The Study On The LncRNAs Related To Cervical Lymph Node Metastasis Of Tongue Squamous Cell Carcinoma

BackgroundTongue squamous cell carcinoma（TSCC）has the highest incidence in malignant tumors of oral squamous cell carcinoma.It is prone to regional cervical lymph node metastasis in early stage tumor and affects the treatment and prognosis of patients critically.According to statistics,the 5-year survival rate of the patients without cervical metastasis is more than 50%while the 5-year survival rate of the patients with cervical metastasis is less than 30%.In present days,the mechanisms of cervical lymph node metastasis of TSCC is still unclear,and early diagnosis and intervention still lack effective means clinically.Therefore,to further understand the mechanisms of cervical lymph node metastasis of TSCC and find new effective ways in early diagnosis and intervention of cervical lymph node metastasis of TSCC have great significant influence in improving prognosis and the quality of patients’ lives.Long non-coding RNA（1ncRNA）is an RNA molecule longer than 200 nucleotides and is not translated into protein.Increasing reports have shown that 1ncRNAs can regulate gene expression on various levels,including chromatin modification,transcription,and posttranscriptional processing.It has been reported that 1ncRNAs control various cellular processes,such as proliferation,apoptosis and invasion,and are implicated in human diseases,including cancer.The research of 1ncRNA in head and neck cancer has just started at present.The reports about 1ncRNA in TSCC are rare and the mechanism is still unclear.So far,there are no researches specializing in 1ncRNA in relation to cervical lymph node metastasis of TSCC.This research uses gene chips to screen out the differential IncRNAs related to cervical lymph node metastasis of TSCC for further study.It will provide a new direction for the diagnosis and treatment of TSCC.This paper in devided into three parts:1.Identification the 1ncRNAs related to cervical lymph node metastasis of TSCC Objective.Explore the IncRNA and regulatory pathway related to cervical lymph node metastasis of TSCC through IncRNA expression profile chip.This provides the direction for further studies.Methods1.Gene chips were used to detect the differential IncRNAs in TSCC tissue.2.Explore the regulatory pathway related to cervical lymph node metastasis of TSCC by bioinformatics analysis.Results1.There were 2210 IncRNAs with a clear difference between metastasis group and non-metastasis group detected by IncRNA expression profile chips.1188 of these IncRNA were up-regulated and the other 1022 were down-regulated.It was screened out that NR₀02819 and NR₀02323 might be related to cervical lymph node metastasis of TSCC.2.We structured the controlling mode and key molecules which may affect cervical lymph node metastasis of TSCC.ConclusionsIncRNAs were differentially expressed in TSCC metastasis group and non-metastasis group.IncRNA may participate in regulating cervical lymph node metastasis of TSCC directly or indirectly by regulating associated factors.The relationship between MALAT1/TUG1 and TSCC is worthy of further studying.2.Verified the expression of MALAT1 and TUG1 in TSCC tissueObjectiveTo further verify the expression of MALAT1 and TUG1 betweenTSCC metastasis and non-metastasis group,and extracting the significantly different IncRNA for further study.MethodsQ-PCR was used to validate the expression of MALAT1 and TUG1 in TSCC tissue and paracancer normal tissue.Results1.In the metastasis group,Q-PCR showed that MALAT1 was significantly down-regulated in cancer tissues compared to paracancer normal tissue（P<0.001）.2.In the non-metastasis group,Q-PCR showed that MALAT1 was significantly up-regulated in cancer tissues compared to paracancer normal tissue（P<0.001）.3.Contrast with the metastasis group and non-metastasis group,Q-PCR showed that MALAT1 was significantly down-regulated in metastasis group cancer tissues compared to non-metastasis cancer tissues（P<0.001）,and the result is consistent with gene chips;Compared to non-metastasis group paracancer normal tissue,Q-PCR showed that MALAT1 was significantly up-regulated in metastasis group paracancer normal tissue（P<0.001）.4.Compared to non-metastasis group cancer tissues,Q-PCR showed that TUG1 was slightly down-regulated in metastasis group cancer tissues,but it was no significant difference（P>0.05）.ConclusionsAccording the results of Q-PCR,MALAT1 was significantly different in cancer tissues compared to paracancer normal tissues in both metastasis group and non-metastasis group.This suggests that MALAT1 plays an inportant role in the occurrence of the tongue squamous carcinoma.The expression of MALAT1 in non-metastasis cancer tissues apparently higher than the paracancer normal tissues,while the expression of MALAT1 in metastasis group cancer tissues significantly lower than the paracancer normal tissues.It suggest that the expression of MALAT1 has undergone significant changes in the process of cervical lymph node metastasis of TSCC.In contrast with the metastasis group and non-metastasis cancer tissues,the expression of MALAT1 in metastasis group was significantly lower than the non-metastasis group,suggesting that the expression of MALATI may related to cervical lymph node metastasis of TSCC.The effects and mechanisms of MALAT1 in cervical lymph node metastasis of TSCC are worthy further studies.3.The effect of MALAT1 on TSCC cell proliferation,migration and invasion ObjectiveTo detect the MALAT1 expression level in TSCC cell lines,and its effects on TSCC cell proliferation,migration and invasion in vitro.Methods1.Q-PCR was used to detect the expression level of MALAT1 in TSCC cell lines.In addition,the cell scratch adhesion test was done to evaluate the migration ability of TSCC cell lines.2.Construction of MALATI lentivirus with effective interference vector and negative control lentivirus.3.CCK8 cell proliferation assay was done to detect the effect of TSCC cell proliferation ability,when down-regulating the expression of MALAT1 in vitro.4.Cell scratch adhesion test and invasion chamber test were done to detect the effect of TSCC cell migration and invasion ability when down-regulating the expression of MALAT1 in vitro.Results1.MALATI was found with higher expression in CAL-27,SCC15 and Tca8113 cell lines（△Ct≤12）.The results of cell scratch adhesion test showed that the mobility of CAL-27 cells was 57%while SCC15 cells was 63%and Tca-8113 cells at 24%.It is feasible for CAL-27 and SCC15 cells to do cell scratch adhesion test,but infeasible for Tca-8113 cells.2.LV-MALAT1-RNAi（25688-1）lentivirus with effective vector and control lentivirus CON077 were successfully established.3.Down-regulation the expression of MALAT1 sighificantly suppressed TSCC cell proliferation ability in vitro:the proliferation rate of CAL-27 and Tca8113 cells were sighificantly suppressed（P<0.01）,while the proliferation rate of SCC15 cells was slightly decreased but showed no significantly difference（P>0.05）.4.Down-regulating the expression of MALAT1 sighificantly suppressed CAL-27 and SCC15 cells migration ability（P<0.05）and invasion ability in vitro（P<00.01）in vitro.Conclusions1.MALAT1 was found with the highest expression level in SCC15 cell line which had high mobility,with the middle expression level in CAL-27 cell line which had mediocre mobility and with the lowest expression level in Tca-8113 cell line which had low mobility.2.MALAT1 can express low level in TSCC cell lines when MALAT1 lentivirus with effective interference vector（MALAT1-shRNA）is transfered.3.Down-regulation the expression of MALAT1 can suppress TSCC cell proliferation,migration and invasion ability in vitro.Suggest that MALAT1 may play an important role in the cervical lymph node metastasis of TSCC.It may be potential target of the diagnosis and treatment target for the cervical lymph node metastasis of TSCC.Its regulation mechanism in the cervical lymph node metastasis of TSCC is worthy of further studies.