancer tissues was significantly lower than that in adjacent tissues(P < 0.01),and was negatively correlated with the expression level of miR-21-5p(r=-0.710;P < 0.01),and HAND2-AS1 were positively correlated(r=0.539;P < 0.01).The dual-luciferase reporter gene experiment results showed that miR-21-5p could significantly change the activity of wild-type TIMP3 luciferase(p < 0.01),while there was no statistical significance for the change of mutant TIMP3 luciferase(p > 0.05),thus proving the target determination relationship between miR-21-5p and TIMP3.In further experiments,The results of qRT-PCR and Western blot showed that:Compared with the control group(pc DNA+ mimic-NC),the expression level of TIMP3 in cervical cancer cells was increased by upregulation of HAND2-AS1(pc DNA+ mimic-NC)and decreased by upregulation of miR-21-5p(pc DNA+miR-21-5p mimic)(P < 0.05).After upregulation of HAND2-AS1 and miR-21-5p mimic,the expression level of TIMP3 was increased by upregulation of miR-21-5p mimic.The decreased expression level of TIMP3 could be restored(P < 0.05).On the contrary,compared with the control group(sh-NC +inhibitor NC),downregulation of HAND2-AS1(sh-HAND2-AS1 +inhibitor NC)could decrease the expression level of TIMP3 in cervical cancer cells(P < 0.05),and downregulation of miR-21-5p(sh-NC+miR-21-5p inhibitor)could increase the expression level of TIMP3(P <0.05).After the simultaneous down-regulation of HAND2-AS1 and miR-21-5p(sh-HAND2-AS1 + miR-21-5p inhibitor),the increased expression level of TIMP3 could be restored(P < 0.05).The above rescue experiments demonstrated that HAND2-AS1 regulates the target gene TIMP3 by competitively binding with miR-21-5p.5.The results of scratch test and Transwell test confirmed that compared with the control group(pc DNA+ sh-NC),the up-regulation of migration and invasion ability of HAND2-AS1(HAND2-AS1+ sh-NC)cervical cancer cells was significantly decreased(P < 0.05).After the up-regulation of HAND2-AS1 and down-regulation of TIMP3(HAND2-AS1+ sh-TIMP3),the reduced migration and invasion ability of cervical cancer cells could be restored(P < 0.05).CCK-8 results showed that the proliferation ability of HAND2-AS1 cells was significantly reduced compared with the control cells(P < 0.05),and the reduced proliferation ability of HAND2-AS1 cells could be restored after the up-regulation of HAND2-AS1 and the down-regulation of TIMP3(P < 0.05).Flow cytometry results showed that the number of apoptosis in HAND2-AS1+ sh-NC group was significantly higher than that in control group(P < 0.05).When HAND2-AS1 was up-regulated and TIMP3 was down-regulated,the number of cell apoptosis was significantly decreased(P <0.05).The above experiments further confirmed that HAND2-AS1 regulates TIMP3,which is a downstream effector molecule of HAND2-AS1 and can block the effect of HAND2-AS1 downstream,jointly affecting the proliferation,migration,invasion and apoptosis of cervical cancer cells.KEGG database shows that TIMP3 is located in the upstream of VEGF signaling pathway,which affects biological behaviors such as tumor migration and apoptosis.Therefore,it is considered that the regulation of TIMP3 by the competitive binding of HAND2-AS1 with miR-21-5p may be through the VEGF signaling pathway to inhibit the migration,invasion and apoptosis of cervical cancer.qRT-PCR and Western blot results showed that the overexpression of HAND2-AS1 significantly reduced the expression levels of Src and e NOS in the VEGF signaling pathway(P < 0.05),and the down-regulation of TIMP3 could restore the effect of the overexpression of HAND2-AS1 on Src and e NOS,and increase the expression levels of Src and e NOS(P < 0.05).Similarly,the overexpression of HAND2-AS1 significantly upregulated E-cadherin(P < 0.05),and significantly down-regulated N-cadherin and MMP-9(P < 0.05)in EMT.The down-regulation of TIMP3 can restore the effects of the overexpression of HAND2-AS1 on E-cadherin,N-cadherin and MMP-9,and increase the expression of E-cadherin and decrease the expression of N-cadherin and MMP-9(P < 0.05).The above experiments proved that HAND2-AS1/miR-21-5p/TIMP3 may regulate the proliferation,migration and invasion of cervical cancer cells through the VEGF signaling pathway,and lead to the occurrence of EMT.6.In the model of subcutaneous metastasis in nude mice,the size,volume and weight of subcutaneous metastases formed by Si Ha and Ca Ski cells in HAND2-AS1+ sh-NC group were significantly smaller than those in the control group(pc DNA+ sh-NC)(P < 0.05),while the volume and weight of metastases formed by cells in HAND2-AS1+ sh-TIMP3 group were significantly higher than those in HAND2-AS1+ sh-NC group(P < 0.05).The expression levels of E-cadherin,N-cadherin and MMP-9 in metastatic tumors were detected by qRT-PCR,immunohistochemistry and Western blot.Compared with the control group,the expression levels of E-cadherin and TIMP3 in HAND2-AS1+ sh-NC group were increased(P < 0.05),while the expression levels of N-cadherin,MMP-9 and VEGFA were decreased(P < 0.05).The expression levels of TIMP3 and E-cadherin in HAND2-AS1+ sh-TIMP3 group were decreased(P < 0.05),while the expression levels of VEGFA,N-cadherin and MMP-9 were increased(P < 0.05).It was further confirmed that the regulation of the downstream target gene TIMP3 by HAND2-AS1 affects the occurrence and development of cervical cancer,and the upregulation of HAND2-AS1 can inhibit the EMT of cervical cancer cells,and may play a role through the VEGF signaling pathway.Conclusion:1.The low expression of HAND2-AS1 in cervical squamous cell carcinoma is related to the lymph node metastasis and the degree of tissue differentiation of the patients.The reduced expression level of HAND2-AS1 indicates that the overall survival of the patients is shortened.2.HAND2-AS1 inhibits the proliferation,invasion and migration of cervical cancer cells,and promotes the apoptosis of cervical cancer cells.3.HAND2-AS1 can inhibit the proliferation,migration and invasion of cervical cancer cells and promote the apoptosis of cervical cancer cells through the competitive binding of miR-21-5p to regulate the target gene TIMP3.Its mechanism of action is related to the inhibition of the occurrence of EMT,and may inhibit the occurrence and development of cervical cancer through the inactivation of VEGF signaling pathway. |
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