| Background:HCC is the sixth most commonly diagnosed cancer and the third leading cause of cancer death in the world,and ranks fifth in the global incidence rate.Tumor metastasis is the main reason that affects the treatment and survival of liver cancer patients,so it is of great significance to study the mechanism of liver cancer metastasis.mi RNA is a single-stranded non-coding RNA with a length of 18-24 nucleotides.mi R-330-3p is located at 19Q12.32,and is abnormally expressed in a variety of tumors,and is related to the occurrence,development,and metastasis of tumors.Mi RNA is a type of RNA with a length of 18-24 nucleotides that binds to the 3?UTR of the target gene to induce translational inhibition or m RNA degradation.mi R-330-3p is located in 19Q12.3.EREG is a member of the EGF.It is significantly differentially expressed in kidney cancer,blood cancer,gastric cancer,and breast cancer,and is related to tumor cell growth,metastasis and prognosis.Longer than 200 nucleotides are long-chain non-coding RNAs(lnc RNA).Lnc RNAs do not have protein-coding capabilities,but they can regulate gene expression at the levels of transcription,post-transcription,and translation.lnc RNA021545 is located on human chromosome 11 and is a long non-coding RNA with a length of 3149 bp.In the early stage of this group,it was found that lnc RNA021545,EREG,mi R-330-3p were differentially expressed significantly.Meanwhile mi R-330-3p may have potential targets for EREG and lnc RNA021545.lnc RNA021545 may compete to bind mi R-330-3p,prevent it from binding to EREG and regulate its level changes to regulate liver cancer metastasis.However,the specific molecular mechanism of lnc RNA021545-mi R-330-3p-EREG in liver cancer is unclear.This paper mainly studies the mechanism of lnc RNA021545-mi R-330-3p-EREG in regulating the malignant behavior of liver cancer cells.Objective:1.To investigate the expression levels of mi R-330-3p,EREG,lnc RNA021545 in clinical samples of HCC patients;2.To explore the effect of mi R-330-3p expression level changes on the expression of EREG and lnc RNA021545;3.To explore the effect of down-regulation of lnc RNA021545 on the expression of EREG and mi R-330-3p;4.To definite the influence of mi R-330-3p,EREG,lnc RNA021545 on proliferation,migration and invasion of HCCLM3和Hu H7 cells;4.To explore the molecular mechanism of lnc RNA021545-mi R-330-3p-EREG regulate the HCC cell biological behavior.Methods:1.WB and q RT-PCR were used to detect the expression of EREG,lnc RNA021545,mi R-330-3p in clinical samples of HCC patients,and analyze the correlation and progress;2.Transfection the mi R-330-3p mimic and mi R-NC or mi R-330-3p inhibitor and mi R-NC,q RT-PCR and WB to detect the effect of mi R-330-3p on the expression level of EREG and lnc RNA021545;3.Transiently transfected si-lnc RNA021545 and si-NC,q RT-PCR and WB to detect the influence of lnc RNA021545 on the expression level of EREG and mi R-330-3p;4.The dual luciferase reporter experiment were used to checked the affects of mi R-330-3p on EREG-3’-UTR and lnc RNA021545;5.Amplify the full-length sequence of the EREG CDS region,construct the PCDH-EREG,and PCDH-EREG and PCDH-NC respectively transfect into 293T cells,collect the virus liquid,obtain a cell line with stable up-regulation of EREG through puromycin screening,and detect the expression level of EREG by q RT-PCR and WB;6.MTT method to detect the effects of mi R-330-3p,EREG,lnc RNA021545 on the proliferation of HCCLM3 and Hu H7 cells;measured the influence of mi R-330-3p,EREG,lnc RNA021545 on HCCLM3和Hu H7 cells proliferation ability in vitro;7.Transwell method to detect the influence of mi R-330-3p,EREG,lnc RNA021545 on HCCLM3和Hu H7 cells migration and invasion ability in vitro;8.WB method to detect the influence of mi R-330-3p,EREG,lnc RNA021545 on EMT-related molecules in HCCLM3和Hu H7 cells;9.Rescue experiments to verify whether the upregulation of EREG reverses the effect of mi R-330-3p on EREG expression levels,HCCLM3 and Hu H7 cell migration and invasion,and EMT-related molecules;10.Inguinal injection of nude mice was used to detect the up-and down-regulation of mi R-330-3p on the tumorigenesis of HCCLM3 cells in vivo,and IHC was used to detect the EREG,snail,slug,E-cadherin,N-cadherin,vimentin expressions in tumors;HE was used to detect the LNM;Results:1.The expression level of mi R-330-3p in HCC samples was increased by70.8%(P=0.0359),the overall expression level of EREG decreased by 32.7%(P=0.0029),and the overall expression level of lnc RNA021545 decreased by 68.7%(P<0.0001);In the clinical tumor tissue samples of liver cancer patients,the expression trends of EREG and lnc RNA021545 are consistent,and the two are positively correlated(R2=0.4542,P=0.002),and the expression trends of mi R-330-3p and EREG are opposite and negative Correlation(R2=0.2227,P=0.0199);meanwhile,the expression trends of mi R-330-3p and lnc RNA021545 are opposite and negatively correlated(R~2=0.3489,P=0.0024).The results are consistent with liver cancer samples.2.The test of dual luciferase report experiment found that compared with mi R-NC,mi R-330-3p mimic and pmir Gl O-EREG-3’-UTR-wt co-transfected luciferase activity by29.9%(P=0.0142),when this binding site is mutated,mi R-330-3p has no inhibitory effect on luciferase activity;at the same time,compared with mi R-NC,mi R-330-3p co-transforms with pmir Gl O-lnc RNA021545-wt,the relative activity of luciferase decreased by 58.9%(P=0.0004).When this binding site is mutated,mi R-330-3p has no inhibitory effect on luciferase activity;3.In HCCLM3 cells,upregulation of mi R-330-3p reduced EREG m RNA expression by53.5%(P=0.0083),protein expression by 65.0%(P=0.0003),and lnc RNA021545 expression by 40.6%(P=0.044)In Hu H7 cells,up-regulation of mi R-330-3p reduced endogenous EREG m RNA expression by 94.3%(P=0.0012),protein expression by 57.7%(P=0.0009),and lnc RNA021545 expression by 45.9%(P=0.0007);in HCCLM3 cells,mi R-330-3p downregulation increased EREG m RNA expression by 46.6%(P=0.0071),protein expression increased by 145.1%(P=0.0003),and lnc RNA021545 expression increased by 52.9%(P=0.0029).In Hu H7 cells,q RT-PCR results showed that after mi R-330-3p was down-regulated,EREG m RNA expression increased by 51.4%(P=0.0357),and protein expression increased by 94.3%(P=0.0357).The expression of lnc RNA021545 increased by79.3%(P=0.0012);4.In HCCLM3 cells,after lnc RNA021545 was down-regulated,EREG m RNA expression was down-regulated by 45.8%(P=0.0001),protein expression was down-regulated by 33.3%(P=0.0023),and mi R-330-3p expression was up-regulated by60.6%(P=0.0097)In Hu H7 cells,EREG m RNA expression was down-regulated by 80.9%(P=0.0007)after lnc RNA021545 was down-regulated,protein expression was down-regulated by 31.0%(P=0.0006),and the expression of mi R-330-3p was increased by52.0%(P<0.0001);5.Obtained a cell line with stable up-regulation of EREG expression.The m RNA and protein expression levels increased by 317.10%(P<0.0001)and 59.33%(P=0.032)respectively;and transiently obtained a cell line with down-regulated EREG.And protein expression levels were reduced by 69.63%(P=0.0007),39.00%(P=0.0010);6.MTT results showed that:overexpression or silencing of mi R-330-3p regulation had no effect on proliferation capacities of HCCLM3和Hu H7 cells;overexpression or silencing EREG u had no effect on proliferation capacities of HCCLM3和Hu H7 cells;downregulation of lnc RNA021545 had no effect on proliferation capacities of HCCLM3和Hu H7 cells.7.Transwell showed the increased of mi R-330-3p promoted the HCCLM3 and Hu H7cells migration and invasion.Compared with HCCLM3-mi R-NC,the migration and invasion capabilities of HCCLM3-mi R-330-3p mimic group were increased respectively.98.93%(P=0.0008)and 84.98%(P=0.0037).The migration and invasion ability of Hu H7-mi R-330-3p mimic group were increased by 66.91%(P=0.0050),70.39%(P=0.0074);Compared with Hu H7-mi R-NC,the migration and invasion abilities of Hu H7-mi R-330-3p inhibitor group were reduced by 52.01%(P=0.0006)and 25.27%(P=0.0334).Compared to HCCLM3-PCDH-NC,the migration and invasion of HCCLM3-PCDH-EREG group decreased by 54.27%(P=0.0005),59.21%(P=0.0116);compared to Hu H7-si-NC,the migration and invasion ability of Hu H7-si-EREG increased by 46.07%(P=0.0376)and65.68%(P=0.0194),respectively.Compared to HCCLM3-si-NC,the migration and invasion ability of HCCLM3-si-lnc RNA021545 group increased by 68.52%(P=0.0020)and 62.66%(P=0.0042)respectively.Compared to Hu H7-si-NC,the migration and invasion ability of Hu H7-si-lnc RNA021545 group increased by 70.88%(P=0.0003)and 107.15%(P=0.0044),respectively.8.mi R-330-3p,EREG,lnc RNA021545 affect the expression of EMT-related molecules:In HCCLM3 and Hu H7 cells,the snail expression level increased by 46.0%(P=0.0048)and45.0%(P=0.0024),slug expression levels increased by 57.0%(P=0.0442),59.3%(P=0.0182),E-cadherin expression levels decreased by 47.0%(P=0.0044),33.0%(P=0.0008),N-cadherin expression levels increased by 96.7%(P=0.0168)and 71.7%(P=0.0084),respectively,vimentin expression levels increased by 49.0%(P=0.0062),56.3%(P<0.0001);in HCCLM3 and Hu H7 cells In,down-regulated the expression of mi R-330-3p,the expression of snail was reduced by 46.7%(P=0.0069),36.0%(P=0.0124),slug was reduced by 44.3%(P=0.0446),45.7%(P=0.0388),E-cadherin increased by 63.3%(P=0.0332),72.0%(P=0.0070),N-cadherin decreased by 38.7%(P=0.0130),66.0%,respectively(P<0.0001),vimentin were reduced by 33.0%(P=0.0002),31.7%(P=0.0009);after down-regulating the expression of EREG in Hu H7 cells,the expression levels of snail and slug increased by 46.7%(P=0.0136),35.0%(P=0.0137),E-cadherin decreased by 41.5%(P<0.0001),N-cadherin and vimentin increased by 51.0%(P=0.0148),77.7%(P=0.0339)).Overexpression of EREG in HCCLM3 cells resulted in a decrease in the expression levels of snail and slug by 38.3%(P<0.0001)and 39.3%(P=0.0013),respectively.The expression level of E-cadherin increased by 33.5%(P=0.0012),while N-cadherin and vimentin decreased by 44.0%(P=0.0013)and 38.7%(P<0.0001),respectively;down-regulating lnc RNA021545 in Hu H7 and HCCLM3 cells,the expression levels of snail increased by53.0%(P=0.0198)and 50.3%,respectively(P=0.005),the slug increased by 47.3%(P=0.013),55.3%(P=0.075),the E-cadherin decreased by 44.0%(P=0.009),31.0%(P=0.034),N-cadherin increased by 58.0%(P=0.0450),72.3%(P=0.0346),and vimentin increased by 71.7%(P=0.0108),56.3%(P=0.0353),respectively;9.Rescue:co-transfection of mi R-330-3p mimic and PCDH-EREG in HCCLM3 cells for 48 h,compared with mi R-330-3p mimic group,the level of EREG m RNA in mi R-330-3p mimic+PCDH-EREG group.The expression increased by 43.0 fold(P<0.0001),and the protein expression level increased by 139.8%(P=0.0004).Compared with the PCDH-EREG group,the expression of EREG m RNA in the mi R-330-3p mimic+PCDH-EREG group decreased 95.80%(P=0.0001),protein expression level was reduced by 16.56%(P=0.0459);and compared with the PCDH-EREG group,migration and invasion ability were enhanced in the mi R-330-3p mimic+PCDH-EREG group;compared with the HCCLM3-mi R-330-3p group,snail and slug expressions decreased by 42.4%(P=0.0017),50.3%(P=0.0007),E-cadherin increased by 159.3%(P=0.0031),and N-cadherin and vimentin were respectively reduced by 43.8%(P=0.0027),43.9%(P=0.0007)in HCCLM3-mi R-330-3p+PCDH-EREG;Compared with the PCDH-EREG group,snail and slug expressions increased by 35.1%(P=0.0257),24.6%(P=0.0489),E-cadherin decreased by 31.4%(P=0.0337),N-cadherin and vimentin increased by 30.1%(P=0.0225),53.7%(P=0.0496)in HCCLM3-mi R-330-3p+PCDH-EREG;10.In the nude mouse tumor formation experiment,21 mice formed solid tumors.Compared with the control group,the mass of the mi R-330-3p up-regulated group increased by 148.3%(P=0.0358),and the mi R-330-3p down-regulated group There was no significant change in quality;compared with the control group,the volume of the mi R-330-3p up-regulated group increased by 148.3%(P=0.0358),and the volume of the mi R-330-3p down-regulated group had no significant change.Compared to the control,the LN in the mi R-330-3p-mimic are diffusely distributed,large in size,round,and have large nuclei and basophilic staining,while the mi R-330-3p down-regulated group has deep and clear staining Cell nucleus;compared with HCCLM3-mi R-NC,the expression level of mi R-330-3p in solid tumors in the mi R-330-3p up-regulation group was increased by 1195.3%(P=0.0209),and the m RNA expression of EREG was reduced by 81.4%(P=0.0249),lnc RNA021545expression was reduced by 66.1%(P=0.0384).Compared with HCCLM3-mi R-NC,the expression level of mi R-330-3p in solid tumors in the mi R-330-3p down-regulation group was down-regulated by 26.5%(P=0.0231),the expression of EREG at the m RNA level increased by 147.0%(P=0.0025),and the expression level of lnc RNA021545 increased by151.8%(P=0.0005);the tumor formation in vitro of each experimental tissue was subjected to immunohistochemical staining and H-Score score,compared with the HCCLM3-mi R-NC group,the expression levels of EREG and E-cadherin in the HCCLM3-mi R-330-3p-mimic group were reduced by 42.9%(P<0.0001)and 82.8%(P<0.0001),respectively,the N-cadherin expression was increased by 43.0%(P<0.0001),vimentin expression was increased by4 4.9%(P=0.0002),snail expression was increased by 31.5%(P<0.0001),slug expression was increased by 30.2%(P=0.0019),and ki67 expression did not changed significantly;compared with the HCCLM3-mi R-NC group,EREG and E-cadherin expression were increased by 45.1%(P<0.0001),101.42%(P<0.0001),and the N-cadherin expression were decreased by 52.86%(P<0.0001),vimentin expression were decreased by 37.04%(P=0.0002),snail expression were decreased by 42.89%(P<0.0001),slug expression were decreased by 37.53%(P=0.0009),and ki67 did not changed significantly.Conclusion:1.The expression levels of mi R-330-3p was increased in HCC samples,while EREG and lnc RNA021545 were decreased in liver cancer samples,and EREG was positively correlated with lnc RNA021545;and mi R-330-3p was negatively correlated with EREG,lnc RNA021545in HCC samples;2.In HCCLM3 and Hu H7 cells,mi R-330-3p inhibit EREG and lnc RNA021545expressions,while down-regulation promotes.3.In HCCLM3 and Hu H7 cells,the down-regulation of lnc RNA021545 expression promotes EREG and inhibits the expression of mi R-330-3p.4.Up-regulation of mi R-330-3p can promote the migration and invasion ability of HCCLM3 and Hu H7 cells,while down-regulation inhibited;EREG inhibits the HCCLM3cells migration and invasion ability,down-regulation promotes the Hu H7 cells migration and invasion ability;down-regulation of lnc RNA021545 promotes the HCCLM3 and Hu H7 cells migration and invasion capabilitie,no effect on the proliferation ability.5.lnc RNA021545-mi R-330-3p-EREG regulates the malignant behavior of HCC cells by regulating EMT-related proteins. |
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