Screening And Identification Of Anoikis-resistant Gene UBCH7in Esophageal Squamous Cell Carcinoma

Objective Esophageal Squamous Cell Carcinoma (ESCC) is one of the top tenmalignant tumors detrimental to health, which morality ranks the forth place among thedeath cause of malignant tumors in China. Anoikis is a kind of programmed cell deathinduced by loss of extracellular matrix (ECM) adhesion, which is one of key factors forhomestasis. Resistance to anoikis is required for tumor cell metastasis. Although variantgenetic expressions of Maspin, SKP2, xCT, PTTG1, TM4SF3, ECRG2, CTTN, CyclinB1and etc. have been reported to be associated with in vivo carcinogenicity andmetastatic ability of Esophageal Squamous Cell Carcinoma, the study group stilldiscovered several anti-anoikis related oncogenes from our previous studies on ESCC.Although all of these oncogenes may take part in the regulation of anoikis resistance inESCC, the different signaling pathways they employed indicated the presence ofcomplicated signaling network in ESCC, for better adaption to the micro-environmentof the host, and the consequential enhancement of the metastatic potential of ESCC.This study mainly aimed to explore more anoikis-resist genes in ESCC so as to furtherelaborate the metastatic mechanism of ESCC at a molecular level, as well as to developan effective strategy for successful induction of anoikis in tumor cellsfor a purpose ofinhibiting distant metastasis on a basis of blocking its dissemination pathway, and thusestablishing an effective approach of preventing and controlling tumor againstmetastasis. Methods By using the functional screening mean of the retrovirus library,the retrovirus library of esophageal squamous cell carcinoma cell lines was establishedat first, followed by viral infection of anoikis-sensitive NIH3T3cells. Then, soft agarcolony formation assay was performed by using hybrid cell lines from infected cDNAlibrary. The monoclonal cells able to from larger colonies under suspension condition (i.e. cells with potential of anoikis resistance) were selected. At last, the exogenouslyinserted fragments of monoclonal anoikis-resist gene lines were amplified through PCRamplification with retrovirus vectors and specific primers, so as to obtain the genes withanoikis-resist potential from the ESCC cell lines cDNA library. Results After soft agarsuspension cultivation was performed on the NIH3T3cells that infected by retroviruscDNA library of ESCC, most of the clones were observed to form multipleanoikis-resist colonies. In addition, the NIH3T3cells that infected by pMSCV-GFPdemonstrated significant variation. Among these cells, those with relatively largerdiameters comparing to the controls were selected for conducting amplified cultivationof these resultant anoikis-resist clones, so as to extract genome DNA. The exogenouslyinserted fragment of target cellular genome was amplified by using retrovirus-specificprimers pMSCV5’ and pMSCV3’. From the results, no amplified product was observedin the parent NIH3T3group, and the target genome that infected by pMSCV-cDNAsignificantly amplified the variant-lengthed cDNA from ESCC hybrid cDNA library.Through sequencing and database comparison, one of the clones was successfullyintegrated with UBE2L3/UBCH7gene. For testing the accuracy of the retrovirus libraryfunctional screening experiment,forced expression of UBCH7in NIH3T3cells byretrovirus encoding UBCH7cDNA (pMSCV-UBCH7) infection increases itsanoikis-resistance. Furthermore, the ESCC cell line MLuC1, which had high metastaticpotential, high anoikis resistance and high expression of UBCH7protein, was used asthe model, and two specific siRNA sequences were used to knockdownUBE2L3/UBCH7in MluC1cells. Knock down of UBCH7expression by siRNAtransfection reduced its anoikis-resistant ability.Conclusion Identification of anoikis resistance gene UBCH7through retroviral cDNAlibrary screening is a anoikis-resistance gene in ESCC.