Hsa-miR-328 And Its Target Gene PLCE1 Play Great Roles In The Development Of Esophageal Squamous Cell Carcinoma And The Underlying Mechanisms

IntroductionEsophageal carcinoma, which is characterized by poor prognosis and high mortality rate, is the fifth most common malignant tumor and ranks fourth as a cause of death from malignant tumor in China.Esophageal squamous cell carcinoma(ESCC) and esophageal adenocarcinoma are identified two histological types of esophageal carcinoma according to pathological properties and about 90% esophageal carcinoma cases are ESCC in China. In spite of extensive studies on the mechanisms responsible for ESCC, the underlying mechanisms of ESCC development are still unclear. Most ESCC cases failed to be diagnosed until in late stage and five-year survival rate is very poor.Therefore, further studies on the mechanisms accounting for ESCC development are necessary for improving diagnosis and prognosis.Micro RNA(miRNA), which is a kind of endogeneous non-coding and single-stranded RNA, is composed of 21-25 nucleotides and plays a great role in the development of many types of tumors. miRNA has the ability to negatively regulate expression of target gene by binding to 3'-untranslated region(3'-UTR) or5'-untranslated region(5'-UTR) of m RNA of target gene.Hsa-miR-328, which is located at 16q22.1, has the ability to regulate proliferation and invasion of many different types of tumors. It is demonstrated that hsa-miR-328 plays a great role in the development of many tumors, including non-small cell lung cancer, gastric cancer, prostate cancer, melanoma, glioma and so on. However, the expression of hsa-miR-328 in ESCC tissues and its role in ESCCdevelopment are not investigated by now.ObjectiveTo investigated the roles of hsa-miR-328 and its target gene PLCE1 in the development and prognosis of ESCC and the underlying mechanisms.Methods1. The expression of hsa-miR-328 in ESCC primary tumors and paired paracancerous normal esophageal tissues from 82 ESCC patients was detected and compared using quantitative real-time PCR(QRT-PCR). The clinical and prognostic significance of hsa-miR-328 expression was analyzed statistically.2. The expression of hsa-miR-328 in different esophageal cell lines was detected by using QRT-PCR. Proliferation test, flow cytometry technique and transwell were used to investigate the effect of hsa-miR-328 on proliferation, apotosis, migration and invasion in human esophageal squamous carcinoma cells EC109 and EC9706.QRT-PCR was employed to study the effect of hsa-miR-328 on the expressions of epithelial-mesenchymal transition markers in EC109 and EC9706. Effects of hsa-miR-328 expression in EC109 and EC9706 on tube formation in HUVEC were stdudied. Proliferation test and transwell were used to investigate the effect of hsa-miR-328 expression in EC109 and EC9706 on migration and invasion in HUVEC.3. Bioinformatics, western blot and QRT-PCR were used for prediction and identification of target gene of hsa-miR-328. Proliferation test, flow cytometry technique and transwell were used to investigate the effect of PLCE1 on proliferation,apoptosis,migration and invasion in EC109 and EC9706.4. The expression of PLCE1 in ESCC primary tumors and paired paracancerous normal esophageal tissues from 82 ESCC patients was detected and compared using QRT-PCR and immunohistochemistry. The clinical and prognostic significance of PLCE1 expression was analyzed statistically. The correlation between hsa-miR-328 and PLCE1 expression in ESCC primary tumors and paracancerous normalesophageal tissues was analyzed.Results1. Compared with paired paracancerous tissues, hsa-miR-328 expression in ESCC primary tumors decreased significantly(P<0.05). ESCC patients with decreased hsa-miR-328 expression showed poor survival(P<0.05). Hsa-miR-328 expression was negatively related to tumor size, infiltration depth, lymph node metastasis, differentiation grade and TNM stage(P<0.05).2. EC109 and EC9706 showed decreased hsa-miR-328 expression compared with human normal esophageal epithelial cells(P<0.05). Proliferation, migration and invasion of EC109 and EC9706 decreased in LV-miR-328 group and increased in LV-anti-miR-328 group compared with LV-NC group significantly(P<0.05). EC109 and EC9706 showed increased apoptosis in LV-miR-328 group and decreased apoptosis in LV-anti-miR-328 group compared with LV-NC group significantly(P<0.05). There was no difference in expressions of E-cadherin, N-cadherin, vimentin in EC109 and EC9706 among LV-NC, LV-miR-328 and LV-anti-miR-328 groups(P>0.05). There was no difference in tube formation, migration and invasion of HUVEC among LV-NC, LV-miR-328 and LV-anti-miR-328 groups(P>0.05).3. Bioinformatics method showed that PLCE1 is a latent target of hsa-miR-328,which was identified by luciferase test, western blot and QRT-PCR. p CDNA3/PLCE1 overexpression vector was constructed successfully. EC109 and EC9706 showed increased proliferation, migration and invasion and decreased apoptosis in p CDNA3/ PLCE1 group compared with control group(P<0.05). EC109 and EC9706 showed increased proliferation, migration and invasion and decreased apoptosis in LV-miR-328+p CDNA3/PLCE1 group compared with LV-miR-328 group(P<0.05).4. Compared with paired paracancerous tissues, PLCE1 expression in ESCC primary tumors increased significantly(P<0.05). ESCC patients with increased PLCE1 expression showed poor survival(P<0.05). PLCE1 expression was positively related to tumor size, infiltration depth, lymph node metastasis and TNM stage(P<0.05). Negative correlation between hsa-miR-328 and PLCE1 expression wasobserved in ESCC primary tumors and paracancerous normal esophageal tissues(P<0.01).ConclusionsDecreased expression of hsa-miR-328 in ESCC cancer tissues has the ability to promote proliferation, migration and invasion and attenuate apoptosis of ESCC cells via enhance PLCE1 expression, which is a possible mechanism responsible for development of ESCC.