Effect And Mechanism Of Dysregulation Of HOXA13 Expression On Proliferation,Migration And Invasion Of HNE1 And CNE1 Cells In Nasopharyngeal Carcinoma

Objective: NPC(Nasopharyngeal Carcinoma)is one of the common malignant tumors of the head and neck in my country.Most of them are poorly differentiated squamous cell carcinoma with high malignancy.The incidence of NPC in China ranks first in the world,and new cases each year account for about65% of new cases in the world.With the improvement of radiotherapy technology and the implementation of adjuvant chemotherapy,the overall 5-year survival rate after NPC treatment is about 80%,but the main cause of death in NPC patients is still recurrence and distant metastasis after treatment.To further explore the molecular mechanism of NPC recurrence and metastasis and to find new tumor molecular markers is the focus of current research,which has important clinical significance to improve the survival rate of patients.HOXA13(Homeobox A13)gene is a member of A cluster of the nuclear transcription factor HOX(Homeobox Gene)family.Current studies have found that HOXA13 is abnormally expressed in tumors and may be involved in regulating the malignant biology of tumor cells behavior.However,whether HOXA13 affects the occurrence and development of NPC has not yet been reported.Lnc RNA(Long Non-coding RNA)HOTTIP(HOXA Transcript At The Distal Tip)is the transcript of the 5’end of the HOXA gene.Some studies have confirmed that HOTTIP can affect tumor progression by targeting the expression of HOXA13.The previous research of our research group found that HOTTIP plays a role as an oncogene in NPC,and the expression of HOXA13 was significantly reduced after HOTTIP in NPC cells was silenced.By consulting the literature and previous studies,we speculate that HOTTIP promotes the malignant biological behavior of NPC cells by regulating the expression of HOXA13.This experiment is based on the previous experiments and the research status of HOXA13,HOXA13 is taken as the research object and the expression of HOXA13 in NPC is analyzed by using public database and clinical samples.The expression of HOXA13 in NPC HNE1 and CNE1 cells was up-regulated and inhibited by lentivirus transfection technique to analyze the expression differences of metastasis and invasion related molecules Snail(Zinc-finger Transcription Factor)and MMP-2(Matrix metallopeptidase-2)in each group of cells,exploring the influence and molecular mechanism of HOXA13 dysexpression on NPC proliferation,clone formation,invasion and metastasis,and providing theoretical basis and experimental basis for the future study of new clinical therapeutic targets for NPC patients.Methods:(1)The expression of HOXA13 gene in NPC was analyzed using public database and verified in clinical samples;(2)NPC HNE1 and CNE1 cell lines with stable overexpression and silencing of HOXA13 and their corresponding control groups were constructed by lentivirus method: HNE1(wild-type HNE1 cells),CNE1(wild-type CNE1 cells),Ctrl group(cells with overexpression of empty vector only),OEHOXA13 group(cells with stable overexpression of HOXA13),sh Ctrl group(cells with inhibition of empty vector),and sh HOXA13 group(cells with stable silence of HOXA13 expression only).The transfection efficiency of each group was detected by fluorescence microscopy.The m RNA content of HOXA13 in each group was detected by RTq PCR(Realtime Fluorescence Quantitative PCR),and the protein content of HOXA13 in each group was detected by Western-Blot assay.(3)MTT assay was used to detect the effects of stable overexpression and silencing of HOXA13 on the proliferation ability of HNE1 and CNE1 cells;(4)The influence of stable overexpression and silencing of HOXA13 on the clonogenic ability of HNE1 and CNE1 cells was detected by plate clone formation assay.(5)The effects of stable overexpression and silencing of HOXA13 on the migration ability of HNE1 and CNE1 cells were detected by Transwell chamber assay.(6)The effect of overexpression and silencing of HOXA13 on the invasion ability of HNE1 and CNE1 cells was detected by Transwell chamber experiment with matrix gel.(7)RT-q PCR and Western-Blot were used to detect the expression of Snail,MMP-2m RNA and protein in HNE1 and CNE1 groups,respectively.Results:(1)(1)The differential expression of HOXA13 in HNSCC(Head and Neck Squamous Cell Carcinoma)was found in TCGA database.Compared with paracancerous tissues,the expression of HOXA13 was significantly upregulatated in HNSCC(Head and Neck Squamous Cell Carcinoma),and positively correlated with HOXA13 expression.(2)HOXA13 was found to be highly expressed in NPC tissues from Oncomine and GEO databases;(3)The expression of HOXA13 in clinical samples was detected,and HOXA13 was found to be highly expressed in tumor tissues of NPC patients.(2)Establishment and validation of HNE1 and CNE1 cell lines of nasopharyngeal carcinoma with overexpression and silencing of HOXA13:(1)Compared with HNE1-Ctrl group,the m RNA and protein expression of HOXA13 in HNE1-OEHOXA13 group were significantly up-regulated;(2)Compared with the HNE1-sh Ctrl group,the m RNA and protein expression of HOXA13 in the HNE1-sh HOXA13 group were significantly decreased;(3)Compared with CNE1-Ctrl group,m RNA and protein expression of HOXA13 in CNE1-OEHOXA13 group were significantly up-regulated;(4)Compared with the CNE1-sh Ctrl group,the m RNA and protein expressions of HOXA13 in the CNE1-sh HOXA13 group were significantly decreased.(3)MTT experiment results showed that:(1)Compared with the HNE1 and HNE1-Ctrl group,the OD value of HNE1-OEHOXA13 group was significantly increased on the second,third and fourth days;(2)Compared with the HNE1 and HNE1-sh Ctrl group,the OD value of HNE1-sh HOXA13 group was significantly decreased on the second,third and fourth days;(3)Compared with the CNE1 and CNE1-Ctrl group,the OD value of CNE1-OEHOXA13 group was significantly increased on the first,second,third and fourth days;(4)Compared with the CNE1 and CNE1-sh Ctrl group,the OD value of CNE1-sh HOXA13 group was significantly decreased on the second,third and fourth days.(4)The results of clone formation experiment showed that:(1)Compared with the HNE1 and HNE1-Ctrl group,the clone formation rate of HNE1-OEHOXA13 group was significantly increased;(2)Compared with the HNE1 and HNE1-sh Ctrl group,the clone formation rate of HNE1-sh HOXA13 group was significantly decreased.(3)Compared with the CNE1 and CNE1-Ctrl group,the clone formation rate of CNE1-OEHOXA13 group was significantly increased;(4)Compared with the CNE1 and CNE1-sh Ctrl group,the clone formation rate of CNE1-sh HOXA13 group was significantly increased.(5)The results of migration experiment showed that:(1)Compared with the HNE1 and HNE1-Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly increased;(2)Compared with the HNE1 and HNE1-sh Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly decreased;(3)Compared with the CNE1 and CNE1-Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly increased;(4)Compared with the CNE1 and CNE1-sh Ctrl group,the number of cells passed through in CNE1-OEHOXA13 group was significantly decrease.(6)The results of invasion experiment showed that:(1)Compared with the HNE1 and HNE1-Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly increased;(2)Compared with the HNE1 and HNE1-sh Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly decreased;(3)Compared with the CNE1 and CNE1-Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly increased;(4)Compared with the CNE1 and CNE1-sh Ctrl group,the number of cells passed through in HNE1-OEHOXA13 group was significantly decreased.(7)The expression of invasion indexes Snail and MMP-2 in HNE1 and CNE1 groups:(1)Compared with the HNE1 group and the HNE1-Ctrl group,the m RNA and protein expressions of Snail and MMP-2 in the HNE1-OEHOXA13 group were significantly upregulated.(2)Compared with HNE1 group and HNE1-sh Ctrl group,the m RNA and protein expressions of Snail and MMP-2 in HNE1-sh HOXA13 group were significantly decreased.(3)Compared with the CNE1 group and the CNE1-Ctrl group,the m RNA and protein expressions of Snails and MMP-2 in the CNE1-OEHOXA13 group were significantly up-regulated.(4)Compared with the CNE1 group and the CNE1-sh Ctrl group,the m RNA and protein expressions of Snail and MMP-2 in the CNE1-sh HOXA13 group were significantly decreased.Conclusion :(1)HOXA13 is highly expressed in NPC tissues;(2)NPC HNE1 and CNE1 cell lines with stable overexpression and silencing of HOXA13 were successfully constructed by lentivirus method;(3)Overexpression of HOXA13 can enhance the proliferation and clone formation ability of HNE1 and CNE1 cells;Silencing HOXA13 can inhibit the proliferation and clone formation of HNE1 and CNE1 cells.(4)Overexpression of HOXA13 can promote the migration and invasion ability of HNE1 and CNE1 cells;Silencing HOXA13 can inhibit the migration and invasion ability of HNE1 and CNE1 cells.(5)HOXA13may affect invasion and metastasis of NPC cells by regulating the expression of Snail and MMP-2;(6)HOXA13 may act as an oncopromoter gene in NPC,and is expected to become a new target for the pathogenesis and treatment of NPC.