Mechanism Of MiR-93 Regulating Liver Cancer Stem Cells And Inducing Drug Resistance And Its Clinical Relevance

Research background and purpose Hepatocellular carcinoma(abbr.as HCC)is one of the most common malignant tumors in the digestive system.According to the latest epidemiological survey,the rate of HCC incidence and mortality ranks sixth and third respectively in all malignant tumors.The number of new cases is nearly 800 thousand each year,of which more than 50% occur in China.Since no obvious symptoms in the early stage,tumor have developed into middle or late stage when HCC are first diagnosed.Therefore,usually 2/3 of these patients lose the opportunity of radical treatment.Another 1/3 of these patients can be treated by surgical resection or liver transplantation,but the recurrence rate within 5 years is higher than 75%.Transcatheter arterial chemoembolization(abbr.as TACE),FOLFOX chemotherapy and sorafenib targeted therapy have become the most important first-line treatment for unresectable HCC patients.Unfortunately,only a small number of patients benefit from this and the rest not only have poor curative effect,but also have serious complications or adverse reactions.Among them,drug resistance is an important factor affecting the overall prognosis of HCC patients.According to the epidemiological data statistics,the rate of standard chemotherapy resistance is about 50%,sorafenib resistance is more than 70%,and drug resistance related mortality is as high as 90%.Therefore,it is urgent to reflect on the shortcoming of traditional HCC therapy,re-examine HCC pathogenesis and drug resistance mechanism,and expand the treatment ideas during this process.Many studies have confirmed that there are a small number of cells with characteristics of self-renewal,unlimited proliferation and multi-directional differentiation in tumor tissues.These cells are defined as cancer stem cells(abbr.as CSCs)or tumor initiating cells(abbr.as TICs).CSCs were first found in hematopoietic system tumors,and then confirmed in many solid tumor tissues.It is generally believed that CSCs play a decisive role in many biological processes,such as tumorigenesis,proliferation,differentiation,recurrence,metastasis and drug resistance.Reflecting on the traditional therapy,it only killed a large number of rapidly proliferating cancer cells but not kill the CSCs.This may be the essential reason of tumor recurrence! The theory of CSCs has gradually changed our concept to traditional cancer treatment,and further provided a unique perspective for tumor etiology research.In addition,other studies have found that there are a small group of cells with selfrenewal characteristic and multi-directional differentiation potential in HCC tissue,which are named as liver cancer stem cells(abbr.as LCSCs).LCSCs not only participate in the occurrence and development of HCC,but also play an important role in tumor metastasis,recurrence and drug resistance.Therefore,in-depth analysis of the occurrence and drug resistance mechanism of LCSCs from the molecular biological level can find potential target molecules or markers with clinical transformation value,which is of great significance for the accurate diagnosis and treatment of HCC.MicroRNA(abbr.as miRNA)is a small non codingRNA with length of 19-25 nucleotides.miRNA binds to the 3'-untranslated region(abbr.as 3'-UTR)of target mRNA via Waston-Crick complementary condition to regulate the translation of target gene.At present,about 2600 miRNAs has found in human genome,which regulate the expression and translation of nearly 60% protein.Most miRNAs are formed through gene transcription,processing,transportation and splicing of primary miRNA.Abnormal expression of miRNA can affect lots of biological processes,including cell proliferation,apoptosis,cycle regulation,differentiation,angiogenesis,autophagy,invasion and migration.miRNAs have differential expression in different types of tumor tissues,and play the role of carcinogenesis or carcinostasis.Previous studies reported that low expression of miR-93 in glioma can inhibit the growth and metastasis of tumor cells.Meanwhile,high expression of miR-93 can promote the proliferation and invasion of breast cancer cells.In addition,miR-93 has abnormal expression in HCC,esophageal cancer,gastric cancer,pancreatic cancer and colorectal cancer and other gastrointestinal cancer,which is involved in the regulation of tumor occurrence,proliferation,invasion,metastasis and drug resistance.However,what is the operating mechanism of miR-93 in LCSCs? Can miR-93 induce cisplatin and sorafenib resistance in HCC,and how it is? What is the correlation between the expression of miR-93 and clinical prognosis? These questions have not been demonstrated by previous researches.This paper will explain the above problems through two parts.In the first part,we separated LCSCs from primary HCC tissue and HCC cell lines,respectively.Then,the high expression of miR-93 was clarified in LCSCs.The HCC cell line models with miR-93 low expression or high expression were subsequently constructed.Based on these cell line,we find that miR-93 has the function of regulating LCSCs surface antigens expression,self- renewal and tumorigenicity.Using TargetScan online tool,we find MTMR3 may be the target gene of miR-93.Further confirmed by gene interference experiments,miR-93 regulates the self-renewal and tumorigenicity of LCSCs by targeted binding MTMR3.In the second part,we established PDX drug resistance model,and found miR-93 high expression in tissues with cisplatin or sorafenib resistant.Then,the HCC cell lines with low or high expression of miR-93 was used to confirm that miR-93 can regulate the drug tolerance of HCC cell lines to cisplatin or sorafenib.Finally,through a retrospective clinical cohort studies,we revealed that the difference of miR-93 expression in tumor tissue is related to the survival and prognosis of patients who accepted TACE or sorafenib treatment.This study will further enrich the theoretical basis of LCSCs and provide new potential targets for patients with TACE or sorafenib resistance.Research method 1.Fresh HCC tissues were collected,then primary HCC cells were isolated from it,and subsequently LCSCs with CD133+ or Ep CAM+ were selected using flow cytometry.2.Fresh HCC tissues were collected,then primary HCC cells were isolated from it,and subsequently LCSCs were enriched by low adhesion culture.3.Cells with CD133+ or Ep CAM+ were isolated form HCCLM3 and CSQT-2 cell lines by flow cytometry.4.Cells with CD133+ or Ep CAM+ were isolated form HCCLM3 and CSQT-2 cell lines by low adhesion culture.5.Real-time PCR was used to detect the expression of miR-93 in LCSCs.6.Establish stable cell lines with high or low expression of miR-93 by lentivirus infecting HCC cell lines.7.Detect the expression of Ep CAM,CD133,CD90,CD24,Sox2,Oct4,c-myc and Nanog in HCC cell lines with miR-93 high,low,or normal expression.8.The self-renewal ability of LCSCs in HCC cell lines was detected by suspension culture.9.The proportion of LCSCs in miR-93 high,low or normal expression cell lines were detected by limited dilution test in vitro.10.The tumorigenesis rate of LCSCs was detected using NOD-SCID mice by in vivo limited dilution test.11.The downstream binding genes of miR-93 were predicted by bioinformatics methods.The 3'-UTR terminal wild-or mutant-type reporter plasmids of MTMR3 were constructed and transfected into miR-93 low expressed or normally expressed HCC cell lines.Then,the combination of miR-93 and MTMR3 was confirmed using luciferase reporter assay.12.The expression of miR-93 and MTMR3 in fresh HCC tissues was detected by realtime PCR to confirm the correlation between them.13.LCSCs with CD133+,Ep CAM+ or enriched from low adhesion culture were obtained for confirming the high expression of MTMR3 in them.14.si MTMR3 or si NC was transfected into HCC cell lines with miR-93 low or normal expression.The malignant phenotypes of transfected cells,including MTMR3 expression,LCSCs proportion under low adhesion culture and tumorigenesis rate after injection into NOD-SCID mice,was detected.15.The expression of miR-93 in cisplatin or sorafenib resistant HCC tissues was detected using real-time PCR.16.The effect of cisplatin or sorafenib on the expression of miR-93 in HCC cell lines was detected using real-time PCR.17.The apoptosis rate and cleaved-PARP expression of HCC cell lines with high or low expression of miR-93 were detected after cisplatin or sorafenib treatment.18.Tumor tissue and clinical data of 60 patients with HCC undergoing TACE treatment was collected.The expression of miR-93 in pathological tissues was detected.Then,the correlation between miR-93 expression and patient prognosis was confirmed by 50-months follow-up.19.Tumor tissue and clinical data of 80 patients with HCC undergoing sorafenib treatment was collected.The expression of miR-93 in pathological tissues was detected.Then,the correlation between miR-93 expression and patient prognosis was confirmed by 30-months follow-up.Research results The first part:(1)miR-93 highly expressed in LCSCs with CD133+or Ep CAM+.miR-93 highly expressed in spheroid colony of LCSCs formed under low adhesion culture.(2)miR-93 low expression cell line,including HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge,is difficult to form spheroid colony under low adhesion culture,indicating a weak self-renewal ability.(3)The expression of Ep CAM,CD133,CD90,CD24,SOX2,OCT4,c- Myc and Nanog down-regulated in HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge,indicating a positive correlation between the expression of miR-93 and tumor markers.(4)Down-regulating the miR-93 expression in cell lines of HCCLM3 and CSQT-2 can reduce their proportion of LCSCs.(5)Down-regulating the miR-93 expression in cell lines of HCCLM3 and CSQT-2 can reduce their tumorigenicity in NOD-SCID mice.(6)miR-93 high expression cell line,including HCCLM3 miR-93 mimic and CSQT-2 miR-93 mimic,is easy to form spheroid colony under low adhesion culture,indicating a strong self-renewal ability.(7)The expression of Ep CAM,CD133,CD90,CD24,SOX2,OCT4,c-Myc and Nanog upregulated in HCCLM3 miR-93 mimic and CSQT-2 miR-93 mimic,indicating a positive correlation between the expression of miR-93 and tumor markers.(8)Up-regulating the miR-93 expression in cell lines of HCCLM3 and CSQT-2 can increase their proportion of LCSCs.(9)Up-regulating the miR-93 expression in cell lines of HCCLM3 and CSQT-2 can enhance their tumorigenicity in NOD-SCID mice.(10)Compared to HCCLM3 and CSQT-2,HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge have higher luciferase activity during luciferase report assay,suggesting the targeted binding between miR-93 and MTMR3.(11)MTMR3 lowly expressed in LCSCs with CD133+or Ep CAM+.MTMR3 lowly expressed in spheroid colony of LCSCs formed under low adhesion culture.(12)Negative correlation between the expression of miR-93 and MTMR3 in HCC.(13)MTMR3 highly expressed in spheroid colony of HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge under low adhesion culture.(14)si MTMR3 transfection can inhibit the expression of MTMR3 in HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge.(15)miR-93 low expression cell line,including HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge,is easy to form spheroid colony under low adhesion culture after si MTMR3 transfection,indicating MTMR3 low expression can enhance self-renewal ability of HCC cell.(16)After si MTMR3 transfection,the proportion of LCSCs in HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge increased to normal level,indicating MTMR3 low expression can promote LCSCs forming.(17)Interfering MTMR3 expression can eliminate the tumorigenesis difference between miR-93 low-and normal-expression cells.The second part:(1)miR-93 highly expressed in cisplatin resistant HCC tissues.(2)After cisplatin treatment,the miR-93 expression of HCC cell lines,including HCCLM3,CSQT-2,Hep G2,Hep3 B and Huh7,increased significantly,indicating cisplatin can induce miR-93 high expression in HCC cells.(3)After cisplatin treatment,the apoptosis of HCCLM3 miR-93 mimic and CSQT-2 miR-93 mimic lower than miR-93 normal expression cell lines,indicating miR-93 can enhance the tolerance of HCC cells to cisplatin.(4)After cisplatin treatment,the apoptosis of HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge higher than miR-93 normal expression cell lines,further confirming miR-93 can enhance the tolerance of HCC cells to cisplatin.(5)miR-93 highly expressed in sorafenib resistant HCC tissues.(6)After sorafenib treatment,the miR-93 expression of HCC cell lines,including HCCLM3,CSQT-2,Hep G2,Hep3 B and Huh7,increased significantly,indicating sorafenib can induce miR-93 high expression in HCC cells.(7)After sorafenib treatment,the apoptosis of HCCLM3 miR-93 mimic and CSQT-2 miR-93 mimic lower than miR-93 normal expression cell lines,indicating miR-93 can enhance the tolerance of HCC cells to sorafenib.(8)After sorafenib treatment,the apoptosis of HCCLM3 miR-93 sponge and CSQT-2 miR-93 sponge higher than miR-93 normal expression cell lines,further confirming miR-93 can enhance the tolerance of HCC cells to sorafenib.(9)After TACE treatment,HCC patients with miR-93 low expression have better prognosis than their high expression counterparts.(10)After sorafenib treatment,HCC patients with miR-93 low expression have better prognosis than their high expression counterparts.conclusion miR-93 is highly expressed in LCSCs.It regulates the expression of phenotype moleculars,self-renewal and tumorigenicity of LCSCs by targeted binding MTMR3.miR-93 is also highly expressed in cisplatin or sorafenib resistant HCC tissues,which has the function of regulating the tolerance of HCC cell lines to cisplatin or sorafenib.The miR-93 expression was related to the survival and prognosis of patients undergoing TACE or sorafenib treatment.Previous studies have shown that autophagy is involved in regulating the tolerance HCC cell to cisplatin and sorafenib,and MTMR3 is the key molecule to initiate this process.Therefore,we will further explore whether miR-93 can mediate autophagy through targeting MTMR3 to influence the resistance of LCSCs or HCC cells to cisplatin and sorafenib.This study enriches the theoretical basis of LCSCs,provides potential predictive molecules for HCC patients with drug resistance,and is of great significance to improve the current diagnosis and treatment of HCC.