| Background and purposeEsophageal cancer is the most common malignant tumor of the digestive system.It has high incidence and mortality rate,and is extremely harmful to human health and life safety.With the development of surgery,radiotherapy and chemotherapy and other ancillary methods,the clinical treatment of esophageal cancer has made significant progress,which has certain effects on delaying tumor progression,improving life quality and prolonging patient survival,but the overall long-term prognosis is still poor,with a 5-year survival rate of less than 30%.MicroRNA(miRNA)has been shown to have important regulatory effects on tumors.With the finding and identification of specifically expressed miRNAs,dozens of miRNAs associated with human esophageal cancer have been exposed.In our previous studies,we found that miR-4286 was elevated in esophageal cancer.It was reported that miR-4286 was associated with melanoma,squamous cell carcinoma,and prostate cancer.We predicted that INPP4A was a target gene of miR-4286 through software,but no report of miR-4286 and INPP4A involved in esophageal cancer has been reported yet.We intended to take the high expression of miR-4286 as an entry point to explore the relationship between miR-4286 and esophageal cancer,investigate its influence on the biological behavior of esophageal cancer through INPP4A and JAK2/STAT3 signaling pathway.Exploring the pathogenesis of esophageal cancer from miRNAs will help to deepen the understanding of complex regulatory networks in the development of esophageal cancer,and provide a new perspective for the research and treatment.The results of the study help to find new biomarkers of esophageal cancer,which are of great value for finding targeted treatments for esophageal cancer and improving the effectiveness of diagnosis and treatment.Chapter 1 The expression of miR-4286 and INPP4A in esophageal cancer tissue samples and cell linesResearch methodsEsophageal tumor tissues and surrounding non-tumor tissues of 75 cases of confirmed esophageal cancer patients were collected,esophageal squamous cell carcinoma cell lines(TE-1,HCE-4 and HCE-7),esophageal adenocarcinoma cell line(SKGT-4 and Bic-1),and normal esophageal epithelial cell line(HEEC)were cultured in vitro.Quantitative PCR or Western Blot was used to detect the expression of miR-4286 and INPP4A in esophageal cancer tissue samples and cell lines.The relationship between miR-4286 and INPP4A expression levels in esophageal cancer was analyzed.Results1.The expression of miR-4286 in esophageal cancer tissues was 3.15 folds higher than that of normal esophageal tissues(p<0.001).The expression levels of miR-4286 in TE-1,HCE-4,HCE-7,SKGT-4 and Bic-1 cell lines were 2.854(p<0.01),2.721(p<0.01),3.273(p<0.001),1.915(p<0.05)and 2.421(p<0.01)folds higher than that of HEEC.2.The expression level of INPP4A mRNA in esophageal cancer tissues was significantly lower,which was 0.487 times than that of normal esophageal tissues(p<0.01).The expression level of INPP4A mRNA in esophageal cancer cells was significantly lower than that in normal epithelial cells,which was 0.676,0.589,0.613,0.575 and 0.622 times than that of normal esophageal epithelial cells in TE-1?HCE-4?HCE-7?SKGT-4 and Bic-1 cells respectively(p<0.01).3.The expression level of INPP4A protein in esophageal cancer tissues was significantly lower than that in distal normal tissues(p<0.01).The expression of INPP4A protein in TE-1,HCE-4,HCE-7 and SKGT-4 cell lines was significantly lower than that in HEEC cells(p<0.01).4.There was a negative correlation between the expression of miR-4286 and INPP4A mRNA in esophageal carcinoma tissues,|r|=0.675,p<0.001.Conclusions1.MiR-4286 is an esophageal cancer related factor,which is highly expressed in esophageal cancer tissues and cell lines.2.INPP4A is a downstream target gene of miR-4286,which is lowly expressed in esophageal cancer tissues and cell lines and is negatively correlated with miR-4286 leves.Chapter 2 MiR-4286 promotes esophageal carcinoma development by targeting INPP4A to evoke the JAK/STAT3 pathway activationResearch methods1.The dual luciferase reporter assay system was carried out in the esophageal cancer HCE-7 cell line to investigate the interaction between miR-4286 and INPP4A and whether INPP4A was the target gene of miR-4286.miR-4286 minic/inhibitor was transfected into esophageal cancer cells,and the expression of INPP4A,JAK2 and STAT3 was detected by quantitative PCR and Western blot.The expressions of factors related to cell proliferation,apoptosis and invasiveness such as BCL-2,BAX,Caspase,E-cadherin and N-cadherin were also detected.Cell survival was detected by MTT assay,apoptosis was detected by flow cytometry,cell invasion and migration was examined by Transwell assay.2.INPP4A siRNA was transfected into esophageal cancer cells alone or in combination with miR-4286 inhibitor.The expression levels of INPP4A,JAK2 and STAT3 were detected by Western Blot.Cell viability was detected by MTT assay,apoptosis was detected by flow cytometry,and cell invasion and migration was detected by Transwell assay.Results1.The luciferase reporter assay showed that after co-transfected with miR-4286 and 3'-UTR wild type recombinant reporter plasmid of INPP4A,the luciferase activity was significantly decreased,indicating that INPP4A was a direct target of miR-4286.2.The expression of INPP4A mRNA in miR-4286 mimic group was 0.37 times than that of the control group(p<0.001).In miR-4286 inhibitor group it was 2.88 times higher than that of the control(p<0.001).The expression of INPP4A mRNA in mimic NC group and inhibitor NC group had no significant difference with the control group(p>0.05).The expression level of INPP4A protein in miR-4286 mimic group was lower than that of the control group.The expression level of INPP4A protein in miR-4286 inhibitor group was higher than that of the control group.The level of INPP4A protein in mimic NC group and inhibitor NC group was not significantly different from that in control group(p>0.05)3.The survival rate of miR-4286 mimic group in TE-1,HCE-7 and Bic-1 esophageal cancer cell lines was 172.12%,170.53%and 144.38%,which was higher than that in the control group(p<0.05).The survival rate of miR-4286 inhibitor group in TE-1,HCE-7 and Bic-1 esophageal cancer cell lines was 71.38%.56.28%? 72.83%,which was much lower than that of the control group(p<0.05).The cell viability of mimic NC group and inhibitor NC group was not significantly different from that of the control group(p>0.05),indicating that miR-4286 can promote cell proliferation.4.The apoptosis rate of miR-4286 mimic group in TE-1,HCE-7 and Bic-1 esophageal cancer cell lines was 4.67%,4.67%and 4.13%,which was lower than that of the control group(p<0.01).The apoptosis rate of miR-4286 inhibitor group in TE-1,HCE-7 and Bic-1 esophageal cancer cell lines was 14.22%,13.51%and 8.64%.which was higher compared with that of the control group(p<0.05).The apoptosis rate of mimic NC group and inhibitor NC group was not significantly different from that of the control group(p>0.05),indicating that miR-4286 can inhibit apoptosis.5.The migration cells of miR-4286 mimic group in TE-1,HCE-7 and Bic-1 were 137,165 and 147 and the invasion cells of miR-4286 mimic group in TE-1,HCE-7 and Bic-1 were 142,184 and 146.which were both higher than those of the control group(p<0.05).The migration cells of miR-4286 inhibitor group in TE-1,HCE-7 and Bic-1 were 76,75 and 57,and the invasion cells of miR-4286 inhibitor group in TE-1,HCE-7 and Bic-1 were 66,44 and 78,which were both lower than those of the control group(p<0.05).The cell migration number of the mimic NC group and the inhibitor NC group was not significantly different from that of the control group(p>0.05),indicating that miR-4286 can enhance the migration and invasion of esophageal cancer cells.6.The expression levels of BCL-2,N-cadherin,Vimentin,ZEB1,Snail,JAK2 and STAT3 in miR-4286 mimic group were higher than those in control group(p<0.05),while BAX,cleaved-Caspase-3,cleaved-Caspase-9 and E-cadherin was lower than that of the control group(p<0.05).The expression of BCL-2,N-cadherin,Vimentin,ZEB1,Snail,JAK2 and STAT3 in the miR-4286 inhibitor group was lower than that of the control group(p<0.05),while BAX,cleaved-Caspase-3,cleaved-Caspase-9 and E-cadherin was higher than that of the control group(p<0.05).The expression levels of the above proteins in the mimic NC group and the inhibitor NC group were not significantly different from those in the control group(p>0.05).7.The mRNA and protein levels of INPP4A in si-INPP4A group were significantly lower than those in the control group(p<0.01),which were significantly higher in si-INPP4A+miR-4286 inhibitor group compared with those in si-INPP4A group(p<0.01).The mRNA and protein levels of INPP4A in si-NC group were not significantly different from those in the control group(p>0.05),and the mRNA and protein levels of INPP4A in si-INPP4A+miR-4286 NC group were not significantly different from those in si-INPP4A group(p>0.05).8.The cell survival rate of si-INPP4A group was 170.34%,which was significantly higher than that of the control group(p<0.01).The cell survival rate of si-INPP4A+miR-4286 inhibitor group was 102.05%,which was significantly lower than that of si-INPP4A group(p<0.01).There was no significant difference of the survival rate between si-NC group and the control group(p>0.05).There was no significantly difference of the survival rate between the si-INPP4A+miR-4286 NC group and the si-INPP4A group(p>0.05).9.The total apoptosis rate of si-INPP4A group was 2.66%,which was significantly lower than that of the control group(p<0.01).The apoptosis rate of si-INPP4A+miR-4286 inhibitor group was 7.78%,which was significantly higher than that of si-INPP4A group(p<0.01).There was no significant difference of the apoptosis rate between si-NC group and the control group(p>0.05).There was no significant difference of the apoptosis rate between si-INPP4A+ miR-4286 NC group and si-INPP4A group(p>0.05).10.The relative migration and invasion cells of si-INPP4A group was 147.26%and 158.72%,which was significantly higher than that of the control group(p<0.01).The relative migration and invasion cells of si-INPP4A+miR-4286 inhibitor group was 89.77%? 89.89%,which were significantly lower than that of si-INPP4A group(p<0.01).There was no significant difference of the relative migration and invasion numbers between si-NC group and the control group(p>0.05).There was no significant difference of the relative migration and invasion numbers between the si-INPP4A+ miR-4286 NC group and the si-INPP4A group(p>0.05).11.The protein levels of JAK2 and STAT3 in the si-INPP4A group were significantly higher than those in the control group(p<0.01),which were lower in the si-INPP4A+miR-4286 inhibitor group than that in the si-INPP4A group(p<0.05).There was no significant difference of JAK2 and STAT3 between the si-NC group and the control group(p>0.05).There was no significant difference of JAK2 and STAT3 between the si-INPP4A+miR-4286 NC group and si-INPP4A group(p>0.05).Conclusions1.The high expression of miR-4286 in esophageal cancer can promote cell proliferation,inhibit apoptosis,enhance migration and invasion,and activate JAK2/STAT3 pathway;the low expression of miR-4286 in esophageal cancer can inhibit cell proliferation,promote apoptosis,inhibit migration and invasion,and inhibit JAK2/STAT3 pathway,which indicates miR-4286 could regulate the malignant appearance of esophageal cancer.2.miR-4286 inhibitor can reverse the effect caused by si-INPP4A,that is the promotion of cell proliferation,the inhibition of apoptosis,the promotion of migration and invasion for esophageal cancer.miR-4286 regulate the malignant appearance of esophageal cancer by targeting INPP4A.miR-4286 activates JAK2/STAT3 signaling pathway by targeting INPP4A to regulate the growth,migration and invasion of esophageal carcinoma. |
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