Mechanism Research Of HOTAIR-miR-326-MTA Pathway On Invasion And Metastasis Of Oral Squamous Cell Carcinoma

Oral squamous cell carcinoma(OSCC)is the most common malignancy in oral and maxillofacial regions.Invasion and metastasis are the most important biological factors affecting prognosis.However,the mechanism of OSCC invasion and metastasis has not been elucidated totally.Current studies have shown that non-coding RNA plays a critical role in the regulation of tumor biological behavior,but studies in oral cancer are relatively few.Homeobox gene(HOX)encodes transcription factors have a characteristic role in the regulation of different parts of the biological behavior on cancer cells.HOX transcript antisense RNA(HOTAIR)is a long non-coding RNA of about 2.2 kb,which is derived from the HOXC locus and is initially found to inhibit the transcription of HOXD gene,thereby regulating the proliferation and invasion of head and neck cancer cells' biological behavior.Recent studies found that HOTAIR has the effect of adsorbing microRNAs through the complementary sponge mechanism,which regulates the expression of cancer-related target genes and realizes its biological regulation effect.Objective:On the basis of detecting the expression of HOTAIR in oral cancer,this study explored the effect of HOTAIR on the biological behavior of OSCC invasion and metastasis.To investigate the role of HOTAIR-miR-326-metastatic tumor antigen(MTA)regulatory axis on the invasion and metastasis of oral cancer cells and Epithelial-mesenchymal transition(EMT).Methods:(1)Fresh tissue specimens from clinical oral cancer were collected,5 normal squamous cell carcinoma cell lines of HN4,HN6,CAL27,SCC9 and SCC25,and total RNA was extracted.Real-time quantitative PCR was performed to detect the expression of HOTAIR in specimens and cells.To analyze the correlation and significance of HOTAIR expression level with lesion size,pathological grade,lymph node metastasis and clinical stage.(2)HN4 and CAL27 cell lines were selected because of HOTAIR expression highly.A small interfering RNA was designed for the specific sequence of HOTAIR,a lentiviral vector(LV3-HOTAIR)and an untransformed control vector(LV3-NC)were constructed.Lentiviral vector transfected HN4 and CAL27 cells,detection HOTAIR knockout efficiency with real-time quantitative PCR.TRANSWELL chamber was used to detect the migration and invasion of cancer cells after HOTAIR knockdown.Western blot was performed to detect the changes of interstitial marker(Vimentin)and Epithelium marker(E-cadherin)after HOTAIR knockdown of HN4 and CAL27 cells.(3)Find out the possible target miRNAs for HOTAIR by bioinformatics predicts.Real-time quantitative PCR was used to detect the expression level of these predicted miRNAs after HOTAIR interference,and the most obvious miRNAs were selected,miR-326.The miR-326 target gene was predicted by targetscan website software,and the target gene MTA family was screened because of close relationship with tumor metastasis.The expression of MTA1,MTA2 and MTA3 protein was detected by immunofluorescence and Western blot after HOTAIR knockdown.The relationship between HOTAIR-miR-326-MTA was analyzed.Construction of miR-326 inhibitor and simulant,transfection of HN4 and CAL27 cancer cells,the expression of MTA1,MTA2 and MTA3 was detected by Western blot,and the relationship between HOTAIR-miR-326-MTA was further analyzed.Using means of HOTAIR knockdown and miR-326 inhibition,TRANS WELL chamber was used to detect the ability of migration and invasion of cancer cells.Results:(1)HOTAIR was highly expressed in oral cancer tissues and HN4 and CAL27 cell lines(P<0.01),There was no statistically significant difference in the expression level between oral cancer tissues and adjacent tissues(P>0.05).The relationship between the expression level of HOTAIR and clinic opathological features is as follows:The grade ?-? of pathological grade was statistically significant compared with grade ?(P<0.05),there was a significant difference between stage?-? and stage ?-? in clinical stage(P<0.05),the size of T3-T4 was statistically significant relative to T1-T2(P<0.05),there was a statistically significant difference between the lymph node metastasis group and the non-lymph node metastasis group(P<0.05).(2)After HOTAIR knock down,the expression of E-cad were significantly enhanced in OSCC cells(P<0.05),While Vimentin expression was significantly reduced(P<0.05).Transwell membrane test showed that the number of transmembrane cells in CAL27 cells was significantly lower than that control group(P<0.01),HN4 cells were more effective(P<0.001).Transwell invasion assay showed that the number of transmembrane cells in CAL27 group was significantly lower than that in control group(P<0.001),HN4 cell membrane number was also significantly reduced(P<0.05).(3)Most of the miRNAs predicted by bioinformatics are manifested elevation at different levels of,the expression of miR-326 was the most obvious,which was 20.9 times of that of the control group(P<0.05).The expression of MTA1,MTA2 and MTA3 in CAL27 and HN4 cells was significantly decreased after HOTAIR knockdown,while the expression of MTA2 mRNA did not change significantly afterHOTAIR knockdown(P>0.05).After HOTAIR knockdown,using miR-326 inhibitor rescue experiments,found HN4 and CAL27 cells migration and invasive ability was significantly enhanced(P<0.05).Conclusion:HOTAIR is highly expressed in squamous cell carcinoma cell lines and is associated with clinical stage of oral cancer and cervical lymph node metastasis.HOTAIR may affect the invasion and metastasis of oral squamous cell carcinoma by HOTAIR-miR-326-MTA pathway,and which can promote EMT.HOTAIR may be a new molecular target for diagnosis and treatment of OSCC.