The Function And Mechanism Of High Mobility Group At-hook 1 (HMGA1) On Proliferation And Lymph Node Metastasis Of Cervical Cancer

Objective: The growth of tumors is mainly due to the malignant proliferation of tumor cells.Aberrant regulation of the cell cycle is one of the major factors of unlimited proliferation of tumor cells.It has been reported that High mobility group AT-hook 1(HMGA1)is involved in cell proliferation in a variety of cancers,but the related research in cervical cancer is less The purpose of this study was to investigate the effects of high mobility group AT-hook 1(HMGA1)on cell cycle,cell proliferation and tumor growth of cervical cancer cells and their corresponding mechanisms.Materials and Methods: Using oncomine Database analyzed the expression of HMGA1 in Cervical Squamous Cell Carcinoma and Cervical Squamous Cell.Gene's m RNA expression was measured by real-time PCR(RT-PCR).Collecting the cancer tissues and adjacent normal tissues of cervical cancer diagnosed by pathologists as well as the normal cervical tissues resected due to gynecological benign diseases.The protein expression of studied genes was detected by western blotting and immunohistochemistry.Constructing the overexpressing HMGA1 lentivirus as well as the knockdown of HMGA1 sh RNA lentivirus,infecting of cervical cancer cell,and screening stable cervical cancer cell lines.Flow cytometry was used to measure the cells ratio in different cell cycle phases.The cervical cancer cells proliferation activity was monitored by Cell Counting kit-8 and clone formation assay in vitro.Nude mice subcutaneous tumor formation experiment was used to observed cervical cancer cell tumorigenicity and tumor growth speeds.Results: Oncomine data showed that the expression of HMGA1 in cervical squamous cell carcinoma was significantly higher than that in cervical squamous epithelium.Both m RNA and protein of HMGA1 was significantly higher in cervical cancer tissues compared with that in adjacent normal tissues.HMGA1 expression was correlated with lymph node metastasis and FIGO in cervical cancer,but it had no relevant with age,histological grade,depth of myometrial invasion,vascular invasion,and histological grade.In vitro,HMGA1 overexpression could accelerate the transition from G1 phase to S phase in cell cycle,promote the expression of key proteins during cell cycle,accelerate cell proliferation and cloning of cervical cancer cells and promote the growth of cervical cancer.Conversely,silencing HMGA1 expression could slow the G1 phase to S phase transition,suppress the expression of related proteins,inhibit the proliferation and clone formation of tumor cells,as well as retard the growth of tumor.Conclusions: HMGA1 could play a key role in the cell cycle and cell proliferation of cervical cancer cells.These findings suggested that the high expression of HMGA1 might promote the development of cervical cancer by accelerating cell cycle progression,indicating that HMGA1 might be a potential target for the treatment of cervical cancer in the further.Objective: The distant metastasis and spread of cancer cells are most important factors affecting the prognosis of cancer patients.The local infiltration and invasion of tumor cells into the surrounding tissues and the sowing of organs from the vasculature to distant organs are the only means for cancer metastasis.Numberous studies have reported that High mobility group AT-hook 1(HMGA1)affects the invasion and metastasis of cancer cells.The purpose of this study was to investigate the effect of HMGA1 on the migration,invasion and lymph node metastasis of cervical cancer as well as its mechanism.Materials and Methods: Collecting the cancer tissues of cervical cancer patients diagnosed by pathologists as well as the normal cervical tissues resected due to gynecological benign diseases.The total RNA was extracted from the tissues and cells and the m RNA expression of the target gene was detected by real-time PCR.The expression of HMGA1 protein was detected by immunohistochemistry in situ cervical carcinoma tissues with lymph node metastasis positive as well as lymph node metastasis negative.Constructing the overexpressing HMGA1 lentivirus and knockdown of HMGA1 sh RNA lentivirus,infecting cervical cancer cell lines,and then screening stable cervical cancer cell lines.Wound healing assay and Transwell assay were used to detect whether HMGA1 had an effect on cervical cancer cell migration and invasion or not.The ALGGEN PROMO website was used to predict the binding site of HMGA1 on its downstream target gene promoter.The miRWalk2.0 website was used to predict the binding site of miR-221 and miR-222 on the 3'UTR of TIMP3?The wild-type TIMP3 3'UTR and the mutant TIMP3 3'UTR were inserted into the pmir GLO vector.Chromatin immunoprecipitation technique was used to find out the biological process how HMGA1 promoted the invasion and metastasis of cervical cancer cell,the related pathways as well as the downstream target genes of HMGA1.Nude mouse paw lymph node metastasis model was used to investigate the effect of HMGA1 on cervical cancer lymph node metastasis.Results: Both HMGA1 m RNA and HMGA1 protein expression was significantly higher in cervical cancer in situ with lymph node metastasis of carcinoma in situ than that in cervical cancer in situ with lymph node metastasis negative.Both miR-221 and miR-222 were highly expressed in cervical cancer tissues,and their expression was positively correlated with the expression of HMGA1 in cervical cancer.HMGA1 promoted cervical cancer cell migration and invasion in vitro.HMGA1 could regulate the expression of miR-221/222 gene cluster by directly binding to the common promoter of miR-221/222 gene cluster,therefore enhanced its promoter activity and promoted their transcription.Both miR-221 and miR-222 could also promote the migration and invasion of cervical cancer cells,and they could disrupt the effects of HMGA1 on the migration and invasion of cervical cancer cells in vitro.Further study found that TIM3 was a common downstream target gene of miR-221/222 gene cluster,and miR-221/222 gene cluster could affect the expression of TIMP3,MMP2 and MMP9 protein levels.HMGA1 also regulated the expression of TIMP3,MMP2 and MMP9 protein levels.Nude mice paw lymph node metastasis model results showed that HMGA1 could accelerate cervical cancer cell lymph node metastasis.Conclusions: HMGA1 might play a key role in cervical cancer lymph node metastasis.These findings suggested that HMGA1 might be involved in cervical cancer lymph node metastasis through the HMGA1-miR-221/222-TIMP3-MMP2 / MMP9 pathway.This uncovers that HMGA1 can be used as an indicator to predict lymph node metastasis in cervical cancer.