| BackgroundEsophageal Squamous Cell Cancer(ESCC)has lately emerging symptoms,high malignant degree.It is easy to occur drug resistance and recurrence.In China,the morbidity and mortality of esophageal cancer ranked the sixth and the fourth respectively,and the mortality rate of ESCC was higher than that of Esophageal Adenocarcinoma(EAC).Cancer Stem Cells(CSCs)in tumors act as important factors leading to tumor metastasis,recurrence and eventual treatment failure.For cancer prevention and treatment,screening for common clinical tumor-related indicators and esophageal precancerous diseases,as well as screening for specific molecules targeting ESCC and CSCs,are of great significance for auxiliary diagnosis,treatment and prognosis evaluation.The abnormal expression or function of Long non-coding RNAs(LncRNAs)induce occurrence and progression of human ESCC and other tumors.LncRNAs not only regulate tumor growth and metastasis as a pro-cancer or anti-cancer regulatory molecule,but also participate in regulation of tumorigenicity,pluripotency and differentiation of CSCs.FER1L6-AS1 is an antisense lnc RNA transcribed by the antisense chain of FER1L6,a member of the Ferlins vesicle fusion protein family.In our previous study,an online database was used to screen out the low expression of FER1L6-AS1 in ESCC tissues.Patients with low expression had a shorter survival time,which may be an important molecule affecting the prognosis of ESCC.At present,the specific role of this gene in ESCC is not clear,so this study will test and verify the results of the above prediction in the tissues of Asian ESCC patients,and clarify its effects on cancer cell growth,metastasis,apoptosis and CSCs by upregulating the expression level of FER1L6-AS1.Objectives1.To verify the expression level of FER1L6-AS1 in ESCC tissues and cells and its subcellular localization.2.To investigate the effect of FER1L6-AS1 on the proliferation,metastasis and apoptosis of EC109 cells;3.To investigate the effect of FER1L6-AS1 on the stemness of EC109 CSCs.Methods1.The tissue specimens of cancer and paracancerous tissues of 32 ESCC patients newly diagnosed were collected,and the ESCC cell lines(KYSE450,EC109,EC9706)and normal esophageal cell line(Het-1A)were cultured.Real-time qRT-PCR was used to verify the difference in FER1L6-AS1 expression between cancer and para-cancerous tissues.2.Subcellular localization assays(Nucleoplasmic separation,FISH)was used to detect the localization of FER1L6-AS1 in EC109 cells.3.EC109 cell lines overexpressing FER1L6-AS1 were constructed with lentiviral vector,and the over-expression level of FER1L6-AS1 gene was verified by qRT-PCR.4.CCK8 assay and clone formation assay were used to observe the proliferation of EC109 after up-regulating FER1L6-AS1.The changes of migration and invasion ability of EC109 were verified by scratch test and Transwell assay.Apoptosis was detected by flow cytometry.Western blot assay was used to detect the expression of invasive protein MMP9 and anti-apoptotic protein Bcl-2.5.EC109 cells with stable overexpression of FER1L6-AS1 were cultured by suspension culture method,and tumor spheres formation(number and diameter)was detected.Stem cell markers after overexpression of FER1L6-AS1 were detected by qRT-PCR.Results1.Real-time qRT-PCR validation results showed that expression of FER1L6-AS1 was lower in the 32 ESCC tissues than that in para-cancerous tissues.FER1L6-AS1 expression was significantly reduced in patients with over 63 years of age,tumors located in the middle thoracic segment and with positive lymph node metastases.Its expression in ESCC cell lines(KYSE450,EC109,EC9706)was also lower than that in normal esophageal epithelial cell line Het-1A.2.The results of nucleoplasmic separation and FISH assays showed that FER1L6-AS1 was distributed in both the nucleus and cytoplasm,but expressed higher in the nucleus.3.The RNA expression level of FER1L6-AS1 in the EC109 stable infected FER1L6-AS1 cell line was significantly increased,FER1L6-AS1 was effectively overexpressed.4.Proliferation experiments showed that the growth curve of EC109-FER1L6-AS1 cells increased slowly and the number of clone formations decreased than EC109-control.Migration and invasion assay showed that FER1L6-AS1 inhibited the wound healing,migrate and invade cells number of EC109.Western Blot assay confirmed that the expression of invasion-related protein MMP9 was reduced.Apoptosis experiments showed that the early apoptosis rate of EC109 cells was increased.In addition,the protein expression of anti-apoptotic protein Bcl-2 was decreased.5.The number and diameter of tumor spheres were significantly reduced,the mRNA expression level of stem cell-related genes(CD44,SOX2,p75NTR)was decreased in EC109-FER1L6-AS1 culture.ConclusionsLncRNAFER1L6-AS1 is mainly distributed in the nucleus of EC109 and is low expressed in ESCC tissues and cell lines.Its low expression is associated with age,tumor location and lymph node metastasis.FER1L6-AS1 inhibits proliferation and migration of EC109 cells,self-renewal and stemness of EC109 CSCs,and promotes early apoptosis to a certain extent.FER1L6-AS1 could is a tumor suppressor for ESCC progression. |
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