| BackgroundRecent statistics show that half of the new cases of Hepatocellular carcinoma(HCC)in the world are in China.Because of its insidious onset,rapid development and frequent recurrence,it poses a serious threat to the life and health of Chinese citizens.Therefore,it is particularly important to clarify the specific regulatory mechanisms affecting the progression of HCC and to find more and more accurate molecular targets in the current individualized treatment of clinical HCC under the comprehensive concept.In recent years,metastasis associated lung adenocarcinoma(MALAT1)transcript has become one of the most popular lnc RNAs studied due to its overexpression in a variety of human solid tumors or lymphomas,acting as miRNA"molecular sponge"to play the role of oncogene.In addition,inflammation-related miR-146a has been found to be dysregulated in HCC and to affect the occurrence and development of HCC through multiple pathways.Based on the fact that lnc RNAs and miRNAs can bind to each other through multiple sites,regulate each other and affect each other’s functions,and a variety of lnc RNAs or miRNAs have been confirmed to regulate the malignant biological behavior of HCC cells through the PI3K pathway.Therefore,we speculated whether there is a MALAT1-miR-146a-PI3K/Akt/m TOR regulatory network that affects the progression of HCC,which is worthy of further investigation in order to provide an experimental basis for clinical screening of combined therapeutic targets for HCC.PART I:The expression of key nodes of MALAT1/miR-146a/PI3K/Akt/m TOR axis in HCC tissues and cells and their targeted regulation relationshipOBJECTIVE:To investigate the expression correlation of MALAT1,miR-146a,PI3K in HCC tissue,normal liver tissue,HCC cells,and normal hepatocytes.To explore MALAT1 The relationship between the expression level and the clinicopathological characteristics of HCC.To investigate the targeting and regulatory relationship of MALAT1 to miR-146a and PI3K/Akt/m TOR axis.METHODS:40 human HCC tissues and 12 normal liver tissue samples were collected,and HCC cell lines and human normal liver cell lines in the logarithmic growth phase were used to detect the expression levels of MALAT1,miR-146a,and PI3K by q RT-PCR,and the expression correlations between MALAT1 and miR-146a,miR-146a and PI3K m RNA in HCC tissues were described by regression analysis;the relationship between MALAT1 and HCC-related clinicopathological characteristics was detected by chi-square test or Fisher’s exact test.Target Scan(http://www.targetscan.org)and Lnc Tar(http://www.cuilab.cn),an online network tool,predicted the possible potential binding sites between MALAT1 and miR-146a,and between miR-146a and PI3K 3’-UTR;DLRA further confirmed the targeted regulatory relationship between the three.si MALAT1/si NC and miR-146a inhibitors/NC inhibitors were simultaneously transfected into HCC cell lines to construct cell models with different MALAT1 and miR-146a expression levels,and q RT-PCR and Western blot were used to detect the regulation of MALAT1 on the expression of miR-146a,PI3K and its downstream molecules Akt,p-Akt,and m TOR.RESULTS:Compared with normal liver tissue,m RNA expression of MALAT1 and PI3K were up-regulated in HCC tissue(all P<0.05),and miR-146a expression was down-regulated(P<0.01).Hep G2 and Huh7 cells showed higher levels of MALAT1 expression(all P<0.01),higher levels of PI3K m RNA expression(all P<0.001),and lower levels of miR-146a expression(all P<0.001;P<0.01);The expression levels of MALAT1 and miR-146a were negatively correlated in HCC tissues(R square=0.6243,P<0.001).The expression levels of miR-146a and PI3K m RNA in HCC tissues were negatively correlated(R square=0.4827,P<0.001).The expression level of MALAT1 was not significantly correlated with age,gender,serum AFP level,HBV,cirrhosis and other parameters(P>0.05),but was closely correlated with tumor size(X~2=6.094,P=0.014)and TNM stage(X~2=11.918,P=0.001).There are negative targeted regulatory relationships between MALAT1 and miR-146a,and between miR-146a and the 3’-UTR region of PI3K.MALAT1 acts as a"molecular sponge"of miRNA,targets and down-regulates the expression of miR-146a,and then upregulates the expression levels of key signaling molecules in the PI3K/Akt/m TOR signaling pathway.CONCLUSIONS:Compared with normal liver tissues and cells,the expression of MALAT1 and PI3K in HCC tissues and cells is up-regulated,and the expression of miR-146a is down-regulated.MALAT1 is negatively correlated with miR-146a in HCC tissues,and miR-146a is negatively correlated with PI3K expression.The high expression of MALAT1 is related to the malignant degree of the tumor and is expected to be a potential biomarker for evaluating the prognosis of HCC patients.MALAT1 acts as a"molecular sponge"of miRNA in HCC,targeting and down-regulating the expression of miR-146a,and subsequently upregulating the expression levels of PI3K,Akt,p-Akt and m TOR,so as to realize the positive regulation of PI3K/Akt/m TOR pathway,and ultimately may affect the progression of HCC.PART II:The mechanism of MALAT1/miR-146a/PI3K/Akt/m TOR signaling axis regulating the malignant biological behavior of HCC cellsOBJECTIVE:To investigate the specific mechanism of MALAT1/miR-146a/PI3K/Akt/m TOR signaling axis regulating HCC cell proliferation.METHODS:si MALAT1 was transfected with Hep G2/Huh-7 cells to construct MALAT1 knockdown cell model.si MALAT1/si NC+miR-146a inhibitors/NC inhibitors were transfected to construct models of different expression levels of MALAT1 and miR-146a.The effect of MALAT1/miR-146a on HCC cell proliferation was detected by MTT assay and colony formation assay,and the effect of MALAT1/miR-146 on cell apoptosis was detected by flow cytometry.The transfected cells were treated with baflomycin A1,and the formation of autophagy markers was detected by Western blot.RESULTS:The results of MTT assay showed that there was no significant difference in the cell proliferation rate after transfection with si MALAT for 124h compared with the si NC transfection group(P>0.05),and the cell proliferation rate decreased significantly after transfection for 48h,72h,and 96h(P<0.05).Compared with the si NC+NC inhibitors transfection group,the proliferation rate of Hep G2 cells transfected with si MALAT1+NC inhibitors for48h,72h,and 96h was significantly decreased(P<0.05);the proliferation rate of Huh-7 cells transfected with si MALAT1+NC inhibitors for 72h and 96h was significantly decreased(P<0.05).Compared with the si MALAT1+NC inhibitors transfection group,the proliferation rate of Hep G2 cells transfected with si MALAT1+miR-146a inhibitors 48h,72h,and 96h increased significantly(P<0.05);the proliferation rate of Huh-7 cells transfected with si MALAT1+miR-146a inhibitors 72h and 96h also showed a significant difference(P<0.05).However,there was no significant difference in the proliferation rate of the two cell lines between the si NC+miR-146a inhibitors transfection group and the si MALAT1+miR-146a inhibitors transfection group,the si NC+NC inhibitors transfection group and the si NC+miR-146a inhibitors transfection group,the si NC+NC inhibitors transfection group and si MALAT1+miR-146a inhibitors transfection group(P>0.05);colony formation assay results showed that compared with si NC transfection group,the number of colonies in Hep G2 and Huh7 cells transfected with si MALAT148 h was significantly reduced(P<0.01,P<0.05);flow cytometry results showed that in Hep G2 cells,the number of apoptosis in si MALAT1 transfection group was significantly increased(5.1%vs 21.2%)compared with si NC transfection group(P<0.05);in Huh7 cells,the number of apoptosis induced by MALAT1 silencing was significantly increased(3.5%vs 23.6%)compared with si NC transfection group(P<0.05).Compared with the si MALAT1+NC inhibitors transfection group,the number of apoptosis in the two HCC cells transfected with si MALAT1+miR-146a inhibitors was significantly reduced(all P<0.05);Western blot results showed that the si MALAT1-transfected LC3-II expression level gradually increased within 0 to 6 hours after 50 n M Baflomycin A1treatment compared with the two cells in the si NC transfection group.Compared with two HCC cells transfected with si MALAT1+miR-146a inhibitors,LC3-II expression levels were increased in si MALAT1+NC inhibitors transfected group.CONCLUSIONS:MALAT1 and miR-146a in HCC act as pro-cancer factors and anti-cancer factors,respectively,that is,they regulate the proliferation of tumor cells by regulating apoptosis and autophagy.MALAT1acts as a"molecular sponge"of miRNA,downregulates the expression of miR-146a,and upregulates the expression levels of key signaling molecules in the PI3K/Akt/m TOR signaling pathway,thereby regulating the apoptosis and autophagy of HCC cells and affecting the progression of HCC. |
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