Expression And Effect Of LncRNA TP73-AS1 On The Proliferation And Apoptosis In Esophageal Squamous Cell Carcinomas

Background and objective Esophageal squamous cell carcinoma（ESCC） is one of the most common malignant diseases. Esophageal squamous cell carcinoma（ESCC） and esophageal adenocarcinoma（EA） are the two main types of esophageal carcinoma. Esophageal adenocarcinoma is more in western countries, but esophageal squamous carcinoma is more popular in developing countries, including east Asia and the Caspian sea belt. Each year more than 300 thousand of newly diagnosed patients with esophageal cancer, more than half are in China. In the Taihang mountains near the area and Linzhou city, there are high incidence. EC is difficult to cure unless it is diagnosed at a very early stage, before metastasis. The five-year survival rate of EC patients remains very poor despite rapid advances in surgical techniques and therapies. Thus it is important to investigate the mechanism and to explore the effective therapeutic strategies of ESCC. lnc RNAs（Long non-coding RNAs） are non-coding RNAs of more than 200 nucleotides（nt） in length and are characterized by diverse and complex sequences and mechanisms of action. Recent studies indicate that lnc RNAs are involved in a variety of biological processes and diseases in humans, such as tumor. lnc RNA TP73-AS1 is located on human chromosomal band 1p36.32. There are few researchs about lnc RNA TP73-AS1. Only Yu, et al. reported that lnc RNA TP73-AS1 is significantly downregulated in non-small cell lung cancer as compared to normal lung tissues（P<0.001）, but it is strongly upregulated in large-cell carcinoma specimens compared to adenocarcinoma, small-cell lung cancer and squamous cell carcinomatissues. These findings indicate that lncRNA TP73-AS1 may play an important role in the development and progression of various tumors.Ours is the first comprehensive analysis of lnc RNA TP73-AS1 in EC. We utilized lnc RNA microarrays to analyze the expression profile of lnc RNAs in ESCC. We investigated the function of lnc RNA TP73-AS1 in the proliferation and apoptosis of ESCC cell lines, and we evaluated the expression and relevance of lnc RNA TP73 AS1 in clinical ESCC. The contribution of lnc RNA TP73-AS1 to ESCC malignancy and the molecular mechanisms underlying its action were also investigated. The research includes three parts. The first part is altered lnc RNA expression in esophageal cancers and correlation with clinicopathological characteristics of ESCC patients. The second part is effect of lnc RNA TP73-AS1 on proliferation and apoptosis of the esophageal carcinoma cell line EC9706 and KYSE30. The last part is preliminary molecular mechanism of lnc RNATP73-AS1.Part One Altered lnc RNA expression in esophageal cancers and correlation with clinicopathological characteristics of ESCC patients.Methods 1. Sixty pairs of esophageal squamous cell cancer tissues and corresponding adjacent normal esophageal tissues specimen were collected. 2. In order to analyze the lnc RNA expression profile, we used lnc RNA microarray. 3. lnc RNA TP73-AS1, lnc RNA LOC345051, lnc RNA XLOC₀08700 and lnc RNA TMEM71 were validated by performing quantitative RT-PCR. 4. Analyse the relation of lnc RNA TP73-AS1 expression levels and tumor location, TNM stage with bivariate correlation analysis.Results 1. lnc RNA microarray results showed that there were 89 differentially expressed lnc RNAs, including 29 lnc RNAs upregulated and 60 lnc RNAs downregulated in ESCC tissues. Lnc RNA TP73-AS1 is upregulated in ESCC tissues. 2. Only lnc RNA TMEM71 was not matched（P >0.05）, lnc RNA TP73-AS1, lnc RNA LOC345051 and lnc RNA XLOC₀08700 were matched by quantitative RT-PCR and Agilent microarray lnc RNA profiles. 3. lnc RNA TP73-AS1 expression levels in ESCC tissues was associated with tumorlocation and TNM stage（P<0.05）. There was no statistically significant correlation between lnc RNA TP73-AS1 expression and either gender, age, lymph node metastases or differentiation status（P>0.05）.Part Two Effect of lnc RNA TP73-AS1 on proliferation and apoptosis of the esophageal carcinoma cell line EC9706 and KYSE30.Methods 1. Construct lentiviral vector with lnc RNATP73-AS1 si RNA, which were transfected into EC9706 and KYSE30 cells. 2. CCK-8 assay and colony formation assay were used to assess the effect lnc RNATP73-AS1 on EC9706 and KYSE30 cell proliferation. 3. Transwell assay was used to assess the effect of lnc RNATP73-AS1 on EC9706 and KYSE30 cell invasion and metastasis. 4. FACS and Hoechst 33342 were used to assess the effect lnc RNATP73-AS1 on EC9706 and KYSE30 cell apoptosis. 5. To confirm the growth inhibitory effect of lnc RNATP73-AS1 si RNA on EC in vivo, a xenograft tumor growth assay was performed.Results 1. The expression of lnc RNATP73-AS1 was significantly decreased in lnc RNA TP73-AS1 si RNA group compared to the control group（P<0.05）. 2. The results of CCK8 assay showed that the proliferation was significantly suppressed 24 hours later in lnc RNA TP73-AS1 knockdown cells（P <0.05）. 3. Compared with blank group（162.0±19.3） and non-sense group（167.4±18.3）, the colony formation number of EC9706 were reduced in lnc RNA TP73-AS1 si RNA1（47.3±7.8） and lnc RNA TP73-AS1 si RNA2 group（56.3±7.8）. The colony formation number of KYSE30 were also reduced in lnc RNA TP73-AS1 si RNA1（43.5±7.5） and lnc RNA TP73-AS1 si RNA2 group（49.5±7.5） than blank group（155.0±17.2） and non-sense group（159.2±18.6）. The results showed that lnc RNA TP73-AS1 knockdown inhibited EC9706 and KYSE30 cell proliferation.4. FITC Annexin V Apoptosis results showed that the number of early and late apoptotic cells at 48–96 hours post-si RNA1-transfection and post-si RNA2-transfection were significantly higher in EC9706 and KYSE30 cells. The results showed that lnc RNA TP73-AS1 knockdown induced EC9706 and KYSE30 cell apoptosis. 5. Hoechst 33342 staining results showed that the percent number of apoptotic cells were（20.72±3.28）% and（18.70±3.10）% in lnc RNA TP73-AS1 si RNA1 and lnc RNA TP73-AS1 si RNA2 group in EC9706, which is significantly higher than blank（5.21±0.61）% and nonsense groups（5.42±0.72）%. In KYSE30 cells, the percent number of apoptotic cells were also significantly higher in lnc RNA TP73-AS1 si RNA1（23.36±4.23）% and lnc RNA TP73-AS1 si RNA2 groups（24.20±4.30）% compared to the control groups. The results showed that lnc RNA TP73-AS1 knockdown induced EC9706 and KYSE30 cell apoptosis. 6. Transwell assay results showed that there was no significant difference between lnc RNA TP73-AS1 si RNA groups and control groups. 7. To confirm the growth inhibitory effect of lnc RNATP73-AS1 si RNA on EC in vivo, a xenograft tumor growth assay was performed. Tumor size and luciferase signal were significantly reduced in the lnc RNATP73-AS1 si RNA mice group（EC9706 and KYSE30 cells transfected with lnc RNATP73-AS1 si RNA1） as compared to control mice（NC and Blank groups） at the fourth week（P<0.05）.Part Three Preliminary molecular mechanism of lnc RNATP73-AS1Methods 1. m RNA microarray was used to analyze the m RNA expression profile in EC9706 and KYSE30 cells transfected with lnc RNA TP73-AS1 si RNA or nonsense RNA. 2. Construct lentiviral vector with BDH2 si RNA, which were transfected into EC9706 and KYSE30 cells. 3. q RT-PCR and Western Blot were used to investigate the relative expression of BDH2 in lnc RNA TP73-AS1 knockdown EC9706 and KYSE30 cells. 4. CCK-8 assay, Hoechst 33342 staining assay and Western Blot were used to assess the effect of BDH2 on EC9706 and KYSE30 cell proliferation and apoptosis.5. q RT-PCR were used to investigate the expression of BDH2. 6. Construct the mutant and wild vector of lnc RNA TP73-AS1. q RT-PCR were used to assess the relative expression of mi R-141-3p. 7. Dual-luciferase assay were used to assess the target gene of mi R-141-3p. 8. Colony formation assay and FACS were used to assess the effect of lnc RNATP73-AS1 on EC9706 and KYSE30 cell proliferation. 9. Transwell assay was used to assess the effect of mi R-141-3p mimic on EC9706 and KYSE30 cell invasion and metastasis.Results 1. To explore the molecular mechanisms of lnc RNA TP73-AS1 in tumorigenesis, m RNA microarray analysis was performed using EC9706 and KYSE30 cells transfected with lnc RNATP73-AS1 si RNA or nonsense si RNA. BDH2 expression was significantly decreased in EC9706 and KYSE30 cells transfected with lnc RNATP73-AS1 si RNA compared to the controls（P<0.05）. 2. BDH2 expression was reduced in lnc RNA TP73-AS1 si RNA1- or si RNA2-transfected cells（P<0.05）. Western blot analysis showed that the BDH2 protein levels were also significantly reduced（P<0.05） in si RNA-transfected cells. 3. BDH2 protein and m RNA levels were significantly decreased in BDH2 si RNA1- or si RNA2-transfected EC9706 and KYSE30 cells as compared to the controls（P <0.05）. 4. CCK-8 assay results showed that at 24, 48 and 72 h hours post-transfection, BDH2 si RNA-transfected cells exhibited significantly reduced proliferation.These results suggested that BDH2 might function as a tumor suppressor in EC cells in vitro. 5. Hoechst 33342 staining results showed that the number of apoptotic cells were 17.70±1.80 and 16.11±1.75 in BDH2 si RNA1 and BDH2 si RNA2 group in EC9706, which is significantly higher than blank（4.22±0.45） and nonsense group（4.31±0.47）. In KYSE30 cells, the number of apoptotic cells were also significantly higher in lnc RNA TP73-AS1 si RNA1（19.71±2.10） and lnc RNA TP73-AS1 si RNA2 group（17.20±1.80） compared to the control groups（P <0.05）。 6. BDH2 expression significantly decreased in EC9706 and KYSE30 cells after BDH2 si RNA1 or lnc RNATP73-AS1 si RNA1 transfection. Levels of cleaved caspase-3 andcleaved caspase-9 proteins were enhanced in BDH2 si RNA1 or lnc RNATP73-AS1 si RNA1-transfected cells. However, levels of Bcl-2, Bax and pro-caspase-9 were not significantly different after transfection. 7. BDH2 levels were significantly upregulated in EC tissues. BDH2 expression was strongly correlated to TNM stage. BDH2 expression was lower during stage I, whereas stages II–III showed higher levels, indicating a correlation between BDH2 expression and TNM stage. There was no statistically significant correlation between BDH2 expression and either gender, age, lymph node metastases or differentiation location（P<0.05）. lnc RNA TP73- AS1 expression and BDH2 m RNA expression is significant positive correlation in esophageal tissues 8. Among these targets of lnc RNA TP73-AS1 predicted by the bioinformatics algorithms, we focused on mi R-141-3p. The 3’ untranslated region（3’UTR） of mi R-141-3p contains two seed regions for lnc RNA TP73-AS1（chr1:3655109-3655129[-] and chr1:3662504-3662525[-]）. 9. lnc RNA TP73-AS1 regulated negatively mi R-141-3p by binding the two seed regions. 10. BDH2 is the target gene of mi R-141-3p. mi R-141-3p mimics inhibited ESCC cell proliferation and induces the apoptosis, which were caused by lnc RNA TP73-AS1 increasing BDH2 expression by regulating mi R-141-3p.Conclusions 1. There are 89 differentially expressed lnc RNAs, including 29 lnc RNAs upregulated（32.6%） and 60 lnc RNAs downregulated（67.4%） in tumor tissues. Lnc RNA TP73-AS1 is upregulated in ESCC tissues. lnc RNA TP73-AS1 expression levels were associated with tumor location and TNM stage in ESCC tissues. 2. Lnc RNA TP73-AS1 knockdown inhibits EC cell proliferation and induces apoptosis. 3. lnc RNA TP73-AS1 regulated negatively mi R-141-3p by binding the two seed regions（chr1:3655109-3655129[-] and chr1:3662504-3662525[-]）. BDH2 is the target gene of mi R-141-3p. mi R-141-3p inhibites ESCC cell proliferation and induces the apoptosis, which were perhaps caused by lnc RNA TP73-AS1 increasing BDH2 expression by regulating mi R-141-3p.